2011 Mechanisms Controlling Hematopoietic Stem Cell Functions During Normal Hematopoiesis and...

7/28/2019 2011 Mechanisms Controlling Hematopoietic Stem Cell Functions During Normal Hematopoiesis and Hematologica

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Advanced Review

Mechanisms controllinghematopoietic stem cell functions

during normal hematopoiesisand hematological malignanciesMatthew R. Warr, Eric M. Pietras and Emmanuelle Passegue

Hematopoiesis, the process by which all mature blood cells are generated frommultipotent hematopoietic stem cells (HSCs), is a finely tuned balancing act inwhich HSCs must constantly decide between different cell fates: to proliferate, toself-renew or differentiate, to stay quiescent in the bone marrow niche or migrateto the periphery, to live or die. These fates are regulated by a complex interplay

between cell-extrinsic cues and cell-intrinsic regulatory pathways whose functionis to maintain a homeostatic balance between HSC self-renewal and life-longreplenishment of lost blood cells. Improper regulation of these competing cellularprograms can transform HSCs and progenitor cells into disease-initiating leukemicstem cells (LSCs). Strikingly, many of the mechanisms required for maintenance ofnormal HSC fate decisions are equally critical for the aberrant functions of LSCs.Because of the inherent complexities of these molecular mechanisms, a systematicapproach to understanding the regulatory networks underlying HSC self-renewalis critical for uncovering the similarities and differences between HSCs and LSCs.In this review, we focus on recent developments in elucidating the regulatorynetworks governing normal HSC self-renewal programs and their implications forleukemic transformation. We describe the current technical and methodologicallimitations in isolating and characterizing HSCs and LSCs, and the emerging

approaches that may afford a better understanding of the regulation of normaland leukemic hematopoiesis. Finally, we discuss how such basic mechanisticinformation may be of use for the design of novel therapies that will selectivelyreprogram and/or eliminate LSCs. 2011 John Wiley & Sons, Inc. WIREs Syst Biol Med 2011DOI: 10.1002/wsbm.145

INTRODUCTION

Hematopoiesis is the process by which all lineagesof blood cells are generated in a hierarchical andstepwise manner from immature cells present in the

bone marrow (BM) and subsequently released intocirculating blood and peripheral organs for furthermaturation steps and/or effector function.1 At theapex of this hierarchy are hematopoietic stem cells

Authors contributed equally to this manuscript.Correspondence to: [email protected]

The Eli and Edythe Broad Center for Regenerative Medicine andStem Cell Research, Division of Hematology/Oncology, Departmentof Medicine, University of California San Francisco, San Francisco,CA, USA

DOI: 10.1002/wsbm.145

(HSCs), which are the only self-renewing cells capable

of life-long production of all lineages of blood cells

(Figure 1). HSCs reside primarily in endosteal regions

of bone within highly regulated niches consisting of

multiple cell types that directly and indirectly regulate

HSC localization, self-renewal, and differentiation.2

Under steady-state conditions, HSCs are maintained

as a quiescent population and their numbers in the

BM and circulation are tightly regulated.3 In response

to differentiation cues, HSCs give rise to non-self-

renewing multipotent progenitors (MPPs), which then

progressively differentiate into an array of more

lineage-committed progenitors and ultimately produce

all of the highly specialized and differentiated mature

blood cells (Figure 1). Regulation of HSC numbers

2011 John Wiley & Son s, Inc.


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Advanced Review www.wiley.com/wires/sysbio

Self-renewal HSC

MPP

CLP

Pro-T

Pro-B

Pro-NK MEP

CMP

GMP

GrMo/MfPtlENK CellB CellT Cell

Differentiation

Lineage commitment

FIGU RE 1 | Hematopoietic cell differentiation is organized in ahierarchical fashion.

can be accomplished through direct modulationof proliferation, apoptosis, and/or retention in theBM microenvironment. While the precise molecularcircuitry controlling HSC behavior still remains to befully elucidated, some of the mechanisms by whichhomeostatic production of blood cells is ensured havenow been uncovered. A large body of work has shownessential roles for two major categories of molecularnetworks in the maintenance of HSC functions: cell-intrinsic processes (i.e., cell cycle regulators andthe PI3-kinase signaling pathway) and cell-extrinsicdevelopmental pathways [i.e., transforming growthfactor- (TGF-), Wnt, Hedgehog, and Notch]. As

essential components of these regulatory networksare often deregulated in cancer, it is not surprisingthat improper control of HSC maintenance isintimately tied to the development of hematologicalmalignancies.

Cancer of the blood system usually results frompathogenic events that disrupt the normal homeostaticbalance between the turnover of mature hematopoieticcells and their replenishment by HSCs and progenitorcells, either due to deregulated proliferation leadingto increased output of progenitor/mature cells, orto the acquisition of self-renewal characteristics in a

progenitor compartment that normally does not self-renew (Figure 2).4 Hematological malignancies are aheterogeneous group of diseases that can globallybe viewed as aberrant hematopoietic processesinitiated by a population of leukemic stem cells(LSCs) that is able, like normal HSCs, to bothself-renew and differentiate, albeit in an aberrantfashion, thereby propagating themselves and givingrise to differentiated progeny that represent thebulk of the cells found in the tumor. Functionally,LSCs are defined by their unique ability to serially

I. Normal hematopoiesis

HSC MP

MP

Granulocytes

Granulocytes

BlastsHSC LSC

LSC

II. Chronic phase CML

III. AML or blast phase CML

FIGU RE 2 | Leukemic stem cells (LSCs) may reside in distinctcompartments and have both common and distinct features.

propagate the disease in transplanted mice. A numberof studies have shown that LSCs can originateeither from rare transformed HSCs or from moreabundant transformed progenitor cells dependingon the stage of the disease (chronic neoplasmvs acute leukemia), its immunophenotype (myeloidvs lymphoid) and the nature of the transformingevent [chromatin remodeling factors such as MLLfusion genes involved in pediatric acute myeloidleukemia (AML) vs activated tyrosine kinases suchas BCR/ABL involved in chronic myelogenousleukemia (CML)].5 While the tumor burden is likelyresponsible for the clinical symptoms, mountingevidence suggests that LSC persistence is responsiblefor disease maintenance and underlies recurrence.6

Thus, failure to achieve long-term cures may directlyrelate to a failure to eradicate these rare disease-causing cells. Developing interventions that willspecifically target LSCs is therefore an appealingapproach to improve cancer treatment. However,much remains to be learned about LSC biology,

including how deregulation of the mechanismsrequired for maintenance of normal HSC functionsare involved in LSC generation and evolution duringdisease development.

In this review, we provide an up-to-datedescription of the surface markers used for identifyingHSCs in mice and humans, and of their limitationsin terms of investigating HSC biological properties.We describe the current understanding for the roleof key components of both cell autonomous andnon-cell autonomous pathways and their function



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WIREs Systems Biology and Medicine Mechanisms controlling HSC functions

in HSC maintenance. Moreover, we discuss howderegulation of these molecular networks impacton the development of LSCs and hematologicalmalignancies.

HSC IDENTIFICATION: AN EVOLVINGPROCESS

The hematopoietic system provides a compellingmodel to study tissue development as HSCs, pro-genitors, and mature cells can be accurately purifiedand functionally tested using a variety of in vitro andin vivo approaches (Figure 1).7 Functionally, HSCsare defined by their ability to serially reconstitute theentire blood system of lethally irradiated recipientmice. The stringency of the reconstitution analysis(i.e., time after transplantation, level of engraftment,

multilineage nature of the reconstitution, etc.) allowsone to discriminate between long-term engraftingHSCs, short-term engrafting MPPs, and non-self-renewing lineage-committed progenitors. The ability

to provide sustained multilineage reconstitution for4 months in transplanted recipients has long been

considered a reflection of long-term HSC activity.However, the recent discovery of an intermediate-termprogenitor population able to provide a multilineage

readout for up to 6 months8 has recently challengedthis assumption and might lead to a re-evaluation ofthe timing of these transplantation assays.9 This noteof caution is particularly important for transplanta-tion of human HSC-enriched populations in xenograftmice since the kinetics of lineage production by human

stem and progenitor cells is less well-established butis likely to be far longer than for mouse cells. In

TABLE 1 Mouse HSC Purification Schemes

Markers (on Adult Bone Marrow Cells) HSC Frequency Notes References

None


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TABLE 2 Human HSC Purification Schemes

Markers (on Cord Blood, Bone MarrowCells, and Mobilized Pheripheral Blood) HSC Frequency Notes References

CD34+ Transplantation in SCID/beige/XID,

NOD/SCID and NOD/SCID/IL2R-null(NOG) mice

29

30

Lin-CD34+CD90+ CD90 (Thy1)Transplantation in SCID-hu mice

3132

CD34+CD38 1:600 Transplantation in NOD/SCID mice 3334

LinCD34+CD38Rholow 1:30 Transplantation in SCID mice 35

Lin-CD34+CD38CD90+CD45RA 1:10 Current standard for human HSC

isolation

Transplantation in newbornRag//c/

11

HSC, hematopoietic stem cell.

the mouse, numerous cell surface markers have beenidentified that can distinguish HSCs from the bulkof the BM cells, with recent staining combinationsachieving exquisitely high purity.10,11 For instance,using a lineagecKit+Sca1+(Flk2)CD150+CD48

staining scheme one can isolate mouse HSCs that haverobust (90%) cloning efficiency in vitro and singlecell transplantability into lethally irradiated congenicrecipients with a purity of 1 in 1.3 cells.10,12 As theadvent of new surface markers allows for the studyof increasingly pure HSCs, it raises issues pertainingto previous studies performed on more heterogeneous

stem and progenitor populations. For decades, markercombinations such as lineage (Lin), linSca1+ (LS),lincKit+Sca1+ (LSK), lincKit+Sca1+Flk2 (LSKFor HSPC), lincKit+Sca1+Flk2Thy1.1low (LSKFT)and lincKit+Sca1+Flk2CD34 (LSKF34), as wellas vital dye exclusion techniques using Hoechst andRodamine-123, have been used to investigate mouseHSC functions.13 It is now clear that a large fraction (ifnot most) of the cells present in these populations donot have HSC properties (Table 1). Similar issues arisefor the study of human HSCs (Table 2). Mouse genet-ics has also shown that the deregulation of numerous

genes and regulatory processes can affect the fre-quency of HSCs and early progenitor cells present inthese enriched fractions. Unfortunately, this skewingin cell composition can be inaccurately read as changesin HSC functional and biological readouts if less strin-gent HSC surface markers are used.14 Therefore, itshould be noted that some of the initial observationsmade on genetically modified mouse models mightnot stand the test of time and future investigationwith more stringent HSC markers and longer trans-plantation assays may provide novel conclusions.

CELL INTRINSIC MOLECULARNETWORKS AND SIGNALINGPATHWAYS

Control of cell proliferation, growth, and survivalis vital for the maintenance of homeostasis in everytissue. During adult life, HSCs are usually kept ina quiescent state (G0 phase of the cell cycle), whichcontributes to their long-term maintenance and allowsthem to rapidly enter the cell cycle in response toa variety of signals. Two major intrinsic cellularnetworks have been shown to play essential roles in

the maintenance of quiescence in HSCs: the Bmi1-p53axis of cell cycle regulators (Figure 3) and the PI3kinase (PI3K) signaling pathway (Figure 4).

Cell Cycle RegulatorsBmi1 is a member of the Polycomb Group genefamily that functions in multimeric protein com-plexes to repress gene expression through addition

Bmi1

p16INK4a p19ARF

RB p53

DNA damageOxidativestress

ApoptosisDNA repairSenescence

Cell cycle arrest

FIGU RE 3 | Bmi1-p53 cell intrinsic pathway.



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Growth factorsnutrients

oxygen status

PI3-kinase

Akt

PTEN

PML

FOXO Tsc1/2

mTORC1

Apoptosis Cell growth

Translation

Cell cycleprogression

Cell cyclearrest

Protection fromoxidative stress

FIGU RE 4 | PI3-kinase signaling pathway.

of epigenetic marks at genetic loci.36 Bmi1 controlscell proliferation specifically through its repressionof the Ink4a/Arf locus, which encodes two struc-turally distinct proteins,37 the CDK-inhibitor p16Ink4a

and the tumor suppressor gene p19ARF. p16Ink4a

expression activates RB, through its repression ofCyclinDCDK4/6 complexes. RB in turn negativelyregulates the expression of E2F target genes includ-ing Cyclin E, thereby restricting cell-cycle entry. Onthe other hand, p19ARF activates p53 by inhibit-ing its degradation by the E3-ligase Mdm2, leadingto the induction of p53 target genes that functionto protect cellular integrity either by inducing cellcycle arrest, DNA repair, initiation of apoptosis,or senescence.38,39 A plethora of p53 target genesinvolved in the mediation of these cellular processeshave been identified and include the CDK-inhibitor

p21 and the pro-apoptotic genes Puma, Noxa, andBax. Of note, in addition to p19ARFmediated activa-tion, p53 can also be activated following a large rangeof cellular insults including DNA-damage and oxida-tive stress.39 Numerous studies have demonstratedthat cell cycle deregulation can lead to profounddefects in the maintenance of HSCs and normalhematopoiesis, and can generate LSCs that driveleukemia development. Below, we describe the per-tinent studies that outline the role of several cell cycleregulators in normal and abnormal HSC functions.

Bmi1Bmi1 is an essential regulator of adult HSC mainte-nance. Mice deficient in Bmi1 develop hematopoieticfailure in early adulthood40 because of a dramaticloss of HSCs.41,42 Bmi1 is expressed in both murine(LSKT) and human (linCD34+CD90+CD38) HSCs

with its expression declining upon hematopoieticdifferentiation.4143 Although fetal HSCs appear todevelop normally in the absence of Bmi1, adult HSCsare rapidly depleted postnatally.41,42 Furthermore,transplanted Bmi1/ BM cells can only transientlyreconstitute host BM.41,42 Bmi1 depletion results inincreased p16Ink4a and p19ARF expression in adultBM and infection of wild-type LSKF cells with eitherp16Ink4a and p19ARF retroviral vectors results ingrowth arrest and cell death, respectively, in vitro.41

This suggests that the loss of HSCs in the absence ofBmi1 may be due to unchecked levels of the Bmi1targets p16Ink4a and p19ARF. However, although dele-tion ofp16Ink4a and p19ARF in Bmi1/ mice restoredthe defective capacity of Bmi1/ BM, their com-bined deletion only partially rescued the loss of HSCs(LSK34) in Bmi1-deficient mice, perhaps suggestinga contribution of Bmi1 in the BM microenvironmentthat is independent of its cell cycle targets.44 Recently,the deletion ofp16Ink4a alone has been shown to rescuethe engraftment defect seen in old purified HSCs,12,45

suggesting that p16Ink4a expression is responsible forlimiting HSC function with age. Whether this waspreceded by a decrease in the expression of Bmi1with age was not shown. However, another study was

unable to detectp16Ink4a expression in either young orold HSCs with no change in the expression of Bmi1.46

Thus, whether the Bmi1-p16Ink4a axis directly controlsHSC aging is still unclear.

Bmi1 was initially identified as an oncogene thatcan cooperate with c-Myc to drive lymphoma47,48 andhas since been shown to be elevated in certain hemato-logical malignancies.49 In addition to its role in normalHSCs, Bmi1 has also been shown to be essential for theself-renewal of LSCs. In a retroviral transplantationmodel of AML utilizing Hoxa9 and Meis1 oncogenes,the absence of Bmi1 did not affect leukemogenesis in

primary mice but Bmi1/

LSCs were unable to trans-fer the disease to secondary recipients, suggesting LSCdepletion.42 Furthermore, Bmi1/ leukemic blastsdisplayed increased accumulation in G1 and elevatedapoptosis, suggesting unchecked levels of p16Ink4a

and p19ARF, respectively.42 Interestingly, the status ofp19ARF has been shown to mediate the transformativeability of defined hematopoietic subsets in a BCR/ABLretroviral transplantation model. In the presence ofintact p19ARF only the HSC compartment could bedirectly transformed by enforced BCR/ABL expression



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resulting in CML, while loss ofp19ARF resulted in thetransformation of more committed lymphoid progeni-tors and B-ALL development.50 Thus, while Bmi1 maybe critical for LSC self-renewal, p19ARF may safeguardthe lymphoid lineage against oncogenic insults.

p53 and p21Considerable evidence suggests that p53 acts as a neg-ative regulator of HSC self-renewal. Mice deficientin p53 have a larger HSC compartment as deter-mined both by phenotyping and transplantation ofp53/ BM cells or purified LSK cells, which showincreased engraftment in competitive reconstitutionassays.51,52 However, transplantation of more purifiedp53/ HSCs (LinCD41CD150+CD48) actuallydemonstrated lower levels of engraftment in a simi-lar competitive repopulation assay.51 Thus, althoughthe loss of p53 can increase HSC frequency in theBM, the functional capacity of each HSC appears tobe decreased. Recently, p53 has been shown to reg-ulate HSC quiescence, as p53/ LSK34 cells showincreased incorporation of BrdU and decreased num-bers of cells in the Go phase of the cell cycle.

53 Thisincreased HSC cycling in p53/ mice may explainthe increased frequency of HSCs and contribute tothe decreased engraftment ofp53/ HSCs, as cyclingHSCs have been shown to have decreased long-termreconstitution potential.54 Lastly, p53 deletion hasalso been shown to rescue the engraftment defectof Bmi1-deficient cells.55 Interestingly, deletion of

p19ARF does not have a similar effect, suggesting thatin the absence of Bmi1, p53 expression levels can becontrolled in a p19ARF-independent manner.

Mice deficient in p21 have been shown tohave increased cycling stem and progenitor cells,as demonstrated by less Lin cells in Go, and anincreased frequency of BM cells able to form long-term colonies in vitro.56 Despite this increase incell numbers, p21/ HSCs displayed self-renewaldefects characterized by early exhaustion followingserial BM transfer and repeated 5-FU treatments.56

Thus, p21 expression may promote HSC stemness

by restricting cell-cycle entry. However, a recentwork using more purified HSCs (LSKCD48CD150+)showed no change in HSC quiescence in the absence ofp21 expression.57 Furthermore, another study showeda limited role for p21 in steady-state maintenance ofHSCs, and only noted a deficiency in competitiverepopulation when the transplanted BM was firsttreated with 2Gy irradiation.58 Thus, the role of p21in regulating quiescence in HSCs, although tempting,is likely minimal. In addition, these contradictoryfindings provide a clear example of how improved

surface markers can change the understanding of genefunction in HSC biology.

Although the role of p53 in cancer is firmlyestablished, how it contributes to the maintenanceof primitive leukemic cells is not yet established.p53 is mutated or deleted in various hematological

malignancies59

; however, whether this is an earlyevent that drives leukemia initiation is unknown.Interestingly, p21 has been proposed to be essentialfor the maintenance of PML/RAR expressing LSCs ina p53-independent manner. PML/RAR induces theaccumulation of p21, which in turn limits DNA-damage foci and proliferation in HSCs (LSKF34)and not more committed downstream progenitors.60

Although PML/RAR-p21/ mice develop acutepromyelocitic leukemia (APL) with similar latenciesas those expressing p21, their disease is nottransplantable, suggesting that p21 may promote LSCself-renewal by restricting cell-cycle entry and limitingexcessive DNA damage.60 Does p21 expressioncreate a permissive environment for the developmentof other leukemias? This is unclear. Althoughp21 expression was also shown to promote theinitiation of AML1/ETO-driven leukemia,60 anothergroup demonstrated that AML/ETO leukemiasdevelop only in the absence of p21.61 Furthermore,p21 deletion has been shown to improve HSCfunction in mice with dysfunctional telomereswithout accelerating cancer formation.62 Therefore,the role of p21 in the initiation and maintenanceof hematological malignancies may be particularly

context dependent.

PI3K Signaling PathwayThe PI3-kinase signaling pathway controls cell prolif-eration, growth, and survival by integrating numerousupstream signals, including growth factors, nutrients,and oxygen status63 (Figure 4). Critical downstreameffectors of PI3-kinase signaling include the ser-ine/threonine kinases Akt and mTorc1, which in turnare negatively regulated by a variety of tumor sup-pressor proteins. These include Pten and Pml, whichnegatively regulate Akt activation, and Tsc1/2, which

inhibit the activation of mTorc1. Activated Akt alsoinhibits the activity of the FoxO family of transcrip-tion factors, which are critical mediators of oxidativestress, by leading to their nuclear exclusion. Numerousstudies have demonstrated that improper Akt-mTorc1signaling can lead to profound defects in HSC main-tenance, suggesting that a tight regulation of thissignaling pathway is essential to maintain normalhematopoiesis. Below, we describe the pertinent stud-ies that outline the role of PI3-kinase signaling in HSCfunction.



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Pten and AktAberrant activation of the PI3K pathway hasbeen shown to lead to substantial hematopoieticphenotypes. Conditional deletion of Pten in adultHSCs using the IFN-inducible Mx1-Cre results inrapid myeloproliferative neoplasm (MPN) shortly

after Poly I:C (PI:C) treatment.64,65 These micequickly progress to frank AML and die within 46weeks.65 Analysis of HSC (LSKFCD48) showed thatalthough there was no evidence of increased apoptosis,there was approximately a threefold increase in thefrequency of cycling HSCs, suggesting that Ptennormally acts to restrain cell-cycle entry in the HSCcompartment.65 Although this led to a transientincrease in HSCs, over time the absolute numbersof HSC decreased. Furthermore, Pten/ HSCs couldnot persist in host mice following transplantation,suggesting that the absence of Pten results in HSC

depletion.

64,65

A similar observation was made in amyr-Akt retroviral transplantation model, in whichAkt is held in a constitutively active state. Mostof the mice transplanted with myr-Akt-infected,5-FU-treated BM developed rapid MPN, T celllymphoblastic leukemia (T-ALL) or AML within 68weeks posttransplantation.66 However, because of thedesign of these experiments and the early diseaseonset, it is difficult to determine whether the effects ofmyr-Akt were indeed mediated through the long-termengrafting HSC compartment. Nevertheless, short-term induction of myr-Akt resulted in a transientincrease in LSK cells that was accompanied by

increased cycling and a marked deficit in the abilityof myr-Akt-expressing BM cells to form long-termcolonies in culture.66 Interestingly, administration ofrapamycin (an inhibitor of mTorc1) following PI:Ctreatment was able to restore normal proliferation andlong-term engraftment potential of Pten/ HSCs,and at least partially rescue the formation of long-term colonies in the presence of myr-Akt.65,66 Thissuggests that the observed deficiencies of hyperactiveAkt signaling are predominantly the result of increasedmTorc1 activity in the hematopoietic system. If this isindeed the case one would expect similar phenotypes

upon Tsc1/2 deletion, which works downstream ofAkt and acts to restrain mTorc1 activity. Indeed, HSCs(LSKFCD48) from mice conditionally deficient forTsc1 in the BM show a rapid exodus from quiescenceand a transient increase in absolute numbers.67,68

Furthermore, Tsc1/ BM display a decreased abilityto competitively repopulate host mice, suggesting adefect in engraftment.67,68 However, unlike Pten/

or myr-Akt mice, Tsc1/ mice do not develophematological malignancies.67,68 Instead, Tsc1/

mice show BM hypocellularity that is likely caused by

increased levels of apoptosis in mature Tsc1/ cells.67

Thus, although there are considerable similaritiesbetween increased mTorc1 and Akt activity, thepathway is understandably more complex. This canlikely be explained by downstream targets of Akt otherthan mTorc1 and the fact that constitutive mTorc1

activation actually results in the inhibition of Aktthrough a negative feedback loop involving S6K.69

FoxOOne difference between Akt and mTorc1 activity isthe fact that Akt signals can also inhibit the activationof FoxO family of transcription factors. FoxO3a hasbeen shown to exhibit strong nuclear localizationin purified HSCs (LSK34), whereas it is primarilycytosolic in other hematopoietic compartments.70

Although mice deficient in FoxO3a exhibit normalhematopoietic differentiation, they have a profound

deficit in HSC self-renewal as evidenced by earlystem cell exhaustion following serial BM transfersand repeated 5-FU treatments.71 There is likelyconsiderable redundancy between FoxO familymembers, as mice triply deficient for FoxO1, FoxO3a,and FoxO4 show more pronounced hematopoieticdefects. FoxO1/3a/4/ mice exhibit an early lossof HSCs (LSKF) in the BM and extramedullaryhematopoiesis in the spleen resulting in myeloidexpansion.72 Both FoxO3a and FoxO1/3a/4 deficientmice show increased HSC cycling and higher levelsof reactive oxygen species (ROS) specifically in theHSC compartment.71,72 Lastly, in vivo treatment

of FoxO1/3a/4/ mice with the antioxidant NACreversed many of the hematopoietic phenotypes.Interestingly, although ROS levels were not examinedin the Pten/ mice, no changes in ROS levels wereobserved in myr-Aktmice.66

Overall PI3K PathwayThe above hematopoietic abnormalities in thepresence of hyperactive Akt-mTorc1 and loss ofFoxO1/3a/4 suggest that HSCs critically depend on acellular environment that minimizes signaling throughthe PI3K pathway. However, as Akt-mTorc1 sig-

nals are essential for numerous cellular processes onewould envision that HSCs do need some, albeit weakor transient, activation of PI3K signaling. Interest-ingly, although transplantation ofAkt1/2/ (Akt iso-forms most expressed in hematopoietic cells) fetal livercells showed increased HSC (LSKCD150+CD48)engraftment, they produced markedly decreased num-bers of mature myeloid and lymphoid cells.73 Long-term culture ofAkt1/2/ HSCs also showed a deficitin colony formation, indicating a likely defect in HSCproliferation/differentiation. Interestingly, analysis of



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engrafted Akt1/2/ HSCs revealed decreased levelsof ROS which, when elevated using pharmacolog-ical modulation, partially restored their long-termcolony-forming activity.73 Thus, it may be summa-rized that hyperactivation of the PI3K pathway resultsin decreased HSC quiescence, higher levels of ROS and

HSC exhaustion, whereas hypoactivation results indecreased HSC proliferation/differentiation, perhapsmediated through insufficient ROS levels.

Do LSCs equally depend on the PI3K pathwayfor their maintenance? As mentioned above, Pten/

mice develop rapid MPN, which progresses toAML because of genomic instability.65 Interestingly,although Pten/ HSCs have an inability to stablyreconstitute recipient mice, Pten/ AML are fullytransplantable, with the majority of the LSCs beingenriched in the HSC (LSKFCD48) compartment.Remarkably, rapamycin not only prevented thegeneration of LSCs, it actually rescued normal HSCfunction.65 This suggests that LSCs may be criticallydependent on high Akt activity, whereas normalHSCs may require Pten to minimize Akt signalsand maintain self-renewal. However, this does notuniversally appear to be the case. Pml was recentlyidentified as a negative regulator of Akt and mTorc1activity.74 Like Pten/ HSCs, the absence of Pmlleads to increased HSC (LSK34) cycling and adeficit in long-term reconstitution in transplantationassays,75 suggesting an essential role for Pml in HSCmaintenance. Pml is often elevated in CML patientsand correlates with a poor clinical outcome. Retroviral

expression of BCR/ABL in Pml/ LSK also leads toincreased cycling and a deficit in their ability to seriallytransplant the disease to recipient mice, suggestingthat Pml may play a critical role in maintaining CMLLSCs in a quiescent state.75 Interestingly, treatmentof BCR/ABL-expressing Pml+/+ LSK transplantedmice with arsenic trioxide (As2O3) resulted in thedegradation of Pml and cell-cycle entry, whichsynergized with AraC to eliminate quiescent LSCs.75

Although As2O3 also led to the cycling of wild-type HSCs, they were less affected than BCR/ABL-expressing Pml+/+ LSCs, suggesting a therapeutic

window to eliminate quiescent LSCs. Although thesetwo examples are conceptually rather different, theyboth demonstrate that it may be possible to interferewith the regulatory framework driving LSC self-renewal while sparing normal HSCs.

EXTRINSIC SIGNALING AND THE BMNICHE

Broadly speaking, the BM niche describes both thephysical association between HSCs and the specialized

cells that support them, as well as a signalingmicroenvironment consisting of growth factors,cytokines, chemokines, adhesion molecules, and theircognate receptors created by the association betweenHSCs and cells in the niche.76,77 Several studieshave defined endosteal and vascular compartments

intertwined within the BM niche that may servedistinct functions in HSC maintenance, as well asseveral cell populations of hematopoietic and non-hematopoietic origin residing in these compartmentsincluding megakaryocytes, osteoblasts, endothelialcells, and CXCL12-abundant reticular (CAR) cells(Figure 5).78 Recent work has also suggested thatthe BM niche is a hypoxic environment, andthat low oxygen concentration contributes to HSCmaintenance and protection against oxidative stress.79

Cells of the osteoblastic lineage were the first describedniche component based on coculture experiments andin vivo work in mice.80,81 More recent work hasalso shown that transplantation of CD105+Thy1

mesenchymal cells from murine fetal bone into thekidney capsule of adult mice can generate ectopicniches supporting HSC activity.82 Although the fullcomplement of signals exchanged between HSCs andtheir niche cells has not been fully elucidated, studiesperformed ex vivo and in mice have identified anumber of critical extrinsic cues required for HSChomeostasis. These include cytokines, chemokines,and growth factors such as SDF-1/CXCL12,Angiopoietin-1, IL-3, Flt3L, SCF, and the myeloidcolony stimulating factors G-CSF and GM-CSF.83 The

BM niche has also been shown to express a numberof key developmental factors that are essential forHSC self-renewal, such as TGF-, Wnt, Hedgehog,and Notch pathway components (Figure 5).

TGF- FamilyThe TGF- family of cytokines includes threemammalian isoforms of TGF- (numbered 13),approximately 20 isoforms of bone morphogenicprotein (BMP), and Activin that have been shownto play critical roles in development of a broad array

of cell types.84,85 TGF- is expressed by both primitivehematopoietic cells and BM stromal cells, suggestingthat HSCs may express/respond to TGF- by a wayof autoregulatory feedback mechanism.86 TGF- issecreted as an inactive peptide, which upon activationby protease cleavage in the extracellular matrix(ECM) binds to an arrangement of three surfacereceptors, TRI/II/III. TRI and II are expressedon primitive hematopoietic cells, and mediate thedownstream activation of Smad family transcriptionfactors, predominantly Smads 2, 3, and 4, and result



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Notch Wnt(Canonical)

Hedgehog TGF

TGFRII

TGFRI

Niche

TGF

Hh

Smo

Wnt3a

Notch

TACE

Numb

NICD

Jagged

LRP5/6

Frz

-catenin

-catenin

-catenin

GSK3APC

Gli

Su(Fu)Smad2

TCF/LEF

TCF/LEF

Cytoplasm

NucleusNICD

Ptc

Gli

Smad4HSC

Smad2

Smad3

Smad3

Smad3

Smad4

Smad4

Smad2

Mib

MAML

NICD

MAML

HSC

Osteob

lasts

EndothelialCells

CARCell

Blood

Vessel

Stroma

HSC

Bone

Osteoclast

Granulocytes

Endosteal

Niche

Vascular

Niche

FIGU RE 5 | Multiple signaling pathways from the niche are integrated in hematopoietic stem cell (HSC) to regulate self-renewal.

in the activation of essential target genes such as Hes1,c-myc, Pdgfra, p21, p15Ink4b, and junB.87,88

Early in vitro studies in both human and mousesuggested that TGF-1 and 3 mediated antipro-liferative effects on hematopoietic stem/progenitorpopulations.89 However, a precise understanding ofthe role of TGF- signaling in vivo has been diffi-cult to obtain due to the complexity of the pheno-types exhibited by mice deficient in TGF- signalingcomponents. The majority of Tgf-1/ mice diein utero at day E10.5 from multiple developmentaldefects, whereas the few that survive to adulthooddevelop a wasting disease due to an autoinflammatorycondition.9092 A similar embryonic lethal phenotypeis observed in TRII/ mice.93 Conditional deletionof TRI or TRII using Mx1-Cre yielded a similarautoimmune manifestation, but had no overt effecton numbers, cell cycle distribution, or self-renewal

characteristics of LSK, suggesting the existence ofcompensatory mechanisms or redundant signals forthe absence of TGF- signaling in vivo.9498 Interest-ingly, Mx1-Cre mediated deletion ofSmad4 impairedHSC self-renewal properties in competitive transplan-tation assays and decreased expression of c-myc andNotch-1 in LSK.87 However, these phenotypes werenot observed in TRI/II/ mice, thereby imply-ing the existence of additional signaling pathwaysoperating via Smad4 but independently of TGF-.Analysis of LSK34 cells treated ex vivo with TGF-

reveals attenuation of mitogen-mediated Akt and Srcactivation via inhibition of lipid raft clustering.71,99

Strikingly, TGF--mediated attenuation of Akt sig-naling increased activation and nuclear accumulationof FoxO3a, which is critical for limiting ROS levelsand maintaining HSC quiescence in vivo. Collectively,these data indicate that TGF- signaling may governROS levels in HSC, which in turn may be criticalfor their maintenance in the BM niche and protectionagainst oxidative stress.

Based on its antiproliferative effect inhematopoietic cells and other tissues, TGF- has ini-tially been thought to act as a tumor suppressor gene.Indeed, TGF- has been shown to repress the growthof both secondary myeloid leukemia cell lines andBCR/ABL-expressing CD34+ cells from human CMLpatients.100,101 Furthermore, BCR/ABL has been sug-gested to prevent TGF--mediated upregulation of the

CDK inhibitor p27, thus preventing inhibition of pro-liferation mediated by TGF-.101 More recent workusing purified HSCs (LSKFlk2CD48CD150+) ina mouse model of human CML demonstrated thatTGF--mediated inhibition of HSC expansion abso-lutely requires the AP-1 transcription factor JunB toregulate the expression of one of its key effectorgenes, the transcription factor Hes1, and prevent LSCgeneration.14 Given that decreased junB expressionhas been observed in human CML,102 these find-ings highlight a critical molecular circuit required



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for TGF--mediated regulation of hematopoiesis.Another study suggested that activation ofFoxO3a byTGF- might in fact play a positive role in the main-tenance of LSCs in vivo.103 Mice transplanted withBCR/ABL-expressing FoxO3a/ BM have decreasedincidence of CML disease relative to mice transplanted

with BCR/ABL-expressing wild-type cells, and inhibi-tion of TGF- signaling also decreased in vitro colonyformation of BCR/ABL-transduced LSK cells and pri-mary human LinCD34+CD38 CML cells. Theseresults suggest that the role of TGF- in maintainingHSC quiescence, while masked during steady-statehematopoiesis, may be critical in cases of stress orleukemia, when HSCs are driven toward an LSCproliferative phenotype. Collectively, these studieshighlight the complexity of the TGF- regulatorymechanism in normal and diseased hematopoiesis,and the challenge in attempting to generate a unifyingmodel. One possible explanation for these disparateobservations may be based on technical considerationssuch as the range of TGF- concentrations used inex vivo culture and in vivo experiments, as well as theheterogeneity of the hematopoietic populations exam-ined. Alternatively, recent evidence points toward theexistence of functionally distinct HSC subpopulationsthat are intrinsically biased toward the generationof myeloid or lymphoid progenies, with low levelsof TGF-1 inducing proliferation in the myeloid-biased population but not in the lymphoid-biasedpopulation.25 Whether the existence of such HSC sub-compartments may in part explain the seemingly con-

tradictory roles of TGF- in normal hematopoiesis,and whether a relationship exists between these HSCsubpopulations and leukemia development warrantfurther investigation.

Wnt/-Catenin PathwayThe Wnt proteins, named in reference to theDrosophila gene Wingless and its mouse homologIntegrase-1, are a large family of 19 solubleglycoproteins that exert a broad range of effects onmature and immature hematopoietic cells.104 While

Wnt proteins activate gene regulation events via atleast three distinct signaling pathways downstreamof their receptor complex, the majority of signalingin immature hematopoietic cells appears to occur viathe canonical Wnt pathway, in which Wnt proteinsexpressed in the BM stroma interact with a cellsurface receptor complex consisting of Frizzled (Frz)and two co-receptors Lrp5/6, resulting in activationof the transcription factor -catenin. In the absenceof receptor activation, -catenin is constitutivelyphosphorylated by a multiprotein complex including

the scaffold proteins Apc and Axin1 and thekinases Gsk3 and Ck1, resulting in proteosomaldegradation. In the presence of Wnt proteins, Lrp5/6are phosphorylated by Ck1 and Gsk3, resulting inrelocalization of Axin1 to the plasma membrane andrelease of-catenin that subsequently translocates to

the nucleus and interacts with the nuclear factors Tcfand Lef, resulting in the activation of essential targetgenes such as cyclinD1 and c-Myc.

In vitro investigations have revealed a potentialrole for Wnt proteins in HSC maintenance andself-renewal, as bcl2-transgenic LSK were shownto expand and maintain their immature phenotypeafter 7 days of ex vivo exposure to Wnt5a.105

Furthermore, retroviral transduction of wild-typeLSK with a constitutively active form of -cateninyielded a similar result in the presence of SCF,and inhibition of Wnt signaling by receptorinhibition or Axin1 overexpression led to decreasedHSC growth. Importantly, LSK expressing active-catenin showed increased expression of Notch1and HoxB4, further suggesting that Wnt signalingpromotes the preservation of an undifferentiatedHSC pool. Interestingly, expression of constitutivelyactive -catenin in lineage-committed myeloid andlymphoid progenitors also conferred multilineagepotential to these cells.106 Collectively, these datasuggest that Wnt signaling proceeding through -catenin plays a significant role in the maintenanceof multipotent HSCs in vitro. Examination of therole of Wnt signaling in vivo, however, has yielded

less clear results, at least initially. Studies using Mx1-Cre to delete -catenin or both -catenin and itshomolog -catenin in mice found no difference inHSC self-renewal as measured by transplantationassays.107,108 However, a later study using Vav-Credeletion of -catenin showed equivalent frequenciesof LSKFlk2 cells and downstream progenitors inadult BM, but deficiencies in HSC self-renewal basedon transplantation assays. These results highlight animportant limitation of mouse genetic experimentsand the fact that Mx1-Cre and Vav-Cre are notequivalent for the study of hematopoiesis with

respect to: (1) the tissue specificity of the deletion;(2) the timing of deletion (embryonic stage forVav-Cre vs adult stage for Mx1-Cre); and (3) therequirement for IFN signaling (for Mx1-Cre) thatexerts significant effects on HSC biology.109 Furtherclarification of the role of Wnt proteins in thehematopoietic system was provided by a studyin which the canonical Wnt signaling inhibitorDickkopf1 (Dkk) was expressed in mice using theosteoblast-specific collagen1 promoter.110 Underthese conditions, increased cell cycling and decreased



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HSC self-renewal by transplantation assay wereobserved in Dkk-expressing mice. Similar loss of HSCself-renewal was observed in Wnt3a/ fetal liverLSK.111 Studies using gain-of-function approachesfurther confirmed a role for Wnt signaling inHSC self-renewal. In vivo administration of the

GSK3 inhibitor CHIR-911 increased the repopulatingability of murine LSK and human CD34+CD45+

stem/progenitor cells in NOD/SCID mice.112 Inaddition, LSKCD48CD150+ cells from Apcmin mice,which carry a single allele containing a prematurestop codon in the Apc gene, showed increasedrepopulation potential in competitive transplantationassays, suggesting that the partial loss of -cateninnegative regulatory complex leads to increasedHSC self-renewal.113 Taken together, these studiesdemonstrate that canonical Wnt pathway is a bonafide regulator of HSC self-renewal.

Multiple lines of evidence indicate that Wnt/-catenin signaling also plays a critical role in leuke-mogenesis and progression of both CML and AML.CML disease in mice transplanted with BCR/ABL-transduced -catenin/ cells was significantly lessaggressive than in mice transplanted with BCR/ABL-expressing wild-type cells, and exhibited decreasedStat5 phosphorylation (a hallmark of BCR/ABL activ-ity) despite equivalent BCR/ABL expression in bothcontrol and -catenin-deficient cells.109 These resultsconfirm that BCR/ABL absolutely requires mainte-nance of the molecular pathways essential for HSCself-renewal to exert its transforming activity.114

Importantly, Wnt signaling has been shown to conferself-renewal properties to otherwise highly prolifer-ative, non-self-renewing committed myeloid progen-itors, and thus may play an important role in theleukemia progression. Granulocyte/macrophage pro-genitors (GMPs) from patients with blast-crisis CMLwere shown to have increased levels of -cateninexpression and activity, and to behave as LSCs.115

Further investigation revealed that a majority ofblast-crisis GMPs contained a misspliced Gsk3 iso-form, resulting in defective negative regulation of-catenin.116 Wnt signaling components have been

similarly implicated in the genesis and progression ofAML. Increased expression ofLef-1 has been observedin AML patients, and expression of a constitutiveactive form of Lef-1 in mice yielded an AML-likedisease.117 A recent study using a mouse model ofMLL/AF9-induced AML showed that GMPs recov-ered from leukemic mice had increased -cateninactivity and that inhibition of -catenin activity byindomethacin impaired disease progression.118 Takentogether, these findings suggest that Wnt signalingis not only necessary for the transformation of HSCs

into LSCs, but when deregulated may also be critical inconferring self-renewal characteristics and LSC prop-erties upon committed progenitor populations.

Hedgehog Pathway

Hedgehog (Hh) is a family of three secreted proteinstermed Sonic hedgehog (Shh), Indian hedgehog (Ihh),and Desert hedgehog (Dhh), which play significantroles in embryonic development. Deregulation of thissignaling mechanism is associated with oncogenesisin numerous tissues.119 Hh proteins bind to thesurface receptor Patched (Ptch1), which normallyassociates with and negatively regulates a secondsurface protein, Smoothened (Smo). Hh ligand bindingto Ptch1 releases its association with Smo, allowingSmo-mediated activation of three Glioblastoma (Gli)transcription factors, with Gli1 and Gli2 acting as

positive regulators of transcription and leading toactivation of essential target genes such as CyclinD1,CyclinE, c-Myc, Pdgf, Ptch1, and Vegf.112

Expression of Ihh in human stromal cells hasbeen shown to increase both colony formation andengraftment potential of human CD34+ progenitors,suggesting that presentation of Hh ligand to HSCsby BM niche cells is important in the regulation ofhematopoiesis.120 Furthermore, Ihh has been shown toplay a role in the development of endochondrial bone,and Shh has been suggested to activate proliferationof human CD34+ cord blood cells, likely via BMPsignaling.121,122 However, examination of the role

of Hh ligand and signaling components in vivo hasbeen complicated by the seemingly contradictoryfindings obtained from conditional knockout mice.Transplantation studies using Smo/ fetal liver cellsor Vav-Cre-mediated Smo-deleted cells demonstratedno overall defects in lineage production but decreasedreconstitution following serial transplantation andimpaired colony formation in serial replating assays,which suggest impaired HSC function in the absenceof Smo signaling.123,124 In contrast, Gli1-deficientmice had twofold more LSKCD34 cells than wild-type mice, and transplantation of Gli1-deficient

LSK resulted in increased reconstitution, likely dueto the increased size of the HSC compartmentobserved in these mice.125 Taken together, theseresults suggest that Gli1 and Gli2 may haveantagonistic effects in mediating Smo signaling inHSC maintenance, although an eventual role forGli2 in HSC function has not yet been formallyaddressed. Another study using mice heterozygous forPtch1 found that decreased Ptch1 expression led toincreased numbers of LSK, increased proliferation,and increased short-term reconstitution efficiency



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based on the transplantation of Lin cells, but yieldedpoor long-term reconstitution.112 This result suggeststhat the release of negative inhibition of Smo byPtch1 increases the number of actively cycling MPPsat the expense of the long-term engrafting quiescentHSCs. However, transplantation of unfractionated

Ptch1+/

fetal liver cells into recipients did notyield a similar defect in long-term reconstitutionpotential, though it is unclear to what extent theengraftment properties of fetal and adult HSCscan be directly compared.123 These findings leadto the suggestion that Hh signaling may functionto maintain self-renewal HSCs in conditions ofhigh hematopoietic stress such as transplantation ormyeloablation following 5-FU treatment. However,studies using Mx-Cre to delete Smo in thehematopoietic compartment of adult mice yielded nophenotype either during steady-state hematopoiesis,

transplantation of unfractionated BM, or upon 5-FUtreatment, leading to the opposite conclusion thatHh signaling may in fact not be necessary for themaintenance of the HSC compartment.126,127 Theapparent disagreement between these results mightjust be a byproduct of when and in what tissues Hhpathway components are deleted using the Mx-Creor Vav-Cre systems, and the eventual activation ofcompensatory mechanisms. Determining whether theloss of Smo during embryogenesis versus adulthoodaffects the epigenetic and expression profiles ofHSCs warrants further investigation. Analysis of HSCself-renewal in these models using highly purified

populations and consistent experimental designs mayalso assist in further defining the exact function of theHh pathway in controlling HSC function.

Hh signaling has recently been implicated inthe pathogenesis of CML. Increased mRNA levelsof Gli1 and Ptch1 have been detected in humanCD34+ cells from CML patients in both chronicand blast-crisis phases.123 Furthermore, treatmentof these cells with the Hh inhibitor cyclopaminesignificantly reduced their colony-forming ability,suggesting that Hh signaling is active in LSCsand may be important for disease progression.

Indeed, mice transplanted with either BCR/ABL-expressing Vav-Cre-mediated Smo-deficient BM cellsor BCR/ABL-expressing Smo/ fetal liver cells havedecreased incidence of CML disease relative tomice transplanted with BCR/ABL-expressing wild-type cells.123,124 Furthermore, cyclopamine treatmentof mice transplanted with BCR/ABL-expressing wild-type LSK show a similar decrease in CML incidence,while transplantation of BCR/ABL-transduced LSKcells expressing constitutively active Smo resulted inincreased disease kinetics and incidence.124 In terms

of mechanism, treatment of BCR/ABL-expressing LSKwith cyclopamine results in apoptosis, while thepresence of active Smo signaling leads to decreasedexpression of Numb, a Notch1-interacting proteinthat has been shown required for the retention of anundifferentiated HSC pool.128 Collectively, these two

studies suggest that Hh may play a critical role in LSCsurvival and thereby CML development.

Notch PathwayThe Notch family consists of four proteins (numbered14) expressed on the cell surface that interact withfive ligands (Jagged1 and 2, and Delta 1, 3, and 4)expressed on the surface of a neighboring cell.129 Uponligand binding, the extracellular domain of Notch(NECD) is cleaved by the TNF--converting enzyme(Tace), and the intracellular domain of Notch (NICD)is subsequently cleaved by a -secretase complex,

allowing its translocation to the nucleus, where itinteracts with coactivator proteins such as Cbf-1and Mastermind-like (Maml) to effect transcriptionof essential target genes such as Hes1, Hes5, andHey1, as well as Notch1 itself.130,131 Interestingly,Notch activity also depends on internalization of theligand by the signaling cell via the E3 ubiquitin ligasesMindbomb (Mib)-1/2 and Neuralized (Neur)-1/2.132

Deletion of Notch1, Jagged-1, or Cbf-1 in miceall lead to defects in embryonic HSC development,indicating a critical role for this developmental path-way in the genesis of the hematopoietic system.133135

The Notch pathway has also been implicated in theregulation of adult hematopoiesis, where signalinghas been shown to occur between the immaturehematopoietic compartment and components of theniche, particularly osteoblasts and endothelial cells,which express Jagged and Delta ligands and are capa-ble of supporting the expansion of wild-type but notNotch1/2-deficient LSK cells.80,136,137 Assessment ofNotch activity in vivo using the TNR-Gfp reportermouse, which carries a transgenic Notch reporterconsisting of GFP driven by four Cbf-1 sites, indi-cated that Notch signaling occurs primarily within thehematopoietic stem and progenitor compartment.138

Induction of Notch signaling in hematopoietic stemand progenitor cells in vitro, either via overexpres-sion of NICD, addition of exogenous ligand, orcoculture with ligand-expressing cells, resulted inhematopoietic progenitor expansion.136,139142 Inhi-bition of Notch signaling by retroviral expressionof dominant-negative Cbf-1 or Maml in LSK led toincreased differentiation of these cells and depletion ofLSK in vivo, supporting in vitro evidence that Notchfunctions to maintain an undifferentiated hematopoi-etic stem/progenitor pool.138 Furthermore, retroviral



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expression of activated Notch in LinSca1+ cellsand subsequent competitive transplantation assaysrevealed that Notch signaling expanded LSK popu-lations in vivo while simultaneously decreasing thenumbers of mature cells, particularly toward themyeloid lineage, suggesting that activated Notch-

mediated enhancement of HSC self-renewal may occurat the expense of HSC differentiation.143 In con-trast, Mx1-Cre-induced deletion of Jagged-1 alone ortogether with Notch1 in both hematopoietic and BMstromal cells yielded no clear phenotype in the HSCcompartment under steady state, transplantation, or5-FU treatment conditions.144 However, these exper-iments did not rule out a potential compensation byother Notch receptors expressed on the hematopoieticcells or by other ligands expressed on niche cells, noris it clear that Mx1-Cre-mediated deletion fully elim-inates Jagged-1 from the relevant niche components.

Subsequently, in vivo studies using Mx1-Cre inducibleexpression of dominant-negative forms of Maml andCbf-1, which broadly inhibit canonical Notch signal-ing, have been conducted. Strikingly, hematopoieticstem and progenitor cells from these mice showedno difference in frequency, number, or reconstitutionpotential relative to control mice.145 Given the previ-ously discussed disparities between studies using Mx1and Vav promoters to drive Cre expression, it wouldbe interesting to test whether the use ofVav-Cre woulduncover defects in HSC self-renewal upon transplantor stress. Collectively, these findings suggest thatNotch plays a debatable role in HSC maintenance by

limiting cell proliferation and differentiation, thoughany requirement for Notch in adult hematopoiesis maybe masked by compensatory signaling mechanisms, ormay only be required under certain stress conditions.

Currently, there is a paucity of clinically-derivedevidence describing a role for Notch signaling inCML. However, as Notch signaling regulates HSCself-renewal, it is conceivable that loss of Notchsignaling may promote leukemogenesis due to dereg-ulated HSC proliferation and differentiation. Con-sistent with this reasoning, overexpression of theNICD in the K562 human CML cell line attenuates

cell proliferation and leads to increased expressionof Rb and Hes1.146 Notably, HSCs (LSKFlk2 andLSKFlk2CD150+CD48) from junB-deficient miceexhibit decreased expression of several Notch tar-gets, including Hes1, Hes5, and Notch1, suggestingthat diminished Notch signaling in LSCs contributeto MPD development in this model of CML-likedisease.14 Mice with conditional deletion of Mib-1(MMTV-Cre or Mx1-Cre mediated) also develop afatal MPN and show expansion of the BM LSKcompartment, but without significant increase in

the numbers of LSKCD150+ HSCs.147 However,transplantation of unfractionated Mib-1-deficient BMinto wild-type lethally irradiated recipients did notgenerate disease in the recipients, whereas trans-plantation of wild-type BM into lethally irradiatedMMTV-Cre Mib-1f/f mice recapitulated the MPD

phenotype. This finding suggests that Notchligandinteractions between cells of the BM niche, rather thanor in addition to interactions between HSCs and nichecells, are important for controlling HSC proliferationand could be implicated in leukemogenesis. However,the precise mechanism by which Notch signaling reg-ulates niche cell function, as well as the identity ofthe cells participating in this signaling circuit, remainas yet unknown. Interestingly, while the aforemen-tioned evidence suggests that loss of Notch signalingmay promote the development of well-differentiatedmyeloid malignancies similar to chronic-phase CMLby promoting excessive proliferation and differenti-ation of LSCs arising from the HSC compartment,some evidence from human patient samples indicatesthat, similar to T-ALL, deregulated Notch signalingin myeloid progenitors may promote the acquisi-tion of inappropriate self-renewal and blast formationcharacteristic of AML.148150 Thus, similar to othersignaling mechanisms that confer self-renewal prop-erties, whether Notch signaling plays a positive ornegative role in the development of myeloid leukemiadepends critically on whether the LSC is an aberrantHSC or a committed progenitor cell.

EPIGENETIC REGULATION ANDREPROGRAMMING OF HSCS AND LSCS

The self-renewal and multilineage potential of HSCsmay be governed to a significant extent by factorsthat can regulate proliferation and differentiation viaepigenetic modifications.151 It therefore follows thatthe state of the HSC genome must remain poisedfor efficient differentiation into progenitor cells, yetsufficiently silenced to maintain quiescence and to pre-vent aberrant activation of differentiation programs.While these two objectives may seem contradictory

in the context of HSC biology, experimental workhas shown that such a balance in the epigenetic stateof HSCs may play important functional roles, andthat changes in or deregulation of this state mayaffect the pathogenesis and progression of leukemia.Recent work using mice with Mx1-Cre-inducible dele-tion of the DNA methyltransferase Dnmt1, whichis required to enforce gene silencing by maintainingmethylation of genomic DNA at CpG motifs, hasshown that constitutive DNA methylation is neces-sary for HSC self-renewal, as Dnmt1-deficient mice



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rapidly develop fatal pancytopenia with depletionof the LSK compartment.152 Strikingly, experimentsusing mice with a hypomorphic allele of Dnmt1showed reduced numbers of LSK cells, decreased HSCself-renewal capacity based on serial transplantation,and aberrant expression of myeloid-erythroid genes

in the LSK compartment, with concomitant loss oflymphoid potential. Interestingly, mice transplantedwith MLL/AF9-transduced Dmnt1-hypomorphic BMdeveloped less severe disease than control recipients,suggesting that repression of differentiation-relatedgenes is crucial to the self-renewal of both HSCs andLSCs. These data demonstrate that the mechanisticunderpinnings of HSC and LSC self-renewal are strik-ingly similar, and, importantly, that the epigeneticstate of HSCs and progenitors is highly dynamic andby no means irreversible.

The mutability of hematopoietic stem/progenitorepigenetic programs has been recently demonstratedby their efficient reprogramming into induced pluripo-tent stem (iPS) cells using an inducible secondaryiPS generation system, in which somatic cells fromchimeric mice, generated by injection of primary cellstransduced with Oct4, Sox2, c-myc, and Klf4 underthe control of doxicycline (dox)-inducible promoters,can be reprogrammed into iPS cells in the presenceof dox.153 Whereas terminally differentiated granu-locytes, B and T cells are reprogrammed with verylow efficiency (


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A systems biology approach, specifically the useof high-throughput methodologies such as expressionmicroarrays, ChIP-seq, genomic sequencing, and othertechnologies, is particularly suited for the discoveryof novel druggable targets that are specifically dereg-ulated in LSCs but not in normal HSCs or myeloid

progenitors as they allow for rapid and global analy-sis of differentially regulated genes and gene networksat the transcriptional, epigenetic, and genomic lev-els. However, in order for such comparative analysesto be informative in understanding how LSCs dif-fer from normal HSCs and progenitor cells, as wellas for the rational development of pharmaceuticaltherapies, a number of caveats must be addressed.First, the input cell populations need to be enrichedto high purity to ensure that true HSC and LSCpopulations are being analyzed; in the mouse forinstance, LSK cell populations contain large numbersof contaminating MPPs which may dilute the desiredHSC or LSC gene expression signatures. However,the use of surface marker phenotyping does not guar-antee that true repopulating cells are being isolated;thus, it remains critical that putative normal or LSC

populations, the latter of which may have aberrantsurface marker expression, also be defined based onlimiting-dilution transplantability in order to ensurethat correct and comparable populations are beinganalyzed. Secondly, serious attention must be paid tothe disease state of the isolated cell population, as

this may significantly affect the nature of the LSC andthe hematopoietic compartment in which it resides,as well as the interpretability of the data due to dif-ferences in gene regulation signatures. Finally, a morestandardized set of analyses, using both systems biol-ogy approaches as well as more traditional in vitro andin vivo studies, should be defined to allow for greaterintercomparability of data between studies performedin different laboratories. With such points in mind, theuse of systems biology and traditional approaches toanalyze the mechanisms that regulate self-renewal innormal and leukemic HSCs and myeloid progenitorsstands to yield significant insight into the basic biolog-ical processes underpinning hematopoiesis, as well asthe nature of LSCs and our capacity to design targetedtherapies that selectively eliminate and/or reprogramthem.

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