**2.1.2 Negative regulation**

### **2.1.2.1 E3 ubiquitin ligase**

The E3 ubiquitin ligase, c-Cbl, is a member of the RING finger-type ubiquitin ligase Cbl (casitas B-cell lymphoma) family. The c-Cbl protein is thought to implement the degradation of various cellular proteins, receptors, and signaling molecules including Notch1, STAT5, and c-Kit (Jehn et al., 2002; Goh et al., 2002; Zeng et al., 2005). c-Cbl–deficient mice were used to study the role of c-Cbl in HSCs (Rathinam et al., 2008). The number of HSCs and progenitors was significantly higher in the BM of c-Cbl-null mice due to increased proliferation. Interestingly, detailed analyses revealed augmented STAT5 phosphorylation in *c-Cbl*-/- HSCs in response to TPO/c-MPL signaling which is crucial for the proliferation and self-renewal of HSCs (Kimura et al., 1998), and this led to enhanced c-Myc expression. C-Cbl–deficient HSCs also showed an increased repopulating ability in competitive reconstitution assays, including serial transplantation. These results suggest that c-Cbl acts as a negative regulator of both the size of the HSC pool and self-renewal (Rathinam et al., 2008).

Recently, Itch, another E3 ligase belonging to the HECT family (Bernassola et al., 2008), was also identified as a negative regulator of HSC homeostasis and function. The phenotype of *Itch*-/- HSCs was similar to that of *c-Cbl*-/- HSCs. However, unlike c-Cbl, Itch-deficient HSCs showed augmented Notch1 signaling. Furthermore, knockdown of Notch1 in Itch-null HSCs resulted in the reversion of the phenotype (Rathinam et al., 2011). Taken together, these studies underscore the pivotal roles of E3 ubiquitin ligases and the importance of posttranslational modification of HSCs in the molecular control of HSC self-renewal.

#### **2.1.2.2 Egr1**

Egr1 is a member of the immediate early response gene family (Gashler et al., 1995). Egr1 is highly expressed in LT-HSCs under steady-state conditions and is downregulated upon proliferative stimulation and migration in response to pharmacological mobilization (Min et al., 2008). Egr1-deficient mice show a significant increase in the frequency of cycling HSCs. This phenomenon results in a slightly higher frequency of HSCs in the BM of *Egr1*-/- mice. Interestingly, loss of Egr1 results in a striking increase (up to 10-fold) in the number of circulating HSCs. Importantly, HSCs isolated from both the BM and peripheral blood of *Egr1*-/- mice show a greater degree of long-term multi-lineage repopulation after transplantation, although their life span is slightly reduced. Quantitative RT-PCR analysis shows that Bmi1 is upregulated in *Egr1*-/- HSCs. In addition, *Egr1*-/- HSCs also show the downregulation of p21CIP1/WAF1 and increased expression of cyclin-dependent kinase 4 (cdk4), which is consistent with their increased cell-cycling status (Min et al., 2008). Taken together, the deletion of Egr1 causes an increase in the number of cycling HSCs but does not lead to stem cell exhaustion. This may be due to Bmi1 upregulation.

#### **2.1.2.3 Lnk**

Lnk is a member of an adaptor protein family that possesses a number of protein-protein interaction domains: a proline-rich amino-terminus, a pleckstrin homology (PH) domain, a Src homology 2 (SH2) domain, and many potential tyrosine phosphorylation motifs (Rudd., 2001). Studies using Lnk-deficient mice show that Lnk-null HSCs are expanded during postnatal development (Ema et al., 2005; Buza-Vidas et al., 2006). The *Lnk*-/- HSC population contains an increased proportion of quiescent cells and shows decelerated cell cycle kinetics and enhanced resistance to repeat treatment with 5-FU *in vivo* compared with wild-type HSCs. Genetic evidence demonstrates that Lnk controls HSC self-renewal and quiescence, predominantly through c-Mpl. Furthermore, Lnk-deficient HSCs show higher levels of symmetric proliferation in response to thrombopoietin (TPO) in *ex vivo* culture than wildtype HSCs (Seita et al., 2007). Biochemical analyses revealed that Lnk directly binds to phosphorylated tyrosine residues in JAK2 after TPO stimulation (Bersenev et al., 2008). Therefore, Lnk is a physiologic negative regulator of JAK2 in HSCs, and TPO/c-Mpl/JAK2/Lnk constitute a major regulatory pathway controlling HSC quiescence and selfrenewal.

#### **2.1.2.4 Myc**

46 Advances in Hematopoietic Stem Cell Research

The E3 ubiquitin ligase, c-Cbl, is a member of the RING finger-type ubiquitin ligase Cbl (casitas B-cell lymphoma) family. The c-Cbl protein is thought to implement the degradation of various cellular proteins, receptors, and signaling molecules including Notch1, STAT5, and c-Kit (Jehn et al., 2002; Goh et al., 2002; Zeng et al., 2005). c-Cbl–deficient mice were used to study the role of c-Cbl in HSCs (Rathinam et al., 2008). The number of HSCs and progenitors was significantly higher in the BM of c-Cbl-null mice due to increased proliferation. Interestingly, detailed analyses revealed augmented STAT5 phosphorylation in *c-Cbl*-/- HSCs in response to TPO/c-MPL signaling which is crucial for the proliferation and self-renewal of HSCs (Kimura et al., 1998), and this led to enhanced c-Myc expression. C-Cbl–deficient HSCs also showed an increased repopulating ability in competitive reconstitution assays, including serial transplantation. These results suggest that c-Cbl acts as a negative regulator of both the size of the HSC pool and self-renewal (Rathinam et al.,

Recently, Itch, another E3 ligase belonging to the HECT family (Bernassola et al., 2008), was also identified as a negative regulator of HSC homeostasis and function. The phenotype of *Itch*-/- HSCs was similar to that of *c-Cbl*-/- HSCs. However, unlike c-Cbl, Itch-deficient HSCs showed augmented Notch1 signaling. Furthermore, knockdown of Notch1 in Itch-null HSCs resulted in the reversion of the phenotype (Rathinam et al., 2011). Taken together, these studies underscore the pivotal roles of E3 ubiquitin ligases and the importance of post-

Egr1 is a member of the immediate early response gene family (Gashler et al., 1995). Egr1 is highly expressed in LT-HSCs under steady-state conditions and is downregulated upon proliferative stimulation and migration in response to pharmacological mobilization (Min et al., 2008). Egr1-deficient mice show a significant increase in the frequency of cycling HSCs. This phenomenon results in a slightly higher frequency of HSCs in the BM of *Egr1*-/- mice. Interestingly, loss of Egr1 results in a striking increase (up to 10-fold) in the number of circulating HSCs. Importantly, HSCs isolated from both the BM and peripheral blood of *Egr1*-/- mice show a greater degree of long-term multi-lineage repopulation after transplantation, although their life span is slightly reduced. Quantitative RT-PCR analysis shows that Bmi1 is upregulated in *Egr1*-/- HSCs. In addition, *Egr1*-/- HSCs also show the downregulation of p21CIP1/WAF1 and increased expression of cyclin-dependent kinase 4 (cdk4), which is consistent with their increased cell-cycling status (Min et al., 2008). Taken together, the deletion of Egr1 causes an increase in the number of cycling HSCs but does not

Lnk is a member of an adaptor protein family that possesses a number of protein-protein interaction domains: a proline-rich amino-terminus, a pleckstrin homology (PH) domain, a Src homology 2 (SH2) domain, and many potential tyrosine phosphorylation motifs (Rudd., 2001). Studies using Lnk-deficient mice show that Lnk-null HSCs are expanded during postnatal development (Ema et al., 2005; Buza-Vidas et al., 2006). The *Lnk*-/- HSC population

translational modification of HSCs in the molecular control of HSC self-renewal.

lead to stem cell exhaustion. This may be due to Bmi1 upregulation.

**2.1.2 Negative regulation 2.1.2.1 E3 ubiquitin ligase** 

2008).

**2.1.2.2 Egr1** 

**2.1.2.3 Lnk** 

Human c-MYC was the second proto-oncogene to be identified and encodes a basic helixloop-helix leucine zipper transcription factor (c-Myc) (Sheiness et al., 1978). Overexpression of one of the three family members has been detected in numerous human cancers including Burkitt's lymphoma (c-MYC), neuroblastoma (N-MYC), and small cell lung cancer (L-MYC) (Nesbit et al., 1999). Conditional deletion of c-Myc in the BM results in cytopenia and the accumulation of functionally defective HSCs. In the absence of c-Myc, HSC differentiation into more committed progenitors is inhibited because they upregulate a number of adhesion molecules, such as N-cadherin, that anchor them in the niche. Conversely, enforced c-Myc expression in HSCs causes marked repression of N-cadherin and integrin expression leading to the loss of self-renewal ability at the expense of differentiation (Wilson et al., 2004). These results suggest that c-Myc activity controls the first differentiation step of LT-HSCs *in vivo*. Unexpectedly, conditional ablation of both c-myc and N-myc results in pancytopenia and rapid lethality due to HSC apoptosis via the accumulation of the cytotoxic molecule, Granzyme B (Laurenti et al., 2008). Thus, Myc activity controls important aspects of HSC function such as proliferation, survival and differentiation.

#### **2.1.2.5 MEF/ELF4**

MEF (also known as ELF4), an Ets transcription factor, was identified as a novel component of the transcriptional circuit that dynamically regulates HSC quiescence (Lacorazza et al., 2006). Mef-deficient HSCs grow more slowly than wild-type HSCs in response to cytokine stimulation Pyronin Y staining and BrdU incorporation show increased quiescence. Enhanced HSC quiescence in Mef-null mice also increases HSC resistance to cytotoxic agents that target dividing cells and allows more rapid hematological recovery after chemotherapy or irradiation. These findings suggest that Mef normally functions to induce or facilitate the entry of quiescent HSCs into the cell cycle and imply that Mef expression and/or activity may be dynamically regulated in HSCs. To explain this, Lacorazza et al. proposed a model in which Mef acts at an earlier stage than p18 and antagonizes p21.

#### **2.2 Survival of HSCs**

HSC self-renewal and apoptosis represent major factors that determine the size of the HSC mass. The number of HSCs is also controlled by their capacity to survive during homeostasis or under conditions of stress.

Regulation of Hematopoietic Stem Cell Fate: Self-Renewal, Quiescence and Survival 49

Zfx is a zinc finger protein belonging to the Zfx/ZFy family. Mammalian Zfx is encoded on the X chromosome and contains an acidic transcriptional activation domain, a nuclear localization sequence, and a DNA binding protein domain consisting of 13 C2H2-type zinc fingers (Schneider-Gadicke et al., 1989). Zfx is highly expressed in both HSCs and undifferentiated embryonic stem cells (ESCs). Using conditional gene targeting, Zfx was identified as an essential transcriptional regulator of HSC function (Galan-Caridad et al., 2007). Constitutive or inducible deletion of Zfx in HSCs (using *Tie2-Cre* and *Mx1-Cre* deletion strains, respectively) impairs self-renewal, resulting in increased apoptosis and the

ADAR (adenosine deaminase acting on RNA) catalyzes the deamination of adenosine to inosine in double-stranded RNA. Conventional *Adar*-/- mice die around embryonic day 11.5–12 because of widespread apoptosis and defective hematopoiesis (Hartner et al., 2004; Wang et al., 2004). Conditional deletion of Adar in HSCs shows that ADAR1 is essential for the maintenance of both fetal and adult HSCs, and leads to global upregulation of type I and II interferon-inducible transcripts and rapid apoptosis (Hartner et al., 2009). Interferon regulatory factor-2 (Irf2), a transcriptional suppressor of type I interferon signaling, is a positive regulator of HSC quiescence (Sato et al., 2009). Irf2-deficient HSCs are unable to restore hematopoiesis in irradiated mice, but the reconstituting capacity of *Irf2*-/- HSCs can

Various external stresses, such as myelosuppressive chemotherapy, bleeding, infection, and total body irradiation, put HSCs under stress, as they must proliferate to produce large numbers of primitive progenitor cells, thereby enabling rapid hematologic regeneration. Although this property has long been recognized, the molecular basis underlying the reaction of HSCs to hematologic emergency remains enigmatic. However, some key players

Heme promotes the proliferation and differentiation of hematopoietic progenitor cells (HPCs) (Chertkov et al., 1991) and stimulates hematopoiesis (Porter et al., 1979; Abraham, 1991). The degradation of heme is catalyzed by heme oxygenase (HO). HO-1, encoded by the *Hmox1* gene, is the stress-inducible isozyme of HO and is highly expressed in the spleen and BM (Abraham, 1991). Heterozygous HO-1–deficient mice (*HO-1*+/-) show accelerated hematologic recovery from myelotoxic injury induced by 5-FU treatment, and mice transplanted with *HO-1*+/- BM cells show more rapid hematopoietic repopulation than those transplanted with *Ho-1*+/+ BM cells. However, *HO-1*+/- HSCs show a reduced capacity to rescue lethally irradiated mice and to serially repopulate irradiated recipients (Cao et al., 2008). These results suggest that HO-1 limits the proliferation and differentiation of HPCs under stressful conditions, and that the failure of this mechanism can lead to the premature

**2.2.4 Zfx** 

**2.2.5 ADAR1** 

have been identified.

**2.3.1 Heme oxygenase-1** 

exhaustion of the HSC pool.

upregulation of stress-inducible genes.

be restored in these cells by disabling type I IFN signaling.

**2.3 Response to hematopoietic emergency** 

### **2.2.1 Bcl-2 family**

Accumulating evidence suggests that the suppression of apoptosis is required for HSC survival. Forced expression of Bcl-2 increases the number of HSCs and provides them with enhanced competitive repopulation ability (Domen et al., 1998, 2000), suggesting that cell death plays a role in regulating HSC homeostasis.

Mcl-1, another anti-apoptotic Bcl-2 family member, is also an essential regulator of HSC survival. Mcl-1 is highly expressed in LT-HSCs, and conditional deletion of MCl-1 results in the loss of the early BM progenitor population, including HSCs, leading to fatal hematopoietic failure (Opferman et al., 2005). Recently, it was reported that Mcl-1 is an indispensable regulator of self-renewal in human stem cells and that functional dependence on Mcl-1 defines the human stem cell hierarchy (Campbell et al., 2010).
