3-Phosphoinositide-dependent kinase-1 (PDK1)-mediated signaling regulates hematopoietic stem cell (HSC) quiescence by governing the

oxidative response

Danielle Erina Matsushima

Submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy in the Graduate School of Arts and Sciences

COLUMBIA UNIVERSITY

2016

© 2016

Danielle Erina Matsushima

All rights reserved

ABSTRACT

3-Phosphoinositide-dependent kinase-1 (PDK1)-mediated signaling regulates hematopoietic stem cell (HSC) quiescence by governing the oxidative response

Danielle Erina Matsushima

Regulation of hematopoiesis through the finite control of hematopoietic stem cell (HSC)

quiescence and proliferation is critical to the health of the organism since disturbances in blood

production can lead to clinical malignancies such as anemia or leukemia. Therefore, elucidating the

processes that govern HSCs is vital to advance our understanding of hematological diseases.

Interestingly, HSCs can be regulated through a variety of ways. Extrinsic cues from the niche provide

signals that govern HSC quiescence, proliferation, self-renewal, and differentiation. These external

signals are converted to internal messages through the use of signal transduction pathways that relay

information from the cytoplasm to the nucleus. While many pathways contribute to HSC regulation, the

PI3K/AKT/mTOR-pathway is especially pertinent because it has been implicated in cell survival,

metabolism, proliferation, and death. Many groups have identified key players within PI3K/AKT/mTOR-

signaling that regulate HSC quiescence; however, these studies are hindered by the redundancy of

multiple isoforms and compensatory signaling mechanisms by other members within the pathway. PDK1

is considered to be a master regulator of PI3K-signaling because it is directly activated by PI3Ks and can

govern activity of a variety of substrates within the PI3K-pathway. Because of this, it is an excellent

candidate to fully delineate how PDK1-mediated PI3K-signaling functions to maintain HSC quiescence.

In the current study, conditional deletion of PDK1 in HSCs was achieved through the use of a

hematopoietic specific Vav-Cre line. The loss of PDK1 in HSCs led to reduced numbers and an inability to

provide radioprotection in primary BMTs. Furthermore, PDK1-deficient HSCs exhibit impaired quiescence

and increased cycling. Strikingly, PDK1-mutant HSCs have markedly high levels of reactive oxygen

species (ROS), which led to increased proliferation, DNA damage, and apoptosis in progenitor cells.

Administration of a ROS scavenger, N-acetyl cysteine (NAC) partially rescued the increased proliferation

and differentiation of phenotype Pdk1Hem-KO cells both in vitro and in vivo, suggesting that abnormal HSC

activity in PDK1-deficient cells was in part due to increased ROS. Furthermore, mechanistic studies

identified a remarkable decrease in the levels of nuclear HIF1α in HSCs. Immunoblots and phosflow

studies demonstrated reduced activation of p-p70S6K, a well defined positive regulator, and increased

GSK3β, a key negative regulator of the HIF1α protein. These data suggested that ROS levels are

unrestrained since HIF1α is not present in Pdk1Hem-KO HSCs to activate transcription of genes that

moderate oxidative stress. In addition, Pdk1Hem-KO HSCs also show increased levels of Hif3α and IPAS

mRNA, which are believed to inhibit the transcriptional activation of HIF1α. In essence, the results from

these experiments are the first to implicate PDK1 as a critical regulator of HSC quiescence and as a

moderator of PI3K-signaling to alleviate oxidative stress. In addition, this study is the first to suggest that

HIF3α and IPAS may play a role in HSCs.

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Table of Contents

List of Figures ...……………………………………………………………………………….v

List of Tables …………………………………………………………………………………vii

Chapter 1: General introduction to PI3K-signaling regulates HSC quiescence ....... 1  

The role of HSCs in hematopoiesis ........................................................................................... 1  

Differentiation ....................................................................................................................................... 2  

Self-renewal .......................................................................................................................................... 4  

Quiescence vs. Proliferation ................................................................................................................. 5  

Extrinsic vs. intrinsic regulation of HSCs ................................................................................... 6  

The hematopoietic niche ...................................................................................................................... 6  

Signaling events in HSCs ................................................................................................................... 10  

Post-Translational Modifications (PTMs) regulate HSCs ........................................................ 12  

Ubiquitination and deubiquitination in HSC quiescence ..................................................................... 13  

Phosphorylation events regulate pathways that promote or repress HSC quiescence ...................... 14  

PI3K-signaling regulates quiescence in HSCs ........................................................................ 16  

PI3K-signaling .................................................................................................................................... 16  

Major downstream targets of PI3K-signaling ...................................................................................... 17  

PI3K-signaling in HSC quiescence ..................................................................................................... 19  

PDK1-signaling in hematopoiesis ............................................................................................ 21  

3-phosphoinosidtide-dependent kinase-1 (Pdk1) ............................................................................... 21  

PDK1 in hematopoiesis ...................................................................................................................... 23  

Abnormal levels of PDK1 result in cancer .......................................................................................... 24  

Importance of PDK1-signaling in HSCs .............................................................................................. 24  

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Chapter 2: Pdk1 is needed for proper hematopoiesis. ............................................. 26  

Introduction .............................................................................................................................. 26  

HSCs are finely regulated for proper hematopoiesis .......................................................................... 26  

HSC identification ............................................................................................................................... 27  

PI3K-signaling in HSCs ...................................................................................................................... 28  

Results ..................................................................................................................................... 29  

Pdk1 is expressed in HSCs ................................................................................................................ 29  

PDK1 is important for HSC maintenance in vitro ................................................................................ 30  

Pdk1Hem-KO mice have hematopoietic defects ..................................................................................... 30  

Loss of PDK1 results in improper hematopoietic lineage differentiation ............................................ 34  

Pdk1Hem-KO mice have reduced HSCs in the bone marrow ................................................................. 40  

Discussion ............................................................................................................................... 42  

PDK1 is necessary for proper lineage differentiation ......................................................................... 43  

Loss of PDK1 results in HSPC disturbances and fewer HSCs ........................................................... 44  

Chapter 3: The functional ability of Pdk1Hem-KO HSCs is compromised. ................. 47  

Introduction .............................................................................................................................. 47  

HSC functional assays ....................................................................................................................... 47  

Quiescence maintains adult HSCs ..................................................................................................... 48  

Results ..................................................................................................................................... 49  

Hypotheses for fewer HSCs in the Pdk1Hem-KO mouse ....................................................................... 49  

PDK1-deficient HSCs are unable to provide radioprotection .............................................................. 50  

Increased HSPCs in the Pdk1Hem-KO spleen ....................................................................................... 53  

Loss of PDK1 in HSCs results in increased proliferation and fewer quiescent cells .......................... 55  

HSPCs have increased cell death and lineage differentiation in vitro ................................................ 58  

Discussion ............................................................................................................................... 59  

BMT analysis ...................................................................................................................................... 60  

Pdk1Hem-KO HSPCs are homing to the spleen ..................................................................................... 61  

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Loss of quiescence, hyperproliferation and increased differentiation of PDK1-deficient cells ............ 61  

Chapter 4: PDK1-deficient HSPCs exhibit reduced response to oxidative stress . 63  

Introduction .............................................................................................................................. 63  

Oxidative Stress ................................................................................................................................. 63  

Mitochondrial dynamics ...................................................................................................................... 63  

ROS and HSC maintenance ............................................................................................................... 64  

PI3K-signaling regulates ROS levels .................................................................................................. 65  

Results ..................................................................................................................................... 66  

Microarray analysis reveals Pdk1Hem-KO HSPCs have reduced response to oxidative stress ............. 66  

Deficiency of PDK1 in HSPCs alters the transcription factor networks associated with HSCs and

oxidative stress ................................................................................................................................... 66  

PDK1-deficient cells have increased levels of ROS ........................................................................... 71  

Treatment with NAC suppresses proliferation and differentiation in PDK1-deficient cells ................. 74  

Discussion ............................................................................................................................... 79  

Microarray ........................................................................................................................................... 80  

Mitochondrial bioenergetics ................................................................................................................ 81  

DNA damage/apoptosis assays ......................................................................................................... 84  

ROS reversion .................................................................................................................................... 85  

Chapter 5: Loss in PDK1 results in reduced Hif1α expression. .............................. 88  

Introduction .............................................................................................................................. 88  

HIF1α regulation ................................................................................................................................. 88  

Hif1α regulates ROS expression ........................................................................................................ 89  

PI3K/AKT/mTOR-signaling ................................................................................................................. 90  

Results ..................................................................................................................................... 92  

Pdk1Hem-KO mice have reduced Hif1α expression ............................................................................... 92  

Inhibition of HIF1α in Pdk1Hem-KO cells ................................................................................................ 93  

Pharmacological inhibition of PDK1 provides insights into downstream signaling ............................. 98  

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Reduced AKT-signaling in Pdk1Hem-KO LSK ...................................................................................... 100  

Discussion ............................................................................................................................. 103  

HIF expression.................................................................................................................................. 103  

PI3K/AKT/mTORC-signaling ............................................................................................................ 105  

Consequences of reduced HIF1α expression .................................................................................. 106  

Chapter 6: Discussion and Future Directions ......................................................... 108  

PI3K-signaling in HSCs ......................................................................................................... 108  

ROS impacts HSC quiescence .............................................................................................. 113  

A novel role for HIF3α in HSC maintenance ......................................................................... 116  

Future Directions and perspectives ....................................................................................... 119  

Chapter 7: Experimental Procedures ....................................................................... 122  

References .................................................................................................................. 129  

v

List of Figures

Figure 1: HSCs give rise to all blood cell components. ............................................................................... 4  

Figure 3: Schematic of PI3K/PDK1-signaling. ........................................................................................... 19  

Figure 4: Inhibitors of PI3K-signaling affect HSPC numbers. .................................................................... 30  

Figure 5: Vav-Cre deletes PDK1 in the hematopoietic organs. ................................................................. 32  

Figure 6: Pdk1Hem-KO mice show hematopoietic defects. ........................................................................... 34  

Figure 7: Lymphoid progenitors are reduced in the Pdk1Hem-KO mice. ....................................................... 36  

Figure 8: Pdk1Hem-KO organs show disturbances in differentiation. ............................................................ 37  

Figure 9: Pdk1Hem-KO thymus has an increase in DN cells. ........................................................................ 39  

Figure 10: The Pdk1Hem-KO BM has fewer HSCs. ....................................................................................... 41  

Figure 11: The Pdk1Hem-KO mouse has reduced ST- and LT-HSC numbers. ............................................. 42  

Figure 12: Possible explanations for reduced functionality in Pdk1Hem-KO HSCs. ...................................... 50  

Figure 13: Pdk1Hem-KO HSCs cannot repopulate the blood cell pool after BMT. ........................................ 51  

Figure 14: PDK1-deficient HSCs cannot compete with wild-type HSCs. ................................................... 53  

Figure 15: The Pdk1Hem-KO spleen has more HSPCs but HSCs are not significantly increased. ............... 54  

Figure 16: Pdk1Hem-KO progenitor cells are less quiescent compared to the control. ................................. 56  

Figure 17: Pdk1Hem-KO progenitor cells proliferate more. ............................................................................ 57  

Figure 18: In vitro culture reveals that Pdk1Hem-KO LSK cells are apoptotic. .............................................. 59  

Figure 19: Pdk1Hem-KO LSK cells exhibit reduced response to oxidative stress. ........................................ 68  

Figure 20: PDK1-deficient cells have augmented ROS levels. .................................................................. 72  

Figure 21: Pdk1Hem-KO progenitor cells have increased DNA damage. ...................................................... 74  

Figure 22: NAC treatment suppresses differentiation and proliferation of Pdk1Hem-KO HSPCs. ................. 75  

Figure 23: HIF1α is reduced in Pdk1Hem-KO cells during hypoxic conditions. ............................................. 93  

Figure 24: Hif mRNA expression in Pdk1Hem-KO cells. ................................................................................ 95  

Figure 25: Pdk1Hem-KO cells have greater IPAS mRNA expression. ........................................................... 97  

Figure 26: PI3K-signaling can regulate HIF1α expression. ....................................................................... 99  

Figure 27: Drug inhibition of PDK1 provides insight into downstream signaling. ..................................... 100  

Figure 28: Loss of PDK1 leads to reduced AKT/mTORC-signaling. ....................................................... 102  

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Figure 29: PI3K/PDK1-signaling phosphorylates AKT-dependent and AKT-independent AGC kinases. 112  

Figure 30: Loss of PI3K-signaling mediated by PDK1 causes HSCs to proliferate more and lose

quiescence. ...................................................................................................................................... 121  

vii

List of Tables

Table 1: Haplosufficiency of PDK1 (Pdk1F/+ ; Vav-Cre) is not phenotypically different from Pdk1F/+ or

Pdk1F/F mice. ...................................................................................................................................... 33  

Table 2: Up-regulated genes in the Pdk1Hem-KO microarray. ...................................................................... 69  

Table 3: Down-regulated genes in the Pdk1Hem-KO LSK microarray. .......................................................... 70  

Table 4: Selected transcription factors involved in hematopoiesis or oxidative stress. ............................. 70  

Table 5: In vitro NAC treatments limits differentiation of Pdk1Hem-KO lineage negative cells. ..................... 77  

Table 6: In vivo NAC treatment impedes proliferation of Pdk1Hem-KO progenitors. ..................................... 78  

Table 7: In vivo NAC treatment significantly reduces ROS levels in Pdk1Hem-KO LK. ................................. 79  

Table 8: Antibodies used in this study. .................................................................................................... 125  

Table 9: Primers for qRT-PCR. ................................................................................................................ 126  

viii

ACKNOWLEDGEMENTS

There are so many individuals who have enriched and aided me in my scientific endeavors during

my tenure here at Columbia University. First I would like to thank my advisor, Choz Rathinam, for

accepting me into his lab, lending me his expertise, and allowing me to grow as a scientist. I have truly

enjoyed our conversations regarding this project and I have learned a great deal under his mentorship.

I would also like to thank my committee members, Riccardo Dalla-Favera and Stavroula

Kousteni, for giving me their time and providing critical and helpful feedback during my TRAC meetings

throughout the years. I would especially like to thank Stavroula for kindly agreeing to be the second

reader for my thesis. In addition, I would like to thank Joji Fujisaki and Martin Picard for graciously serving

on my dissertation committee and lending me their expertise from their respective fields. Furthermore,

examiners on my Qualifying Committee, Mimi Shirasu-Hiza, Ginny Papaiannou, and Lori Sussel, provided

initial guidance when I first joined Choz’s lab. Completion of my dissertation work and graduate school

career could not be possible without these professors.

I was lucky to be surrounded by two individuals that enriched my lab experience while at

Columbia. Keyur Thummar aided me in maintaining a mouse colony at Rockefeller as well as kept up

morale with his quiet but incredibly witty comments. I also would not be where I am today without the

guidance and help of Marshall Nakagawa. Marshall is so knowledgeable in the field and helped me

troubleshoot experiments while always putting a smile on my face with his antics.

Furthermore, my scientific colleagues have greatly enriched my experiences during my years at

Columbia. To the Genetics Girls, Lori Khrimian, Junko Shimazu, Angela Pring-Mill Churchill, and Kayla

Hager, I will forever remember discussions in 1609 and late night skit writing. I would also like to thank

Mike Churchill for always attending my student seminars and giving me helpful comments on my project. I

had so much fun envisioning an improved graduate school community and implementing various projects

and events with my fellow Co-President of the Graduate Student Organization, Stephanie Siegmund. We

were able to initiate programs that promoted interactions between graduate students within CUMC as well

increased outreach with the greater New York community. Also, when aspects of this project veered

towards mitochondrial dynamics, discussions with Stephanie were incredibly useful. In addition, thank you

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to Lucja Grajkowska, Tulsi Patel, and John Seeley for offering me wisdom that can only be given from

older students. I have truly enjoyed getting to know all of you over the years. Also, many thanks to

Caroline Ng, Leila Ross, and Becca Lewis for keeping me company at lunch as well as for working in a

malaria lab so that I knew who to turn to when I needed advice on hypoxia experiments. Furthermore,

thank you to Thomas Postler, who has been a wonderful sounding board and kept me smiling and well-

fed during these last few months of thesis writing. I could not have accomplished writing this thesis

without your support.

Lastly, I would like to thank the Genetics Department for accepting me into the program and for

providing me with a resource rich environment. Thank you to the faculty, for offering classes that

promoted discussion on a wide range of topics; to members of the Genetics office, who have been

incredibly helpful from the time I interviewed; and to the students for their encouraging words and helpful

advice. This work would not have been completed without funding from the training grant and support

from all of members in the Department of Genetics and Development.

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DEDICATION

In dedication to my parents, Dr. Glenn and Deborah Matsushima, who have lovingly supported and

encouraged both my personal and academic endeavors, and to my brother Brian, who has patiently

listened to the joys and tribulations of those endeavors.

In addition, to Dr. Larysa Pevny (1965 - 2012), whose mentorship, unwavering encouragement,

professional poise, inquisitiveness, and love for life continues to inspire me to be a better scientist and

person.

1

Chapter 1: General introduction to PI3K-signaling regulates HSC

quiescence

The role of HSCs in hematopoiesis

Throughout the history of mankind, from battle wounds in ancient wars to hemorrhaging during

modern day surgeries, the loss of blood has been associated with the loss of life. Indeed, the blood

system is vital for maintaining homeostasis. Blood delivers oxygen to tissues and fights off foreign

pathogens and without it organisms cannot survive. Understanding the mechanisms that regulate blood

production is vital to scientists and clinicians when treating blood diseases. For instance, a reduction in

blood cells can result in disorders such as anemia and the overabundance of blood cell production can

result in leukemia. Given this, blood equilibrium must be maintained to meet the demands of the

organism.

Blood generation occurs by a process known as hematopoiesis where all the mature cells of the

blood system are produced from a common ancestor cell, known as a hematopoietic stem cell (HSC).

Unlike embryonic stem cells, which are totipotent, HSCs are multipotent and are restricted in terms of

types of cells their progeny can become. The process by which HSCs proliferate and divide into other

more restricted cell types is called differentiation. In order to differentiate, HSCs go from a state of

inactivity, termed quiescence or dormancy, then enter the cell cycle, where they divide, and produce two

daughter cells. Often, one daughter cell is a copy of the HSC, created by a process termed self-renewal,

and the other daughter cell is a more differentiated cell. The stem cell properties of self-renewal,

quiescence, proliferation, and differentiation are vital in maintaining an organism’s blood homeostasis.

These cellular properties of HSCs will be discussed in depth later.

Vertebrate hematopoiesis is thought to occur in successive waves. The first wave, termed

primitive hematopoiesis, occurs around embryonic day (E) 7.5 in the yolk sac and fetal HSCs primarily

function to produce red blood cells, which are necessary for oxygen transport (Dzierzak and Medvinsky,

2

1995; Palis et al., 1999; Pietras et al., 2011). The second wave, or definitive hematopoiesis, provides the

foundation for adult hematopoiesis and is characterized by HSCs migrating to and engrafting into the

bone marrow (BM). It is also at this stage that the output of HSCs switches from the generation of red

blood cells to cells of all blood lineages (Pietras et al., 2011). In definitive hematopoiesis, HSCs first

colonize hematopoietic embryonic tissues around E8.5-E10.5, and then expand to the liver, which is

where the majority of fetal hematopoiesis occurs. Then, from E17.5 until three weeks post-natal, HSCs

migrate to their final niche in the BM. The objectives of fetal and adult hematopoiesis are slightly different.

During fetal hematopoiesis, HSCs primarily serve to: (1) generate the entire blood cell system, the

predominant cell being erythrocytes, which serve to oxygenate growing tissues, and (2) establish the

beginnings of an immune system by generating the first white blood cells. Therefore, almost all fetal

HSCs are active, or cycling, and very few are inactive, or quiescent. In contrast, adult hematopoiesis

functions to maintain balanced blood cell production so the number of actively cycling HSCs is

considerably reduced compared to the fetal stage. However, when there is an immune response, adult

HSCs can activate to supply the immune system with necessary cells and then become dormant when

homeostasis is returned (Orford and Scadden, 2008; Pietras et al., 2011). Taken together, HSCs are able

to respond to hematopoietic needs through dynamic regulation of their opposing hallmark properties:

quiescence and proliferation.

Differentiation

Hematopoiesis occurs based on a hierarchy of cell types (Orkin, 2000), with the most plastic

cells, HSCs, giving rise to the most committed and differentiated cells (Figure 1). During differentiation the

most plastic cells divide and become further restricted in terms of the types they can become. As such, a

terminally differentiated cell cannot become another cell type. As a cell goes from undifferentiated to

terminally differentiated, it progresses through a series of progenitor stages as it commits fully to the

differentiated cell type. In adult hematopoiesis, the long-term (LT)-HSC is the most multipotent and is

characterized by its ability to remain dormant, proliferate, self-renew, and differentiate. LT-HSCs can exit

dormancy and generate short-term (ST)-HSCs, which are more active than LT-HSCs but still retain the

ability to repopulate all blood cell types but only for 3-4 months (Suda et al., 2011; Yang et al., 2005). ST-

3

HSCs can give rise to multipotent progenitors (MPPs), which are distinct in their functional capacity to

give rise to more lineage committed progenitor cell types (Pietras et al., 2011; Wilson et al., 2008). For

instance, Pietras et al. showed that MPP2 is biased towards the megakaryocyte/erythrocyte lineage,

MPP3 towards the granulocyte/monocyte lineage, and MPP4 preferentially differentiates into lymphoid

cells. While MPPs are still able to give rise to more differentiated progeny, they lack the capacity to

engraft, or repopulate the blood cell pool when transplanted. This inability to engraft indicates that they

are in an intermediate differentiation stage. Members of the myeloid and lymphoid lineages are derived

from the MPP stages. Myeloid cells consist predominantly of members of the innate immune system such

as granulocytes, monocytes, macrophages, erythrocytes, megakaryocytes, basophils, eosinophils,

neutrophils, and platelets. B cells, T cells, and natural killer (NK) cells are members of the adaptive

lymphoid immune system. Dendritic cells (DC) arise from progenitors of both the myeloid and lymphoid

lineages. As evidenced by the ability to produce a variety of lineage-specific cells that can fight off

pathogens, HSCs possess a remarkable ability to adapt and respond to urgent requirements of the blood

cell system.

HSCs and progenitor cell types are unable to differentiate without instructive cues to determine

their outcome. Indeed, many transcription factors have been implicated in the differentiation of lineage-

committed cells and these genes were predominantly discovered through genetic knock out studies. For

example, Gata1 controls erythroid differentiation (Kulessa et al., 1995; Orkin, 2000; Pevny et al., 1991),

Notch1 is needed for T cell development (Rothenberg, 2007), and Pax5 is highly important for B cell

differentiation (Nutt et al., 1999; Orkin, 2000). Furthermore, many signals from cells in the niche are

important for governing lineage specification (Morrison and Scadden, 2014). In addition to signals from

the microenvironment, oxidative stress and reactive oxygen species (ROS) levels have been shown to be

necessary during myeloid differentiation (Kohli and Passegue, 2014). Based on the variety of forces that

can influence blood production, it is essential that homeostasis is maintained since a disturbance may

alter HSC output, resulting in an imbalance of immune cell types. This can then result in immune defects,

anemia, or various proliferative disorders that can impact the life of the organism.

4

 

Figure 1: HSCs give rise to all blood cell components.

A schematic of hematopoietic differentiation. Adult LT-HSCs are able to exit quiescence, proliferate, and differentiate to give rise to all cell types in the blood. LT-HSC: long-term HSC, ST-HSC: short-term HSC, MPP: multipotent progenitor: MEP: megakaryocyte/erythroid, CMP: common myeloid progenitor, CLP: common lymphoid progenitor, and NK: natural killer.  

Self-renewal

A defining characteristic of HSCs is that they are able to reconstitute the entire blood supply of a

recipient (Orkin and Zon, 2008). In steady state conditions, HSC division normally yields one HSC and

one differentiated progeny. During regenerative conditions two other possibilities may occur: (1) HSC

expansion, where both daughter cells are HSCs and (2) HSC exhaustion, where both daughter cells are

differentiated progeny (Kohli and Passegue, 2014). One way to test for HSC functional ability is to

perform bone marrow transplantation (BMT) experiments in which donor BM is placed into irradiated

recipients and recipient survival and blood regeneration are monitored. This can only be accomplished if

an HSC is able to both differentiate into various blood cell types as well as actively self-renew so that it

can continue to produce more progeny. HSCs are considered immortal since they have the ability to

successively replicate themselves; however, they do suffer from proliferative stress and show reduced

5

ability to repopulate the blood cell pool after subsequent transplantations. This phenomenon is called

stem cell exhaustion. Interestingly, mouse strains that have different life spans show that the shorter-lived

strains have HSCs that proliferate more than HSCs from longer-lived strains. When HSCs of young

animals from shorter-lived strains were transplanted into recipients, reconstitution capacity was faster

than it was when HSCs from longer-lived strains were transplanted. However, when HSCs of old animals

from shorter-lived strains were transplanted, the cells lost the capacity to quickly reconstitute the blood

cell pool. This suggests that HSCs from shorter-lived strains experience stem cell exhaustion and lose the

ability to self-renewal more quickly than HSCs from longer-lived strains (de Haan et al., 1997; Orford and

Scadden, 2008; Phillips et al., 1992). It is thought that the ability to self-renew is not enough to ensure

HSC survival throughout the lifetime of the organism since the likelihood of stem cell exhaustion

increases with every division. To circumvent this issue, adult HSCs remain in an inactive or quiescent

state for the majority of an organism’s lifetime. This allows the HSC to avoid proliferative activation for

prolonged periods of time, thereby extending the lifespan of the cell (Orford and Scadden, 2008; Suda et

al., 2011).

Quiescence vs. Proliferation

The balance between adult HSC quiescence and proliferation is extremely important to ensure

proper regulation of hematopoiesis as well as to allow HSCs to survive for the lifetime of the organism. In

addition, it is thought that quiescence serves to protect the cell from accumulating damage that can occur

during physiological stress (Kohli and Passegue, 2014; Suda et al., 2011; Wilson et al., 2008). Indeed,

slow cycling HSCs, or LT-HSCs, are more resistant to ultraviolet light, ionizing radiation, and cytotoxic

reagents (Suda et al., 2011). In addition, HSCs and other inactive cell types have highly active ATP-

dependent transport pumps that efflux toxic agents from the cell in order to prevent the accumulation of

hazardous compounds (Challen and Little, 2006; Goodell et al., 1996; Lin and Goodell, 2006). Taken

together, quiescence serves to protect HSCs from deleterious effects that can impact their ability to self-

renew and proliferate when needed. Many studies have investigated numerous biological factors that

regulate HSC quiescence such as signals from the niche, transcriptional factors, and metabolic makeup.

These factors influence HSC quiescence and will be discussed further in the next section.

6

Quiescence in HSCs also serves to promote balanced blood cell production during homeostasis

and after injury. Quiescent murine HSCs divide relatively slowly, once every 145 days or five times

throughout the mouse’s lifetime. Inactive HSCs can be stimulated to initiate hematopoiesis until

homeostasis of the entire blood system is achieved whereupon they return to a quiescent state (Wilson et

al., 2008). This suggests that HSCs are able to dynamically respond to the organism’s immediate

requirements while also ensuring the longevity of the cell.

In summary, HSCs are able to quickly respond to the needs of the organism, such as

replenishment due to blood loss bleeding, battling infections, or defending against environmental trauma.

When necessary, HSCs are able to swiftly activate and produce large quantities of differentiated progeny.

However, to maintain homeostasis, there is a tightly controlled balance between two conflicting forces:

quiescence and proliferation. Without exquisite regulation of these two phenomena, HSCs can be

depleted or expanded, leading to various hematological disorders such as anemia, bone marrow failure,

or leukemia. Therefore, it is critical to have a comprehensive understanding of the mechanisms that

govern quiescence versus proliferation in HSCs.

Extrinsic vs. intrinsic regulation of HSCs

The hematopoietic niche

External cues, including cytokines, chemokines, and growth factors, are vital to maintaining HSC

quiescence in the adult animal. These signaling factors can come from other hematopoietic cell types or

from the cells that compose the HSC niche. Throughout development, the location of the niche changes.

For instance, in the mouse, the first major site of definitive hematopoiesis occurs in the fetal liver while

adult hematopoiesis takes place in the BM. However, in response to hematopoietic stress, the niche can

switch to or be supplemented by other hematopoietic sites such as the spleen or liver (Johns and

Christopher, 2012; Morrison and Scadden, 2014). It is thought that the murine adult BM niche is

composed of two zones: the endosteal niche and the vascular niche. Some evidence suggests that the

location of an HSC, near the endosteal interface or near vasculature, plays a role in its functionality (Orkin

7

and Zon, 2008). For instance, Angiopoeitin-1 is needed to maintain HSC adherence to osteoblasts, which

are located at the endosteal interface. According to this study, close proximity to osteoblasts promotes

HSC quiescence (Arai et al., 2004). This suggests that cell-to-cell contacts may also influence HSC

dynamics (Schofield, 1978). In addition, CXCL12, a chemokine, encourages migration of HSCs to the

vascular niche (Kiel and Morrison, 2006) suggesting that the vascular zone is important for HSC

maintenance. It is important to note that these two regions are very closely situated and that much

controversy surrounds whether or not there are distinct sub-regions within the BM that define HSC

functionality (Orkin and Zon, 2008). For instance, many argue that the vascular niche serves to activate

HSCs while the endosteal niche promotes dormancy (Boulais and Frenette, 2015; Morrison and Scadden,

2014; Orkin and Zon, 2008). In line with the controversy, many genetic studies demonstrate that

osteoblasts are vital for HSC maintenance while recent imaging studies suggest that less than 20% of

HSCs are found within 10uM of the endosteum (Morrison and Scadden, 2014). Despite this discrepancy,

extrinsic signals and the cells that compose the adult BM HSC niche are essential for regulating the

balance between active and quiescent HSCs.

Signaling molecules from the niche are vital to maintain stem cell homeostasis. For instance,

stem cell factor (SCF) and thrombopoietin (TPO) are cytokines produced by immune cells and other cells

in the BM niche. These signaling molecules have been shown to regulate quiescence in adult HSCs

(Wilson et al., 2009; Yamazaki et al., 2006). In addition, the loss of the cytokine receptors Tpo and Kit

(SCF receptor) result in increased HSC cell cycle activity and premature depletion of HSCs (Qian et al.,

2007; Thoren et al., 2008). Interestingly, many of the chemokines and cytokines that are vital for adult

HSC quiescence, are not necessary in fetal hematopoiesis (Wilson et al., 2009), probably because they

are not needed as this is a time when HSCs are highly active. This difference in the requirement of

regulatory molecules at different developmental stages, highlights the importance of identifying

mechanisms that govern the inactive state of adult HSCs.

Many of the signaling molecules that drive quiescence and proliferation in HSCs originate from

cells within the BM niche. Indeed, many of the niche cell types play a positive or negative role in

regulating HSC quiescence and proliferation. It is thought that adipocytes negatively impact HSCs since

HSC and adipocyte numbers are inversely correlated in microenvironments (Naveiras et al., 2009). In

8

contrast, studies demonstrate that osteoblasts, mesenchymal stem cells (MSCs), and endothelial cells

actively contribute to HSC quiescence and cell numbers. For instance, it has been shown that osteoblasts

produce TPO to maintain HSCs in a quiescent state (Yoshihara et al., 2007). Furthermore, co-culturing

osteoblasts with HSCs causes HSC expansion (Lord et al., 1975); however, genetic loss of mature

osteoblasts using target specific Cre-lines have shown that mature osteoblasts may not play a role in

HSC maintenance (Morrison and Scadden, 2014). It is important to note that some of this theory is based

on experiments that deleted SCF from osteoblasts using Col2.3-Cre and showed that HSC frequency was

not changed (Ding and Morrison, 2013). This study is flawed because it assumes that SCF is the only

factor HSCs need; however, other groups have shown that other factors are important for HSC

maintenance including TPO and angiopoietin-1 (Arai et al., 2004; Qian et al., 2007; Yoshihara et al.,

2007). Because the Col2.3-Cre experiments did not evaluate other potential factors released by

osteoblasts, conclusive evidence suggesting osteoblasts do not directly impact HSC quiescence is

lacking.

Despite conflicting evidence regarding whether osteoblasts directly impact HSC maintenance,

there is general agreement that cells of the osteoblastic lineage have a role in progenitor differentiation,

and in particular, development of the B cell lineage (Visnjic et al., 2004; Zhu et al., 2007). Osteoblasts

may not secrete SCF but mesenchymal stem cells (MSCs) and endothelial cells generate this cytokine to

promote HSC maintenance. In addition, these cells are found near sinusoids and other blood vessels,

suggesting that the vascular niche is important for HSC maintenance (Ding et al., 2012). Indeed,

perivascular MSCs are necessary to organize the niche (Sacchetti et al., 2007) and they also express the

chemokine CXCL12 that is needed for HSC retention in the BM (Ding and Morrison, 2013; Greenbaum et

al., 2013). Interestingly, other cell types in the niche produce CXCL12 such as sympathetic nerves

(Katayama et al., 2006). Furthermore, non-myelinating Schwann cells were shown to be responsible for

releasing TGFβ, a cytokine that is necessary for promoting HSC self-renewal (Yamazaki et al., 2011).

Taken together, these experiments demonstrate that different cells in the perivascular and endosteal

zones locate HSCs within the niche and secrete factors that contribute to HSC maintenance and

quiescence (Figure 2).

9

 

In addition to cell-to-cell contacts and signaling events, the metabolic physiology of the BM can

help promote quiescence in HSCs. The BM niche is highly hypoxic compared to other tissues and

mechanisms controlling the hypoxic response in HSCs are vital for their existence under this low oxygen

condition (Suda et al., 2011). Evidence that HSCs reside in a low oxygen environment comes from

studies that stained HSCs with pimonidazole, a dye that recognizes proteins associated with low oxygen

status, or by staining with Hoescht 33342 and then compared the distance between HSPCs and sites

blood flow. These studies found that HSCs were predominantly located at sites within the BM where the

oxygen gradient was the lowest (Parmar et al., 2007; Winkler et al., 2010). Interestingly, culturing human

CD34+ cells in low oxygen levels or transplanting these cells into the murine BM niche promotes a G0 cell

cycle or quiescent state (Hermitte et al., 2006; Shima et al., 2010). Taken together, these experiments

suggest that HSCs reside in low oxygen conditions, which promote quiescence.

Figure 2: The HSC BM niche.

Adult hematopoiesis occurs in the bone marrow (BM), which is composed of a variety of cell types that can promote or inhibit HSC functions such as by releasing cytokines or chemokines. MSC: mesenchymal stem cell, SNS: sympathetic nervous system.

10

HIf1α is a master regulator of hypoxic stress response in HSCs (Lee et al., 2004; Simsek et al.,

2010; Takubo et al., 2010) since the Hif1α driven metabolic program is critical for maintaining HSCs in a

quiescent state. Indeed, genetic inactivation of Hif1α in vivo has been documented to disrupt HSC

quiescence and functions (Takubo et al., 2010). HIF1α is a bHLH-PAS-type transcriptional regulator that

under normal oxygen conditions is degraded when prolyl hydroxylases hydroxylate its oxygen-dependent

degradation (ODD) domain. The ODD domain is recognized by an E3 ligase known as the von Hippel-

Lindau protein (VHL), which will promote HIF1α degradation in normoxic conditions. In low oxygen

conditions, prolyl hydroxylases are inactivated, allowing the stabilization of HIF1α, which forms a

heterodimer with HIF1β and translocates to the nucleus to activate transcription of downstream targets

such as antioxidants (Dengler et al., 2014; Suda et al., 2011). Interestingly, recent evidence suggests

that the regulation of Hif1α is not only dependent on oxygen levels within the niche but also other

signaling mechanisms, such as the cytokines TPO and SCF, that can stabilize HIF1α to promote

quiescence (Nombela-Arrieta et al., 2013; Pedersen et al., 2008). Taken together, these studies suggest

the hypoxic composition of the niche contributes to quiescence in HSCs.

Signaling events in HSCs

Quiescence in HSCs is also governed by a variety of internal signals including: transcription

factors, cell cycle inhibitors, microRNAs, and post-translational modifications (PTMs). These cell-intrinsic

mechanisms function to promote quiescence, or a G0 state, within HSCs, while allowing for immediate

entry into the cell cycle when prompted. Much work has been done to elucidate the genes and signaling

networks that maintain HSC quiescence and some studies are highlighted below.

Transcriptional regulation of HSCs occurs at all stages of the HSC life cycle. For instance, Runx1

is essential for the formation of HSCs in the embryo and without it hematopoietic clusters do not form

(North et al., 1999; Orkin, 2000). With regard to HSC quiescence, genetic loss of Gfi1 (growth factor

independent 1) resulted in increased HSC cycling and loss of long term repopulating ability (Hock et al.,

2004). Notch1, a major regulator of cell specification, is needed to promote HSC expansion and self-

renewal in mice and humans through the transcription of its target genes (Guruharsha et al., 2012).

Interestingly, deletion of the Notch ligand, Jagged-1 in endothelial cells caused reduced LT-HSC numbers

11

and a loss in quiescence (Poulos et al., 2013). In contrast, loss-of-function studies suggest that the

absence of Notch-signaling does not affect HSC maintenance (Cerdan and Bhatia, 2010). The

discrepancy between the two experiments could be a result of the experimental conditions such as

conditional deletion in the mouse versus studies with hematopoietic cells derived from human pluripotent

stem cells. Another transcription factor that mediates quiescence is Pbx1. Pbx1 promotes proliferation

during development and when it is conditionally deleted, quiescent HSCs are reduced and serial

repopulating ability is lost (Ficara et al., 2008). In all, a variety of transcription factors function to mediate

HSC quiescence.

Proteins involved in cell cycle regulation play a role in HSC quiescence and self-renewal. For

example, when all three Retinoblastoma (Rb) family members are conditionally deleted in adult mice,

severe myeloproliferation occurs presumably as a result of increased HSC numbers and proliferation

(Viatour et al., 2008). Furthermore, the loss of cell cycle inhibitors p21, p27, and p57 has been shown to

be critical for the maintenance of HSCs in an inactive state (Cheng et al., 2000; Matsumoto et al., 2011;

Pietras et al., 2011; Zou et al., 2011). Taken together, these studies show how important regulation of

cell cycle proteins are for HSC quiescence.

Proper microRNA processing is also critical to HSC quiescence since conditional deletion of

Dicer, a component in the microRNA processing machinery, causes a reduction of LSK cells (Guo et al.,

2010). In addition, conditional knockouts of Ago2, a slicer endonuclease responsible for cleaving

microRNAs to match their targets, display compromised HSC pool functions (Lu et al., 2016).

Furthermore, miR-125b increases HSC engraftment and overexpression of this microRNA causes a

dose-dependent myeloproliferative disorder suggesting that it encourages HSC proliferation. The same

study identified other microRNAs that were enriched in HSCs, which have positive or negative effects on

HSC proliferation (O'Connell et al., 2010). Other microRNAs that have been identified to promote HSC

expansion are miR-125a, miR-99b, and let-7e (Guo et al., 2010). These studies demonstrate that

mircoRNAs work to aid in the balance of blood production. In addition to the transcriptional and post-

transcriptional mechanisms already discussed, emerging evidence suggests that PTMs of proteins play

key roles in normal HSC and leukemic HSC regulation (Moran-Crusio et al., 2012; Nakagawa et al., 2015;

Rathinam and Flavell, 2010; Rathinam et al., 2011).

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Post-Translational Modifications (PTMs) regulate HSCs

Post-translational modifications (PTMs) are unique in that they allow the cell to modify signaling

events in order to promote cellular changes that meet its physiological needs. A unique property of PTMs

is that they are site specific and can often be reversed (Jensen, 2006; Prabakaran et al., 2012). This

allows for a dynamic response to a change of status within the cell. There are two types of PTMs that can

dictate the function of a protein: (1) polypeptide modifications and (2) addition of small molecular groups

(Prabakaran et al., 2012). These modifications are initially made on the protein by an activating enzyme

(E1). After this, a second conjugating enzyme (E2) adds additional modifiers. Lastly, E3 ligase is needed

to build the modifying polypeptide that can then be targeted for further ubiquitin-like modifications

(Schwartz and Hochstrasser, 2003; Ulrich and Walden, 2010). One of the most well known polypeptide

modifiers is ubiquitin, which is predominantly responsible for tagging a protein for degradation by the

proteasome. However, ubiquitin and ubiquitin-like proteins can also help maintain homeostasis by

contributing to DNA repair (Ulrich and Walden, 2010). Protein substrates may be either be

monoubiquitinated or polyubiquitinated, which dictates the fate of the protein. It is thought that

monoubiquitination serves to alter protein activity whereas polyubiquitination leads to protein termination

via the proteasome. Furthermore, individual ubiquitin substrates can be removed by deubiquitinating

enzymes (DUBs), which allows for precise regulation of the protein (Moran-Crusio et al., 2012).

The second type of PTMs, addition of small molecular groups, adds material from the cell to a

protein to modulate protein expression. Some of the molecular groups that may be added include:

phosphorylation, acetylation, GlcNAcylation, palmitoylation, and methylation (Prabakaran et al., 2012). Of

particular interest are modifications by phosphorylation because they can be added or removed at site-

specific locations on a given protein. Furthermore, activation or partial activation of the protein can occur

depending on how many sites are phosphorylated. The number of phosphorylation events provides an

extra level of regulation that can have important physiological consequences. In this study, I

demonstrated that phosphorylation cascades are vital for the transduction of signals from the external

13

environment to the nucleus of the cell and that this communication is important for maintaining HSC

homeostasis.

Ubiquitination and deubiquitination in HSC quiescence

In the past 20 years, various studies have provided insight into the role of ubiquitination and its

reversal, deubiquitination, on HSC quiescence. Evidence that E3 ligases contribute to HSC self-renewal

was provided through studies with Casitas B-lineage Lymphoma (c-CBL) knockouts, where it was shown

that c-CBL-deficient HSCs were hyperproliferative and had impaired differentiation capabilities.

Interestingly, increased HSC cycling in the c-CBL knockout was due to increased STAT5 phosphorylation

in response the cytokine TPO (Rathinam et al., 2008). Another E3 ligase, Itch, has been shown to be

responsible for promoting HSC quiescence since knockouts display increased proliferation and

competitive BMT experiments show increased self-renewal capacity. Partial rescue in HSC progenitors

occurred when Notch1 was knocked down through shRNA, suggesting that increased Notch-signaling

promoted quiescence (Rathinam et al., 2011). F-box and WD repeat domain-containing 7 (Fbw-7) is also

an E3 ligase, and its loss in the hematopoietic system results in a highly proliferative LSK fraction that led

to reduced HSC numbers and downregulation of genes associated with HSC quiescence (Matsuoka et

al., 2008; Thompson et al., 2008). Furthermore, the von Hippel-Lindau (VHL) domain can be targeted by

RING finger E3 ligases to tag proteins for degradation. Deleting the VHL protein in HSCs led to HIF1α

stabilization, which caused an increase in LSK cells in the G0 phase of the cell cycle (Takubo et al.,

2010). Another example of a RING finger E3 ligase controlling HSC proliferation is Mouse double minute

2 (MDM-2). MDM-2 targets p53 for degradation and MDM2-deficient HSCs in a p53 hypomorphic

background led to stabilized p53, increased cell cycle arrest, increased levels of ROS, and HSC death

(Abbas et al., 2010). Taken together, these studies suggest that E3 ligases function to maintain HSC

quiescence based on the proteins they target.

Deubiquitinases are responsible for decoupling the ubiquitin molecules that ubiquitinases link

together. It has been shown that the loss of Usp1 in HSCs results in greater numbers of HSPCs that are

not as competent at reconstituting the blood pool (Parmar et al., 2010). Furthermore, A20, a dual-function

ubiquitin-editing enzyme, functions to promote HSC quiescence under steady state conditions since

14

without A20, HSCs hyperproliferate and experience exhaustion (Nakagawa et al., 2015). In essence,

PTMs that use polypeptide modifications are critical for HSC maintenance.

Phosphorylation events regulate pathways that promote or repress HSC quiescence

Phosphorylation is a PTM that allows the status of a protein to be changed quickly since

phosphorylation events often act as an “on/off” switch, meaning that an individual protein substrate or

amino acid can either be phosphorylated or unphosphorylated. Threonine, serine, and tyrosine are the

commonly phosphorylated amino acids and although some sites are constitutively phosphorylated, others

are occupied in a binary fashion that can lead to activation or degradation of a protein (Prabakaran et al.,

2012). Intact phosphorylation cascades are critical to transducing external signals from the niche to the

HSC nucleus so that HSCs can dynamically respond to their environment. To date, many signaling

pathways have been implicated in moderating HSC quiescence and a few are discussed below.

There are controversial reports regarding the role of Wnt-signaling, a major regulator in

embryonic development, in HSC quiescence (Boulais and Frenette, 2015; Jagannathan-Bogdan and Zon,

2013). β-catenin gain-of-function studies showed that increased Wnt-signaling correlates with HSC

expansion in vitro. β-catenin over-activation promoted sustained ability to reconstitute lineage colony

formation (Reya et al., 2003; Willert et al., 2003) but in vivo β-catenin over-activation using Mx-Cre led to

early stem cell exhaustion and impaired multilineage reconstitution (Kirstetter et al., 2006; Scheller et al.,

2006). Loss-of-function studies of Wnt-signaling have shown that deleting the Wnt ligand, Wnt3α, led to

decreased HSC numbers and loss of self-renewal in fetal liver HSCs (Luis et al., 2009). Furthermore,

conditionally deleting β-catenin with Vav-Cre showed reduced reconstitution ability (Cobas et al., 2004);

however, deleting β-catenin using Mx-Cre did not affect self-renewal (Koch et al., 2008). The disparate

results can be explained by differences in methods used in the experiments to delete β-catenin. For

instance deleting in fetal (Vav-Cre) versus adult (Mx-Cre) might contribute to the overall phenotype since

fetal HSCs are highly proliferative (Mendelson and Frenette, 2014).

TGFβ-signaling has been shown to be a negative regulator of HSC proliferation. Previous in vitro

reports have implicated TGFβ-signaling in HSC quiescence (Sitnicka et al., 1996) but a genetic in vivo

study has not recapitulated these results. In vivo deletion of the TGFβ cytokine or total knockouts for the

15

two receptors, TGFβ-RI or TGFβ-RII, results in embryonic lethality (Capron et al., 2010), therefore

inducible Cre lines have been used to assess the role of TGFβ-signaling in HSCs. TGFβ-RI or TGFβ-RII

conditional knockouts with Mx-Cre yielded no major HSC phenotype but the mice developed lethal

inflammatory disorder (Larsson et al., 2005). In contrast, the same group showed that in vivo retroviral

overexpression of Smad7, the negative feedback loop inhibitor of TGFβ-signaling, resulted in increased

stem cell proliferation (Blank et al., 2006). Further adding to the confusion, HSCs lacking SMAD4, a

positive downstream TGFβ -signaling regulator, showed reduced ability to repopulate the hematopoietic

system (Karlsson et al., 2007) while the Smad 2/3 total knockouts did not show a noticeable HSC

phenotype (Blank and Karlsson, 2011). Although TGFβ and its downstream components have been well

studied, clearly there are major discrepancies in the field and further in vivo studies are needed to

conclude how TGFβ-signaling impacts HSC quiescence. Interestingly, cytokines use lipid rafts, areas

where there are large receptor concentrations, to stimulate dormant HSCs to enter cell cycle and start

proliferating (Yamazaki et al., 2006). In vitro studies have shown that TGFβ1 acts to repress lipid raft

formation in HSCs and thus promotes HSC dormancy by repressing the AKT/FOXO-pathway (Yamazaki

et al., 2009). This probably occurs through activation of p57 since it has been shown that TGFβ induces

cell cycle arrest in human hematopoietic cells (Scandura et al., 2004).

The MAPK-signaling family has been shown to be important in converting cytokine signals from

the niche to the HSC nucleus to govern HSC cellular events. MAPKs are a group of serine/threonine

kinases that transduce signals from the cell surface to the nucleus through phosphorylation cascades.

They govern the transcription of many target genes involved in proliferation, differentiation, migration, and

apoptosis (Geest and Coffer, 2009). Furthermore, the MAPKs have been implicated in the maintenance

of HSC quiescence. For instance loss of ATM (ataxia telangiectasia mutated), a cell-cycle checkpoint

regulator, caused increased levels of ROS and constitutively active p38 MAPK phosphorylation, which

reduced HSC self-renewal (Ito et al., 2004). Treatment with an inhibitor of p38 or antioxidants reverted the

phenotype. Deletion of p38 MAPK in HSCs also caused increased levels of ROS and p38-deficient HSCs

have reduced quiescence as well as reduced repopulating activity (Ito et al., 2006). In addition, single

knockouts of either ERK1 or ERK2, needed for ERK MAPK-signaling, did not show a hematopoietic

phenotype probably due to genetic redundancy. However, double knockouts of ERK1 and ERK2 using

16

Mx-Cre showed rapid loss of HSPCs that manifested in leukopenia and anemia (Chan et al., 2013; Staser

et al., 2013). To date, the role of the third MAPK pathway, JNK, has not been established in HSCs. Taken

together, this information indicates that MAPK-signaling is important in maintaining HSC quiescence.

PI3K-signaling regulates quiescence in HSCs

PI3K-signaling

Phosphoinositide 3-kinases (PI3K) are a family of lipid kinases that act as intracellular second

messengers that activate downstream AGC kinases, such as AKT. These kinases regulate many

biological processes including: protein synthesis, insulin metabolism, proliferation, differentiation, and

apoptosis (Buitenhuis and Coffer, 2009). In addition, constitutive activation of PI3K-signaling has been

observed in many leukemias including acute myeloid leukemia (Polak and Buitenhuis, 2012) rendering

the study of this signal transduction pathway clinically relevant. Furthermore, PI3K-signaling is highly

conserved in evolution and the role of insulin-PI3K-AKT can be seen in a range of organisms, the

nematode C. elegans, to mammals (Engelman et al., 2006).

The PI3Ks are heteromeric proteins that contain catalytic and regulatory subunits. The eight

catalytic subunits are divided into three different classes and it is the Class I of PI3Ks that generates

PI(3,4,5)P3 in response to external stimuli and receptor activation. In vertebrates, the PI3K catalytic

subunits are further divided into Class 1A (p110a, p110b, and p110d) and class 1B (110y). The SH2

domain of the regulatory subunits (p85 for Class 1A and p101 for Class 1B) will associate with their

catalytic counterparts and recruit them to the plasma membrane. The PI3Ks are responsible for

converting inositol rings into lipid products that can activate other kinases. The inositol rings have a

glycerol backbone with fatty acids located at positions 1 and 2 and a phosphate group at position 3

(PI(3)). Additional phosphate groups at various points in the inositol ring can be phosphorylated when

there is tyrosine kinase activity and in the case of HSCs, this predominantly occurs when cytokines

stimulate receptor tyrosine kinases (Vanhaesebroeck and Alessi, 2000). The important conversion from

the PI(3,4)P2 (PIP2) substrate to PI(3,4,5)P3 (PIP3) is essential because the triphosphorylated

phosphoinositide (PIP3) is a membrane tether for other proteins that have one or more pleckstrin

17

homology (PH) domains. The phosphate and tensin homolog (PTEN) can inhibit PI3K-signaling by

converting PIP3 back to PIP2 via phosphorylation. In addition, SHIP1 (SH2-containing inositol-5’-

phosphate) and SHIP2 can also perform the PIP3 to PIP2 conversion through hydrolyzation. In PI3K-

signaling, the AGC protein, RAC-alpha serine/threonine-protein kinase (AKT or PKB), is one of the most

heavily studied and it is recruited to the plasma membrane for activation through the interaction of its PH

domain with PIP3. Once activated, AKT can go on to phosphorylate a number of targets that can lead to

transcription of genes involved in a variety of cellular processes (Buitenhuis and Coffer, 2009;

Okkenhaug, 2013; Polak and Buitenhuis, 2012).

Major downstream targets of PI3K-signaling

Many groups have investigated the AKT-pathway because of its ability to regulate the mammalian

target of rapamycin (mTOR) complex and the Forkhead box (FOXOs) transcription factors, two proteins

conserved across evolution (Figure 3). Furthermore, AKT targets are involved in many biological

processes such as cell survival and proliferation (Manning and Cantley, 2007). There are three isoforms

of AKT and they are activated when their PH domain binds to PIP3. This causes a conformational

change, which allows for phosphorylation on Ser473, predominantly by mTORC2, and phosphorylation on

Thr308 by 3-phosphoinosidtide-dependent kinase (PDK1) (Calamito et al., 2010; Okkenhaug, 2013). AKT

is constitutively activated at Thr450, which ensures that the hydrophobic motif is protected so that the

protein is active and stabilized (Pearce et al., 2010). The classical AKT-signaling target is considered to

be mTORC1 although there are multiple substrates that are affected by AKT phosphorylation. AKT can

activate mTORC1 by phosphorylating TSC2, which inhibits the TSC2-TSC1 complex from acting as a

GTPase-activating protein for Rheb. This allows Rheb-GTP to accumulate and then activate mTORC1. In

addition, ERK and RSK, can also inhibit TSC2 and therefore activate mTORC1. Once mTORC1 is

activated, it can go on to phosphorylate p70S6K and 4E-BP1, two proteins involved in protein translation

and synthesis. Once active, p70S6K and 4E-BP1 can phosphorylate IRS1, a scaffolding protein that is

needed to bind and activate PI3K to the insulin receptor. The loss IRS1 can reduce PI3K activation at the

receptor thus providing a negative feedback loop (Manning and Cantley, 2007). AKT can also

phosphorylate FOXOs, causing their inhibition. By blocking FOXO-mediated transcription, AKT can

18

impede apoptosis, cell cycle arrest, and other metabolic properties. Furthermore, AKT can phosphorylate

MDM2, an E3 ubiquitin ligase that can degrade p53, and thus further regulate apoptotic events. In

addition, glycogen synthase kinase-3 (GSK3), a signaling protein often associated with Wnt/β-catenin-

signaling, is inhibited when phosphorylated by AKT. GSK3 can also be inhibited by S6K allowing for the

dephosphorylation of GSK3 substrates, glycogen synthase and eIF2b, which are involved in insulin and

protein synthesis (Cohen and Frame, 2001). Taken together, AKT can act on many substrates to control

the regulation of metabolism, protein synthesis, cell survival, and proliferation.

19

 

Figure 3: Schematic of PI3K/PDK1-signaling.

PI3K-signaling is activated by external factors such as cytokines and insulin receptors that phosphorylate PI3K. In turn, PI3K converts PIP2 to PIP3 by phosphorylation, which can be removed by the phosphatase, PTEN, or hydrolyzed by SHIP1. Once PIP3 is activated, it can recruit AGC kinases that have a pleckstrin homology (PH) domain to the plasma membrane for activation. PDK1 phosphorylates AKT at residue Thr308. The mTORC2 complex phosphorylates a second phosphorylation site on AKT. Once AKT is fully active, it can negatively regulate the TSC1/TSC2 complex, which will allow enough RHEB-GTP to accumulate to activate the mTROC1 complex. mTORC1 is upstream of two proteins involved in protein synthesis, p70S6K and 4EBP1. The mTORC1 complex is responsible for phosphorylating S6K at Thr389 while PDK1 can phosphorylate S6K at Thr229. AKT can also inhibit GSK3 and FOXOs through phosphorylation, thus promoting cell survival. PDK1 can also activate PI3K/AKT-independent kinases such as RSK and PKC. RSK is also downstream of ERK-signaling while PKC is needed to recruit IKK to lipid rafts when NEMO is ubiquitinated by the CARD11-Bcl10-MALT complex in T cells. This allows IKK to phosphorylate IκB, allowing for NFκB-activation.  

 

PI3K-signaling in HSC quiescence

Many inhibitors of PI3K, such as wortmannin and LY294002, have been used to investigate how

PI3K impacts hematopoiesis, cell survival, and cell proliferation (Recher et al., 2005; Wandzioch et al.,

2004). Mice lacking the p85a and p85b regulatory subunits of PI3K die a few days after birth.

Furthermore, these mice have decreased fetal HSPC numbers and decreased multilineage repopulating

20

ability (Haneline et al., 2006). However, to date no study has deleted PI3K subunits in adult HSCs.

Conditional loss of PTEN, a negative regulator of PI3K-signaling, in adult HSCs resulted in short term

expansion followed by exhaustion as well as myeloproliferative disorder that can be reverted when

treated with the mTORC1 inhibitor, rapamycin (Yilmaz et al., 2006; Zhang et al., 2006). Another group

suggested that PTEN is not needed for fetal hematopoiesis, but is required in adult HSCs to regulate

mTORC2-signaling (Magee et al., 2012). Similarly, the loss of SHIP1 in HSCs, another inhibitor of PI3K-

signaling, also resulted in increased HSC proliferation and reduced long-term repopulation ability

(Desponts et al., 2006). Furthermore, targeted loss of Itpkb (Inositol(1,4,5)trisphosphate 3-kinase-b), a

competitor that blocks AKT activation by PIP3, reduced HSC quiescence and repopulating capacity

(Siegemund et al., 2015). Taken together, these studies suggest that PI3K-signaling is needed to

promote HSC maintenance.

Many other experiments have investigated the mechanism by which downstream signaling

molecules in the PI3K-pathway affect HSC maintenance. For instance, loss of AKT1 and AKT2 in fetal

HSCs results in a prolonged G0 phase and reduced repopulation capacity (Juntilla et al., 2010). In

addition, over-activating AKT in HSCs caused short-term expansion and increased proliferation, which led

to apoptosis (Kharas et al., 2010). In line with these experiments, loss of FOXO1/3a/4 in adult HSCs led

to increased proliferation, a decrease in HSC numbers, loss of reconstitution ability, and increased levels

of ROS (Miyamoto et al., 2007; Tothova and Gilliland, 2007). Inactivation of GSK3, another AKT target,

by pharmacologic inhibition in HSCs caused an increase of HSPCs that had normal repopulating capacity

(Trowbridge et al., 2006); however, in vivo loss of GSK3 showed a transient increase in HSC cycling that

was due to augmented β-catenin expression. This was followed by a decrease in HSC numbers, which

was mTORC1 mediated (Huang et al., 2009). In summary, negative regulation of AKT substrates is

necessary for proper HSC cycling.

Unlike FOXO or GSK3, which are inhibited by AKT phosphorylation, mTORC is activated by AKT.

Conditional loss of TSC1, a negative regulator of mTORC1, led to increased HSC cycling as well as self-

renewal during serial transplantation experiments (Chen et al., 2008; Gan et al., 2008). One of the main

subunits of mTORC1 is Raptor and conditional deletion of Raptor showed non-lethal pancytopenia;

however, complications arose upon transplantation implicating a role for mTORC1-signaling in HSC self-

21

renewal under regenerative conditions (Kalaitzidis et al., 2012). At the same time, another group deleted

Rictor, a critical factor in mTORC2, which is responsible for phosphorylating AKT at residue Ser473. This

study found that the HSC phenotype was similar to the control. However, when Rictor was deleted in a

PTEN-deficient background, leukemogenesis and HSC depletion were prevented suggesting that

mTORC2-signaling plays a role downstream of PTEN/PI3K mediated self-renewal and proliferation

(Magee et al., 2012). Taken together, members of the PI3K-signaling cascade are vital in HSC

maintenance. Furthermore, PI3K-signaling functions to promote proper regulation of HSC quiescence and

proliferation during normal and regenerative hematopoiesis.

PDK1-signaling in hematopoiesis

3-phosphoinosidtide-dependent kinase-1 (Pdk1)

3-phosphoinosidtide-dependent kinase-1 (Pdk1), also known as protein kinase C (PKC), is a 63

kDa serine/threonine kinase that has a N-terminal kinase domain and a C-terminal pleckstrin homology

(PH) domain (Alessi et al., 1997; Vanhaesebroeck and Alessi, 2000). The PH domain is able to interact

with lipids that cause changes in the proteins subcellular localization, conformation, activation state, and

interaction with other proteins. It is through the PH domain that the lipid PIP3 is able to recruit PDK1 to

the cell membrane for activation. PDK1 is constitutively activated since it can trans-autophosphorylate on

residue Ser241 (Pearce et al., 2010). Once PDK1 is activated in the cell it is able to target up to 23 other

AGC kinases for activation. PDK1 is required to phosphorylate the activation loop of a variety of AGC

kinases within the PI3K-signaling pathway including: protein kinase B (PKB)/AKT, mTORC1, GSK3, and

p70 ribosomal S6K (see Figure 3). Therefore, PDK1 is a master regulator of PI3K-signaling. Furthermore,

PDK1 has other well-known AKT-independent substrates such as: PKCθ, serum- and glucocorticoid-

induced protein kinase (SGK), and p90 ribosomal S6K (RSK) (see Figure 29 in Chapter 6). Taken

together, this information demonstrates that PDK1 is an essential signal transduction regulator that plays

a role in a variety of biological pathways.

PDK1 was first identified as the kinase responsible for phosphorylating AKT at Thr308 (Alessi et

al., 1997; Stokoe et al., 1997). In addition, Pdk1 is evolutionarily conserved. Indeed, genetic studies with

22

C. elegans have placed it downstream of AGE-1, the equivalent PI3K catalytic subunit in the worm.

Furthermore, in both mammals and worms, PDK1 has been found to activate AKT, which led to the

activation of mTOR-signaling (Vanhaesebroeck and Alessi, 2000). Some studies suggest that AKT must

first be localized to the plasma membrane by PtdIns(3,4,5)P3 before it can be activated by PDK1 (Mora et

al., 2004) since interaction with PtdIns lead to a conformational change in AKT that promotes

phosphorylation by PDK1. It is important to note that some studies have shown that AKT can still be

activated when a mutated form of PDK1 is unable to act with the plasma membrane, thus suggesting that

co-localization of both proteins may not be needed for AKT activation (Pearce et al., 2010). Without

phosphorylation at Thr308 by PDK1, it is thought that AKT is not fully activated (Vanhaesebroeck and

Alessi, 2000) although phosphorylation at this site alone can cause partial AKT activation. AKT activation

has been implicated in inhibiting GSK3 as well as FOXOs to promote a variety of cellular events such as

proliferation, survival, metabolism, and growth are affected (Manning 2007). Interestingly, the

PDK1/FOXO pathway has been implicated in energy regulation and glucose metabolism (Kawano et al.,

2012).

In addition, in mice, PDK1 can phosphorylate AKT on Thr308 as well as a variety of other

proteins within PI3K-signaling such as S6K (Pullen et al., 1998). Thus, PDK1 provides multiple sites of

regulation within PI3K-signaling. While the most well known substrate for PDK1 is AKT, PDK1 has also

been implicated in NFκb-signaling. NFkB-signaling is blocked when it is bound to the IκB kinase (IKK)-

NEMO complex. PDK1 phosphorylates PKCθ, and once activated PKCθ interacts and recruits IKKs to

lipid rafts at the cell membrane allowing for NFκB-activation. In addition, PDK1 also interacts with the

CARD11-Bcl10-MALT complex. This causes the ubiquitination and degradation of NEMO allowing NFκB

to be activated. This mechanism is extremely important in T cell signaling (Lee et al., 2005).

The PI3K-pathway is vital for maintaining proper regulation of cell insulin levels, growth, size, and

metabolism. Indeed, many studies involving various organ systems corroborate a role for PDK1 in these

biological processes. PDK1 total knock out mice were embryonic lethal and PDK1 hypomorphic mice

were small with reduced organ size, which was attributed to smaller cell size (Lawlor et al., 2002). This

study also found that cell size in the hypomorphic mice was regulated independently of insulin signaling

(Lawlor et al., 2002). Furthermore, loss of PDK1 in cardiac muscle led to reduced cell size, heart failure,

23

and death, presumably due to increased cardiomyocyte sensitivity to hypoxia (Mora et al., 2003). In

addition, PDK1 was needed for insulin and glycogen synthase tolerance in the heart (Mora et al., 2005b).

Similarly, PDK1-deficient hepatocytes led to glucose intolerance (Mora et al., 2005a) and loss of PDK1 in

pancreatic β cells induces diabetes because β cell mass was reduced. Interestingly, haplosufficiency of

FOXO1 increased β cell number and glucose levels suggesting that β cell mass is regulated through the

PDK1/AKT/FOXO1-pathway (Hashimoto et al., 2006). Taken together, these studies domonstrate that

PDK1 functions in non-hematopoietic organs to regulate metabolic nutrient uptake as well as cell size.

PDK1 in hematopoiesis

Many studies in the past 10 years have focused on the role of PDK1 in hematopoiesis. From

these experiments, it appears that ablation of PDK1 has detrimental effects on the lymphoid lineage. For

instance, ablating PDK1 in T cells using Lck-Cre, which is expressed in T cell progenitors, caused a block

in DN4 T cell differentiation in the thymus. The same study also reduced PDK1 levels to 10% of normal

and found that with low levels of expression, T cell differentiation remained intact; however, T cell

proliferation capacity was hindered (Hinton et al., 2004). In addition, PDK1 was needed to incorporate

CD28 and T cell receptor signaling to activate NFκB, which is needed for proper T cell proliferation and

ability to mount a proper immune response (Hayashi et al., 2007; Lee et al., 2005; Park et al., 2013). In B

cells, PDK1 was needed to promote B cell survival and differentiation. When PDK1 was ablated using B

cell specific Cre lines or the hematopoietic Vav-Cre, B cell antigen receptor expression was lost as well as

VDJ recombination (Venigalla et al., 2013). In NK cells, loss of PDK1 caused fewer cell numbers as well

as reduced mTOR-signaling, which was important for inducing expression of downstream proteins

needed for proper NK development (Yang et al., 2015). Taken together, deletion of PDK1 in lymphoid

progenitors led to reduced NK, T and B cell numbers and decreased capacity to perform as immune cells.

In contrast, overexpression of Ras in human CD34+ progenitor cells was found to stabilize PDK1 and

promote monocyte colony formation (Pearn et al., 2007). Furthermore, loss of PDK1 using a platelet

specific Cre revealed a slight decrease in numbers leading to mild thrombocytopenia and reduced

activation (Chen et al., 2013). In summary, PDK1 is a vital component in the specification of both the

24

lymphoid and myeloid lineages and loss of this kinase leads to reduced survival and loss of immune

functions.

Abnormal levels of PDK1 result in cancer

Many cancers possess mutations in genes associated with the PI3K-pathway. For instance, one

of the most commonly mutated proteins is PTEN. Aberrances in PTEN cause excessive proliferation

through PI3K-signaling (Mora et al., 2004). Furthermore, PDK1 overexpression is seen in a wide variety

of cancers including pancreatic, esophageal, ovarian, and breast cancer (Ahmed et al., 2008; Arsenic,

2014; Eser et al., 2013; Iorns et al., 2009). In the context of hematopoiesis, overexpression of PDK1 is

found in 45% of patients with acute myeloid leukemia and is associated with poorer treatment outcome

(Pearn et al., 2007; Zabkiewicz et al., 2014). Interestingly, many leukemias arise from stem cells that

have become deregulated and proliferate unchecked. There is evidence that targeting PDK1 in cancer

treatment is mechanistically relevant because when PDK1 was ablated in PTEN-deficient glioblastomas it

led to reduced cancer proliferation and survival (Flynn et al., 2000). In addition, many studies have

attempted to target malignant cells with mutations in PDK1 or other PI3K-signaling components through

the use inhibitors (Mora et al., 2004; Najafov et al., 2011) or RNAi against Pdk1 because it is a master

regulator of PI3K-signaling (Iorns et al., 2009; Najafov et al., 2011; Yu et al., 2012; Zabkiewicz et al.,

2014). Based on these studies, it is clear that understanding how PDK1 functions in hematopoiesis,

specifically how it regulates HSC quiescence and proliferation, may provide novel mechanistic insight into

clinical treatments for a wide variety of oncogenic malignancies.

Importance of PDK1-signaling in HSCs

In the current study, I hypothesize that the PI3K-pathway plays a role in HSC quiescence since it

is downstream of many cytokines required for HSC maintenance (Pietras et al., 2011; Warr et al., 2011).

Although some information regarding the role of PI3K and AKT in HSC quiescence is already available,

these studies are hindered by redundancy due to multiple PI3K subunits or multiple isoforms of AKT. In

an attempt to validate my hypothesis, I focus on PDK1 because it is a master kinase of PI3K-signaling

and because PDK1 can function beyond the AKT axis to activate downstream targets in HSCs. Through

25

the use of a conditional allele of PDK1 and a hematopoietic specific Vav-Cre line, I can specifically ablate

PDK1 in HSCs in vivo and assess the physiological consequences associated with the loss of PDK1.

While previous studies have looked at the role of PDK1 in the differentiation of myeloid and lymphoid

lineages, this is the first study to assess how PDK1 regulates HSC maintenance. Based on previous

experiments implicating PI3K-signaling in HSC self-renewal and metabolism, I hypothesize that PDK1-

mediated PI3K-signaling is vital for maintaining HSC quiescence and cellular functions.

26

Chapter 2: Pdk1 is needed for proper hematopoiesis.

Introduction

HSCs are finely regulated for proper hematopoiesis

The adult blood system is composed of a variety of cell types that range from undifferentiated to

fully lineage-committed circulating cells. These differentiated cells are responsible for performing

functions necessary for the animal to survive such as oxygenating tissues and fighting off pathogens.

Indeed, the work force of the immune system, the cells that are activated upon invasion or injury, can be

found throughout the blood network and are generated from a common ancestor, known as a

hematopoietic stem cell (HSC). In a process called hematopoiesis, all of the blood cell components of the

organism are generated primarily in the bone marrow. Hematopoiesis is structured as a hierarchy where

the least differentiated cell type, HSCs, are able to give rise to progenitors, cells with varying levels of

abilities to differentiate into various types of fully committed and functionally distinct cells. The

differentiation process is controlled intrinsically by transcription factors and also extrinsically by signals

from the cellular niche. HSCs have the potential to divide and become any of the differentiated blood cell

types; however, they may have various levels of developmental potential based on internal and external

factors. While differentiation is important for generating immune cells and maintaining the numbers of

differentiated cell types, balanced control of the HSCs themselves is of the utmost importance.

The murine adult hematopoietic system is tightly controlled by a variety of external and internal

forces that dictate the maintenance and differentiation of HSCs. External regulation is dictated by cues

from the microenvironment and cell-to-cell contacts. Internal regulation of HSCs can occur at the

transcriptional, post-transcriptional, or post-translational levels. Indeed, a variety of transcription factors

and signaling pathways have been implicated in the process of HSC self-renewal and differentiation. In

addition, signal transduction pathways have been shown to regulate HSC quiescence. The control of cell

cycle quiescence is thought to be important for HSC longevity and long-term function. It is vital to the

hematopoietic system and the overall health of the organism that HSC self-renewal, differentiation, and

27

quiescence are tightly controlled to avoid unwanted consequences, such as anemia and leukemia, that

can arise.

HSC identification

The hematopoietic system is one of the most widely studied and well defined with regards to

identifying the different developmental stages of immune cells. This is because cell surface markers can

be used to uniquely identify the most primitive and multipotent cells, the long-term HSCs (LT-HSCs), and

subsequent stages, from the progenitors to the committed lineage cell types (see Figure 1 in Chapter 1).

All HSPCs are found in a population of cells known as LSK cells that are defined based on their

immunophenotype. LSK stands for Lineage-negative, c-kit receptor positive, and Sca1 (stem cell antigen

1) positive (Ikuta and Weissman, 1992). Of note, in this work, Lineage- denotes lack of expression of:

Ter119+, B220+, CD19+, CD11b+, Ly6G+, CD3e+, and CD11c+. The LSK fraction is further divided into LT-

HSCs, short-term (ST)-HSCs and multipotent progenitors (MPPs). A few groups have identified the

various progenitor stages through functional assays (Morita et al., 2010; Oguro et al., 2013; Pietras et al.,

2015; Wilson et al., 2008; Yamamoto et al., 2013) and there is some degree of variability between the

identification schemes. In this study, all populations analyzed will also be designated by their cell surface

expression. In essence, LT-HSCs are the most primitive and are often considered to have the highest

level of multipotency. They mostly remain quiescent but can enter the cell cycle when necessary. ST-

HSCs are considered to be “active” HSCs and these cells go on to differentiate into the progenitor, MPP,

population. The MPPs have reduced engraftment ability in BMT experiments and are not able to self-

renew. According to the most recent study by Pietras et al., the MPP group is divided into three subsets:

(1) MPP2 (LSKFlt3-CD150+CD48+), (2) MPP3 (LSKFlt3-CD150-CD48+), and (3) MPP4 (LSKFlt3+CD150-

CD48+). These three subsets give rise to specific progenitor lineages that guarantee a balanced output of

differentiated blood cell types. For instance, MPP2 is biased towards the megakaryocyte/erythroid

lineage, MPP3 to the granulocyte/monocyte lineage, and MPP4 to the lymphoid lineage. Therefore, blood

production is mediated by HSCs through the production of functionally distinct subsets of progenitor cells

that favor differentiation of certain lineages. Furthermore, specific cell surface markers can distinguish

progenitor and differentiated cell types. For instance, myeloid progenitors are found in the LK fraction

28

(Lineage-c-Kit+) and most B-cells can be identified based on CD19+B220+ expression. In this work all

immunophenotyping will be referred to by cell type and antigen identity.

PI3K-signaling in HSCs

The phosphatidylinositol-3 kinase (PI3K) signaling pathway incorporates cell extrinsic signals,

such as cytokines, from the niche, with internal processing events that lead to the transcription of various

genes that control HSC functions. PI3Ks are a family of lipid kinases that act as intracellular messengers

that activate downstream AGC kinases, such as AKT, and regulate many biological processes including:

protein synthesis, insulin metabolism, proliferation, differentiation, and apoptosis (Buitenhuis and Coffer,

2009). In addition, constitutive activation of PI3K-signaling has been observed in many leukemias

including acute myeloid leukemia (Polak and Buitenhuis, 2012) rendering the study of this signal

transduction pathway clinically relevant. Upon receptor activation, PI3Ks convert the PI(4,5)P2 substrate

to PI(3,4,5)P3 (PIP3), which can act as a membrane tether for other proteins that have one or more

pleckstrin homology (PH) domains (Buitenhuis and Coffer, 2009; Okkenhaug, 2013). In PI3K-signaling,

the AGC proteins, such as AKT and PDK1, are recruited to the plasma membrane through the interaction

of their PH domains with PIP3. Many groups have investigated the AKT-pathway because it can activate

mTOR and FOXOs, two proteins conserved across evolution, and it is involved in many biological

processes such as cell survival, proliferation, and HSC quiescence (Manning and Cantley, 2007). In the

current study, I hypothesize that the PI3K-pathway plays a crucial role in HSC quiescence since it is

downstream of many cytokines required for HSC maintenance (Pietras et al., 2011; Warr et al., 2011;

Yamazaki et al., 2009).

PIP3, also activates 3-phosphoinosidtide-dependent kinase (Pdk1), which is required to activate a

variety of other AGC kinases, such as AKT, PKCθ and S6K. Because PDK1 can act on many

downstream targets independent of AKT, it is considered to be a master regulator within PI3K-signaling.

In the hematopoietic system, conditional deletion of PDK1 leads to the loss of differentiation and functions

of B cells, T cells, NK cells, and platelets (Baracho et al., 2014; Chen et al., 2013; Kloo et al., 2011; Lee

et al., 2005; Park et al., 2013; Venigalla et al., 2013; Yang et al., 2015). In addition, over-activation of

PDK1 in human CD34+ cells promotes myeloid differentiation (Pearn et al., 2007). These studies illustrate

29

that PDK1 is important for proper end-stage development and function of the immune system; however,

none of the previous reports investigated the importance of PDK1 in early hematopoiesis, specifically in

HSCs. Since a role for PI3K-signaling in HSC quiescence has been demonstrated with the downstream

kinase AKT (Juntilla et al., 2010), it is a likely hypothesis that PDK1 also impacts the maintenance of HSC

cellular functions by governing quiescence.

Results

Pdk1 is expressed in HSCs

Previous investigation into how PI3K/AKT-signaling impacts hematopoiesis has been hindered by

a variety of factors including genetic redundancy between AKT isoforms and embryonic lethality of PI3K

and AKT total knockouts. In this study, I overcome these difficulties by conditionally deleting Pdk1

specifically in the hematopoietic lineages to determine how PDK1 affects hematopoiesis.

Before generation of an in vivo model, it was necessary to assess if Pdk1 is expressed in HSCs.

To check if Pdk1 expression could be detected in HSCs, wild type bone marrow (BM) was sorted by flow

cytometry into hematopoietic cell fractions: LT-HSCs (Lineage-Sca1+c-Kit+CD150+CD48-), ST-HSCs

(LSKFlt3-CD34+Flt3-), MPP (LSKCD34+Flt3+), LK (Lineage-c-Kit+), and total BM. Furthermore, detection of

Pdk1 mRNA by PCR with primers designed to recognize exons 3 and 4 of the Pdk1 coding sequence

revealed detection of Pdk1 mRNA in all of the isolated hematopoietic cell fractions (Figure 4 A). These

results suggested that HSCs express Pdk1; however, what needed to be determined was whether Pdk1

is required for HSC maintenance and function.

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Figure 4: Inhibitors of PI3K-signaling affect HSPC numbers.

(A) cDNA was extracted sorted total BM from wild type animals, converted to cDNA, and Pdk1 mRNA expression was determined by PCR using primers located in exons 3 & 4 (n = 3). (B) Wild type total BM was MACs sorted for lineage negative cells and then cultured in vitro for 48h in 10%FBS/DMEM + 0.5ng/ml cytokines (SCF, TPO, Flt3l, IL6, & IL3) with no inhibitor (1ul/ml DMSO) or 1uM Pdk1 inhibitor. The LK and LSK cell fractions were assessed by flow cytometry and it appears that blocking PDK1 activity reduces HSPC numbers. (C) Quantification of LK and LSK described in (B). LT-HSC = LSKCD150+CD48-, ST-HSC = LSKCD34+Flt3-, MPP = LSKCD34+Flt3+. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).  

 

PDK1 is important for HSC maintenance in vitro

To assess whether Pdk1 has a role in HSC maintenance, I MACS sorted wild type lineage

negative cells and cultured them for 48 hours with 1:1000 DMSO or 1uM PDK1 inhibitor (GSK2334470)

(Najafov et al., 2011) and assessed the relative frequency of the LK and LSK compartments. After 48

hours, the cells treated with the PDK1 inhibitor were significantly reduced in both the LSK (6.8±0.9% with

no inhibitor compared to 2.62±0.7% with PDK1 inhibitor) and LK (28.0±3.2% with no inhibitor compared to

23.6±2.6% with PDK1 inhibitor) compartments compared to the cells that were cultured with DMSO

(Figure 4 B, C). This suggests that in vitro, PDK1 is involved in the maintenance of the hematopoietic

stem and progenitor cells (HSPC); however, the use of a chemical inhibitor may not have accurately

depicted what was happening to HSCs in a biological context so it was important to assess the impact of

deleting Pdk1 in vivo.

Pdk1Hem-KO mice have hematopoietic defects

To address the in vivo function of Pdk1 during hematopoiesis, I conditionally ablated Pdk1 by

breeding Pdk1flox mice (Inoue et al., 2006) (kindly provided by Dr. Sankar Ghosh in the Department of

31

Microbiology and Immunology) to the hematopoietic specific Vav-Cre (Georgiades et al., 2002) to obtain

Pdk1Flox/+, Pdk1Flox/Flox, or Pdk1Flox/+ ; VavCre/+ (referred to as control) and Pdk1Flox/Flox ; VavCre/+ (referred to

as Pdk1Hem-KO) mice (Figure 5 A). Of note, no differences in body weight, cellularity of hematopoietic

organs, and HSC numbers were detected among the control genotypes (Table 1). In the Pdk1Flox mouse,

exons 3 and 4 are flanked by loxP sites so I examined by PCR the expression of Pdk1 mRNA at this

locus in the BM and Spleen and found highly reduced Pdk1 mRNA levels in Pdk1Hem-KO mice when

compared to the controls. This suggested that Pdk1 transcription is abolished in the mutant mouse.

Immunoblots showed loss of PDK1 protein in the BM and spleen of Pdk1Hem-KO mice (Figure 5 B), but not

in the brain, a non-hematopoietic organ (Figure 5 C), suggesting that the deletion is specific to the

hematopoietic lineage. Initial characterization of Pdk1Hem-KO mice showed reduced body weight in

Pdk1Hem-KO mice (Figure 6 A & B; 18.4±1.3g in control compared to 12.1±1.7g in Pdk1Hem-KO). To

determine whether reduced body weight was related to a hematopoietic defect, control and Pdk1Hem-KO

mice were bled and a complete blood count analysis found that the Pdk1Hem-KO peripheral blood (PB) had

reduced white blood cells (WBC) (7.5±0.6 x103/uL in control compared to 1.1±0.2 x103/uL in Pdk1Hem-KO)

and reduced platelets (1062±105.7 x104/uL in control compared to 558±83.6 x104/uL in Pdk1Hem-KO) but

similar numbers of red blood cells (RBC), hemoglobin, and hematocrit (Figure 6). In addition, cell count

analysis of Pdk1 mutants showed ~50% reduction in BM cellularity (34.81.9±x106 cells in control

compared to 16.7±1.7x106 cells in Pdk1Hem-KO), ~78% reduction in the spleen (31.8±3.4x106 cells in

control compared to 7.3±1.7x106 cells in Pdk1Hem-KO), and ~93% reduction in thymus (58.0±4.5x106 cells

in control compared to 3.7±2.2x106 cells in Pdk1Hem-KO; Figure 6 D). Taken together, these results suggest

that the ablation of Pdk1 using Vav-Cre results in hematopoietic abnormalities. Since there was reduced

cellularity in the PDK1-deficient hematopoietic organs, I next investigated whether the loss of Pdk1

caused abnormal proportions of lineage-committed cells or just an overall reduction of cells.

32

 

Figure 5: Vav-Cre deletes PDK1 in the hematopoietic organs.

(A) Breeding scheme to generate Pdk1Hem-KO mice. (B) Pdk1 mRNA, normalized to Hprt and the control, for exons 3-4 is decreased in total BM (n = 4; p <0.0001) and in the spleen (n = 2) of the Pdk1Hem-KO mice. (C) Western Blots for PDK1 reveal reduced protein levels in the Pdk1Hem-KO in the BM and spleen but equal levels in the non-hematopoietic brain (n = 2). For mRNA expression, samples were normalized to Hprt and then to the control. CKO = Pdk1Hem-KO. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).  

 

 

 

 

 

 

 

 

 

 

 

 

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Table 1: Haplosufficiency of PDK1 (Pdk1F/+ ; Vav-Cre) is not phenotypically different from Pdk1F/+ or Pdk1F/F mice.

There are no significant differences between Pdk1F/+, Pdk1F/F, and Pdk1F/+ ; Vav-Cre mice in terms of body weight, BM cellularity, spleen cellularity, thymic cellularity and relative HSC numbers. Data are mean ± SEM and two-tailed unpaired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).  

 

34

 

Figure 6: Pdk1Hem-KO mice show hematopoietic defects.

(A) Pdk1Hem-KO mice have reduced body weight when compared to littermate controls (n = 23). (B) Quantification of body weight shown in (A). (C) Complete Blood Count (CBC) analysis shows hematopoietic disturbances (n = 9) which is also reflected in (D) reduced cellularity of the hematopoietic organs BM, spleen, and thymus (n = 22, 19, 15, respectively) in Pdk1Hem-KO (CKO) mice. Mice analyzed were between 4-8 weeks old. Data are mean ± SEM and two-tailed paired Student’s t-test were used to assess significance (*P<0.05, **P<0.01, ***P<0.001, not significant (ns) = P>0.05).

 

Loss of PDK1 results in improper hematopoietic lineage differentiation

To determine, whether the disturbances in HSPC number affected lineage differentiation in the

BM, I analyzed lymphoid and myeloid progenitors through cell surface immunostaining and flow

cytometry. Analysis of the common lymphoid progenitors (CLP: L-SlowK+, Flt3+, CD127+) showed a

reduction in cells lacking PDK1 (12.2±2.0% in the control compared to 3.1±1.6% in the Pdk1Hem-KO; Figure

7 A & C). Analysis of the myeloid progenitors showed no difference in relative proportions in LK,

granulocyte-monocyte progenitors (Figure 7 B & D; GMP: LK, CD16/32+, CD34+), common myeloid

progenitors (CMP: LK, CD16/32-, CD34+), or in the megakaryocyte-erythroid progenitor population (MEP:

35

LK, CD16/32-, CD34-). However, there were significantly fewer absolute cell numbers of LK (0.65±0.3x106

cells in control compared to 0.46±0.1x106 cells in Pdk1Hem-KO) and GMP (0.28±0.04x106 cells in control

compared to 0.17±0.02x106 cells in the Pdk1Hem-KO) in the Pdk1Hem-KO mice (Figure 2.4B&D). Previous

studies have found that PDK1 plays a role in B cell differentiation. Indeed, my analysis found that B cells

are reduced by 66% in the Pdk1Hem-KO BM (30.5±3.6% in the control compared to 10.0±3.9% in the

Pdk1Hem-KO). Furthermore, the relative frequency of BM Ter119+ erythrocytic lineage cells was the same

between the control and Pdk1Hem-KO, the frequency of granulocytes (CD11b+Ly6G+) was decreased

(58.8±1.3% in the control compared to 26.1±9.5% in the Pdk1Hem-KO), the frequency of monocytes

(CD11b+Ly6G-) was increased (30.0±1.3% in the control compared to 72.4±9.8% in the Pdk1Hem-KO), and

the frequency of CD41+ cells was equivalent (Figure 8 A). Thus, Pdk1 appears to mostly affect the

lymphoid progenitor compartment and partly the absolute numbers of the LK and GMP populations.

36

 

Figure 7: Lymphoid progenitors are reduced in the Pdk1Hem-KO mice.

(A) Gating scheme for isolating the common lymphoid progenitors (CLP) and (B) myeloid progenitors. Relative and absolute cell numbers of the (C) CLP show a decrease in PDK1-deficient cells. (D) Relative and absolute numbers of myeloid progenitors show reduced LK and GMP cells in the Pdk1Hem-KO mice. CKO = Pdk1Hem-KO, GMP: granulocyte/monocyte progenitors, MEP: megakaryocyte/erythrocyte progenitors, CMP: common myeloid progenitors. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).

37

 

Figure 8: Pdk1Hem-KO organs show disturbances in differentiation.

(A) Relative and absolute numbers of lineage committed cells in the BM and (B) spleen show reduced B cell numbers and an increase in monocytes in the Pdk1Hem-KO. (C) Analysis of the spleen for T cells show markedly changes in the Pdk1Hem-KO when compared to the control. (D) Complete blood count analysis of peripheral blood shows a decrease in lymphocytes and an (E) increase in monocytes in the Pdk1Hem-KO. CKO = Pdk1Hem-KO. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).

 

38

The proportions of hematopoietic lineage-derived cells were also altered in the Pdk1Hem-KO spleen

(Figure 8 B). The relative frequency of CD19+ B cells in the spleen was decreased (42.0±2.8% in the

control compared to 4.7±1.4% in the Pdk1Hem-KO), Ter119+ erythroid cells was increased (3.8±0.5% in the

control compared to 19.7±2.5% in the Pdk1Hem-KO), CD11b+ myeloid cells was increased (17.0±1.2% in

the control compared to 50.5±4.2% in the Pdk1Hem-KO), granulocytes (CD11b+Ly6G+) was increased

(16.4±1.6% in the control compared to 33±1.5% in the Pdk1Hem-KO), monocytes (CD11b+Ly6G-) was

decreased (83.1±1.7% in the control compared to 64.6±1.6% in the Pdk1Hem-KO), and CD41+ cells was

increased (1.5±0.3% in the control compared to 6.0±1.2% in the Pdk1Hem-KO) in the absence of PDK1. Of

note, the trends seen in the relative numbers may not be depicted in the absolute values since the spleen

cellularity was incredibly low. The lymphoid compartment was also evaluated in the Pdk1Hem-KO mice and

T cell disturbances are also detected in the spleen. The relative numbers of CD3e+, a general marker for

T cells, cells was decreased (40.1±3.7% in the control compared to 29±1.2% in the Pdk1Hem-KO, Figure 8

C). Corroborating this trend, CD4+ and CD8+ T cells were decreased in Pdk1Hem-KO mice (Figure 8 C).

Furthermore, the altered proportions of lineage cells can be seen with complete blood count analysis of

the Pdk1Hem-KO peripheral blood which showed severe decreases in the relative and absolute number of

lymphocytes (Figure 8 D; 83.6±2.0% in the control compared to 60.8±5.2% in the Pdk1Hem-KO) and

monocytes (Figure 8 E 4.4±0.5% in the control compared to 28.0±6.0% in the Pdk1Hem-KO) in the absence

of PDK1. Thus, the alterations found in the absence of PDK1 in HSCs also affect the blood and peripheral

secondary lymphoid compartment such as the spleen.

39

 

Figure 9: Pdk1Hem-KO thymus has an increase in DN cells.

(A) Surface staining for thymic cell populations reveals that the Pdk1Hem-KO has an increase in immature thymocytes (DN: CD4-CD8-) and a decrease in maturing CD4+CD8+ double-positive (DP) thymocytes. (B) Relative and absolute numbers of thymic cell populations. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).  

 

HSCs provide lymphoid progenitors that migrate to the thymus, which is critical for the

development of T cells. Lymphoid progenitors are devoid of CD4 and CD8 markers and are labeled

double negative (DN) but then progress to an intermediary stage where the thymocytes express both

markers and become double-positive (DP) thymocytes. Once DP thymocytes express a T cell receptor,

they can be further selected to become single positive for either CD4 or CD8. I analyzed the thymic

population to determine whether the absence of PDK1 in HSCs affects the development of T cells. As

shown in Figure 9 A and quantified in Figure 9 B, there is a dramatic decrease in double-positive

(CD4+CD8+) thymocytes when PDK1 is absent. The lack of DP thymocytes is correlated to a greater

number of DN thymocytes suggesting progression into DP thymocytes is severely inhibited in the

absence of PDK1. Furthermore, the DN thymocytes that lack a functional T cell receptor can be divided

into four categories based on their CD44 and CD25 profiles. Thymocytes from control mice display all

four DN populations (Figure 9 A lower left panel): DN1 (CD44-CD25+), DN2 (CD44+CD25+), DN3

(CD44+CD25-), and DN4 (CD44-CD25-). In contrast, Pdk1Hem-KO mice display reduced DN1, DN2, and

40

DN4 thymocytes but have an excess percentage of DN3 thymocytes (Figure 9 A, lower right panel and

Figure 9 B upper panel). Thus, PDK1 has a dramatic effect in regulating maturation of DN thymocytes

from the DN3 stage to the DN4 stage and subsequently to DP thymocytes. Taken together, similar to

decreased B cell differentiation, T cell development in the thymus is also altered by PDK1 depletion in

HSCs.

Pdk1Hem-KO mice have reduced HSCs in the bone marrow

To assess whether the loss of PDK1 impacts HSCs, I dissected BM from femurs and tibias of

control and Pdk1Hem-KO mice, processed the cells to single cell suspension and examined the HSC

compartment through cell surface staining and flow cytometry. Immunophenotyping revealed no

difference in the relative frequency of lineage negative cells (Figure 10 B) but a decrease in the absolute

number (2.6±0.2x106 cells in control compared to 1.6±0.3x106 cells in Pdk1Hem-KO). The relative and

absolute numbers of LSK cells (Figure 10 C) were equivalent in the Pdk1Hem-KO mice compared to the

control group. Similarly, analysis of CD150+CD48-LSK (Wilson et al., 2008) revealed no change in the

relative and absolute numbers (Figure 10 D). Strikingly, HSC analysis, as defined by CD150+CD48-Flt3-

CD34-LSK (Wilson et al., 2008), revealed a large decrease in cell number in the Pdk1Hem-KO (23.9±0.4% in

control compared to 6.8±2.1% in Pdk1Hem-KO; Figure 10 E). Furthermore, MPP analysis revealed a relative

increase in MPP1 (68.4±3.7% in control compared to 86.6±4.7% in the Pdk1Hem-KO) and MPP3

(38.4±4.4% in control compared to 67.5±3.5% in Pdk1Hem-KO) and a decrease in MPP4 (60.4±4.4% in

control compared to 30.5±3.7% in Pdk1Hem-KO; Figure 10 F). Recently, Pietras et al. demonstrated another

strategy to isolate various HSPC fractions based on their functional properties. Using this scheme (Figure

2.8A), HSCs are divided into long-term (Flt3-/lowLSKCD48-CD150+), and short-term fractions (Flt3-

/lowLSKCD48-CD150-), and multipotent progenitors are divided into three subsets: MPP2 (Flt3-

/lowLSKCD48+CD150+), MPP3 (Flt3-/lowLSKCD48+CD150-), and MPP4 (LSKFlt3+). Immunophenotyping

analysis of the Pdk1Hem-KO revealed a striking decrease in LT-HSCs (12.9±1.1% in control and 6.0±1.8%

in Pdk1Hem-KO) and ST-HSCs (25.5±2.6% in control compared to 8.1±2.1% in Pdk1Hem-KO; Figure 11 B).

Analysis of the Pdk1Hem-KO progenitor populations showed an increase in MPP2 (13.0±2.2% in the control

compared to 28.0±3.2% in the Pdk1Hem-KO), no change in MPP3, and a decrease in MPP4 (44.0±1.9% in

41

the control compared to 17.0±5.0% in the Pdk1Hem-KO; Figure 11 C). The aberrant differences in the

Pdk1Hem-KO HSPC populations suggest that PDK1 is necessary for maintaining a proper balance of the

HSPC pool.

 

Figure 10: The Pdk1Hem-KO BM has fewer HSCs.

(A) Gating scheme and analysis according to Wilson et al. 2008. (B-F) Relative and absolute cell numbers of the cell population show disturbances in the HSPCS. CKO = Pdk1Hem-KO. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001)

 

42

 

Figure 11: The Pdk1Hem-KO mouse has reduced ST- and LT-HSC numbers.

(A) Gating scheme and analysis according to Pietras et al. 2015. (B&C) Relative and absolute cell numbers of the cell population show alterations in the HSPCS. CKO = Pdk1Hem-KO. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).  

 

Discussion

While previous studies have used both gain- and loss-of-function approaches to study how PI3K-

signaling affects HSC survival, proliferation, and differentiation, to date no study has looked at how PDK1-

mediated PI3K-signaling influences HSC self-renewal and quiescence. Based on this data, abrogation of

PDK1-signaling through genetic deletion by Vav-Cre leads to hematopoietic disturbances as evidenced

by the reduced cellularity of hematopoietic organs and changes in CBC. Furthermore, cell surface

staining and flow cytometry revealed disturbances in lymphoid lineage differentiation and astonishingly

fewer HSCs in the Pdk1Hem-KO mice. While the results regarding lineage differentiation corroborate findings

43

from other groups, the HSC phenotype is striking and provides evidence that PDK1 is essential for the

maintenance of HSC numbers.

PDK1 is necessary for proper lineage differentiation

Other groups have shown a role for PDK1 in hematopoietic differentiation and the findings from

this study corroborate these results. For instance, these experiments show reduced numbers of B cells in

both the BM and spleen of Pdk1Hem-KO animals (Figure 8) and earlier reports have demonstrated that

PDK1 regulates B cell differentiation and homeostasis (Baracho et al., 2014; Park et al., 2013; Venigalla

et al., 2013). This corroborates an earlier investigation that established a need for PI3K-signaling in early

B cell differentiation (Baracho et al., 2014). Interestingly, two of these groups used two different B cell

specific Cre lines, mb1-Cre and CD19-Cre, therefore indicating that PDK1 has an independent role in

determining B cell survival; however, this study as well as the one conducted by Venigalla et. al, make

use of Vav-Cre which deletes in HSCs suggesting that PDK1 is needed further upstream at the progenitor

or even HSC level in order to determine B cell fate. The assumption that PDK1 is needed at the

progenitor stage to promote B cell survival, would corroborate the findings from Pietras et al. that suggest

the progenitors are biased in their lineage output. It is important to note that Venigalla et al. did not focus

on the role PDK1 in HSC maintenance and that this study was already in progress at the time of

publication. Furthermore, although the same deletion system was used in both studies, Vav-Cre is the

only non-inducible Cre line that faithfully deletes in HSCs at E 10.5 to adulthood, rendering it a powerful

tool to study how PDK1 governs HSC quiescence. In addition to supporting the B cell finding, this study

also demonstrates a role for PDK1 in T cell survival (Figure 9). Other groups have used T cell specific Cre

lines to demonstrate that PDK1 is needed to activate NFκB-signaling to promote T cell proliferation during

the adaptive immune response (Lee et al., 2005; Park et al., 2013; Park et al., 2009). An extremely small

thymus, a reduction in thymic cellularity, a reduction in splenic CD3e+ cells, and a relative increase in the

DN population in the Pdk1Hem-KO suggests that PDK1 is needed before either CD4 or CD8 is expressed,

and that this kinase is necessary at the progenitor stage to promote T cell differentiation and survival.

With regards to the myeloid lineage, there is an increase in the relative numbers of granulocytes and an

increase in the relative numbers of splenic CD11b+ cells in the Pdk1Hem-KO mice. This is interesting

44

considering that overexpression of PDK1 in human CD34+ progenitor cells through a myristolated N-

terminus retroviral construct promoted a 3-fold increase in monocyte colony formation (Pearn et al.,

2007). It is possible that PDK1 is needed for active differentiation of the lymphoid lineage and that high

levels of PDK1 are not needed to specify granulocyte/monocyte cell types. However, results of the

Pdk1Hem-KO CBC and of Chen et al. suggest that PDK1 is needed to specify, at least, some myeloid

lineages such as platelets (Chen et al., 2013). Taken together, the results from this study and previous

studies suggest that PDK1 is needed for balanced proportions of myeloid and lymphoid lineage cell types

and that it plays a major role in HSPC differentiation.

Interestingly, I found that relative numbers of lineage-committed cells in the spleen do not reflect

the composition seen in the BM. For instance, the relative numbers of Ter119+ erythroid progenitors and

CD41+ megakaryocytes are increased in the spleen and are equal in the BM. These discrepancies

suggest that the spleen is acting as a secondary site of hematopoiesis in Pdk1Hem-KO mice. Indeed, during

times of trauma or injury extramedullary hematopoiesis, which occurs outside of the BM, can act as a

supplement to normal hematopoiesis (Kim, 2010). Furthermore, during infection or injury, the spleen is

able to support myelopoiesis thus providing a possible explanation for increased relative numbers of

CD11b+ cells in the Pdk1Hem-KO spleen. Although, extramedullary hematopoiesis can supplement normal

adult BM hematopoiesis, it is clear based on the Pdk1Hem-KO organ cellularity and CBC results that it is not

enough to sustain the organism for prolonged periods of time. Furthermore, it is unclear if the Pdk1Hem-KO

spleen can serve as a niche for mutant HSCs (discussed in Chapter 3). In summary, despite the

possibility that extramedullary hematopoiesis happens in the Pdk1Hem-KO mice, it is evident that it is not

enough to account for the hematopoietic differences seen in the BM between the control and the Pdk1Hem-

KO mice.

Loss of PDK1 results in HSPC disturbances and fewer HSCs

While many of the previous studies have focused on the role of PDK1 in the committed myeloid

and lymphoid cell types, this study also assessed changes at the BM progenitor level. I found no major

differences in the number of myeloid progenitors (LK, GMP, CMP, and MEP); however, there was a

reduction in CLP numbers (Figure 7 A). In addition, the Pdk1Hem-KO mouse has a large reduction in the

45

lymphoid primed MPP4 population and taken together, this suggests that PDK1 is needed in HSCs to

promote lymphoid progenitor specification. It would be interesting to delete Pdk1 at the progenitor MPP

stage to determine if there was an independent role for PDK1 in lymphoid progenitor lineage specification

from the progression of HSC to MPP4. Indeed, crossing the Pdk1Flox mouse with Flt3-Cre (Benz et al.,

2008), which deletes at the progenitor stage, when the Fms-like tyrosine kinase receptor (Flt3 or CD135)

is expressed, would elucidate if there is an independent role for PDK1 in HSCs (Vav-Cre) versus

progenitors (Flt3-Cre). Of note, the deletion efficiency of Pdk1Flox/Flox ; Flt3-Cre mice might be poor since

there is a large reduction in the Fms-like tyrosine kinase receptor (Flt3 or CD135) expression in the

progenitor fractions of Pdk1Hem-KO mice (Figure 11 and data not shown). Low Flt3 expression in HSPCs

also suggests that PDK1 may play a role in this receptor’s expression. Interestingly, Pdk1Hem-KO mice

have reduced expression of other receptors that are important in HSC maintenance. For instance,

expression of c-kit, the receptor for stem cell factor (SCF), is low in all LSK compartments and c-mpl, the

receptor for thrombopoietin (TPO), is reduced in LT- and ST-HSCs (data not shown). Interestingly, a

previous report showed a role for AKT-signaling downstream of lipid rafts in HSCs (Yamazaki et al., 2009)

and another group has implicated a positive feedback loop for AKT/mTORC in maintaining lipid raft

formation in human melanoma cells (Yamauchi et al., 2011). Lipid rafts are areas that allow for the

concentrated formation of cytokine receptors. Since PDK1 mediates AKT phosphorylation as well as a

few other downstream components of PI3K-signaling, it would be interesting to determine whether lipid

raft formation was compromised in the Pdk1Hem-KO mice. However, assessing this connection, and how

PDK1 may regulate expression of cytokine receptors in HSPCs in general, is beyond the scope of this

study.

A major finding of this study is that HSC numbers are fewer when PDK1 is deleted in the most

primitive cells of the hematopoietic system. Reduced HSC number in the Pdk1Hem-KO mice is unexpected

considering that the AKT 1/2 DKO, as described by Juntilla et al., showed increased quiescence and

therefore increased HSC cell numbers. This discrepancy can be explained by the following possibilities:

(1) The AKT mutant used in their study was a double knockout of AKT isoforms 1 and 2 so the third

isoform could have compensated the activity. This isoform could be contributing to some AKT-signaling

and thus allowing for a quiescence signal to be administered; (2) The AKT1/2 DKO used in their study

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were total knockouts and the mice were embryonic lethal. To circumvent this, Juntilla et al. transplanted

fetal liver HSCs into adult recipient mice. Currently, it is widely accepted that the physiology of fetal liver

HSCs are different from adult HSCs and that fetal liver HSCs are controlled by distinct cellular

mechanisms. It is unclear if transplantation of HSCs from fetal liver into the adult BM niche could

recapitulate the physiology of adult HSCs.

Overall, my studies suggest that the numbers of HSCs are clearly affected in Pdk1Hem-KO mice.

Nevertheless, it is unknown if the functions of PDK1-deficient HSCs are impaired. Therefore, what

remains to be determined is whether or not the mutant HSCs are as functional as control HSCs and for

this, future experiments such as bone marrow transplants (BMTs) and cell cycle experiments need to be

performed. It is also important to note that this study was performed with Vav-Cre, which deletes Pdk1

when HSCs first appear, around embryonic day 10. While this ensures a complete understanding of how

PDK1 functions in HSCs, it does not specifically address the role of PDK1 in maintaining adult HSCs. To

answer this question, similar studies will need to be performed using an inducible Cre, such as Mx-Cre, in

which PdkFlox/Flox ; Mx-Cre and control BM can be transplanted into lethally irradiated recipient mice and

then deletion is induced after transplantation. This would ensure accurate depiction of the role for PDK1

once HSCs reside within the BM. Despite these questions, the results from this study establish a novel

role of PDK1 in maintaining HSC numbers and future experiments will elucidate the mechanistic action.

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Chapter 3: The functional ability of Pdk1Hem-KO HSCs is compromised.

Introduction

HSC functional assays

The hallmark of HSCs is that they are able to both self-renew and produce all the blood cell types

of an organism. Therefore, it follows that a single HSC should be able to populate the entire blood

system since it can replenish itself as well as generate progenitors that can go on to differentiate into

committed cells. Although early studies suggested that 200 HSCs are needed to repopulate the BM of an

entire mouse (Becker et al., 1963; Siminovitch et al., 1963; Till and Mc, 1961; Wu et al., 1968), some

evidence suggests that this can be performed by only one HSC (Eaves, 2015). Although, the minimum

number of HSCs needed to repopulate an organism’s blood system is interesting, the more pertinent

question to this study is the functional ability of PDK1-deficient HSCs.

A variety of assays exist to help determine whether a stem cell can perform its responsibilities

appropriately. One is the colony forming cell (CFC) or colony forming unit (CFU) assay (Orford and

Scadden, 2008). CFUs test the ability of a cell to produce more cells as well as reveal the identity of the

progeny. This is especially important when assessing the overall differentiation potential of HSPCs. Other

assays used to assess HSC function involve transplantation studies that allow for the assessment of HSC

migration, implantation and reconstitution of the BM and blood system. One of the most common

transplantations is bone marrow transplantation (BMT) in which total BM or an isolated BM fraction from a

donor is transplanted into a lethally irradiated recipient. Since lethal irradiation kills and depletes the

host’s rapidly dividing BM hematopoietic cells, donor cells can be used to determine repopulation of the

blood system or the function of the BM microenvironment. This concept is also known as radioprotection

since repopulation occurs after irradiation, thereby preventing the organism from dying of bone marrow

failure. Often, HSCs are able to self-renew and regenerate the entire blood system after the first

transplantation; however, if the first transplantation is adequate, then subsequent transplantations may

test the self-renewal capacity of HSCs and provide an understanding of a stem cell’s exhaustion potential.

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In addition, competitive repopulation assays, where a mixture of syngeneic and allogeneic donor cells are

transplanted, test the ability of an experimental cell population to perform at the level of a wild type cell.

This experiment allows for a direct head-to-head comparison (1:1) or can be done at various dilutions to

gain insight into the level of fitness of the experimental population (Eaves, 2015; Orford and Scadden,

2008).

Based on the immunophenotyping results in Chapter 2, HSCs are reduced in Pdk1Hem-KO mice

when compared to control mice. What remains to be determined is whether the HSCs are fully functional

but reduced in numbers or whether the performance of HSCs is compromised. Functional assays such as

total BMT and competitive BMT will shed light on the ability of PDK1-deficient HSCs to perform their

function as the most multipotent hematopoietic cell.

Quiescence maintains adult HSCs

HSCs are one of the longest living cells within the hematopoietic system since they are required

to produce blood cells throughout the lifetime of the organism. Adult HSCs are able to continually

repopulate the blood cell pool through their ability to self-renew; however, proliferation and self-renewal

increase the likelihood of acquiring deleterious mutations, which can cause improper hematopoiesis and

lead to malignancies such as leukemia and anemia. One mechanism HSCs employ to prevent this is

quiescence because cells in the G0 stage of the cell cycle are less likely to accumulate the harmful

mutations that occur during DNA replication in dividing cells (Suda et al., 2011). Indeed, during a 24 hour

period, 5.3-11.1% of adult ST-HSCs and 0.8-1.8% of adult LT-HSCs are actively cycling. Furthermore,

90-95% of all adult HSCs are quiescent since their primary function is to maintain balanced hematopoietic

cell numbers rather than accommodate the growth of an organism as in the case of fetal HSCs where

closer to 100% are cycling (Pietras et al., 2011). In addition to preventing DNA damage, quiescence has

been shown to protect HSCs from immune response such as the killing effect seen with type I interferon

exposure (Pietras et al., 2014; Pietras et al., 2015). In summary, quiescence is vital to HSC survival and

its loss is detrimental to the overall health of the organism. Given this, I wondered whether quiescence

was lost in the Pdk1Hem-KO HSCs and, if so, whether this loss contributed to their reduced HSC numbers in

this model. In order to determine whether PDK1-deficient HSCs have reduced quiescence, a variety of

49

functional assays can be performed. One such assay is challenging the cell to repopulate the entire blood

pool through BMT. Furthermore, if Pdk1Hem-KO cells have reduced quiescence, then it is important to

determine the physiological consequence of this loss: are they proliferating at the same rate as control

HSCs, or are they experiencing other biological phenomenon such as apoptosis? Given that other loss-

of-function mutants, such as TSC1 knockouts (Chen et al., 2008), in PI3K-signaling experience increased

proliferation, I hypothesized that the Pdk1Hem-KO HSCs have reduced quiescence and proliferate more,

and are thus unable to repopulate the blood cell pool during regenerative conditions. The aim of this

chapter is to investigate why there are fewer HSCs in a mutant model and whether the few remaining

HSCs are functioning similar to their wild type counter parts. Since PDK1 is a master regulator of PI3K-

signaling and this pathway has been implicated in cell survival, it is likely that Pdk1Hem-KO HSCs are

experiencing increased rates of cell proliferation and death.

Results

Hypotheses for fewer HSCs in the Pdk1Hem-KO mouse

Immunophenotyping analysis of the Pdk1Hem-KO mouse revealed a reduction in both the quantity

of HSCs and in the numbers of lineage-committed cells (Chapter 2). Lower BM cellularity and an

imbalance of differentiated cell types could be due to: (1) reduced overall HSC numbers since there are

fewer cells that differentiate, (2) the HSCs themselves are functionally compromised and are therefore

unable to perform their responsibilities as a multipotent cell, or (3) a combination of both, fewer and less

competent HSCs. A trademark of HSCs is that they are able to respond to the needs of the organism in

that they have the ability to self-renew, proliferate, and differentiate into progenitors, which can then go on

to repopulate the blood cell pool. The lower lineage-committed numbers found in Pdk1Hem-KO could

suggest that they are unable to perform this repopulating function. Reduced numbers in the Pdk1Hem-KO

mouse could be explained by a variety of reasons: (1) the HSCs could be migrating out of the BM niche to

another site of hematopoiesis, (2) the HSCs could be dying, and (3) the HSCs could be proliferating and

then differentiating into lineage-restricted progenitor cells and simultaneously not self-renewing therefore

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they are not replenishing their numbers (Figure 12). This phenomenon of reduced self-renewal is called

stem cell exhaustion and testing the functional ability of a stem cell can be accomplished with BMT

experiments (Figure 13 A). Total BMT were performed on with the Pdk1Hem-KO mouse model to determine

why there was a reduced number of HSCs and whether the existing HSCs were fully competent. This

model was chosen because it determines the functional ability of Pdk1Hem-KO HSCs to fully repopulate the

blood cell pool.

 

Figure 12: Possible explanations for reduced functionality in Pdk1Hem-KO HSCs.

Pdk1Hem-KO HSCs may have fewer cell numbers if HSCs were migrating into the blood stream to secondary sites of hematopoiesis such as the spleen. Another possibility for fewer HSCs in the BM is increased cell death. Furthermore, reduced numbers or a loss of functionality can be explained by increased proliferation without self-renewal. Increased proliferation would not explain an overall reduction in numbers therefore the active HSCs/HSPCs could be dying or differentiating into terminally differentiated cell types.  

 

PDK1-deficient HSCs are unable to provide radioprotection

To assess the functional ability of Pdk1Hem-KO HSPCs, I performed BMT. Wild type CD45.1+

recipient mice were lethally irradiated (10gy), and then injected intravenous (i.v.) with either control or

PDK1-deficient BM (106). By day 18 post-transplantation, all recipient mice receiving control BM remained

viable; however, 12/13 of the Pdk1Hem-KO BM recipients had died suggesting that PDK1-deficient HSPCs

are unable to provide radioprotection (Figure 13 B). Analysis of BM from the one surviving recipient of

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PDK1-deficient BM, revealed presence of donor cells (CD45.2+) in the BM. Homing, therefore, appears to

be in tact. However, there was a high percentage (99%) of CD45.1+ BM present, which suggests that

survival of this mouse might have been due to the escape of wild type HSCs (Figure 13 C).

 

Figure 13: Pdk1Hem-KO HSCs cannot repopulate the blood cell pool after BMT.

(A) BMT experimental set-up. Control or Pdk1Hem-KO total BM was injected into 10 grey irradiated CD45.1+ mice. (B) Survival rates of recipient mice show 12/13 recipients of Pdk1Hem-KO total BM died before day 20 (n = 13 per genotype). Data points represent one or more deaths logged. (C) Donor cell analysis of peripheral blood (PB), bone marrow (BM), and spleen of recipient mice at day 45 after BMT reveals reduced engraftment of Pdk1Hem-KO cells. CKO = Pdk1Hem-KO. Survival Curve comparison was determined by Log-rank (mantel-Cox) Test (*P<0.05, **P<0.01, ***P<0.001) and are representative of animals from two different experiments. Error bars in C represent mean ± SEM.  

 

To determine whether Pdk1Hem-KO HSPCs could function as well as wild type HSPCs, I performed

competitor BMTs. Donor (CD45.2+) BM from either control or Pdk1Hem-KO mice were mixed 1:1 with wild-

type CD45.1+ BM cells and then injected into lethally irradiated CD45.1+ recipients (10gy) (Figure 14 A).

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Peripheral blood (PB) analysis at 2 weeks, 4 weeks, and 6 weeks revealed that the wild-type, CD45.1+,

BM outcompeted Pdk1Hem-KO cells since PDK1-deficient cells were dramatically reduced in number (Week

2: 16.3±2.5% vs. 2.1±0.6%; Week 4: 28.3±2.6% vs. 1.5±0.4%; Week 6: 25.8±3.0% vs. 0.6±0.1%; control

vs. Pdk1Hem-KO, respectively; Figure 14 B). Flow cytometric multilineage analysis of PB at 4 weeks post-

transplantation showed fewer Pdk1Hem-KO donor derived B cells (23.4±1.7% in control compared to

10±4.8% in Pdk1Hem-KO) and fewer Pdk1Hem-KO donor derived T cells (8.7±3.5% in control compared to

0.9±0.3% in Pdk1Hem-KO) when compared to the control donor derived BM (Figure 14 C & D). Most of the

detectable Pdk1Hem-KO donor derived cells were most likely granulocytes or monocytes since the

proportions of myeloid cells present were similar to those seen in the donor control PB (Figure 14 D). Cell

surface-staining and flow cytometric analysis of hematopoietic organs harvested at 6 weeks post-

transplantation, revealed reduced multilineage contribution of Pdk1Hem-KO cells in the BM (<1%), spleen

(<1%), and the thymus (<1%) (Figure 14 E). Collectively, the results from the BMT experiments

demonstrate that without PDK1, HSPCs are functionally compromised since they are unable to self-renew

and provide radioprotection after irradiation and cannot contribute to multilineage differentiation, two

hallmarks of HSC identity.

53

 

Figure 14: PDK1-deficient HSCs cannot compete with wild-type HSCs.

(A) Schematic of a 1:1 competitor BMT. (B) Analysis of peripheral blood (PB) at 2, 4, and 6 weeks show miniscule numbers of Pdk1Hem-KO donor cells. (C) Flow cytometry of PB at 4 weeks show fewer B cells in Pdk1Hem-KO recipient mice. (D) Quantification of donor cells at 4 weeks shown in (C). (E) Analysis at 6 weeks of the hematopoietic organs show an absence of Pdk1Hem-KO donor cells in recipient BM and spleen. B cells = CD19+, T cells = CD4/8+, granulocytes = CD11b+Ly6G+, and monocytes = CD11b+Ly6G-. Data are of 5 mice per genotype from two independent experiments. CKO = Pdk1Hem-KO. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).  

 

Increased HSPCs in the Pdk1Hem-KO spleen

The Pdk1Hem-KO mice had fewer HSCs present in the BM (Chapter 2) than the control group and

one reason for this could be that the HSCs are exiting the BM niche and moving to a secondary site of

hematopoiesis. Extramedullary hematopoiesis happens when the generation of blood components occurs

outside of the BM. In the adult mouse, the most common alternative location is the spleen and to some

extent the liver (Johns and Christopher, 2012; Kim, 2010). Using immunophenotyping by staining single

cell suspensions, control and Pdk1Hem-KO spleens were assessed and while there is a relative increase in

the LK (3.9±1.8% in control compared to 31.0±7.0% in Pdk1Hem-KO) and LSK (0.2±0.1% in control

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compared to 1.1±0.2% in Pdk1Hem-KO) fractions, the relative and absolute numbers of HSCs

(LSKCD150+CD48-) were not altered (Figure 15).

 

Figure 15: The Pdk1Hem-KO spleen has more HSPCs but HSCs are not significantly increased.

(A) Gating scheme for HSC isolation of LSK populations in the spleen by flow cytometric analysis. (B) Quantification of the relative cell numbers by percentages show an increase in LK and LSK in Pdk1Hem-KO spleens and (C) absolute cell numbers of spleen hematopoietic cell fractions show a decrease in lineage negative cell. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).  

 

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Loss of PDK1 in HSCs results in increased proliferation and fewer quiescent cells

To determine whether Pdk1Hem-KO BM was unable to provide radioprotection because the HSPCs

were not present due to cell death, control and Pdk1Hem-KO BM were stained with Annexin V, a protein that

is labeled with a dye to detect the externalization of phosphatidylserine in apoptosing cells. Analysis of

the control and Pdk1Hem-KO BM revealed slightly fewer but statistically insignificant Annexin V+ cells in both

the LSK (13.0±2.5% in control compared to 10.6±1.8% in Pdk1Hem-KO) and CD150+CD48+LSK (6.9±1.0%

control compared to 4.8±1.8% in Pdk1Hem-KO) compartments suggesting that the PDK1-deficient HSCs are

not dying (Figure 16 A & B).

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Figure 16: Pdk1Hem-KO progenitor cells are less quiescent compared to the control.

(A) Representative flow cytometric plots of LSK and HSC (CD150+CD48-LSK) show no difference in Annexvin V+ cells (n = 11). (B) Representative FACS plots of cells stained for the side population reveals fewer quiescent CD150+SK cells in Pdk1Hem-KO BM (n = 4). (C) LSK cells deficient in Pdk1, have fewer cells in the side population (n = 4) and (D) are less likely to be in G0 (n = 6). CKO = Pdk1Hem-KO. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).  

 

It is possible that Pdk1Hem-KO HSPCs are not as quiescent as control HSPCs and are thus unable

to provide radioprotection due to exhaustion and active proliferation. To determine whether the HSPCs

lacking PDK1 were less quiescent, I performed a Hoescht 33342 dye labeling experiment to identify the

“side population”, or the quiescent cells. According to Goodell et al. 1996, quiescent cells have highly

active ABC transport pumps and therefore, cells that are active retain the dye longer than those that are

quiescent. In the CD150+SK fraction, there was a reduction in the relative percentage of side population

cells (8.5±0.2% in control compared to 3.8±0.9% in Pdk1Hem-KO; Figure 16 C), indicating that these cells

were quiescent and therefore not active. This assay also corroborates that there are reduced HSC

numbers in the Pdk1Hem-KO mice. Pyronin Y is an intercalating dye that is used to measure the separation

between G0 and G1 phases in the cell cycle. Analysis of the Pdk1Hem-KO LSK revealed a reduction in both

the relative and absolute cell numbers in G0 when compared to the control group (14.4±0.9% in control

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compared to 8.6±1.5% in Pdk1Hem-KO; Figure 16 D). Taken together, it appears that fewer Pdk1Hem-KO

HSCs are inactive suggesting that they are proliferating more.

 

Figure 17: Pdk1Hem-KO progenitor cells proliferate more.

(A) Representative flow cytometric plots of LSK and HSC (CD150+CD48-LSK) show increased levels of cell cycle protein, Ki67 (n = 6). (B) Mice pulsed for 16h with a DNA intercalating agent, 5-bromouridine (BrdU), revealed increased incorporation in Pdk1Hem-KO LSK and HSCs (CD150+CD48-LSK) (n = 6). Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).  

 

In order to repopulate the blood cell pool, HSCs need to proliferate and differentiate to meet the

demands of the organism. To determine whether the Pdk1Hem-KO BM had proliferation defects, 5’-bromo-

uridine (BrdU) was administered for a pulse of 16 hours. BrdU is a synthetic thymidine analog that that

intercalates into replicating DNA. Flow cytometric analysis for BrdUhigh cells revealed a significant

increase in the LSK (36.8±4.5% in control compared to 55.9±5.6%in the Pdk1Hem-KO) and CD150+CD48-

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LSK (15.5±2.0% in control compared to 40.9±8.1% in the Pdk1Hem-KO; Figure 17 A) fractions. To

corroborate that the Pdk1Hem-KO HSPCs were cycling more, I next analyzed expression of Ki67, a cell

cycle protein that is present in active phases of the cell cycle. Immuno-staining showed that there were

increased levels of Ki67 in the Pdk1Hem-KO LSK (51.2±3.0% in control compared to 65.1±10% in Pdk1Hem-

KO) and CD150+CD48-LSK (39.5±8.1% in control compared to 66.3±2.8% in Pdk1Hem-KO) fractions (Figure

17 B). Taken together, the increased levels of Ki67 and BrdUhigh cells in Pdk1Hem-KO HSPCs suggested

that these cells were hyperproliferating.

HSPCs have increased cell death and lineage differentiation in vitro

Since Pdk1Hem-KO HPSCs cells were proliferating more, I wanted to determine whether this

resulted in increased differentiation or apoptosis. To determine this, control and Pdk1Hem-KO LSK cells

were stained with a proliferation dye, cultured and assayed for proliferation, cell death, and differentiation

at 24, 48, and 72h. CellTrace is a fluorescent dye that is initially taken up by the cell cytoplasm (Figure 18

A). As the cell divides, the concentration of dye in each cell decreases as it is shared by the daughter

cells. In this way, the amount of dye present in the cell indicates the amount of proliferation cells are

undergoing, i.e. cells with increased proliferation exhibit weaker florescence. At all time points analyzed,

the PDK1-deficient cells showed high mean florescence indicating reduced proliferation, when compared

to the control cells (Figure 18 B & C; 24h: 15876±444 in control compared to 23844±2052 in Pdk1Hem-KO,

48h: 8424±493 in control compared to 14204±382 in Pdk1Hem-KO, 72h: 6572±1266 in control compared to

11288±1909 in Pdk1Hem-KO). Interestingly, cells stained for expression of AnnexinV+7AAD- showed

increased cell death in the Pdk1Hem-KO LSK at all time points (Figure 18 D; 24h: 10.9±1.4% in control and

27±2.2% in Pdk1Hem-KO, 48h: 15.1±5.1% in control and 28.0±2.6% in Pdk1Hem-KO, 72h: 22.1±4.9% in

control compared to 50.6±4.8% in Pdk1Hem-KO). Lastly, Pdk1Hem-KO LSK cells had more cells that were

lineage positive (CD11b+, B220+, CD19+, Ly6G+, Ter119+, CD3e+) at 72h (Figure 18 E; 26.2±9.5% in

control compared to 34.4±7.2% in Pdk1Hem-KO) suggesting these cells differentiated more. In summary,

unlike the in vivo phenotype, the cultured HSPC results suggest that when isolated, PDK1-deficient

HSPCs have increased cell death, do not hyperproliferate, and are more likely to terminally differentiate.

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Figure 18: In vitro culture reveals that Pdk1Hem-KO LSK cells are apoptotic.

(A) Cartoon depicting LSK isolation scheme. LSK cells were cultured at 37C with stem cell cytokines and analyzed for CellTrace expression at 24h, 48h, and 72h. (B) Representative flow cytometric plots (C) and geometric mean florescence intensity (GMI) show increased CellTrace expression meaning cultured Pdk1Hem-KO LSK cells are less proliferative. (D) Cell death analysis at 24h, 48h, and 72h reveals increased Annexin V expression in PDK1-deficient cells. (E) Isolated Pdk1Hem-KO LSK cells significantly differentiate more into lineage-positive cells at 72h. Data are of 6 wells from two independent experiments. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).  

 

Discussion

Lineage disturbances in the Pdk1Hem-KO blood could be due to a variety of reasons including

fewer, less functionally competent HSCs. The results from this chapter shed light on the functional ability

of Pdk1Hem-KO HSCs. Since Pdk1Hem-KO cells could be detected in recipient BM and total BMT was unable

to reconstitute the blood cell pool after transplantation, it suggested that the PDK1-deficient cells were

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able to engraft but are not able to overcome the trauma of transplantation and thus fulfill their role as

donor HSCs. Furthermore, competitive BMTs showed that wild type cells outcompeted mutant cells to

reconstitute the blood supply in recipient mice, further providing evidence that Pdk1Hem-KO HSCs could not

appropriately proliferate, differentiate, and self-renew when stressed. Further experiments showed that

loss of reconstitution ability maybe a result of reduced quiescence and hyperproliferation of Pdk1Hem-KO

cells. Taken together, these results suggest that PDK1 is needed for the maintenance of quiescence and

self-renewal properties of HSCs.

BMT analysis

While the results from the total and competitive BMTs conclusively show that Pdk1Hem-KO BM

cannot provide radioprotection to lethally irradiated recipients, there are a few things to consider when

trying to determine the cause. First, although 1 x 106 total BM cells of control and mutant were injected

into each recipient, the composition of the two types of cells differed so fewer Pdk1Hem-KO HSCs could

have been transplanted per recipient compared to control recipients. This difference, however, seems

negligible since theoretically, only one HSC is needed to repopulate the blood system. However, further

homing experiments are needed to properly determine this. Very severe engraftment defects following the

total and competitive transplantation of Pdk1Hem-KO BM cells can also be explained by increased sensitivity

of HSCs to radiation stress. To address this possibility, further studies including a transplantation assay

following sub-lethal irradiation are needed. Detection of higher chimerism of donor cells after

transplantation, would suggest that Pdk1Hem-KO cells are unable to repopulate the entire blood system but

are able to contribute to partial repopulation of the blood. In essence, Pdk1Hem-KO HSCs require a boost

from the host wild type cells since they cannot fully perform their duties in a stressed environment. This is

logical in the context of survivability of the Pdk1Hem-KO animals since they survive well into adulthood (8+

weeks), after the transition from fetal to adult BM has occurred. Injection of PDK1-deficient BM into

partially irradiated donors seems excessive given that competitive BMTs more or less delineate the same

answer. Furthermore, it would be interesting to see if the Pdk1Hem-KO niche was compromised since a

study (Schepers et al., 2013) reports that hyperproliferative myeloid cells can impact cells of the

endosteal niche and the PDK1-deficient BM has increased numbers of monocytes. To assess this, wild

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type donor BM could be injected into sub-lethally irradiated Pdk1Hem-KO recipients and hematopoietic

output could be assessed. HSCs receive many quiescent and self-renewing signals from the niche so

irradiation would make it necessary for the host and donor cells to repopulate the BM. However, if the

external signals coming from the niche are altered, the wild type HSCs may not be able to provide

radioprotection. While this would be interesting, it is beyond the scope of this paper, which focuses on the

intrinsic role of PDK1 in HSCs.

Pdk1Hem-KO HSPCs are homing to the spleen

Interestingly, immunophentoyping of the spleen suggests that Pdk1Hem-KO HSCs are exiting the

niche and homing to a secondary site of hematopoiesis. This is common when an organism is

experiencing trauma or injury or the BM is in capable of facilitating hematopoiesis. Although, the LK and

LSK fractions are increased in Pdk1Hem-KO mice, the relative and absolute numbers of CD150+CD48-LSK

are not increased, suggesting that the HSCs found in the spleen are unable to compensate for the lack of

HSCs in the BM. Indeed, the hematopoietic output and number of differentiated lymphoid or myeloid cell

types are still skewed in Pdk1Hem-KO mice, which indicates that the HSPCs in the spleen are ineffective.

Loss of quiescence, hyperproliferation and increased differentiation of PDK1-deficient cells

Through multiple assays, it is highly evident that Pdk1Hem-KO HSCs, loose quiescence and self-

renewal ability, so that when they hyperproliferate they are unable to maintain their cell numbers.

Interestingly, the results of the in vivo and in vitro apoptosis and proliferation assays yield opposing

results. Detection of increased apoptosis in vitro but not in vivo can be explained by the fact that there are

clearing mechanisms within the host that could be phagocytosing the dead cells. I tried to stain ex vivo

BM with TUNEL, an apoptosis marker, to corroborate levels of cell death. Preliminary experiments failed

due to lack of detection of BM fractions and it is possible that an enzyme in the assay is cleaving the

conjugated antibodies used to identify different cell populations. Future experiments with sorted cells

using TUNEL or Caspase-3 staining via IHC will be helpful to verify that HSCs are not dying more in vivo.

Furthermore, an artifact of in vitro culture is increased cell death. In addition, cultured HSPCs no longer

receive signals from the in vivo niche, which could include cell survival signals as well as proliferation

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signals. Lack of these signals from the niche, could be a reason why the in vitro CellTrace experiments

suggested reduced LSK proliferation. Regardless of the discrepancies, it is clear based on the BrdU and

Ki67 studies that in vivo HSPCs are cycling more.

The in vitro culture experiments shed light into the ramifications of hyperproliferation. It seems

that after 72 hours, Pdk1Hem-KO LSK cells are more likely to have expression for lineage markers, which

are indicative of specific differentiated cell types. In order to determine the full differentiation capacity of

PDK1-deficient HSCs, the cells could be sorted and differentiated in vitro in either liquid culture or on

semi-solid medium. While an elegant differentiation study would be thorough, the results from this study,

as well as others with lineage specific Cre lines have shown that PDK1 is needed for proper

differentiation of the lymphoid lineage.

The results from this study provide a rationale for reduced HSC numbers in the Pdk1Hem-KO

mouse but the reason for cellular status is affected, loss of quiescence and increased proliferation, is

something that remains to be determined. Exploration into the mechanisms that govern HSC quiescence

and cycling will elucidate what is happening in the Pdk1Hem-KO HSCs at a molecular level.

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Chapter 4: PDK1-deficient HSPCs exhibit reduced response to

oxidative stress

Introduction

Oxidative Stress

When the body is subjected to physical and metabolic stressors, an imbalance of free radicals

accumulates within cells and organs and can cause oxidative stress. The generation of free radicals is

fueled by events outside the cell particularly from environmental insults such as infection and disease that

activate immune responses, exposure to heat or cold that trigger heat shock proteins, oxygen deprivation

or hypoxia possibly from trauma or exercise, metabolic deprivation such as from starvation, and ionizing

radiation from natural and man-made sources (Mendelson and Frenette, 2014). Exposure to these events

can cause the cell to generate excessive amounts of reactive oxygen species (ROS), particularly from the

mitochondria, which can lead to signaling cascades and by-products that can cause DNA damage and

cell death (Bigarella et al., 2014; Wilson et al., 2008). High levels of ROS can interact with proteins

causing amino acid oxidation therefore impacting protein signaling and degradation (Bigarella et al., 2014;

Jensen, 2006). Similarly, undue ER stress induced by unfolded proteins or metabolic perturbations can

also generate ROS and then lead to increased autophagy and increased cell death (Zhang, 2010) (Figure

19 C). Taken together, it is clear that metabolic homeostasis and the regulation of ROS within a cell, such

as HSCs, must be maintained or there will be drastic consequences to HSC viability and production of

lineage-specific cells.

Mitochondrial dynamics

One of the largest contributors to oxidative stress within a cell is the mitochondrion. Indeed,

roughly 2% of the oxygen consumed by mitochondria is estimated to reduce to ROS (Finkel, 2012; Handy

and Loscalzo, 2012). Large amounts of ROS are generated by the mitochondrion when the oxidative

phosphorylation pathway (OXPHOS) is used for energy production. In brief, energy production begins

64

with glycolysis, which is when glucose is converted to pyruvate. The intermediate of glucose, glucose 6-

phosphate, can be bypassed to the pentose phosphate pathway which can regenerate co-factors needed

during glycolysis as well as the antioxidant glutathione which can repress ROS. Once pyruvate is formed,

it can be directed towards the tricarboxylic acid cycle, which powers the electron transport chain to

produce adenosine triphosphate (ATP). Most intracellular ROS is generated from the electron transport

chain (Bigarella et al., 2014; Ito and Suda, 2014; Suda et al., 2011). Interestingly, progenitors and

differentiated cells predominantly use OXPHOS for energy production while stem cells use glycolysis (Ito

and Suda, 2014; Simsek et al., 2010; Zhang and Sadek, 2014). In addition, it has been shown that

quiescent HSCs have fewer mitochondria when compared to differentiated cells (Lewandowski et al.,

2010), since differentiated cells need mitochondria for OXPHOS ATP production. Taken together, this

suggests that in order to maintain homeostasis, HSCs have metabolic mechanisms that encourage low

levels of ROS production. Many dyes exist to detect intracellular ROS, as well as mitochondria mass, and

mitochondria membrane potential. Furthermore, metabolomics can be used to assess energy output

associated with either glycolysis or OXHPOS. For instance, cellular oxygen consumption, lactate

production, ATP production, NADH levels, and drug inhibition to measure basal and maximal cellular

respiration can provide insight into the energetic output of a cell. These assays can provide insight into

the mitochondrial metabolic properties of Pdk1Hem-KO HSCs.

ROS and HSC maintenance

Many avenues of evidence implicate ROS as a regulator in stem cell activation and progenitor

differentiation. ROS can interact with amino acids in proteins through oxidative modification to influence

many of the signaling cascades that are necessary to activate the transcription of differentiation programs

(Bigarella et al., 2014). Furthermore, work by other groups has suggested that ROS regulates the

epigenetic landscape, a vital component in controlling stem cell fate (Bigarella et al., 2014; Challen et al.,

2012). In addition, major sources of ROS generation can impact stem cell properties. For instance, ROS

due to high levels of irradiation can alter HSC repopulating abilities (Cao et al., 2011; Ito et al., 2004; Ito

et al., 2006). In addition, OXPHOS produces ROS, and overactivation of OXPHOS led to loss of stem cell

dynamics and inhibition of OXPHOS by HIF activation improved proliferation and maintenance of

65

embryonic stem cells (Bigarella et al., 2014). Reduced OXPHOS is also seen in HSCs since they have

decreased mitochondrial respiration and are highly dependent on glycolysis (Norddahl et al., 2011;

Unwin, 2006).

Recent work by a number of groups has provided evidence that suggests low ROS levels are vital

to maintain HSCs in an undifferentiated state. Work by Takubo et al. showed that HSCs are highly

positive for pimonidazole, a hypoxic label, and hypoxia has been correlated with lowering levels of ROS

by initiating energy production to glycolysis (Ito and Suda, 2014; Zhang and Sadek, 2014). Furthermore

Jang and Sharkis separated ROShigh and ROSlow HSCs and found that unlike ROSlow HSCs, ROShigh

HSCs undergo stem cell exhaustion much more quickly when serial transplanted. The ROShigh and

ROSlow HSCs were isolated by the same immunophenotype scheme but had different oxidative stress

levels so taken together, this suggests that there is a physiological significance of low levels of ROS in

the maintenance of stem cell self-renewal. For HSCs, the most quiescent and multipotent cell within the

hematopoietic system, a minimal amount of oxidative stress is vital therefore the HSC must remain in an

inactive cycling state that protects the cell from acquiring undue mutations and potential cell death (Naka

and Hirao, 2011; Takubo et al., 2010).

PI3K-signaling regulates ROS levels

Interestingly, genetic knockouts of members in the PI3K/AKT/mTOR-pathway that have

disturbances in HSC quiescence often have an imbalance in oxidative stress. For instance AKT1/2 DKO

HSCs show increased quiescence and decreased levels of ROS (Juntilla et al., 2010). Mutants for TSC,

which is inhibited by AKT activation and negatively regulates mTORC1, have high levels of ROS and

reduced quiescence (Chen et al., 2009). In addition, mice lacking FoxO1/3a/4 show an increase in LSK

cells, which correlates with an increase in ROS and a decrease in quiescence (Miyamoto et al., 2007;

Rimmele et al., 2015; Tothova and Gilliland, 2007). The Pdk1Hem-KO HSCs are proliferating more and are

less quiescent. Therefore, I hypothesize that the loss of a regulator, PDK1, upstream of the AKT/mTOR-

pathway, changes the oxidative stress in HSCs.

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Results

Microarray analysis reveals Pdk1Hem-KO HSPCs have reduced response to oxidative stress

The results in Chapter 3 suggest that HSCs lacking PDK1 loose quiescence, have impaired self-

renewal, and are hyperproliferative. To gain an understanding of why Pdk1Hem-KO HSCs elicit this aberrant

phenotype, I wanted to know which transcriptional targets were deregulated in PDK1-deficient cells. This

would provide insight into the molecular mechanisms that were governing PDK1-mediated HSC

quiescence. To determine the transcriptional regulation differences between control and Pdk1Hem-KO

HSCs, RNA was extracted from sorted LSK cells, converted to cDNA, and then processed for microarray

analysis. Microarray data are useful since they provide a broad overview of gene expression data within a

sample. Using Gene Set Enrichment Analysis (GSEA), I first checked to see whether genes associated

with the HSC profile were affected. GSEA compares the gene clusters associated with our data with data

sets that are publically available. As expected, there was a loss of genes associated with HSC identity in

Pdk1Hem-KO cells (Figure 19 A). In addition, the GSEA indicated that PDK1-deficient HSPCs have reduced

ability to respond to oxidative stress (Figure 19 B). This intrigued me considering other mutants of the

AKT/mTOR pathway experience differences in ROS expression while also seeing differences in HSC

quiescence. Taken together, the microarray data indicate that the loss of PDK1-signaling does result in

different transcriptional programs compared to normal HSCs, and that Pdk1Hem-KO HSPCs may have

changes in metabolic activity.

Deficiency of PDK1 in HSPCs alters the transcription factor networks associated with HSCs and

oxidative stress

The results from the GSEA analysis suggested that the transcription networks between the

Pdk1Hem-KO mice were altered in HSC signature and in the machinery to oxidative stress when compared

to the control mice. To understand the molecular basis for decreased HSC quiescence in PDK1-deficient

HSCs I selected transcription factors (TFs) that were differentially up- or down-regulated in the conditional

knockout. I focused on TFs since their activity is required to promote HSC quiescence, self-renewal, and

differentiation (Orkin, 2000). After I selected any gene higher than a 1.5 expression ratio (Pdk1Hem-KO

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expression/control expression) for up-regulated genes and any gene lower than 0.5 (Pdk1Hem-KO

expression/control expression) for down-regulated genes, there was a list of 422 up-regulated TFs and

128 down-regulated TFs (Table 2 and Table 3). I further narrowed this list by selecting for genes known to

be involved in hematopoiesis, the hypoxic response, or oxidative stress (Table 4). Interestingly, many of

the differentially regulated genes correlated with the Pdk1Hem-KO BM phenotype (Agrawal et al., 2008;

Buscarlet et al., 2014; Carreras et al., 2010; Chan et al., 2001; Elvert et al., 2003; Feng et al., 2010;

Finlay, 2014; Fox et al., 2004; Gross et al., 2008; Jin et al., 2008; Kawanami et al., 2009; Kuure et al.,

2010; Leiherer et al., 2014; Lukin et al., 2010; McKinney-Freeman et al., 2012; Morosetti et al., 1997;

Passegue and Ernst, 2009; Pevny et al., 1991; Schuettpelz et al., 2012; So et al., 2004; Tian et al., 2012;

Ubeda et al., 1999; van der Veer et al., 2014; Wolfler et al., 2010; Xu et al., 2005; Yamamoto et al., 2011;

Yap et al., 2005; Zhou et al., 2015). For example, Klf7, whose overexpression suppresses HSPC

functions (Schuettpelz et al., 2012), is up-regulated, This corroborates with the loss of expression of HSC

maintenance and differentiation genes: Hopx, Fos, Fosb Klf5, and Tal1 (McKinney-Freeman et al., 2012;

Zhou et al., 2015). In addition, Cebpa and Cebpe, two genes that direct myeloid differentiation (Morosetti

et al., 1997; Wolfler et al., 2010), were increased. The data also suggested that inhibitors of HIF1a were

increased, Cited2 and HoxA7 (Agrawal et al., 2008; So et al., 2004), while a downstream target of Hif1,

Fosl2 (Leiherer et al., 2014), was decreased. Furthermore, genes involved in oxidative metabolism, such

as Ncor1 and Nfe2l2 (Chan et al., 2001; Yamamoto et al., 2011), were deregulated. Taken together, this

data suggests that many of the differentially regulated TFs in the Pdk1Hem-KO LSK may explain why PDK1-

deficient HSCs lose quiescence and are unable to respond to oxidative stress. However, further qRT-

PCR experiments are needed to validate the results described in the microarray.

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Figure 19: Pdk1Hem-KO LSK cells exhibit reduced response to oxidative stress.

(A) Microarray GSEA analysis of control and Pdk1Hem-KO LSK cells indicate that the HSC signature is lost and (B) that there is reduced ability to respond to oxidative stress in Pdk1-deficient cells. (C) Schematic representing how oxidative stress can affect an HSC cell.  

 

69

Table 2: Up-regulated genes in the Pdk1Hem-KO microarray.

 

 

                         

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Table 3: Down-regulated genes in the Pdk1Hem-KO LSK microarray.

 

Table 4: Selected transcription factors involved in hematopoiesis or oxidative stress.

 

71

PDK1-deficient cells have increased levels of ROS

One of the hallmarks of oxidative stress is the presence of reactive oxygen species (ROS; Figure

19 C). To determine whether the Pdk1Hem-KO HSCs exhibited increased ROS-related oxygen stress, I

stained freshly isolated BM from control and Pdk1Hem-KO mice with a dye that measures acetate

intracellular esterases and oxidation, CM-H2DCFDA. I found increased levels of ROShigh expressing cells

in all of the blood cell populations analyzed (Figure 20 A & B) including the CD150+CD48-LSK HSC

fraction (4.2±0.9% in control compared to 10.4±2.4% in Pdk1Hem-KO), the progenitor LSK fraction

(8.2±1.2% in control compared to 28.8±6.5% in Pdk1Hem-KO), the progenitor LK fraction (35.4±3.0% in

control compared to 49.1±6.3% in Pdk1Hem-KO) and the lineage negative fraction (27.7±4.2% in control

compared to 40.5±4.6% in Pdk1Hem-KO). CM-H2DCFDA is considered to be a general oxidative stress

indicator; however, other specific forms of oxidative stress can be found within the cell. To assay for

oxidative stress due to mitochondria specific superoxide, I stained control and Pdk1Hem-KO BM with

MitoSOX Red. Although I did not detect significant increases in superoxide levels in the HSPC fractions, I

did detect significant increases in oxidative stress due to superoxide in the PDK1-deficient lineage

negative fraction (Figure 20 C & D; 7.6±2.3% in control compared to 17.6±4.4% in Pdk1Hem-KO).

Interestingly, previous reports have shown that high levels of ROS can activate the p38 MAPK pathway,

which then promotes HSC exhaustion and loss of HSC quiescence (Ito et al., 2006; Jang and Sharkis,

2007). To determine whether this pathway was activated in the absence of PDK1, protein was extracted

from wild-type total BM that was either treated with or without 1uM PDK1 inhibitor. Immunoblots showed

an increase of phosphorylated p38 in the absence of PDK1-signaling (Figure 20 E). To corroborate this

with the in vivo model, I next looked at the p38 GSEA from the microarray and indeed, there was

increased transcription of p38 targets in Pdk1Hem-KO LSK cells (Figure 20 F). These experiments

suggested that Pdk1-deficient cells have high levels of ROS that trigger aberrant signaling in associated

pathways. Consequently, a high level of oxidative stress can have detrimental effects on the cell such as

increased proliferation, providing opportunities for DNA damage, apoptosis, and eventually HSC

exhaustion (Figure 19 C).

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Figure 20: PDK1-deficient cells have augmented ROS levels.

(A) Representative flow cytometry plots of cells labeled with CM-H2DCFDA, a dye that measures reactive oxygen species. (B) Analysis of CM-H2DCFDA high cells showed increased ROS in the Pdk1Hem-KO BM (n = 13). (C&D) Cells labeled for mitoSOX, which measures ROS due to mitochondrial superoxide levels, showed an increase in the lineage negative fraction in the mutant (n = 5). (E) Immunoblots of total BM treated with and without 1uM PDK1 inhibitor showed increased p-p38 expression when Pdk1 activity is blocked (n = 2). (F) Microarray analysis revealed that p38 transcriptional targets were up in the Pdk1Hem-KO LSK cells (pool of 5 mice). Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).  

 

To determine the functional consequences of increased ROS levels, I first analyzed the

progenitor populations for proliferation. Ki67 is expressed during active phases of the cell cycle and the

Pdk1Hem-KO LK (51.2±2.9% in control compared to 70.8±4.6% in Pdk1Hem-KO) and lineage-negative

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(47.3±4.3% in control compared to 64.5±3.9% in Pdk1Hem-KO) fractions have more Ki67high cells (Figure

4.3A). These results followed the trend I saw with the BrdU and Ki67 analysis of the LSK and

CD150+CD48-LSK in Figure 17. Increased cell cycling exposes the cells’ DNA to mutagens and other

harmful events that can cause damage. To assess whether increased levels of ROS resulted in increased

levels of DNA damage, we assayed for double stranded breaks. Double stranded breaks are

phosphorylated upon cleavage; therefore, control and Pdk1Hem-KO BM were stained with antibody against

phospho-H2A.X. Although there was a trending increase in cells with high levels of DNA damage in all

BM populations analyzed, only the lineage negative fraction was at significant levels (Figure 21 B & C;

13.6±3.6% in control compared to 32.4±4.4% in Pdk1Hem-KO). Cell death analysis of the LSK and

CD150+CD48-LSK showed no significant difference in apoptosis in vivo (Figure 16 A & B); however,

analyzing the progenitor populations revealed that the PDK1-deficient LK (2.4±0.3% in control compared

to 3.6±0.3% in Pdk1Hem-KO) and lineage-negative (6.4±0.7% in control compared to 8.0±0.7% in Pdk1Hem-

KO) fractions have more AnnexinV+7AAD- cells (Figure 21 D). Taken together, perturbations in PDK1-

mediated signaling lead to higher levels of oxidative stress that results in increased cell proliferation, DNA

damage, and cell death.

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Figure 21: Pdk1Hem-KO progenitor cells have increased DNA damage.

(A) Quantification of Ki67 expression shows that Pdk1Hem-KO LK and Lineage negative populations are proliferating more (n = 6). (B) Representative flow cytometry plots and (C) quantification of cells stained for phosphorylation of double stranded breaks, p-H2A.X, shows increased DNA damage in the Pdk1Hem-KO (n = 5 except for CD150+CD48-LSK: n = 3). (D) Quantification of AnnexinV+7AAD- expression shows increased cell death in the Pdk1Hem-KO LK and Lineage negative cells (n = 11). Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).  

 

Treatment with NAC suppresses proliferation and differentiation in PDK1-deficient cells

High levels of oxidative stress can be mediated by antioxidants, which terminate the oxidative

chain reactions that lead to ROS generation. N-acetyl cysteine (NAC) is a free radical scavenger that has

been shown to decrease ROS levels and revert hematopoietic physiologic pathologies due to oxidative

stress (Tothova et al., 2007). To determine whether NAC can rescue hematopoietic disturbances in

PDK1-deficient cells, lineage depleted control and Pdk1Hem-KO total BM were cultured with or without NAC.

After 24 hours the cells were harvested and immunophenotyped. Pdk1Hem-KO cells treated with NAC had

decreased levels of lineage positive cells (Figure 22 B; 81.5±18.3% in Pdk1Hem-KO compared to 67.2±3.3%

in Pdk1Hem-KO with NAC) and increased numbers of lineage negative cells (Figure 22 C; 18.0±1.8% in

Pdk1Hem-KO compared to 30.9±3.1% in Pdk1Hem-KO with NAC) when compared to the untreated mutant

cells. These results suggest that treatment with NAC was able to block lineage negative cells from

differentiating into lineage positive cells (Table 5).

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Figure 22: NAC treatment suppresses differentiation and proliferation of Pdk1Hem-KO HSPCs.

(A) Representative flow cytometry plots of lineage negative cells cultured for 24h with PBS or NAC. (B) Culturing Pdk1Hem-KO lineage negative cells with NAC in vitro for 24h resulted in reduced lineage positive cells (C) and increased lineage negative cells when compared to Pdk1Hem-KO cells cultured with PBS. (D) Representative flow cytometry blots of HSCs (CD150+CD48-LSK) from mice treated with PBS or NAC. (E) Pdk1Hem-KO mice treated with NAC retained lineage negative cells although not to significance. (F) Pdk1Hem-KO mice treated with NAC have increased LK cells, (G) fewer LSK cells, and (H) increased HSCs (CD150+CD48-LSK) when compared to Pdk1Hem-KO mice treated with PBS. In vitro data are representative triplicate wells from two independent experiments. In vivo data are a merge of three independent experiments and are represented as mean ± SEM. Control: n = 4; control + NAC: n = 6; Pdk1Hem-KO: n = 5; Pdk1Hem-KO + NAC: n = 6. Two-tailed paired (in vitro) and unpaired (in vivo) Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).

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The in vitro NAC results were promising but it still did not provide evidence whether this

mechanism worked in vivo as well. To assess whether BM lineage defects could be rescued in vivo, I

injected control and Pdk1Hem-KO 2-week-old mice with 100mg/kg NAC or PBS daily for 4 weeks. After

treatment, BM was harvested and immunophenotyped for HSPCs. Compared to untreated Pdk1Hem-KO

mice, mutant mice treated with NAC had increased numbers of lineage negative cells, increased numbers

of LK cells, and reduced LSK cells (Figure 22 E – G, Table 6). Most importantly, treating Pdk1Hem-KO mice

with NAC increased the numbers of CD150+CD48-LSK (Figure 22 D & H, Table 6). This suggests that

NAC is able to prevent hyperproliferation and increased differentiation of Pdk1Hem-KO cells. Since NAC is

an antioxidant, I wanted to know whether the rescue of HSPC numbers was due to ROS scavenging by

the drug. To determine whether retention of CD150+CD48-LSK numbers in Pdk1Hem-KO treated with NAC

were due to reduced ROS levels, BM cells were stained with CM-H2DCFDA, that detects ROS as a

reflection of oxidative stress. The results revealed that while the mean florescence values of both control

and Pdk1Hem-KO cells were reduced compared to the untreated mice in all BM fractions, only ROShigh cells

in LK were decreased at significant levels (Table 7). This implies that NAC treatment is able to partially

rescue the detrimental ROS phenotype. Therefore, ROS appears to increase in Pdk1Hem-KO HSCs and

accounts for the loss in quiescence and subsequent increase in proliferate.

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Table 5: In vitro NAC treatments limits differentiation of Pdk1Hem-KO lineage negative cells.

Sorted control and Pdk1Hem-KO lineage negative cells were cultured with PBS or NAC for 24h and then stained for cell-surface markers to assess hematopoietic fractions. Data are representative of triplicate wells from two independent experiments. std = standard deviation, SEM = standard error margin. Paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).

 

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Table 6: In vivo NAC treatment impedes proliferation of Pdk1Hem-KO progenitors.

Control and Pdk1Hem-KO mice were either treated with PBS or 100mg/kg NAC for 4 weeks and then dissected and BM was stained for cell-surface markers to assess hematopoietic fractions. Data are representative of 3 independent groups (control: n = 4, control + NAC: n = 6, Pdk1Hem-KO: n = 5, Pdk1Hem-KO + NAC: n = 6). std = standard deviation, SEM = standard error margin. Unpaired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).

 

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Table 7: In vivo NAC treatment significantly reduces ROS levels in Pdk1Hem-KO LK.

Control and Pdk1Hem-KO mice were either treated with PBS or 100mg/kg NAC for 4 weeks. Values represent the mean percentage of gated ROShigh cells. Data are representative of 3 independent groups (control: n = 4, control + NAC: n = 6, Pdk1Hem-KO: n = 5, Pdk1Hem-KO + NAC: n = 6). std = standard deviation, SEM = standard error margin. Unpaired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).

 

Discussion

In summary, microarray data of Pdk1Hem-KO HSPCs suggest that they are unable to respond to

oxidative stress and indeed, there is accumulation of ROS in these cells. Furthermore, progenitor cells

are more proliferative, have higher levels of DNA damage, and increased cell death in the absence of

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PDK1. Interestingly, in vivo NAC treatment aimed to inhibit ROS is only able to partially rescue the ROS

phenotype; however, NAC treatment increases retention of HSPC populations both in vitro and in vivo.

Microarray

While the microarray led to exciting insight on the physiological status of PDK1-deficient cells,

regrettably, the results of the microarray are not from a pure HSC population. The LSK fraction is

composed of both HSCs and MPPs so the gene expression data is representative of numerous cell

populations rather than purified ones. This could allow for misinterpretation of which gene sets are

actually differential in the Pdk1Hem-KO mouse. For instance, a set of genes could be increased in the HSCs

but severely decreased in MPPs and the microarray would not be able to discriminate separate profiles

because my samples contained both populations. Unfortunately, due to the number of available mice and

extremely low HSC count in the Pdk1Hem-KO mouse, I was unable to obtain enough RNA when I isolated

only HSCs (LSKF-). Indeed, the results from this one microarray are a pool of enriched LSK that were

sorted from 5 animals in each group. Currently, a more accurate method for determining broad scale

gene expression levels as well as other components of the cellular transcriptome is through RNA-

sequencing. Performing this assay would be contingent on obtaining enough material; however, with the

latest technologies in single cell RNA-seq, RNA-seq on LSKF- or even LSKF-CD34-CD150+CD48- cells

would provide a more accurate representation of Pdk1Hem-KO HSC gene expression which could then be

validated at the protein and signaling levels. Despite the limitations, the microarray did serve to provide

an answer regarding why Pdk1Hem-KO mice were loosing quiescence: low response to oxidative stress.

This was corroborated by subsequent experiments measuring ROS and superoxide ROS levels.

PDK1 is an AGC kinase that is capable of activating a variety of downstream signaling pathways

to govern cellular processes. Earlier studies have shown that PDK1 is important for NFkB activation in T

cells (Lee et al., 2005). In addition, loss of the NFkB subunit p65 (RelA) causes increased HSPC cycling,

loss of HSPC differentiation ability (Stein and Baldwin, 2013). Microarray analysis of NFkB targets in

Pdk1Hem-KO LSK cells yielded no significant changes compared to the control (data not shown). Based on

this, it is possible that in HSCs, PDK1 is indispensible for NFkB activation; however, more extensive

studies assessing NFkB-signaling in the Pdk1Hem-KO BM need to be performed in order to determine this.

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Furthermore, other groups as well as this study (Chapter 5) have shown that the loss of PDK1 also leads

to a reduction in AKT-signaling. In addition, loss of the AKT/FOXO-signaling pathway in HSCs has been

shown to cause a decrease in HSPCs due to increased cycling and apoptosis. Furthermore,

FOXO1/3a/4-deficient HSCs were found to have increased ROS levels that could be reverted with the

addition of an antioxidant drug (Miyamoto et al., 2007; Tothova and Gilliland, 2007). Given that many of

the stem cell physiological properties are altered in both Pdk1Hem-KO HSCs and FOXO-deficient HSCs and

that Pdk1Hem-KO HSCs have reduced AKT-signaling, I also looked at FOXO targets with the microarray

and did not see any differences between control and Pdk1Hem-KO LSK. Although it is believed that FOXO is

highly regulated by AKT, there are other non-AKT-mediated PTMs that can regulate FOXO activity in the

nucleus and it is possible that FOXO is being regulated by a PDK1/AKT-independent mechanism (Obsil

and Obsilova, 2008). Thus would make sense that a loss of AKT-signaling should lead to an increase in

FOXO activity; however, I do not see an increase in FOXO targets. While the microarray provides

insights, further experiments immunoblotting for FOXO with Pdk1Hem-KO cells or phosflow of HSCs would

be useful to determine a comprehensive understanding of the downstream consequences of the loss of

PDK1.

Mitochondrial bioenergetics

The results from labeling HSCs with CM-H2DCFDA provided a physiological explanation for the

Pdk1Hem-KO phenotype. Using CM-H2DCFDA, I was able to assess that there are more cells that are

considered to be expressing high levels of ROS. Interestingly, various dyes exist that target organelle

specific ROS as well as the different chemical components responsible for ROS, H2O2 and NO. It is

important to note that there are genetic methods to alter cells so that they emit florescence (Woolley et

al., 2013); however, in the HSC field, the most commonly used method to detect ROS levels is staining

cells with the florescent CM-H2DCFDA dye which is activated upon reduction and it is detected via flow

cytometry (Chen et al., 2008; Ito et al., 2004; Ito et al., 2006; Jang and Sharkis, 2007; Juntilla et al., 2010;

Kocabas et al., 2012; Lee et al., 2010; Miyamoto et al., 2007; Nakada et al., 2010; Qian et al., 2016;

Rimmele et al., 2015; Tothova and Gilliland, 2007; Warr et al., 2013; Yu et al., 2013). Employing this

method makes use of the fact that an isolated population of cells can be identified either by cell surface

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markers or by purification via cell sorting and the overall florescence within a population can be measured

by mean florescence intensity. This provides a quantifiable measure of the number of cells expressing a

certain intensity, based on the florescence of a particular dye, which in this case is a readout of ROS. In

addition, unlike many other cell types, hematopoietic cells are free floating allowing them to easily be

studied by flow cytometry rather than adhering, fixing, and then staining the cells in preparation for

analysis by another methods such as confocal analysis of immunohistochemistry, which is more time

consuming, more labor intensive, and more expensive. Furthermore, using flow cytometry analysis, high

ROS expressing cells can be distinguished from low ROS expressing cells based on the level of

florescence intensity. This is particularly useful given that some studies have shown that the level of HSC

quiescence is based on the amount of ROS within the niche (Jang and Sharkis, 2007; Ludin et al., 2014).

Furthermore, since ROS has been shown to cause a variety of cellular events, such as protein

modification and DNA damage, I verified this in the Pdk1Hem-KO cells. The purpose of this study was to

determine why PDK1-deficient HSCs are no longer quiescent with a focus on what the cellular

consequences are for increased ROS, and not the exact location where ROS generation occurs.

However, it should be noted that there are reports that mitochondrial ROS can be measured by staining

with MitoTracker and performing confocal microscopy (Li et al., 2013).

Since CM-H2DCFDA measures general ROS levels, I also additionally assayed for ROS

generated by superoxide. Although the trend seen with mitoSOX staining was similar to the CM-

H2DCFDA staining, it was only significant in the lineage-negative population. It is possible that assaying a

greater number of animals would provide a more accurate assessment and may eventually lead to

significant differences. Conversely, there may actually be less superoxide ROS in the HSPC fractions.

Interestingly, ROS and superoxide-specific ROS have been shown to promote differentiation (Sauer et

al., 2001; St Clair et al., 1994; Zhang et al., 2013); therefore, significant expression in the lineage-

negative fraction could be indicative of increased differentiation. Furthermore, it has been shown that

myeloid cells need higher levels of ROS in order to differentiate properly (Tothova and Gilliland, 2007;

Zhang et al., 2013). Thus, it is plausible that detection of superoxide ROS is highest in the lineage-

negative fraction because Pdk1Hem-KO BM is predominately composed of myeloid progenitors that are

known to readily generate ROS during differentiation (Chapter 2). In this study, I did not assay for free

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radical production due to H2O2. However, when I incubated control and Pdk1Hem-KO BM with H2O2 as a

positive control and then stained with CM-H2DCFDA, I saw significantly increased ROShigh cells in the

control and a slight shift in the Pdk1Hem-KO BM (data not shown). Firstly, this suggests that the Pdk1Hem-KO

cells were already producing almost maximum ROS levels. Secondly, CM-H2DCFDA detects free radicals

produced by H2O2 so it is not necessary to measure what percentage of ROS is due specifically to H2O2

especially since the mitoSOX staining is in line with the CM-H2DCFDA staining. Taken together, detecting

ROS levels with CM-H2DCFDA provided novel insight into why Pdk1Hem-KO HSCs were cycling more.

Within a cell, one of the primary sites of ROS generation is in the mitochondria. ROS is typically

generated when there is mitochondrial distress. Analysis of Pdk1Hem-KO mitochondrial biogenesis would be

interesting. Preliminary results suggest that Pdk1Hem-KO BM populations have greater mitochondrial mass

when assayed via flow cytometry with a fluorescent dye, MitotrackerGreen. Although this method can

determine the relative mass per cell, it cannot distinguish between the number mitochondrial species

within a cell. Nonetheless, the amount of mitochondrial mass could be explanation for increased

metabolic differences between the control and Pdk1Hem-KO mice and can be used in combination with

other assays to determine the energetic consequences in the mutant cells. A greater number or larger

mitochondria would provide an explanation for increased ROS levels or delineate that the Pdk1Hem-KO

mitochondria are able to generate higher levels ROS per equivalent amount of mitochondria.

Interestingly, treating cells with an electron transport chain inhibitor, Antimycin A, reverted the

mitochondria mass levels back to the control. I did not see a correlated reduction in ROS in this

experiment but that could be due to Antimycin A that is known to cause high levels of H2O2, which can

lead to more ROS. I have only performed this experiment once so further replicates are needed for

verification and statistics. An alternative to assess for mitochondrial mass would be to look for the amount

of mtDNA per cell or to assess for TAM20 or TFAM by protein expression. Similar to the signaling studies,

protein expression by immunoblots is difficult given the number of HSCs per mouse. Furthermore,

assaying for mitochondrial membrane potential through the use of available dyes such as MitotrackerRed

would provide insights into the ability for ATP generation and OXPHOS activity, a major producer of ROS

(Bigarella et al., 2014; Suda et al., 2011). In addition, stress tests can be performed using drugs to target

the oxidation chain to measure cellular respiration or promote and inhibit glycolysis to determine the

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glycolytic output of a cell. These studies would provide an in depth understanding of how loss of PDK1

impacts mitochondrial processes.

Another readout of oxidative stress is autophagy. Autophagy is a part of the lysosome pathway

whereby components within the cell are degraded to prevent cellular damage. Interestingly, loss of ATG7,

a component of the autophagosome causes loss of reconstitution ability of HSCs in transplantation, loss

of lymphoid and myeloid production, increased ROS, and increased mitochondrial mass (Mortensen et

al., 2011). A specific form of autophagy, known as mitophagy, is instrumental in ensuring that aberrantly

active mitochondrial are inactivated. If Pdk1Hem-KO mitochondria are malfunctioning and the machinery to

maintain mitochondrial quality control is affected, then this would provide an explanation for the buildup of

ROS levels. Taken together, further experiments into mitochondria bioenergetics and mitophagy may

provide a rationale for why ROS is elevated in Pdk1Hem-KO cells and may further provide insights for

PDK1-regulation in metabolism.

DNA damage/apoptosis assays

While the results of the p-H2A.X staining showed increased phosphorylation of double stranded

breaks in the lineage negative fraction, the trend is not significant in the HPSC fractions. One reason may

be due to the lower numbers of cells. Only three animals were useable to assess for CD150+CD48-LSK

cells while these cells were not detected in two additional animals. Although PDK1 ablation showed

dramatic effects in the BM, unfortunately the Pdk1Hem-KO mice have very few HSCs, thus limiting the

number of cells we can work with. I assessed p-H2A.X levels via flow cytometry and an alternative

method would be to perform IHC and count the number of p-H2A.X foci per cell (Flach et al., 2014). This

may provide visual representation of p-H2A.X foci in each HSC and this can be counted by a researcher

for statistical numbers; however, since HSCs are hematopoietic cells and are thus free floating, it is just

as easy to stain and asses by flow cytometry in order to generate a readout of the number of cells that

express a certain level of florescence associated with positive stain. Furthermore, since cell surface

markers can be used to distinguish subpopulations, data can be quickly and simultaneously obtained

regarding a variety of cell types proving this method cost and time effective. Since p-H2A.X stain was not

statistically significant in the LT-HSC population, a possible explanation is that DNA damage is not

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increased in PDK1-deficient HSCs. This would suggest that Pdk1Hem-KO HSCs instead proliferate into

progenitors where consequences manifest themselves, such as greater numbers of DSBs (lineage

negative) and apoptosis (LK and lineage negative).

ROS reversion

Intriguingly, I saw an increase of phosphorylated p38 expression by immunoblots from treated

with PDK1 inhibitor and an increase in p38 transcriptional targets in Pdk1Hem-KO LSK from the microarray

analysis. Interestingly, a pilot study where I cultured sorted LK and LSK from control and Pdk1Hem-KO mice

with 10uM p38 inhibitor (data not shown, SB203590, Selleckchem). This treatment did not lower ROS

levels as shown in other studies (Jang and Sharkis, 2007). This suggests that the increase of p38 activity

is a readout of ROS rather than a cause of ROS. This also suggests that there is another target that is

responsible for reducing ROS levels in the PDK1-pathway, which I will expand upon in Chapter 5.

While the results of the in vitro and in vivo NAC treatment studies show that antioxidant exposure

results in retention of HSPC numbers, it was not able to significantly decreases ROS levels as seen in

other studies (Tothova and Gilliland, 2007). Interestingly, other groups have used an inducible deletion

model (Mx-Cre) so that treatment of NAC was administered the day after deletion. In this study, I used

Vav-Cre, which is a temporal and spatially specific hematopoietic marker where vav expression occurs as

early as embryonic day 10.5. The mice in this study were treated with NAC on post-natal day 14 when

there was a large time period in which the Pdk1Hem-KO mice were not treated and this could explain the

partial rescue phenotype. To circumvent this, pregnant mothers could be treated as well as administered

NAC in their drinking water. In addition, pups can be treated from birth so that oxidative stress can be

suppressed during critical periods of lymphoid development. Furthermore, crossing the Pdk1Flox mouse

with Mx-Cre would allow me to treat animals with NAC from the onset of deletion.

It is important to note that the cell has a variety of mechanisms to counteract the accumulation of

ROS. One mechanism is that cell has antioxidants and there are many that have been studied such as:

vitamins, inorganic antioxidants, synthetic antioxidants, and plant-derived polyphenols. One is the

enzyme superoxide dismutase (SOD), which catalyzes superoxide anions into H2O2 and oxygen.

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Catalase is also a ROS scavenger that is found in the cell (Li et al., 2013). In addition, a group of

enzymes, gluthathione peroxidase, degrades H2O2 and peroxides into alcohols. Vitamin C and E have

also been suggested to limit the consequences of an abundance of ROS (Gorrini et al., 2013). Since

some studies need ROS scavengers to cross the blood brain barrier and some biological compounds

cannot cross the mitochondrial membrane, many synthetic forms of antioxidants have been created to

directly target mitochondria. For instance MitoVit-E (Vitamin E) has been shown to decrease ROS

production and limit apoptosis in endothelial cells and it was created to selective inhibit oxidative damage

due to mitochondria (Sheu et al., 2006). Another drug, MitoQ, makes use of the fact that ubiquitnone and

its derivatives have known antioxidant effects. MitoQ is specifically taken up by energized mitochondria

and specifically blocks H2O2 activation of apoptosis (Li et al., 2013; Sheu et al., 2006). Furthermore,

MitoPBN specifically targets ROS due to the uncoupling of proteins within the electron transport chain.

PBN does not react with superoxide so when used in combination with MitoVitE and MitoQ, which do

block superoxide, inferences can occur about what types of ROS are being generated from the

mitochondria (Sheu et al., 2006). Another drug, MitoPeroxidase, when used in combination with the

glutathione peroxidase-like ebselen, can inhibit mitochondrial respiration, decrease the mitochondrial

membrane potential, leading to reduced ROS (Sheu et al., 2006). Since, in this study, it is unclear where

the source of ROS is, either from mitochondria or other sites within the cell, I chose to use a general ROS

scavenger drug, NAC, to revert the effects of ROS in HSCs. It is important to note that some studies

show that NAC was created to specifically target mitochondria (Sheu et al., 2006) while it has also been

reported to be a general scavenger (Tothova et al., 2007). In HSCs, NAC has commonly been used to

rescue detrimental effects of ROS in a variety of different studies (Chen et al., 2008; Gurumurthy et al.,

2010; Ito et al., 2004; Ito et al., 2006; Kocabas et al., 2012; Lee et al., 2010; Miyamoto et al., 2007; Qian

et al., 2016; Rimmele et al., 2015; Tothova and Gilliland, 2007), many of which find that the loss of certain

genes associated with various pathways including PI3K/mTOR have increased ROS levels due to

mitochondrial dysfunction. Further experiments using drugs to directly inhibit mitochondrial ROS in

Pdk1Hem-KO cells are needed to attribute elevated ROS levels solely to mitochondrial causes. Taken

together, the results from these experiments show that Pdk1Hem-KO HSCs have increased oxidative stress

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which can be partially reverted through NAC treatment and thus provide a cellular explanation for why

PDK1-deficent HSCs loose quiescence.

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Chapter 5: Loss in PDK1 results in reduced Hif1α expression.

Introduction

HIF1α regulation

The bone marrow niche is naturally a hypoxic environment; therefore, HSCs must have

intracellular machinery that allows for their survival in low oxygenic conditions. A group of DNA-binding

proteins, known as Hypoxia Inducible Factors (HIFs), orchestrates the expression of several genes that

allow the cell to cope with a hypoxic environment such as controlling energy production, antioxidant

transcription, angiogenesis, oxygen supply, proliferation, and apoptosis (Dengler et al., 2014). These

mechanisms provide the basis for quiescence as a means for HSCs to survive throughout the lifetime of

mammals.

HIFs are transcription factors that heterodimerize with each other in order to regulate transcription

of target genes. They are basic helix-loop-helix DNA-binding proteins and in mammals, they are

separated into alpha and beta subunits. In mice, there are three genes that encode the alpha subunits:

Hif1α, Hif2α (EPAS), and Hif3α. The beta subunit, HIF1β or aryl hydrocarbon receptor 1 (ARNT), is

constitutively expressed and can bind to any of the alpha subunits with varying effects on nuclear

translocation and transcription. Heterodimerization only occurs if the alpha subunit is stabilized, which

occurs during hypoxic conditions. In normoxia, the alpha subunits are hydroxylated at specific proline and

asparagine residues by prolyl hydroxylases, inhibiting their transactivation functions and targeting them

for degradation. When oxygen is low, hypoxia inhibits prolyl hydroxylases allowing HIFαs to accumulate

and dimerize with HIF1β to initiate transcription (Dengler et al., 2014; Lee et al., 2004). HIF1α is

expressed in all cell types whereas HIF2α and HIF3α appear to be tissue-specific. HIF1α and HIF2α are

structurally similar and appear to activate similar targets (Dengler et al., 2014). Not much is known about

HIF3α, but evidence suggests that it exists in many variant isoforms, some of which act to inhibit HIF1α

and HIF2α (Kaelin and Ratcliffe, 2008). The most well known negative regulator of HIF1α is inhibitory

PAS domain (IPAS) domain protein and it is thought that HIF1α can regulate IPAS transcription, which

IPAS can then, in turn, generate a negative feedback loop by inhibiting HIF1α (Makino et al., 2001;

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Makino et al., 2007). Taken together, the HIF family works in both in concert and opposition to each other

to respond to low oxygen conditions.

HIF1α activity can be regulated through various mechanisms that act to ensure HSC survival in

the niche. First, other transcription factors, such as Meis1, can alter the transcription of HIF1α (Kocabas

et al., 2012). Second, translation of HIF1α protein could be affected. Third, oxygen levels can impact

HIF1a stabilization (Dengler et al., 2014; Suda et al., 2011). In addition, modifications of redox conditions

can activate HIF1α expression (Fukuda et al., 2007; Suda et al., 2011). Also, interactions with co-

activators, such as ARNT or HIF3α, can affect HIF1α translocation into the nucleus (Dengler et al., 2014;

Makino et al., 2007). Furthermore, other transcription factors, such as Cited2, can compete with HIF1α for

the p300 subunit to prevent DNA binding (Bakker et al., 2007; Kranc et al., 2009; Majmundar et al., 2010).

Lastly, HIF1α expression can be regulated by phosphorylation from other signaling molecules, such as

members of the PI3K/AKT-pathway (Flugel et al., 2007; Mottet et al., 2003). Thus, the regulation of HIFα

occurs from the transcriptional level with feedback loops to the protein interactions and signal

transduction pathways within the HSC.

Hif1α regulates ROS expression

Regulation of metabolic activity of HSCs is vital for the health of the cell because it is required to

maintain self-renewal and multipotent differentiation abilities, two disparate functions. In order to protect

HSCs from a variety of physiological stresses, including oxidative stress, there are cellular regulators that

help maintain homeostasis. One defense mechanism is that the cell can remain in quiescence as it

protects a cell from accumulating mutations that can occur during division. It has been shown that

hypoxia induces quiescence in cultured HSCs (Hermitte et al., 2006; Shima et al., 2010) and it has been

suggested that most LT-HSCs reside within the hypoxic endosteal BM niche (Arai et al., 2004; Calvi et al.,

2003; Zhang et al., 2003). Taken together, it appears that HSCs are able to use hypoxia as a mechanism

to maintain quiescence.

Response to oxygen levels and hypoxic conditions within a cell is predominantly governed by the

hypoxia inducible factor-1 (HIF1) transcription factor. In low oxygenic conditions, the HIF1α isoform is

stabilized through its VHL domain and can then form a heterodimer with HIF1β and then translocate to

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the nucleus to initiate transcription of hypoxic response genes (Kaelin and Ratcliffe, 2008; Semenza,

2010). HIF1α is highly expressed in HSCs and conditional knockouts of HIF1α in HSCs have reduced

quiescence (Simsek et al., 2010; Takubo et al., 2010). Furthermore, deletion of ARNT caused increased

HSC apoptosis and loss of long-term reconstitution ability and double knockouts of HIF1α and HIF2α

phenocopied the ARNT phenotype (Krock et al., 2015). However, excessive levels of HIF1α led to HSC

exhaustion suggesting that a fine balance must be maintained (Suda et al., 2011). Nonetheless, these

experiments demonstrated that the role of HIF1α in HSCs is essential because it is needed to reprogram

energy output from oxidative to glycolytic pathways (Lou et al., 2011; Semenza, 2009). This is interesting

because oxidative phosphorylation by mitochondria tend to generate high levels of ROS, which mandates

intracellular responses that can cause destabilization of the cell. Indeed, loss of HIF1α in mouse

embryonic fibroblasts (MEFs) resulted in increased ROS production in hypoxic conditions (Fukuda et al.,

2007; Suda et al., 2011). Furthermore, it has been shown that ROSlow HSCs retain self-renewal properties

while ROShigh HSCs experience exhaustion (Jang and Sharkis, 2007). In addition, ROS expression has

been shown to inactivate the destabilization enzymes that tag HIF1α for degradation therefore, causing

HIF1α transcriptional activation, which can then regulate ROS levels (Bonello et al., 2007; Kaelin, 2005;

Klimova and Chandel, 2008). Given the link between hypoxia, quiescence and ROS, I speculated that

defects in HIF1α may affect the PDK1-signaling pathway and this may explain the high levels of ROS and

loss of quiescence found in the Pdk1Hem-KO HSCs.

PI3K/AKT/mTOR-signaling

Extrinsic cues from the BM niche are transduced primarily from membrane cytokine receptors to

the nucleus through intermediary signaling components. The PI3K/AKT-signaling pathway is a major

pathway involved in the regulation of cell growth, survival, and proliferation. This pathway is activated

when cytokines or other stimuli, such as growth factors, bind to receptor tyrosine kinases or when

intracellular scaffolding proteins, such as IRS1, that are bound to the receptor cytosolic tails help recruit

and activate PI3K. PI3K can then phosphorylate PIP2 to PIP3, and thus allowing for either PDK1 or AKT1

recruitment to the plasma membrane. Of note, PTEN, a phosphatase, can revert PIP3 to PIP2 therefore

acting as a negative regulator of the pathway. Although AKT can be directly activated by PIP3, one of the

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prevailing dogmas is that PDK1 is needed to phosphorylate the activation loop of AKT at Thr308. For full

AKT activation, the second phosphorylation site, Ser473, must be phosphorylated, which is predominantly

accomplished by the mTOR2 complex. Once AKT is phosphorylated, it can lead to activation of the

mTOR1 complex by inhibition of TSC1/2 that then releases Rheb from inhibiting mTOR1, and thereby

leading to the activation of downstream S6K by phosphorylation at Thr389. Interestingly, PDK1 is also

responsible for phosphorylating S6K at Thr229 indicating that PDK1 regulates PI3K-signaling at multiple

levels and can regulate targets independently from AKT (Pullen et al., 1998). Furthermore, it is well

established that AKT phosphorylates glycogen synthase kinase-3 (GSK3), causing the inhibition of GSK3

that then regulates several pathways including limiting apoptosis (Cross et al., 1995). Interestingly, GSK3

has been shown to phosphorylate the VHL domain on HIF1α, thus causing its degradation (Flugel et al.,

2007; Mottet et al., 2003). To prevent over-activation of the PI3K/AKT-pathway, active mTORC1 and S6K

can phosphorylate the scaffolding protein IRS1, impeding PI3K-signaling (Manning and Cantley, 2007).

While many of the signaling events in this cascade have been well established, I wanted to determine

whether the loss of PDK1 was perturbing AKT-signaling in the Pdk1Hem-KO mice. This would allow me to

gain insight into the molecular mechanisms that cause reduced quiescence and increased proliferation in

the mutant HSCs.

Members of the PI3K/AKT/mTOR-pathway can regulate HIF1α expression, which can support

HSC quiescence. For instance, the inhibition or loss of GSK3 induced HIF1α expression and the over-

activation of GSK3 caused HIF1α depletion in HepG2 cells (Flugel et al., 2007). Furthermore, a role for

mTORC1 and its downstream substrate, S6K has been linked to increased HIF1α expression by

regulating its transcription and translation (Duvel et al., 2010; Hudson et al., 2002; Laplante and Sabatini,

2012). Interestingly, both GSK3 and mTORC1 are separately activated by the AGC kinase AKT therefore,

the loss of AKT could lead to decreased HIF1α expression. Given that PDK1 is responsible for

phosphorylating the activation loop on AKT, I hypothesized that PDK1 may mediate HIF1α expression by

modulating the downstream signaling events in the PI3K-pathway. My approach to determine the role of

PDK1 in HIF1α expression will use in vitro and in vivo approaches taking advantage of PDK1-deficient

mice.

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Results

Pdk1Hem-KO mice have reduced Hif1α expression

While partial rescue of HSC cell numbers through NAC treatment was encouraging (Chapter 4),

it did not explain why NAC-treated animals still maintained high levels of ROS (Table 7). During steady-

state conditions, ROS is predominantly produced by oxidative phosphorylation (OXPHOS) during

mitochondrial energy production. When there is excessive oxidative stress, negative feedback

mechanisms activate HIF1α, a transcription factor that responds to hypoxic stress, to reduce ROS by: (1)

activating transcription of antioxidants and (2) shunting the primary mode of energy production from

OXPHOS to glycolysis, which produces less ROS. To determine whether this feedback system was

abrogated in the Pdk1Hem-KO mouse, I first assessed whether PDK1-deficient cells had abnormal levels of

HIF1α that translocated to the nucleus. Sorted control or Pdk1Hem-KO HSCs (LSKF-) and progenitor (LK)

cells were pipetted onto poly-D-lysine coated plates and cultured for 4 hours in hypoxic conditions

(5%O2/5%CO2/90%N2). HIF1α nuclear expression is associated with transcriptional activation, therefore

HIF1α was evaluated by immunohistochemistry and captured images showed the mean florescence

intensity (MFI) levels were reduced in Pdk1Hem-KO HSCs (Figure 23 A & C; 2.4±0.3 in control compared to

1.2±0.1 in Pdk1Hem-KO) and the progenitor cells (Figure 23 B & C: 3.0±0.2 in control compared to 0.8±0.03

in Pdk1Hem-KO). These results suggest that under hypoxic conditions, PDK1-deficient cells have reduced

levels of HIF1α in the nucleus and are thus unable to initiate the transcriptional machinery that would lead

to reduced levels of ROS expression.

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Figure 23: HIF1α is reduced in Pdk1Hem-KO cells during hypoxic conditions.

(A) Representative confocal images of IHC for HIF1α expression in HSCs (LSKF-) and (B) myeloid progenitors (LK) that have been incubated in 5% O2 for 4h show reduced HIF1α in Pdk1Hem-KO cells. (C) Quantification of mean florescencent intensity (MFI) of HIF1α reveals reduced expression in Pdk1Hem-KO LSKF- (control: n = 18, Pdk1Hem-KO: n = 19) and LK (control: n = 45, Pdk1Hem-KO: n = 70) fractions. Scale bars indicate 10um. Data are a merge of two independent experiments and are represented as mean ± SEM. Two-tailed unpaired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).

Inhibition of HIF1α in Pdk1Hem-KO cells

The reduced HIF1α expression in the Pdk1Hem-KO cells was intriguing but the consequence of

limited nuclear expression of HIF1α was not clear. There are a few possible explanations for reduced

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nuclear HIF1α expression in PDK1-deficient HSCs: (1) there is less transcription of the Hif1α gene, (2)

Hif1α is transcribed but not translated, and (3) HIF1α is sequestered in the cytoplasm for degradation and

therefore cannot activate its downstream promoter targets in the nucleus.

To investigate why HIF1α was decreased in the mutant, I first quantified Hif1α mRNA in the BM.

In total BM, qRT-PCR revealed that Pdk1Hem-KO Hif1a mRNA is present at low levels similar to the control,

whereas expression of Arnt is slightly, yet significantly, reduced, and that Hif2α mRNA is increased

(Figure 24 B). Because Pdk1Hem-KO BM is composed of an abnormal proportion of cell types, the low

abundance of Hif1α mRNA may be due to the limited cells that express Hifs. I next examined Hifs in

sorted LSK and LK fractions and I found increased levels of Hif1α mRNA (Figure 24 C & D). These

results suggest that transcription of Hif1α is unaffected or even increased in the LSK and LK fractions

suggesting that there is a possible disturbance in translation of the transcript or post-translational

problems. It is possible that there is reduced nuclear HIF1α in the Pdk1Hem-KO mouse because HIF1α

protein is not being translated. Of note, some of the translational machinery must be in tact since there

translation of HIF1α is occurring because HIF1α expression is detectable in the IHC confocal images in

HSCs (LSKF-) and progenitors (LK) (Figure 23 and data not shown). Because detecting HIF1α protein

levels by IHC implies that there must be translation of mRNA protein, I speculated that HIF1α is present in

the cytosol but it is being hindered from entering the nucleus. In assessing other HIFs in cell fractions of

the BM, analysis of Hif3α mRNA in LSK and LK showed large increases compared to the control (Figure

24 B & C). In contrast, Hif2α did not reveal a large difference and there was no difference between control

and Pdk1Hem-KO mRNA. This result was fascinating because in other systems, such as eye

vascularization, HIF3α has been shown to inhibit HIF1α expression (Makino et al. 2001, 2007)

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Figure 24: Hif mRNA expression in Pdk1Hem-KO cells.

(A) Schematic of HIF1α activation by ROS. Oxidative stress causes increased HIF1α protein, promoting binding to ARNT. The HIF1α/ARNT heterodimer translocates to the nucleus were it transcriptionally activates hypoxic response genes. (B) qRT-PCR showed reduced Arnt, no change in Hif1α, and increased Hif2α mRNA in Pdk1Hem-KO total BM. (C) qRT-CR of Hif1α, Hif2α, and Hif3α in LSK and (D) LK revealed increased Hif3α mRNA expression. qRT-PCR expression was normalized to Hprt and control. Total BM: n = 3, LSK: n = 2, LK: n = 2. Data are representative of two-wells from each experiment. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).

This finding led me to consider three possibilities that could explain the results (Figure 25 A). The

first is that HIF1α is able to bind to its coactivator, ARNT, heterodimerize and translocate to the nucleus to

activate Hif transcriptional targets. This scenario may explain what is occruing in the control BM. The

second scenario is that HIF3α is directly competing with HIF1α for binding with ARNT (Dengler et al.

2014). If this happens, then HIF3α/ARNT can translocate to the nucleus altering Hif targets resulting in

low transcription. Based on reduced Arnt mRNA in total BM (Figure 24 B) this seems unlikely, although

Arnt mRNA levels and ARNT protein expression must be verified in Pdk1Hem-KO HSCs. The third possibility

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is that HIF3α, specifically the inhibitory pas domain (IPAS) isoform, is directly binding to HIF1α and

sequestering it in the cytosol for degradation (Makino et al. 2001). The later scenario, is an attractive

hypothesis because it explains why nuclear HIF1α is lower in PDK1-deficient cells and as well, implies

that there are downstream consequences to the loss of HIF1α-signaling, such as reduced transcription of

antioxidant genes.

The previous experiment reveals that the levels of Hif3α mRNA is up in Pdk1Hem-KO LSK and LK;

however, these results were using primers that did not distinguish between IPAS and other isoforms of

Hif3α. To resolve which mechanism inhibits Hif1α function in PDK1-deficient cells, qRT-PCR was

performed with total BM using primers specifically designed to recognize the IPAS isoform, forward primer

in exon 4a and reverse primer in exon 6 (Figure 25 B), and the full length Hif3α (Hif3αFL) isoform, forward

primer in exon 11 and reverse primer in exon 13. Independent experiments from three different pairs of

mice show increased Hif3αFL and IPAS (Figure 25 C) mRNA. To confirm that the primers were amplifying

Hif3αFL and IPAS, sequencing of PCR amplification product was performed, with the aforementioned

primers, on both control and Pdk1Hem-KO BM. Amplicons were then gel-purified and the extracted DNA

was sent for sequencing (Figure 25 D). NCBI BLAST of sequencing results revealed that both the control

and Pdk1Hem-KO samples align with known sequences of Hif3αFL (data not shown) and Hif3α isoform1/2 or

IPAS (Figure 25 E). This data show that transcription of Hif3αFL and IPAS are increased and therefore, if

translated appropriately, IPAS could be acting to inhibit HIF1α. Future experiments to elucidate whether

HIF3α and IPAS protein expression are increased in Pdk1Hem-KO cells, and if either HIF3α or IPAS are

hindering HIF1α expression, will elucidate which scenario is occurring.

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Figure 25: Pdk1Hem-KO cells have greater IPAS mRNA expression.

(A) Schematic demonstrating how HIF3α can inhibit HIF1α. (B) Schematic of IPAS and HIF3α CDS and primer design. (C) Full length Hif3α (Hif3αFL) and IPAS mRNA transcription is increased in the Pdk1Hem-KO BM (n = 3). (D) PCR amplification using primers for Hif3αFL and IPAS of control and Pdk1Hem-KO total BM cDNA show increased expression in Pdk1Hem-KO cells. (E) BLAST of sequencing results of samples from (D) show that the primers are amplifying Hif3α isoforms 1 and 2 (n = 1). qRT-PCR expression was normalized to Hprt and control. Data are of two-wells from three independent experiments. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).  

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Pharmacological inhibition of PDK1 provides insights into downstream signaling

While the hypothesis that IPAS may bind to and inhibit the function of HIF1α is an attractive

model that explains a plausible regulatory mechanism, I wanted to determine whether there were other

post-translational events that could be disrupting HIF1α expression. The AKT/mTOR-pathway has been

implicated in the regulation of HIF1α expression (Figure 26). Indeed, PDK1 is considered to be a master

regulator of PI3K-signaling and it is thought that the primary role of PDK1 is to activate AKT via

phosphorylation. To determine whether AKT-signaling was reduced in the absence of PDK1-signaling, I

decided to treat cells with a PDK1-specific inhibitor and perform immunoblot analysis. Other groups have

demonstrated that pharmacological inhibition of PDK1 can be used to recapitulate the downstream-

signaling events (Park et al., 2013). Furthermore, due to the small number of HSCs within a mouse it is

difficult to perform immunoblots on such few cells. In addition, the Pdk1Hem-KO has reduced B cells so

protein extraction from total BM would be representative of a different composition of cell types. To

circumvent these issues and to delineate which signaling events were abrogated in Pdk1Hem-KO mice, I

used a highly specific small molecule inhibitor of PDK1, GSK2334470 (Najafov et al. 2011) in my studies.

I chose this inhibitor because GSK2334470 does not block activity of 93 other AGC kinases while also

inhibiting T-loop activation in AKT, the classical target of PDK1. Total BM was treated with or without 1uM

PDK1 inhibitor, starved for 5.5 hours or starved for 5 hours and then stimulated with 10%FBS/DMEM and

0.5ug cytokines (IL6, IL3, SCF, TPO, Flt3l) for 30 minutes. Protein cell lysates were extracted and

immunoblotting for components of the AKT/mTOR-signaling pathway were assessed for phosphorylation.

Similar to the Pdk1Hem-KO total BM immunoblot, there was reduced AKT phosphorylation at residues

Thr308 and Ser473 as well as reduced downstream p70S6K phosphorylation (Figure 27 A). Furthermore,

immunoblots of protein from wild type total BM treated with and without PDK1 inhibitor show loss of

phosphorylation of GSK3b at residue Ser9 (Figure 27 A). Overall, as expected, PI3K/AKT-signaling is

reduced in the absence of PDK1 activity.

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Figure 26: PI3K-signaling can regulate HIF1α expression.

Schematic shows that PI3K is activated when cytokines or growth factors bind to receptors. PI3K then phosphorylates PIP2 to become PIP3. PIP3 recruits PDK1and AKT to the membrane through their PH domains allowing PDK1 to phosphorylate AKT at Thr308. mTORC2 (not pictured) is needed to phosphorylate AKT at Ser473. AKT signals through mTORC1 by inhibiting TSC1/TSC2 (not pictured). mTORC1 activates p70S6K (S6K) through phosphorylation at Thr389. S6K initiates synthesis of a variety proteins needed for cell survival, such as HIF1α. PDK1 phosphorylates p70S6K at Thr229 independently of AKT activation. In addition to mTORC1 activation, AKT can inhibit GSK3 and FOXO through phosphorylation. FOXO acts to suppress reactive oxygen species (ROS) levels. Elevated ROS levels activate p38, which initiate proliferation and cell death signaling cascades that may cause DNA damage. GSK is able to inhibit HIF1α by phosphorylating the HIF1α VHL domain. During hypoxic conditions, or in response to oxidative stress, HIF1α bind with ARNT to form a heterodimer that will translocate to the nucleus to transcriptonally activate a variety of HIF1α target genes including antioxidants and glycolytic genes. The HIF3α isoform, IPAS can block the HIF1α/ARNT heterodimer by binding to HIF1a and sequestering in the nucleus for degradation.

 

PDK1 is an intermediate signaling kinase, which means that it converts signals from upstream

receptors to activate other downstream-signaling cascades. To determine whether PDK1 inhibition had

an effect on other pathways known to be activated by stem cell cytokine receptors, I examined the MAPK

pathways, which are known for integrating external signals to transcriptional programs and play roles in

proliferation, differentiation, and apoptosis (Geest et al. 2009). I found that there was very little difference

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in p-ERK and p-JNK levels in immunoblots of cells treated with and without PDK1 inhibitor (Figure 27 B).

Taken together, pharmacological inhibition of PDK1 recapitulates the loss of AKT/mTOR-signaling

reported in the literature and shows that the ERK- and JNK-signaling pathways in HSCs remain in tact.

 

Figure 27: Drug inhibition of PDK1 provides insight into downstream signaling.

(A) Total BM with or without 1uM PDK1 inhibitor (GSK2334470) was serum starved for 5.5h or serum starved for 5h and then stimulated for 30 min with 10% FBS and 0.5ug cytokines (IL6, IL3, Flt3l, TPO, SCF). Components of the PI3K/AKT/mTOR-signaling cascade were checked for phosphorylation and there was reduced AKT, p70S6K, and GSK3β phosphorylation indicating reduced signaling via this pathway. (B) Immunoblots of other known signaling pathways downstream of cytokine receptors showed no major differences in the ERK or JNK pathways. Data are representative of two or three independent experiments.

 

Reduced AKT-signaling in Pdk1Hem-KO LSK

To corroborate the findings using PDK1 inhibitor studies that PDK1-signaling affects

AKT/mTORC-signaling in vivo, control and Pdk1Hem-KO total BM were depleted of B cells to ensure similar

cell composition between the two groups and protein was immunoblotted for phosphorylated AKT at

residues Thr308 and Ser473. In both the starved and stimulated conditions, there is reduced

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phosphorylation of AKT at both residues whereas total AKT protein levels appear to be the same (Figure

28 A). Total BM has many differentiated cell types, so to ensure that the progenitor populations also had

reduced AKT activity, phosflow on sorted LK and LSK cells for phospho-AKT at T308 revealed reduced

phosphorylation in the Pdk1Hem-KO cells (Figure 28 B & C; LSK: 77.8±10.1% in control and in Pdk1Hem-KO,

LK: 75.8±18.6% in control compared to 34.9±4.8% in Pdk1Hem-KO). In the PI3K/AKT/mTOR-signaling

cascade, mTOR is activated by AKT and in turn phosphorylates p70S6K. To corroborate that there is

reduced PI3K-signaling in the Pdk1Hem-KO cells, I sorted LSK and LK and found decreased phosho-

p70S6K in the Pdk1Hem-KO cells (Figure 28 D & E; LSK: 67.3±2.4% in control and in 28.2±4.7% Pdk1Hem-

KO, LK: 62.6±4.1% in control compared to 21.8±6.1% in Pdk1Hem-KO). Taken together, loss of PDK1 results

in reduced AKT/mTOR-signaling in the myeloid progenitor fraction, LK, and the HSPC fraction, LSK.

Interestingly, loss of mTOC1 and p70S6K activation can lead to reduced transcription and translation of

HIF1α (Duvel et al., 2010; Hudson et al., 2002; Laplante and Sabatini, 2012). In light of this data, PDK1

appears to be regulating HIF1α through PI3K/AKT/mTORC1-signaling.

Previous reports have shown that activated GSK3β negatively regulates HIF1α (Chan et al.,

2001; Flugel et al., 2007; Mottet et al., 2003). AKT has been shown to inactivate GSK3 by

phosphorylating residues Ser21, GSK3α, and Ser9, GSK3β (Cohen and Frame 2001). Taken together,

this led us to hypothesize that loss of PDK1 leads to reduced AKT-signaling, allowing for the activation of

GSK3, which causes HIF1α protein levels to be downregulated. Phosflow analysis of sorted LSK

(86.9±5.7% in control compared to 40.3±7.8% in Pdk1Hem-KO) and LK (71.2±12.0% in control compared to

34.4±3.5% in Pdk1Hem-KO) for p-GSK3β(Ser9) showed less phosphorylation in Pdk1Hem-KO cells (Figure 28

B & C). These results suggest that GSK3β is activated in PDK1-deficient cells. Taken together, it appears

that HIF1α expression is reduced in Pdk1Hem-KO mice since a positive regulator, p-p70S6K, is reduced,

and the negative regulator, GSK3β, is increased. These experiments provide mechanistic insights into

Pdk1Hem-KO HSCs by showing that increased GSK3β activity and lack of p-p70S6K lead to reduced HIF1α

expression. With HIF1α expression reduced, that ROS levels remain high that causes reduced HSC

numbers because of loss of HSC quiescence and increased HSC exhaustion.

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Figure 28: Loss of PDK1 leads to reduced AKT/mTORC-signaling.

(A) Immunoblot of control and Pdk1Hem-KO (CKO) total BM either starved or starved then stimulated with 10%FBS + cytokines (IL6, SCF, TPO, IL3, Flt3l) for 30 min showed reduced phosphorylation at residues Thr308 and Ser473 on AKT in cells without PDK1 (n = 2). (B) Representative flow cytometry plots and (C) analysis of phosflow of LSK (n = 4) and LK (n = 3; p = 0.0994) BM fractions showed reduced p-AKT at T308 in cells lacking PDK1. (D) Representative flow cytometry plots and (E) quantification showed loss of PDK1 led to reduced phosphorylation of p70S6K at Thr389, a downstream signaling molecule of AKT and mTORC1 (n = 3). (F) Representative flow cytometry plots and (G) quantification of sorted LSK (n = 3) and LK (n = 4) cells showed reduced p-GSKβ(Ser9) in the Pdk1Hem-KO cells. Secondary lines represent secondary antibody controls. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).  

 

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Discussion

In Chapter 4, experimental evidence suggested that Pdk1Hem-KO HSCs have higher levels of ROS

that leads to a loss of quiescence (Chapter 3) as well as increased progenitor proliferation and cell death.

Based on the most recent data, it appears that high levels of ROS is due to loss of HIF1α expression

which can act as a negative feedback loop. Further investigation into the loss of HIF1α expression in

Pdk1Hem-KO cells, suggested that HIF1α might be mediated by the inhibitory isoform IPAS and that loss of

PDK1-mediated AKT/mTOR and AKT/GSK-signaling might cause decreased Hif1α transcription and

translation. Taken together, the results from this study suggest that PDK1 regulates the PI3K-signaling

pathway to provide cellular balance in HSCs through the regulation of ROS and HIF1α. Without PDK1,

HSCs lose quiescence and proliferate more, resulting in the loss of HSC numbers and cellular functions.

HIF expression

Since ROS levels were elevated in the Pdk1Hem-KO mouse, I wanted to know whether there was a

defect in HIF1α expression, a negative regulator of ROS. The results from IHC for the detection of HIF1α

in HSCs showed that indeed, HIF1α expression is reduced in Pdk1Hem-KO cells. As mentioned before,

there are a number of explanations for reduced HIF1α expression: (1) there is less transcription of Hif1α,

(2) there is normal transcription but reduced translation of HIF1α, and (3) there is post-translational

inhibition of HIF1α. The first possibility seems unlikely since RT-PCR of total BM led to the conclusion that

Hif1α is being transcribed at normal levels and preliminary data show a dramatic increase in Hif1α

expression in the LSK and LK fractions. Of note, the LSK experiment was only performed once and the

LK experiment only twice, so it is necessary to further replicate this data. To address the second

possibility, HIF1α protein is detected in LSKF- and LK as evidenced in the IHC results (Figure 23). This

suggests that translation is occurring and thus, perturbations in transcription nor translation cannot

account for diminished HIF1α expression.

With regards to the third explanation for reduced HIF1α, there are many instances where HIF1α

can be affected post-translationally: (3a) ARNT, which can bind with HIF1α, is reduced therefore the

HIF1α/ARNT heterodimer may not be formed and HIF1α targets are not transcribed, (3b) the other HIFs

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may be competing for ARNT binding, (3c) IPAS is inhibiting HIF1α, (3d) other signaling molecules are

inhibiting HIF1α expression. With regards to scenario 3a, if ARNT is decreased in HSCs, then it is

possible that HIF1α is being translated properly but the co-factor ARNT is not at sufficient levels to

translocate the heterodimer into the nucleus. Further experiments to determine Arnt transcriptional levels

in HSCs needs to be performed. To address scenario 3b, Hif2α mRNA is increased in total BM, so it is

plausible that HIF2α competes with HIF1α for ARNT binding; however, this may not be the case in the

LSK fraction because the role for Hif2α may be different in HSCs. Further replicates of Hif2α mRNA in

HSCs need to be addressed. The same could be true for Hif3α. To determine whether IPAS is inhibiting

HIF1α (scenario 3c), mRNA levels of IPAS in HSCs require further investigation. In addition, Hif3αFL

mRNA levels in HSCs should be checked as well because there are multiple isoforms of Hif3α that could

have different functions. Increased IPAS mRNA does not necessarily suggest increased protein

expression so HIF3α/IPAS IHC with LSKF- cells or Western blots with total BM protein should be

performed. In these experiments, I would predict higher HIF3α/IPAS in the Pdk1Hem-KO cells than in the

control. Ideally, IHC colocalization of HIF1α and HIF3α would suggest a direct inhibition of HIF1α by

HIF3α/IPAS. One caveat would be that the HIF3α antibody may not strongly recognize the IPAS isoform.

Alternatively, future experiments could include retroviral shRNA knock down of HIF3α and IPAS in HSCs

to rescue HIF1α expression and this may point to a suppression mechanism by certain isoforms of HIF3α.

In addition, overexpression of HIF1α can be accomplished in Pdk1Hem-KO cells using similar molecular

approaches to determine whether circumventing PDK1-deficiency could decrease high ROS levels when

PDK1 is ablated. Lastly, it would be interesting to determine whether overexpression of IPAS in normal

HSCs would recapitulate the Pdk1Hem-KO phenotype.

It is possible that transcription factors associated with other pathways could also be regulating

HIF1α expression (scenario 3d). For instance, Cited2 can compete with HIF1α for binding at with the

histone acetyltransferase p300/CBP (Bakker et al., 2007; Kranc et al., 2009); however, RT-PCR analysis

of total BM (n = 2, data not shown) does not show an increase in Cited2 levels. In addition, loss of Meis1

can lead to reduced Hif1α and Hif2α transcription (Kocabas et al., 2012). Interestingly, RT-PCR of Meis1

in total BM shows the same or slightly increased levels and we know that transcription of Hif1α and Hif2α

are normal or increased in total BM so Meis1 is not impacting Hif1α transcriptional activity. Taken

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together, these preliminary results further provide evidence that loss of HIF1α expression is due to

aberrant in PDK1-signaling (see next section), direct inhibition by IPAS, or a combination of both.

PI3K/AKT/mTORC-signaling

In addition to the scenarios mentioned in the previous section regarding HIF1α regulation,

evidence from this study suggests that PDK1/AKT/mTORC-signaling can regulate HIF1α (scenario 3d).

The results from the PDK1 inhibitor immunoblots, the B cell depleted Pdk1Hem-KO total BM Western blots,

and phosflow conclusively show that AKT phosphorylation is decreased in the Pdk1Hem-KO mouse. In

addition, loss of mTORC1-signaling is corroborated by loss of p70S6K phosphorylation. To provide a

comprehensive understanding of how PDK1 impacts AKT/mTORC1-signaling, immunoblots of 4EBP,

another downstream target of mTORC1 involved in protein translation, can be performed with the PDK1

inhibitor. Furthermore, loss of AKT-signaling leads to activated FOXO-signaling. FOXOs have been

implicated in HSC quiescence and conditional knockouts show increased levels of ROS (Tothova and

Gilliland, 2007). Theoretically, FOXO expression should be increased in the absence of PDK1, and

preliminary immunoblots showed reduced p-FOXO3a (data not shown). AKT phosphorylates FOXO for

degradation, so this is in line with the current model; however, further experimentation using immunoblots

or IHC for 4EBP or FOXO needs to be duplicated to corroborate that p-4EBP is down and that FOXO

expression is increased.

AKT is also responsible for inhibiting GSK3 via phosphorylation. Results from these experiments

show that GSK3β phosphorylation is down-regulated but there is an alpha subunit of GSK that can also

be phosphorylated. Investigation into p-GSK3α(Ser21) levels need to be assessed although it is thought

that the two subunits do not have compensatory roles since knocking out either one results in embryonic

lethality (reviewed in McCubrey et al., 2014). Furthermore, overexpression of GSK3β in HepG2 cells led

to reduced HIF1α (Flugel et al., 2007; Mottet et al., 2003). This would suggest that loss of GSK3β

phosphorylation might be enough to actively inhibit HIF1α. It is important to note that GSK3 is also

downstream of Wnt/β-catenin signaling; however, the role for Wnt-signaling in HSCs is controversial with

some studies showing that it impacts HSC quiescence and others demonstrating that Wnt-signaling is

negligible for HSC maintenance (Mendelson and Frenette, 2014). Lastly, it has been shown that NFkB

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can activate HIF1α through functional binding to a promoter site in Hif1α (Bonello et al., 2007), therefore

suggesting that NFkB could promote HIF1α expression. However, it has been shown that PDK1 can

activate NFkB-signaling through PKCθ in T cells (Lee et al., 2005). In our model, PDK1 is ablated

suggesting that there is reduced NFkB-signaling and therefore reduced Hif1α transcription. Although, the

microarray showed that NFkB targets were similar in the control and Pdk1Hem-KO groups, further

experiments to verify that NFkB-signaling is decreased, such as IHC to look for p65, the downstream

target of NFkB, are needed to demonstrate that activated GSK3 and reduced p70S6K phosphorylation

are coordinately working to down-regulate HIF1α expression.

Consequences of reduced HIF1α expression

Many studies have suggested that the primary function of HIF1α in HSCs is to shunt energy

production from the high ATP producing but also high ROS producing oxidative phosphorylation

(OXPHOS) pathway in mitochondria to the lower ATP producing and lower ROS generating glycolysis

method in the cytoplasm. In addition, it has been shown that stem cells primarily use glycolysis as their

mode of energy production since their metabolic need is different from progenitor and differentiated cell

types as well as to help prevent some of the deleterious effects that can occur with high amounts of ROS.

Indeed, glycolysis has been implicated in HSC survival in the hypoxic niche as a means to promote

quiescence (Suda et al., 2011). The Pdk1Hem-KO mouse has reduced quiescence and increased ROS

levels; therefore, it would be interesting to determine whether glycolysis was reduced in Pdk1Hem-KO HSCs

and as a consequence OXPHOS was increased. Preliminary results show a reduction in Glut1 mRNA

expression, the major receptor for glucose transport into cell; however, further investigation into glucose

uptake, through either Seahorse assays or with a florescent glucose dye, in Pdk1Hem-KO cells need to be

performed. In addition, RT-PCR of genes associated with these pathways can shed light on the

consequences of reduced HIF1a expression in Pdk1Hem-KO HSCs.

In conclusion, I have determined that PDK1 controls ROS activity by regulating HIF1α expression

and this ultimately has consequences for HSC quiescence and viability. PDK1 appears critical for HSCs

to favor anaerobic glycolysis and maintain low ROS by regulating HIF1α expression. The expression of

HIF1α and low ROS serves to limit HSC cell cycling, to avoid unnecessary mutations, and to promote

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longevity of the HSC pool. Here, I have examined a number of potential mechanisms by which PDK1 may

regulate HIF1α and my data suggests that PDK1 may regulate HIF1α expression by a number of

plausible mechanisms including direct PI3K/AKT/mTORC-signal transduction that results in HIF1α

expression and by suppressing potential binding partners such as IPAS or HIF3α that may limit HIF1α

dimerization with ARNT. Such competitive binding partners that inhibit HIF1α dimerizing with ARNT may

prevent translocation of HIF1α to the nucleus and inhibit transcription of antioxidation genes. Thus, PDK1

promotes HIF1α expression that ultimately serves in HSC maintenance and homeostasis.

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Chapter 6: Discussion and Future Directions

PI3K-signaling in HSCs

The precise balance between HSC quiescence and proliferation is essential for adequate blood

production within an organism. Through instructive external niche signals, such as cytokines, and internal

signals that govern the activation of cellular programs, adult HSCs are able to exit quiescence, and then

self-renew and differentiate to maintain hematopoietic homeostasis. Many signal transduction pathways,

such as MAPKs, TGFβ, NFκB, and Wnt have been implicated in the maintenance of HSCs; however, the

PI3K/AKT/mTORC-signaling cascade is one of the major pathways involved in HSC cell survival,

proliferation, and metabolism. The PI3K/AKT/mTORC-signaling pathway lies downstream of a variety of

growth factors and cytokines that are needed to maintain HSCs during homeostasis as well as under

regenerative conditions. Indeed, TPO has been shown to signal through mTOR in stress conditions (Qian

et al., 2007) and SCF promotes lipid raft clustering and AKT-activation to promote HSC cell cycle entry

(Yamazaki et al., 2006). In the current study, I was interested in unraveling the role of PI3K-signaling in

HSC biology. For this purpose, I decided to focus on PDK1 as this protein has been considered the

“master kinase” of the PI3K-signaling pathway. PDK1 is directly activated by PI3Ks and can initiate the

activity of many other PI3K-signaling members by phosphorylating their T-activation loop such as AKT

and S6K. Furthermore, PDK1 is needed for proper lineage differentiation but a role for PDK1 in HSC

maintenance has not been established. In this study, I show that PDK1 unequivocally functions as a

master regulator of PI3K-signaling to promote HSC quiescence.

While previous groups have explored how PDK1 impacts lymphoid and myeloid lineage

differentiation, this is the first study to investigate how PDK1 governs HSC maintenance. Through the use

of a hematopoietic specific Cre line, I was able to ablate PDK1 in the most multipotent hematopoietic

cells, which unsurprisingly led to aberrant defects in lymphoid differentiation as reported by previous

studies (Baracho et al., 2014; Hinton et al., 2004; Lee et al., 2005; Park et al., 2013; Park et al., 2009;

Yang et al., 2015). Interestingly, myeloid progenitor numbers were not significantly affected; however, the

relative ratio of monocyte and granulocyte levels was changed. Furthermore, CBC analysis of the

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Pdk1Hem-KO BM recapitulated the thrombocytopenia defect reported earlier (Chen et al., 2013). This

suggests that PDK1 may be dispensable for myeloid progenitor specification but necessary for lineage

differentiation of specific myeloid cell types. Consistently, Pearn et al. suggested that PDK1

overexpression promotes monocyte formation. In the Pdk1Hem-KO mice, I see a decrease of monocytes in

the BM and an increase in the spleen. Thus, these studies highlight the importance of PDK1-signaling in

monocyte differentiation. Overall, it appears that the loss of PDK1 expression in Pdk1Hem-KO mice had

detrimental effects on the lymphoid lineage and influenced the differentiation of specific cell types within

the myeloid lineage.

Through the use of genetic gain- and loss-of function studies, many groups have demonstrated

that signaling through the PI3K-pathway is essential for HSC survival, proliferation, and differentiation

during normal and regenerative hematopoiesis (Chen et al., 2008; Juntilla et al., 2010; Kalaitzidis et al.,

2012; Kharas et al., 2010; Magee et al., 2012; Miyamoto et al., 2007; Siegemund et al., 2015; Tothova

and Gilliland, 2007; Yilmaz et al., 2006; Zhang et al., 2006). In this study, immunophenotyping of the

Pdk1Hem-KO BM revealed a striking reduction in the number of HSCs and that PDK1-deficient cells were

unable to provide radioprotection in primary BMTs. Furthermore, Pdk1Hem-KO HSCs had normal levels of

viability staining, fewer cells in the G0 phase, and increased cycling. In Pdk1Hem-KO mice, there was

reduced phosphorylation of AKT at Thr308 and Ser473 as well as reduced phosphorylation of p70S6K at

Thr389, a downstream target of mTORC1-signaling. Thus, these results clearly demonstrate that key

downstream targets of the PI3K-pathway are not activated in the absence of PDK1. It should be noted

that although PDK1 and AKT act in concert to activate mTORC1-signaling, activation of the downstream

targets of PI3K-signaling is not strictly dependent on AKT. This is because PDK1 has been shown to

directly phosphorylate the targets of PI3K, such as RSK and PKC, independently of AKT (Figure 29)

(Najafov et al., 2011; Vanhaesebroeck and Alessi, 2000; Vasudevan et al., 2009). Of note, it is thought

that phosphorylation of AKT at Thr308 is predominantly the responsibility of PDK1 whereas

phosphorylation of AKT at Ser473 is executed via mTORC2 (Vivanco et al., 2002; Sarbassov et al.,

2005). Without proper activation of AKT at both of these phosphorylation sites, there is less activated AKT

to facilitate cell cycle progression (Gao et al., 2014). Interestingly, in the Pdk1Hem-KO BM, I see a decrease

in AKT phosphorylation at both residues rather than just at Thr308. This could be explained by the fact

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that loss of phosphorylation at Thr308 leads to reduced AKT activity, which then subsequently results in

reduced TSC activity. Reduction of TSC activity would cause a decrease in both mTORC1 and mTORC2

activity since TSC is responsible for releasing RHEB from the mTOR complexes. Therefore, a reduction

in mTORC2 activity could cause further reduction of AKT activity by diminishing phosphorylation at

Ser473.

In contrast to the Pdk1Hem-KO results described in this study, genetic knockouts of AKT1 and 2, a

key effector kinase in PI3K-signaling, revealed reduced expansion of transplanted fetal HSCs due to

increased levels of quiescence. AKT1/2 mutant HSCs competed poorly against wild-type HSCs during

competitive repopulation experiments suggesting that they had reduced differentiation capacity (Juntilla et

al., 2010). When AKT is activated, it can sequester FOXOs in the cytoplasm to prevent their transcription

of a variety of genes associated with the cell cycle as well as oxidative stress. Conditional deletion of

FOXO1/3a/4 using Mx-Cre, caused reduced HSC numbers and reduced ability to repopulate the blood

cell pool during BMT (Tothova and Gilliland, 2007). Interestingly, targeted mutants for FoxO3a alone had

HSCs that cycled normally and had proper differentiation capacity. Intriguingly, FOX3a-deficient HSCs

had reduced ability to compete during competitive transplantation experiments (Miyamoto et al., 2007).

The discrepancies could be due to functional redundancy among the different isoforms of FOXO in the

second study as well as the type of deletion system used: Mx-Cre versus targeted deletion, respectively.

Nonetheless, both studies reveal that loss of FoxO transcription can impact HSC functions.

Another target of AKT is the mTOR1 complex. AKT negatively regulates the TSC1/TSC2

complex, which in turn negatively regulates mTORC1 by interacting with RHEB, which is needed for

mTORC1 activation. Conditional loss of TSC1 led to decreased HSC quiescence and increased

proliferation. TSC1-deficient HSCs lost self-renewal capacity and reduced hematopoietic reconstitution

ability (Chen et al., 2008). The TSC1 mutant phenotype is interesting for three reasons: (1) PDK1 is

upstream of AKT/mTORC1-signaling, (2) PDK1 can directly impact mTORC1-mediated protein synthesis

by phosphorylating S6K, and (3) the TSC1 mutant HSC phenotype is similar to the Pdk1Hem-KO HSC

phenotype. Taken together, this suggests that the Pdk1Hem-KO phenotype is partially due to loss of

mTORC1-signaling and indeed, there is reduced phos-p70S6K, a readout of mTORC1-signaling, in

Pdk1Hem-KO LSK and LK (Figure 28). Furthermore, loss of Raptor, a key component of the mTOR1

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complex, resulted in pancytopenia and splenomegaly under normal hematopoiesis and after BMT, or

stress conditions, HSCs were unable self-renew (Kalaitzidis et al., 2012). Another study deleted Rictor, a

key component in the mTOR2 complex, and found very few changes in HSC functions during steady

state conditions. However, loss of Rictor, or mTORC2-signaling, in a PTEN-null background prevented

HSC depletion and leukemogensis that can occur when PI3K-signaling is over-activated (Magee et al.,

2012). Pdk1Hem-KO mice might have a different phenotype from these studies because PI3K/PDK1/AKT-

signaling is not altered in the Raptor or Rictor mutants since these kinases act earlier in the signaling

cascade. Furthermore, the normal HSC phenotype seen in both the Raptor and Rictor mutants during

steady state conditions suggests that there could be compensatory mechanisms that maintain mTORC-

signaling. For instance, PDK1 can phosphorylate S6K at Thr229 independently of mTORC1, which

primarily phosphorylates S6K at Thr389 (Figure 29) (Pullen et al., 1998). Thus, the downstream target of

mTORC1, S6K, can still be activated or at least partially activated. Collectively, these experiments show

that loss of PI3K-signal transduction through PDK1/AKT/mTORC is necessary to maintain HSC

quiescence and repopulating capacity both during normal and regenerative hematopoiesis.

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Figure 29: PI3K/PDK1-signaling phosphorylates AKT-dependent and AKT-independent AGC kinases.

In addition to the ‘classical’ PI3K/AKT/mTORC-pathway, PI3K/PDK1-signaling can activate a variety of other AGC kinases including: SGK, RSK, and PKC. These other kinases need to be phosphorylated on different residues by other signaling components for full activation.

 

Previous studies have shown that increased PI3K-signaling through the loss of PTEN, results in

myeloproliferative disease since HSCs proliferate more. This causes HSC depletion and inability to

repopulate the blood cell pool. Treatment with rapamycin restored HSC function, which suggests that the

loss of quiescence was due to reduced mTORC1-signaling (Yilmaz et al., 2006; Zhang et al., 2006). The

loss of SHIP1 should also lead to increased ATK/mTOR-signaling. However, unlike the PTEN mutant,

SHIP1 deficient mice have increased HSC numbers and fewer apoptotic cells. In addition, SHIP1-

deficient HSCs have reduced reconstitution ability due to decreased expression of CXCR4 and VCAM,

which are needed for engraftment (Desponts et al., 2006). Both PTEN and SHIP1 function to convert

PIP3 to PIP2, thus negatively regulating PI3K-signaling; therefore, it is odd that the HSC phenotype

reported in the PTEN and SHIP1 mutants are different. One possible explanation is that the mechanistic

action is different between the two proteins: PTEN dephosphorylates PIP3 while SHIP1 hydrolyzes PIP3.

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Depending on the receptor or signaling cascade, PTEN or SHIP might be preferentially regulated. In

addition, another explanation for different phenotypes is that the PTEN mutants were conditionally

ablated using Mx-Cre in adult HSCs and the SHIP1 mice were total knockouts. As discussed earlier, the

mechanisms that govern fetal versus adult quiescence are different so the experimental conditions

employed might account for the discrepancy. Another group over-activated PI3K-signaling by performing

BMT with an overexpression allele of myristylated Akt (myr-Akt). The data revealed that HSPCs

transiently expanded and had increased cycling that hindered engraftment. These mice also exhibited

myeloproliferative disease and rapamycin treatment was able to elongate their lifespan by limiting

mTORC1 expression (Kharas et al., 2010). In addition, loss of Itpkb, a competitor of AKT for PIP3

activation, should also result in greater PI3K-signaling. Indeed, Itpkb mutants displayed fewer HSC

numbers, loss of HSC quiescence, increased proliferation, and reduced HSC reconstitution ability. Itpkb-

deficient HSCs had augmented mTOR-signaling as evidenced by S6K phosphorylation and rapamycin

treatment reduced HSC hyperactivity (Siegemund et al., 2015). The HSC phenotypes of the myr-Akt and

Itpkb mutants were similar to the PTEN mutant data reported by Yilmaz et al., suggesting that excessive

PI3K-signaling lead reduced HSC self-renewal. Collectively, these studies demonstrate that increased

AKT/mTORC-activity leads to reduced quiescence and loss of reconstitution ability of the blood cell pool.

Taken together, through the use of genetic knockouts the PI3K/AKT/mTORC-signaling pathway

can either be amplified or diminished in HSCs. Based on the previous experiments, regardless of the

level of PI3K-signaling, HSC quiescence and hematopoietic repopulating ability is compromised

suggesting that this pathway must be precisely regulated to maintain proper HSC numbers and

physiology. The results from the current study also suggest that precise regulation of PI3K-signaling

through PDK1 must be maintained to promote HSC quiescence and long-term repopulating ability to

ensure a balanced hematopoietic output.

ROS impacts HSC quiescence

Through recent studies, ROS has been implicated as a major intracellular signal mediator that

causes the destabilization of HSCs by promoting proliferation and differentiation as well as increased

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DNA damage that can lead to apoptosis. Many studies have implicated various cellular pathways in

maintaining oxidative balance such as the p38 MAPK pathway and hypoxia inducible factors (Hifs);

however, the precise mechanisms that regulate oxidative stress in HSCs are still being elucidated.

Previous experiments show that aberrant ROS signaling results from perturbed PI3K-signaling. Indeed

AKT1/2 DKO fetal liver HSCs have reduced levels of ROS and normal expression can be restored by in

vivo treatment with buthionine sulfoximine (BSO) (Juntilla et al., 2010). In line with this observation, the

loss of FOXO1/3a/4 transcription factors caused increased ROS levels that were reduced upon in vivo

NAC treatment (Miyamoto et al., 2007; Tothova and Gilliland, 2007). Interestingly, conditional deletion of

TSC1 results in elevated HSC ROS levels and mitochondrial biogenesis presumably due to the increase

in mTOR-signaling. Treating the TSC1 mutant with NAC restored ROS levels to normal and rescued BM

cellularity (Chen et al., 2008). Collectively, these results suggest that PI3K-signaling is a positive

regulator of the ROS pathway. In sharp contrast, this study posits that PDK1-mediated PI3K-signaling

functions to limit excessive ROS levels.

In the current study, although it was evident from BMT and cell cycle experiments that PDK1-

deficient HSCs loose quiescence and proliferate more, it was unclear why this was occurring. To

investigate possible causes of HSC instability, microarray analysis was performed on control and

Pdk1Hem-KO LSK cells, and GSEA analysis revealed that PDK1-deficient cells had lost the HSC signature

as well as were compromised in their response to oxidative stress conditions. To verify this, I checked for

increased ROS expression in these mice and found that indeed, ROS was augmented in Pdk1Hem-KO

HSCs. Further experiments suggested that increased ROS expression led to more proliferation, DNA

damage, and apoptosis in progenitor cells. NAC administration partially rescued HSC hyperproliferation

and differentiation of Pdk1Hem-KO cells both in vitro and in vivo. This suggested that abnormal HSC activity

in PDK1-deficient cells was largely due to elevated levels of ROS. These data indicate that incredibly

elevated levels of ROS are toxic for the cells. It is important to note that since PI3K-signaling has

pleiotropic effects, it is unlikely that ROS is the only mediator acting in Pdk1Hem-KO HSCs. Further

experiments investigating other mechanistic modes of action, such as the role of cell cycle regulators, are

needed to provide a comprehensive understanding of PDK1-mediated HSC quiescence. Despite this,

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ROS is responsible for a portion of the Pdk1Hem-KO HSC phenotype since NAC treatment was able to

partially rescue the aberrant HSC changes.

In contrast to the previously reported studies that utilized AKT1/2, FOXO1/3a/4, or TSC1 mutants,

Pdk1Hem-KO mice have reduced AKT/mTORC1-signaling and increased levels of ROS, which, superficially,

does not fit into the paradigm. However, previous studies have demonstrated that mTORC1 mutants,

through ablation of Raptor, that ROS levels are normal (Kalaitzidis et al., 2012). There could be a few

explanations for this: (1) The deletion systems of the knockouts are different and therefore, the mode of

deletion as well as the time of deletion, fetal versus adult, could impact the physiological significance

attributed to the deleted gene. This study ablated PDK1 during fetal hematopoiesis (Vav-Cre) and

measured disturbances in adult HSCs. In contrast, the AKT1/2 DKO study used total knockout fetal HSCs

(Juntilla et al., 2010) and assessed fetal HSC physiological differences in the context of adult BMT, while

the TSC1 and FOXO study used Mx-Cre to ablate in adult HSCs (Chen et al., 2008; Tothova and

Gilliland, 2007). Furthermore, Pdk1Hem-KO ROS levels were only partially rescued with NAC treatment

while treating the TSC1 and FOXO1/3a/4 mutants with NAC completely reverted ROS levels as well as

rescued HSC numbers. The mode of deletion as well as the time of NAC administration after deletion

could influence the level of rescue seen in each mutant. Therefore, further experiments utilizing

Pdk1Flox/Flox ; Mx-Cre mice are needed to determine whether loss of PDK1 during adult hematopoiesis has

the same consequences for HSCs and if complete rescue by NAC treatment is possible. In addition,

extensive studies using proper controls, such as performing knockout studies of all these genes in parallel

with the same Cre line, are required to comprehensively explain the discrepancies in these models. (2)

Another rationale for opposite levels of ROS between Pdk1Hem-KO mice and the TSC1 or AKT1/2 or

FOXO1/3a/4 mutants could be that PDK1-signaling is normal in the other three mutant mice. The FOXOs

are transcription factors so loss of transcriptional activation of a variety of cell survival genes could impact

levels of ROS. The other three proteins, PDK1, AKT, and TSC1, are signaling molecules in the “classical”

PI3K-pathway so I will focus on these proteins for the remainder of the discussion. Unlike AKT or TSC1,

PDK1 can phosphorylate other members in the PI3K/mTORC-pathway, such as S6K, independently of

AKT/mTORC-activation and in addition, activate AKT-independent targets. Therefore, it is tempting to

speculate that PDK1 may be playing a compensatory role and there may be a residual mTORC1-

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signaling or signaling through other AKT-independent PDK1-dependent substrates resulting in ROS

production or repression depending on the context. Overall, the data from this study, as well as results

from others, demonstrate that: 1) the PI3K-signal transduction pathway needs to be tightly controlled to

moderate ROS levels in HSCs, and (2) our studies demonstrate that each individual signaling member of

the PI3K-pathway has a distinct role in activating or restricting ROS levels in HSCs. While elevated ROS

levels provided an explanation for increased cycling in Pdk1Hem-KO HSCs, I next wanted to explore the

mechanistic rationale for augmented ROS expression in these cells.

A novel role for HIF3α in HSC maintenance

Earlier studies depicting a critical role for cell cycle regulation by HIF1α to promote HSC

quiescence have highlighted the importance of maintaining proper metabolic homeostasis within the HSC

(Majmundar et al., 2010; Takubo et al., 2010). Furthermore, HSCs reside in a hypoxic BM niche and their

metabolic makeup is distinctly different from progenitor and differentiated cells. Recent evidence

suggested that HSCs use low-oxygen metabolism and a high rate of glycolysis to promote HSC

quiescence by limiting the generation of ROS (Ito and Suda, 2014; Kohli and Passegue, 2014). As

discussed earlier, high levels of ROS can have detrimental effects on the cell leading to increased

proliferation, DNA damage, and cell death. Given that the Pdk1Hem-KO mouse had elevated levels of ROS,

I wanted to ascertain whether this was caused by a defect in PDK1-mediated HIF1α expression. Confocal

images of control and Pdk1Hem-KO HSCs showed reduced nuclear HIF1α expression in PDK1-deficient

cells. Analysis of downstream PI3K-signaling through phosflow assays found that AKT phosphorylation

was reduced and as a consequence, activity of a positive regulator of HIF1α, S6K, was decreased and a

negative regulator of HIF1α, GSK3, was increased. Diminished p-70S6K activity suggests that HIF1α

protein synthesis is less (Koh et al., 2008), which can explain why Pdk1Hem-KO HSCs have reduced

nuclear HIF1α expression. Interestingly, in CD8+ T cells, the PDK1-mTORC-HIF1α pathway is AKT-

independent therefore suggesting that PDK1 can directly regulate S6K-HIF1α (Finlay, 2014); however, it

is unclear if this is happening in Pdk1Hem-KO HSCs since I also see a reduction in AKT phosphorylation. In

addition to loss of HIF1α protein translation by S6K, active GSK3β can interact with the VHL domain on

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HIF1a to promote degradation (Flugel et al., 2007; Mottet et al., 2003), thus suggesting that there are two

mechanisms in Pdk1Hem-KO HSCs that promote the loss of HIF1α activity. HIF1α is vital to maintain normal

levels of ROS in HSCs to initiate transcription of antioxidants as well as direct metabolic processes to

methods that are low oxidative stress generators (Jang and Sharkis, 2007; Suda et al., 2011). Taken

together, these experiments suggested that ROS levels are unrestrained since HIF1α is not present in

Pdk1Hem-KO cells to moderate oxidative stress. Although, loss of AKT-signaling, and therefore the

simultaneous loss of S6K and gain of GSK activity, explains a post-translational rationale for reduced

HIF1α expression, another explanation could be that Hif1α transcription was reduced in Pdk1Hem-KO mice.

It is important to note that the role for HIF1α in HSC maintenance may be different in fetal and

adult HSCs. A recent study deleted Hif1α with Mx-Cre and found that HSCs were able to self-renew and

provide long-term reconstitution during serial transplantation experiments, which contradict the results

described by Takubo et al. (Vukovic et al., 2016). Furthermore, this study did not report on the metabolic

state of the HSCs so it is unclear if ROS levels are normal in these mice. Given that it is accepted that

HIF1α controls energy output towards glycolysis to reduce ROS production and that mTORC-signaling is

a major regulator of metabolism, it is possible that HIF1α is dispensable as long as mTORC-signaling is

intact. However, in this study, mTORC-signaling is abrogated in the Pdk1Hem-KO HSCs and in addition,

HIF1α activity appears to be reduced therefore two metabolic mechanisms are affected, which may

explain the elevated ROS levels in PDK1-deficient cell.

Interestingly, my investigation into the transcriptional regulation of Hif1α in the Pdk1Hem-KO

revealed some fascinating insights regarding Hif transcription in PDK1-deficient cells. In Pdk1Hem-KO total

BM, there was a decrease in Arnt mRNA, normal levels of Hif1α mRNA, an increase in Hif2α mRNA, and

remarkable increases of Hif3αFL and IPAS mRNA expression. Increased Hif3αFL and IPAS mRNA

expression was corroborated by sequencing and BLAST analysis of total BM. In addition, preliminary RT-

PCR analysis of Pdk1Hem-KO LSK and LK cells showed increased Hif1α mRNA as well as increased Hif3α

mRNA but whether Hif3α mRNA was predominantly Hif3αFL or IPAS needs to be validated. Taken

together, RT-PCR analysis of the Hifs revealed a potential novel explanation for reduced HIF1α protein

expression in the Pdk1Hem-KO HSCs: (1) HIF3α is competing against HIF1α for binding to ARNT and/or (2)

increased IPAS is binding to HIF1α and sequestering it in the cytoplasm. Although, before coming to any

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conclusions, it is first important to first ascertain if HIF3α/IPAS protein expression is increased in Pdk1Hem-

KO HSCs (See Chapter 5 Discussion), I can discuss the likelihood and ramifications of each scenario.

The prevailing dogma is that HIF1α binding to ARNT or HIF2α/ARNT are transcriptional activators

and HIF3α primarily serves as an inhibitor of HIF1/2 (Dengler et al., 2014); however, emerging evidence

in the zebrafish suggests that HIF3α might act as a transcriptional activator and have its own set of

distinct target genes (Zhang et al., 2014). Zhang et al. showed, through transcriptomics and chromatin

immunoprecipitation assays, that HIF1α and HIF3α can activate the same set of genes as well as

distinctly separate genes in response to hypoxia. Zhang et al. corroborated these results in human cells

by overexpressing zebrafish Hif3α, containing the HRE-dependent transactivation activity, and detected

HIF3α in the nucleus. Stabilized HIF3α resulted in increased expression of HIF3α endogenous target

genes whose expression was unchanged when HIF1α was stabilized. Given that HIF3α can

transcriptionally activate a set of genes unique from HIF1α in zebrafish, HIF3α in murine HSCs might

have an additional role besides inhibiting the activity of HIF1/2α. To date, no study has reported HIF3α as

a transcriptional activator in murine hematopoiesis therefore, experiments to further elucidate HIF3α

target genes could provide novel insight into HSC quiescence mediated by hypoxia or even provide

unexplored regulators of HSC maintenance.

While the necessity for HIF3α transcriptional activation is one plausible explanation for increased

HIF3α in Pdk1Hem-KO BM, I also saw upregulation of a specific isoform of Hif3α, IPAS, which is known to

directly inhibit HIF1α expression (Makino et al., 2001). This suggested that the second scenario, where

IPAS could sequester HIF1α in the cytoplasm, may also be occurring to inhibit HIF1α expression and

transcription in Pdk1Hem-KO HSCs. Before making any assumptions, the levels of IPAS protein expression

in the Pdk1Hem-KO HSCs must be verified. However, IPAS inhibition of HIF1α provides an additional

explanation for why HIF1α expression is decreased in PDK1-deficient HSCs besides the loss of

PDK1/AKT/mTORC1-signaling and an increase in GSK3 activity.

Interestingly, there are other Hif3α splice variants besides IPAS. Indeed, there are up to six

reported isoforms of Hif3α (Lee et al., 2004) and so far a comprehensive understanding of whether or not

these isoforms are expressed in HSCs and if they serve a function in HSCs is lacking. For example,

knockouts for NEPAS (neonatal embryonic PAS), another Hif3α isoform, were found have defects in

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embryonic heart and lung development. It was shown that NEPAS primarily functioned to block HIF1α

function (Yamashita et al., 2008). Whether NEPAS is expressed in hematopoietic cells and functions to

inhibit HIF1α remains to be determined. Further analysis regarding which Hif3α isoforms are present in

Pdk1Hem-KO HSCs may shed light into which isoforms have roles in hematopoiesis. In addition, it would be

interesting to ascertain why Hif3α and IPAS are increased in the Pdk1Hem-KO BM? Furthermore, does

PDK1 regulate the transcription of Hifs and control Hif3α isoform splicing? These are questions that still

remain to be answered. Regardless, this study is the first to suggest that Hif3α may have a functional role

in HSC quiescence. Further investigation into the Hif mechanism will broaden our understanding of how

hypoxia and PDK1-mediated PI3K-signaling regulate quiescence.

Future Directions and perspectives

In the past 10 years, studies elucidating the metabolic regulation of HSCs have provided insight

into the mechanisms that govern quiescence. It is thought that quiescence serves to limit the amount of

cellular damage from cytotoxic agents and mitochondria respiration (Ito and Suda, 2014; Kohli and

Passegue, 2014; Takubo et al., 2010). In addition, it appears that quiescence has an anaerobic bias since

HSCs reside in a hypoxic niche and their primary mode of energy production is glycolysis, which

predominantly occurs under low oxygenic conditions and produces less oxidative stress. Given that the

Pdk1Hem-KO HSCs have increased levels of ROS and reduced HIF1α expression, future experiments to

assess the metabolic profile of Pdk1Hem-KO HSCs to gain a comprehensive understanding of how PDK1-

deficiency impacts HSC quiescence are needed. Based on the current data and what is reported in the

literature, I expect to see decreased glycolysis in Pdk1Hem-KO HSCs, which can be assessed via RT-PCR

of glycolytic and oxidative phosphorylation genes are needed to assess if this is true.

Many hematological malignancies show mutations in the PI3K-pathway. Indeed, many cancers

have mutations in PTEN and augmented PDK1 expression is seen in a variety of other cancer types

(Ahmed et al., 2008; Arsenic, 2014; Eser et al., 2013; Iorns et al., 2009). Furthermore, 40% of acute

myeloid leukemia (AML) patients have mutations that overexpress PDK1 (Mora et al., 2004; Pearn et al.,

2007; Zabkiewicz et al., 2014) and it has been suggested chronic or acute myeloid leukemias develop

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from transformed HSCs that exhibit leukemia-initiating stem cell properties (Passegue et al., 2003).

Thus, understanding the biology behind PDK1-mediated signaling in HSCs to maintain quiescence as

well as promote balanced proliferation is highly relevant to our understanding of clinical diseases. This

study, along with other studies of genetic mutants in the PI3K-pathway, suggests a link between HSC

metabolism and the onset of leukemic-like properties in HSCs. Interestingly, cancer cells use glycolysis

even in the presence of oxygen in what is known as the ‘Warburg effect’ (Kohli and Passegue, 2014).

Therefore, elucidating how mitochondrial biogenesis and energy production are affected in Pdk1Hem-KO

could provide novel insight into cancer therapies.

The results from this study are the first to suggest that PDK1 is responsible for maintaining HSC

quiescence (Figure 30). Without proper PDK1-signaling, HSCs hyperproliferate, loose self-renewal

capacity, and are unable to provide radioprotection presumably because of augmented ROS expression.

This caused increased differentiation and apoptosis in the progenitor cells. NAC treatment partially

rescued HSC hyperproliferation, differentiation, and increased ROS levels in the Pdk1Hem-KO mice. Further

experiments suggested that elevated ROS levels were due to decreased HIF1α protein expression. There

are two possible explanations for decreased HIF1α: (1) A coordinated loss of PDK1/AKT/mTOR1-

signaling and increased GSK3 activity, which results in less HIF1α translation and promotes HIF1α

degradation, respectively; and (2) high levels of Hif3α and IPAS mRNA expression in total BM could

indicate inhibition of HIF1α at the protein level. While further experiments are needed, taken together, the

results from this body of work suggest that PI3K-signlaing mediated by PDK1 is essential for promoting

HSC quiescence.

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 Figure 30: Loss of PI3K-signaling mediated by PDK1 causes HSCs to proliferate more and lose quiescence.

Schematic shows that reduced PI3K-signaling can cause reduced S6K activity and increased GSK3 activity. Thus, a positive and negative regulator of HIF1α expression and a negative regulator of HIF1α expression.

 

122

Chapter 7: Experimental Procedures

Mouse Generation

Pdk1Flox mice have previously described (Inoue et al., 2006) and were crossed to Vav-Cre (Georgiades et

al., 2002) mice. Pdk1Flox/+ ; VavCre/+ mice were then crossed to Pdk1Flox/+ or Pdk1Flox/Flox to generate control

(Pdk1Flox/+, Pdk1Flox/Flox, or Pdk1Flox/+ ; VavCre/+) or Pdk1Hem-KO (Pdk1Flox/Flox ; VavCre/+) mice. For a schematic

of the breeding scheme, see Figure 5. Sex matched littermate 4-8 week old mice were used for all

experiments.

Cell Preparation

Single cell suspension of total bone marrow (BM) was created after flushing the tibias and femurs from

mice ages 4-8 weeks and lysing RBCs with ammonium chloride (STEMCELL Technologies) for two

minutes at RT, washing with PBS and then straining through 100um mesh. Live cell counts were negative

for Trypan Blue staining (Amresco). Single cell suspensions were cell surface stained for 10 min on ice,

washed with PBS, spun at 1200rpm for 5 minutes at 4C, reconstituted in PBS, and then acquired by flow

cytometry. Lineage positive markers were CD11b, CD19, B220, CD3e, CD11c, Ter119, and Ly6G and

were obtained from BD biosciences.

Bone Marrow Transplantation Experiments

For radioprotection assessment, 106 total BM cells were injected into lethally irradiated (10 Gy) congenic

wild-type (CD45.1+) recipient mice. For competitive repopulation experiments, 0.5 x 106 total BM from

either control of Pdk1Hem-KO mice were mixed 1:1 with WT (CD45.1+) total BM cells and injected into

lethally irradiated congenic wild-type (CD45.1+) recipients.

Apoptosis Assay

Apoptotic cells were detected with the Annexin V PE Apoptosis Detection Kit (BD559763). Briefly, 2.5-3 x

106 total BM cells were stained for cell surface markers on ice for 10 minutes, washed with PBS, and spun

at 1200rpm for 5 minutes at 4C. For each sample, 5ul of Annexin V was added to 100ul of 1X buffer and

123

incubated at room temperature for 15 minutes. 200-400ul of 1X buffer was added to dilute out the stain

and 1ul of 7AAD was added to denote live cells. Cells were acquired by flow cytometry immediately.

Cell Proliferation/Cell Cycle Measurements

For BrdU incorporation experiments, 6-week old mice were i.p. injected with 3.33 mg of BrdU (Sigma

B5002) and drinking water was treated with 0.8mg/ml of BrdU. At 16 hours after injection, mice were

sacrificed and BM cells were stained for cell surface markers for 10 minutes on ice, washed with PBS,

and spun down at 1200rpm for 5 minutes at 4C. Cells were then prepped using BD fix/perm Buffer kit by

adding 100ul of fix/perm buffer and incubating at 4C for 20 minutes. Cells were washed with 1ml of 1X

perm/wash buffer and then digested with DNaseI for 1 hour at 37C. Cells were washed with PBS and

then stained with anti-BrdU-FITC (BD347583) for 80 minutes at room temperature (RT) before detection

by flow cytometry. For Ki67 analysis, BM cells were stained for cell surface markers, fixed, and

permeabilized with BD Fix/Perm kit. Cells were stained with anti-Ki67—APC (BD558615) for 30 minutes

on ice and analyzed by flow cytometry. For Pyronin Y analysis, cells were surface stained, incubated with

5ug/ml of Hoechst 33342 at 37C for 45 minutes and then 1ug/ml Pyronin Y (Sigma) was added for an

additional 45 minutes at 37C before acquisition by flow cytometry.

Side Population Analysis

BM cells were stained for cell surface markers, incubated with 5ug/ml of Hoechst 33342 (Life

Technologies) in DMEM (+ 2% FBS + 1% HEPES) for 90 minutes at 37C and washed twice with HBSS (+

2% FBS + 1% HEPES). 1ul of 7AAD was added to each sample before flow cytometry acquisition.

Measuring ROS levels

BM cells were stained with cell surface markers and then incubated with 2mM CM-H2DCFDA (Life

Technologies C6827) in pre-warmed HBSS at 37C for 15 minutes. The cells were then pelleted and

resuspended in PBS before acquisition. For positive controls, cells were incubated with 100uM H2O2

while staining with CM-H2DCFDA.

124

mitoSOX staining

BM cells were surface stained and then incubated with 5uM MitoSOX Red (Life Technologies M36008) in

HBSS for 30 minutes at 37C. Cells were then pelleted and resuspended in PBS before FACS acquisition.

DNA damage assessment

BM cells were surface stained and processed according to the BD cyto fix/perm kit instructions 554714.

Cells were stained with rabbit anti-phos-H2A.X (Cell Signaling, 2718) diluted 1:300 in perm/wash buffer

for 1 hour at RT. Cells were washed in perm/wash buffer and then stained with Alexa 488 anti-rabbit (Life

Technologies, 0.2ul/100ul of perm/wash buffer) for 1 hour at RT. Cells were washed and then

resuspended in PBS before acquisition. For positive controls, BM cells were either irradiated at 2 grey or

incubated with 200uM H2O2 in HBSS for 3 hours at 37C.

Western Blot Analysis

For immunoblot analysis, control or Pdk1HemKO BM cells were MACS B cell depleted (CD19, B220),

starved for 5.5 hours in 1%BSA/DMEM or starved for 5 hours and then stimulated for 30 minutes. with

10%FBS/DMEM with 50ng/ml stem cell cytokines (Stemgent: IL6, IL3, TPO, SCF, Flt3l) before being

lysed with 1X Cell Lysis Buffer (Cell Signaling) with 1:25 protease inhibitor cocktail (Complete; Roche)

and 1mM PMSF (Santa Cruz). Cell lysates were incubated at 95C for 5 minutes with sample buffer

(NuPAGE; Life Technologies) containing 1% 2-Mercaptoethanol (Sigma). For inhibitor WB, wild type total

BM was incubated with either no inhibitor or 1uM PDK1 inhibitor (Sigma GSK2334470) while being

starved for 5.5 hours in 1%BSA/DMEM or starved for 5 hours and then stimulated with 10%FBS/DMEM

with stem cell cytokines for 30 minutes. before being lysed. Proteins were run on an 8-10% SDS-PAGE

and transferred to PVDF membrane (BioRad Laboratories). The membranes were blocked with 5% BSA

(Fisher) and then treated with primary and secondary antibodies. The blots were visualized using

ProtoGlow ECL (National Diagnostics) or SuperSignal West Pico Chemiluminescent Substrate (Life

Technologies, 34080) and detected on Image Station 440 (Kodak). Antibodies used can be found in

Table 8.

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Phosflow

BM cells were starved in 1% BSA/DMEM for 2 hours, stained for cell surface markers, and then

stimulated for 30 min with 10%FBS/DMEM and 50 ng/ml stem cell cytokines (Stemgent: Flt3l, SCF, TPO,

IL3, IL6). Cells were then fixed (BD558049) for 10 min at 37C and incubated with primary antibodies in

permeabilization buffer (BD558050) for 1 hour on ice and then with secondary antibodies for 30 minutes

on ice. Cells stained only with secondary were used as a control. Antibodies used can be found in Table

8.

Table 8: Antibodies used in this study.

 

126

RNA Extraction and Microarray Analysis

Total RNA was isolated with RNeasy Mini kit (Qiagen). cDNA was synthesized with Oligo(dT) primer and

Maxima reverse transcription (Thermo Fisher). For transcriptional profiling studies, HSPCs (Lin-Sca1+c-

kit+) cells were sorted using BD FACS Aria from both control and Pdk1Hem-KO mice (n = 5 and 10,

respectively) before RNA extraction. RNA samples taken from three independent sorts were pooled for

microarray studies. Expression profiling was performed suing Illumina’s MouseWG-6 v2.0 Expression

BeadChip at Yale Center for Genome Analysis with max probes by CollapseDataset modeule in

Genepattern (Reich et al., 2006). These genes were preranked for log2 fold change and analyzed for

gene enrichment set analysis (GSEA) (Mootha et al., 2003; Subramanian et al., 2005). The list of

transcription factors described in this study was selected using GO0003700 (sequence-specific DNA

binding transcription factor activity) from Quickgo using its default behavior (is_a, part_of and occurs_in

relationships only) (Binns et al., 2009; Huang da et al., 2009a, b).

Table 9: Primers for qRT-PCR.

 

127

In vitro culture

Lineage depleted BM cells were placed in culture with 10%FBS/DMEM + 50ng/ml stem cell cytokines

(Flt3l, SCF, TPO, IL3, IL6; STEMGENT). Cells were cultured in the presence of no drug or 1uM PDK1

inhibitor (GSK2334470; Sigma). Cells were harvested and stained for LSK or lineage positive markers

(CD11b, CD19, B220, CD3e, CD11c, Ter119, Ly6G) and acquired by flow cytometry.

Cell Trace staining

In vitro cells were stained for proliferation with 5uM Cell Trace CFSE (Life Technologies C34554) or 5uM

Cell Trace Violet (C34557) in PBS for 30 minutes at 37C. Cells were washed twice and proliferation was

assessed at 24, 48, or 72 hours.

In vivo NAC treatment

Two-week-old mice were i.p. injected with PBS (vehicle) or with 100mg/kg N-acetyl cysteine (Sigma)

diluted in PBS and pH adjusted to 7.4 everyday for 4 weeks.

In vitro NAC treatment

Lineage depleted BM cells from either control of Pdk1Hem-KO mice were placed in culture with

10%FBS/DMEM + 50ng/ml stem cell cytokines (Flt3l, SCF, TPO, IL3, IL6; STEMGENT). After 24 hours,

cells were harvested and stained for LSK.

In vitro rescue with p38 Inhibitor and NAC

Control and Pdk1Hem-KO BM was sorted by FACS Aria for LSK. 50,000 LSK cells were plated per condition

in 10%FBS/DMEM + 50ng/ml stem cell cytokines (IL3, SCF, TPO, Flt3l, IL6) with vehicle, 100uM NAC

(Sigma A7250), or 10uM 38 inhibitor (SB203580; Selleckchem). Cells were harvested at 24 and 48 hours,

stained for cell surface markers and assessed for ROS levels using CM-H2DCFDA.

128

IHC staining and Microscopy

Freshly sorted cells, typically 500-2000, were plated onto poly-D-lysine (Sigma) coated chamber slides

and incubated at 37C in 10%FBS/DMEM + 50ng/ml stem cell cytokines (IL3, SCF, TPO, Flt3l, IL6) for 2h

before fixing for 10 minutes with 4%PFA at RT. Cells were permeabilized in 0.15% Triton-X100 for 2

minutes at RT and then blocked overnight in 1%BSA/PBS at 4C. Cells were incubated with primary in

blocking solution for 2 hours at 37C and then with secondary for 1 hour at 37C. For nuclear stain, 5ug/ml

Hoescht/PBS was added for 6 minutes at RT and then slides were mounted with VectaShield and imaged

on a Zeiss 710 Confocal using a 100x objective. Antibodies used can be found in Table 8. Florescence

quantification was performed by ImageJ (NIH) analysis similar to McCloy et al.

(http://www.tandfonline.com/doi/abs/10.4161/cc.28401). In summary, Mean Florescence Intensity (MFI)

was calculated by determining the corrected total cellular fluorescence (CTCF).

CTCF = integrated density – (area of selected cell X mean florescence of background)

For these experiments, the background is an average of three measurements taken from the surrounding

cell. For statistical analysis, two-tailed unpaired Student’s t-test were performed using Graphpad Prism

v4.

Statistical analysis

Statistics were calculated using Graphpad Prism v4. Two-tailed, paired Student’s t-tests were performed,

unless otherwise specified, to determine how different the means of the control and Pdk1Hem-KO groups

were with a one star significant p-value set at less than 0.05. Two stars is equivalent to p <0.01 and three

stars p<0.001.

129

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