Acute myelogenous leukemia stem cells (AML–LSC) give rise to the leukemic bulk population and maintain disease. Relapse can arise from residual LSCs that have distinct sensitivity and dependencies when compared with the AML bulk. AML–LSCs are driven by genetic and epigenomic changes, and these alterations influence prognosis and clonal selection. Therapies targeting these molecular aberrations have been developed and show promising responses in advanced clinical trials; however, so far success with LSCs has been limited. Besides the genetic diversity, AML–LSCs are critically influenced by the microenvironment, and a third crucial aspect has recently come to the fore: A group of evolutionarily conserved signaling pathways such as canonical Wnt signaling, Notch signaling, or the Hedgehog pathway can be essential for maintenance of AML–LSC but may be redundant for normal hematopoietic stem cells. In addition, early reports suggest also regulators of cell polarity may also influence hematopoietic stem cells and AML biology. Interactions between these pathways have been investigated recently and suggest a network of signaling pathways involved in regulation of self-renewal and response to oncogenic stress. Here, we review how recent discoveries on regulation of AML–LSC-relevant evolutionarily conserved pathways may open opportunities for novel treatment approaches eradicating residual disease. Clin Cancer Res; 21(2); 240–8. ©2015 AACR.

F.H. Heidel is a consultant/advisory board member for Novartis. No potential conflicts of interest were disclosed by the other authors.

The following editor(s) reported relevant financial relationships: J.R. Grandis—None.

The members of the planning committee have no real or apparent conflicts of interest to disclose.

Upon completion of this activity, the participant should be able to understand the roles of Notch–, Wnt–, and Hedgehog–signaling in the development and maintenance of AML stem cells and their potential applications in the new generation of targeted therapeutics for AML.

This activity does not receive commercial support.

Development of next-generation sequencing (NGS) techniques has facilitated detailed analysis of genetic heterogeneity in acute myelogenous leukemia (AML) and enabled detection of novel mutations with distinct functional properties (1–3). Genetic diversity, however, is not the only determinant of relapse, drug resistance, and aggressiveness of leukemia biology. Multiple determinants shape hierarchical organization of the leukemic bulk, where a subpopulation of leukemia stem cells (LSC) maintains disease.

Aspects that influence leukemia stem cell biology and self-renewal include (i) genetic and epigenomic alterations, (ii) alterations of the bone marrow niche, and (ii) nongenetic determinants, such as evolutionarily conserved signaling pathways (ECSP; Fig. 1).

Figure 1.

Determinants of heterogeneity in AML biology. LSCs are influenced by exogenous and endogenous factors: 1. genetic and epigenetic alterations, 2. the bone marrow niche and stroma, and 3. ESCPs. These determinants of LSC self-renewal interact with each other at multiple levels and contribute to heterogeneity of disease biology.

Figure 1.

Determinants of heterogeneity in AML biology. LSCs are influenced by exogenous and endogenous factors: 1. genetic and epigenetic alterations, 2. the bone marrow niche and stroma, and 3. ESCPs. These determinants of LSC self-renewal interact with each other at multiple levels and contribute to heterogeneity of disease biology.

Close modal

Genetic alterations are not homogeneously present within a single individual. AML presents rather as a heterogeneous mixture of genetically distinct subclones arising through branching evolution (4, 5), and minor resistant subclones may emerge as the origin of relapse after discontinuation of chemotherapy.

Location of hematopoietic stem cells (HSC) and LSCs in the bone marrow niche is able to modify its function by cross-talk of the stem cell with the stromal cells forming the niche.

Adhesion molecules, membrane receptors, and secretion of chemokines influence proliferation and drug resistance irrespective of the underlying genetic diversity. Microenvironmental changes may even drive malignant transformation of HSCs within the niche and lead to activation of signaling pathways as well as secondary genetic alterations. Interaction with the niche may also influence the third determinant of AML heterogeneity: ECSPs.

Although some signaling pathways are frequently mutated in AML and act as drivers of malignant transformation for HSCs, progenitor cells, and the bulk population, other ECSPs are neither frequently mutated nor necessarily relevant for the AML bulk. These ECSPs modulate self-renewal capacity of stem cells. Self-renewal is considered to be the integral property of both HSC and LSC, and its deregulation is known to affect development and maintenance of AML–LSC (6). Recent data indicate that ECSPs play an important role in stem cell development, with differential requirement of these signaling pathways in maintenance of stem cell hierarchies. Importantly, some ECSPs seem dispensable for maintenance of normal adult HSC, whereas LSC seems to retain dependency on specific signaling nodes (6, 7). Another important aspect is based on the underlying genetic diversity: Expression of specific oncogenes such as MLL fusions (fusions affecting the mixed-lineage-leukemia gene on chromosome 11q23) may create new dependencies on specific ESPCs in LSCs. This concept of acquired vulnerability may create novel therapeutic target structures in AML. Targeting dependency on ESPCs that are otherwise dispensable for maintenance of adult hematopoiesis may offer a therapeutic window to eradicate minimal residual disease (MRD).

Although the role of various ESCPs has been described in ontogenesis and stem cell development, this review focuses on a group of ESPCs that have been described in the context of disease development, biology, and prognosis of AML: They represent a unique class of AML-relevant signaling pathways acting in the shadows of genetic diversity and cell–niche interaction.

Notch signaling

Notch receptors are an evolutionarily conserved family of transmembrane proteins that are expressed and active in normal HSC (8, 9). The Notch pathway is highly regulated and requires a specific cell–cell interaction between Notch and its ligand (Fig. 2; refs. 10, 11). In lymphoid malignancies, an oncogenic role of Notch was anticipated, and several groups provided evidence that activating mutations of Notch contributed to development of T-cell acute lymphoblastic leukemia (T-ALL; ref. 12) or chronic lymphocytic leukemia (13).

Figure 2.

Canonical Notch (A) and Wnt signaling (B, simplified view). A, inactive Notch receptor is known to promote ubiquitylation and proteasomal degradation of phosphorylated β-catenin (the central signaling node of the canonical Wnt pathway), indicating interaction between these evolutionarily conserved pathways. B, delta/Jagged ligands expressed on neighboring cells bind to the extracellular domains of Notch receptor. In mammalians, four receptor isoforms (Notch1–4) exist with five canonical notch ligands being classified into Jagged and delta-like families (Jag1, Jad2, Dll1, Dll3, and Dll4; ref. 9). Upon ligand binding, the pathway is activated through double proteolytic cleavage of the receptor. First, the receptor becomes extracellularly modified (S2 site) by an A-Disintegrin-and-Metalloprotease (ADAM), and subsequently, the notch intracellular domain (NICD) is released by a γ-secretase/presenilin complex. The NICD travels from the cytoplasm to the nucleus, forming an active transcription complex with the DNA-binding transcription factor CSL/RBP-Jk (9). The complex is stabilized by the coactivator mastermind-like (MAML1–3), among others, switching on the transcription of target genes, such as Hes family genes, c-Myc, cyclins D1 and D3, or Notch 1 and 3 (10). C, in the absence of Wnt ligand (inactive), β-catenin is recruited by a multifactor complex formed by glycogen synthase kinase-3β (GSK-3β; ref. 65), casein kinase 1 (CK1), adenomatous polyposis coli (APC) a tumor-suppressor protein, and the scaffold protein Axin (66). Axin promotes GSK-3β–dependent phosphorylation of β-catenin for the ubiquitin-proteasome pathway, maintaining low levels of the protein. D, upon Wnt binding to the frizzled (FZD) and LRP5/6 coreceptor complex (active), β-catenin phosphorylation is blocked. This leads to cytoplasmatic accumulation of unphosphorylated β-catenin that can shuttle to the nucleus. Nuclear β-catenin binds to T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors to promote gene expression of the respective target genes and others such as c-myc, cyclin D1, or c-jun (67). Recently, detailed investigation of canonical Wnt signaling revealed a novel mechanism of Ctnnb1 stabilization. Here, β-catenin requires the association of Axin with the phosphorylated LRP6-receptor complex, followed by a destruction of the repressor complex and an accumulation of newly translated β-catenin in the cytosol (68). Prostaglandin (PGE2) signaling is known to modulate Wnt pathway activity through PKA/cAMP–mediated signaling.

Figure 2.

Canonical Notch (A) and Wnt signaling (B, simplified view). A, inactive Notch receptor is known to promote ubiquitylation and proteasomal degradation of phosphorylated β-catenin (the central signaling node of the canonical Wnt pathway), indicating interaction between these evolutionarily conserved pathways. B, delta/Jagged ligands expressed on neighboring cells bind to the extracellular domains of Notch receptor. In mammalians, four receptor isoforms (Notch1–4) exist with five canonical notch ligands being classified into Jagged and delta-like families (Jag1, Jad2, Dll1, Dll3, and Dll4; ref. 9). Upon ligand binding, the pathway is activated through double proteolytic cleavage of the receptor. First, the receptor becomes extracellularly modified (S2 site) by an A-Disintegrin-and-Metalloprotease (ADAM), and subsequently, the notch intracellular domain (NICD) is released by a γ-secretase/presenilin complex. The NICD travels from the cytoplasm to the nucleus, forming an active transcription complex with the DNA-binding transcription factor CSL/RBP-Jk (9). The complex is stabilized by the coactivator mastermind-like (MAML1–3), among others, switching on the transcription of target genes, such as Hes family genes, c-Myc, cyclins D1 and D3, or Notch 1 and 3 (10). C, in the absence of Wnt ligand (inactive), β-catenin is recruited by a multifactor complex formed by glycogen synthase kinase-3β (GSK-3β; ref. 65), casein kinase 1 (CK1), adenomatous polyposis coli (APC) a tumor-suppressor protein, and the scaffold protein Axin (66). Axin promotes GSK-3β–dependent phosphorylation of β-catenin for the ubiquitin-proteasome pathway, maintaining low levels of the protein. D, upon Wnt binding to the frizzled (FZD) and LRP5/6 coreceptor complex (active), β-catenin phosphorylation is blocked. This leads to cytoplasmatic accumulation of unphosphorylated β-catenin that can shuttle to the nucleus. Nuclear β-catenin binds to T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors to promote gene expression of the respective target genes and others such as c-myc, cyclin D1, or c-jun (67). Recently, detailed investigation of canonical Wnt signaling revealed a novel mechanism of Ctnnb1 stabilization. Here, β-catenin requires the association of Axin with the phosphorylated LRP6-receptor complex, followed by a destruction of the repressor complex and an accumulation of newly translated β-catenin in the cytosol (68). Prostaglandin (PGE2) signaling is known to modulate Wnt pathway activity through PKA/cAMP–mediated signaling.

Close modal

Notch is essential for the development of HSCs during embryonic blood formation (Table 1; ref. 14). There are conflicting data on the role of Notch signaling in the function of adult HSCs and myeloid differentiation. These differences may be explained by the experimental approach and the extent of Notch modulation. Genetic deletion of Notch receptors or modulation of ligands and complex members does not impair adult HSC function (15, 16). However, abrogation of Notch signaling by conditional deletion of Nicastrin—a crucial component of the γ-secretase complex—led to transformation of adult HSCs into a preleukemic state (17). Cross-talk between Wnt and Notch signaling pathways as described in colon cancer models may also predict potential influence of Notch signaling on Wnt-dependent AML (Fig. 2; ref. 18). Ex vivo investigation of AML cell lines and primary patient blasts revealed downregulation of Notch1 expression to be associated with a decrease in PU.1-mediated differentiation capacity. This indicated for the first time a crucial role in maintenance of an immature state (19). Until most recently, the relevance of Notch signaling in AML, especially in self-renewal of AML–LSC, remained elusive. Recently, publications demonstrated a role for Notch expression in AML and activation of Notch signaling as a potential therapeutic opportunity (17, 20): Notch receptors have been shown to be expressed in AML; however, expression of Notch downstream targets was extremely low, indicating silencing of Notch signaling both in both human and murine AML, which may be influenced by underlying genetic alterations. In contrast, activation of Notch signaling either by activating mutations in vivo or ligand stimulation in vitro led to significant induction of differentiation, apoptosis, and cycle arrest in AML–LSC (17). Inactivation of Notch signaling has been described to initiate a chronic myelomonocytic leukemia (CMML)–like phenotype in vivo (21) comparable with the phenotype achieved by deletion of the hydroxymethylation pathway regulator Tet2. Combination of Notch inactivation with Tet2 deletion led to overt AML, indicating cooperation in AML development. This suggests a role of reduced Notch target gene expression in early development of AML generating a preleukemic state.

Table 1.

Requirement of evolutionarily conserved pathways in normal HSC and AML–LSC

NotchWntHh
HSC development (+) 
Adult HSC maintenance (+) − − 
AML–LSC development + tumor suppressor − 
AML–LSC maintenance + tumor suppressor 
NotchWntHh
HSC development (+) 
Adult HSC maintenance (+) − − 
AML–LSC development + tumor suppressor − 
AML–LSC maintenance + tumor suppressor 

NOTE: +, required; (+), context-dependent requirement; −, not required.

As outlined before, cell intrinsic mutations that activate Notch signaling have been found in lymphoid neoplasia, while not being reported in myeloid malignancies. Functional investigation in experimental mouse models confirmed that activation of Notch signaling is thought to contribute to acute lymphoblastic leukemia (ALL), whereas abrogation of Notch signaling may contribute to myelodysplasia (MDS) or eventually AML. Most recently, activation of Notch through the bone marrow microenvironment has been shown to promote leukemogenesis in AML. Interestingly, Notch activity is triggered by constitutive activation of the Wnt signaling molecule β-catenin (Ctnnb1) caused by an activating mutation of Ctnnb1 in bone marrow osteoblasts (22). This constitutive activation of Ctnnb1 leads to overexpression of the Notch-ligand jagged 1 and results in exogenous activation of Notch signaling in hematopoietic stem/progenitor cell (HSPC). This activating mutation of Ctnnb1 has been reported in up to 38% of patients with MDS or AML. This adds another layer of complexity to the dual role of Notch in myeloid malignancies.

Inhibitors of Notch signaling have already been developed and tested in T-cell leukemia, in which gain-of-function mutations are frequent. γ-secretase inhibitors (GSI) MK-0752 and PF03084014 have, therefore, been tested in T-ALL, whereas others are currently being investigated in early-phase clinical trials for solid tumors (Table 2; ref. 23). Monoclonal Notch-receptor antibodies (24), soluble receptor decoys, or RNAi are still under development in preclinical stages.

Table 2.

Gamma-secretase inhibitors (GSI) and Hedgehog/Smoothened inhibitors (HhI) currently investigated in clinical trials

Indications in malignant diseaseCompanyPhase
GSI    
 RO4929097 Leukemia (ALL), lymphoma, sarcomas, solid cancers Roche I–II 
 MK0752 Leukemia (ALL), breast cancer Merck I–II 
 PF03084014 Leukemia (ALL) Pfizer I–II 
HhI 
 lde225 (Sonidegib) Leukemia (AML), myeloproliferative neoplasia (MPN), solid cancers Novartis I–III 
 PF04449913 Leukemia (AML), MDS, MPN Pfizer I–II 
 GDC0449 (Vismodegib) MPN, multiple myeloma, solid cancers Genentech I–II 
 BMS833923 MPN (CML), multiple myeloma, solid cancers Exilixis I–II 
 IPI-926 MPN (PMF), solid cancers Infinity I–II 
 LY2940680 Solid cancers Eli Lilly I–II 
Indications in malignant diseaseCompanyPhase
GSI    
 RO4929097 Leukemia (ALL), lymphoma, sarcomas, solid cancers Roche I–II 
 MK0752 Leukemia (ALL), breast cancer Merck I–II 
 PF03084014 Leukemia (ALL) Pfizer I–II 
HhI 
 lde225 (Sonidegib) Leukemia (AML), myeloproliferative neoplasia (MPN), solid cancers Novartis I–III 
 PF04449913 Leukemia (AML), MDS, MPN Pfizer I–II 
 GDC0449 (Vismodegib) MPN, multiple myeloma, solid cancers Genentech I–II 
 BMS833923 MPN (CML), multiple myeloma, solid cancers Exilixis I–II 
 IPI-926 MPN (PMF), solid cancers Infinity I–II 
 LY2940680 Solid cancers Eli Lilly I–II 

Described as an oncogene in ALL, Notch1 seems to exert tumor-suppressor activity in AML. This dual role is highly cell context dependent and may be influenced by cell-type–specific genetic alterations. The potential suitability of Notch activation to serve as a target in AML–LSC now adds another layer of complexity to the development of Notch-targeted therapies. Activating agents of the Notch pathway such as Notch1 agonistic antibodies have been successfully tested in animal models of a different biologic context (25). Others, such as activating Notch ligand or small-molecule agonists, could be developed for targeting MRD in subtypes of AML (17). However, given the dual role of Notch signaling in hematopoiesis (Table 1), it is difficult to predict the effects of novel therapeutic approaches. Thus, while targeting the malignant clone in one lineage, one might cause (pre-) malignant transformation in another cell type or lineage.

Canonical Wnt signaling

Wnt signaling plays a critical role in embryonic and hematopoietic development. Wnt ligands are secreted glycoproteins, which can be released or presented on the cell surface and induce different pathways. The canonical Wnt pathway has been studied in great detail with β-catenin (Ctnnb1) being the central player of the signaling cascade (Fig. 2A and B).

During development, canonical Wnt signaling plays an important role in generation of normal HSPCs (Table 1; refs. 26, 27). Ctnnb1 deletion in early HSC development does not affect HSC establishment, but HSCs are impaired in long-term growth and maintenance following transplantation (27). In contrast, conditional inactivation of both, β- and γ-catenin in adult, steady-state HSC does not cause significant perturbation of hematopoiesis (28). This indicates differential requirement for canonical Wnt signaling in development versus maintenance of adult HSCs.

Activation of Ctnnb1 has variable effects on HSCs depending on the magnitude of activation (29). These dose-dependent effects may explain the variability of Ctnnb1 requirement in different models (30). Mild activation of Wnt signaling modified HSC function toward self-renewal, resulting in improved maintenance and reconstitution of HSC. Modest activation of canonical Wnt signaling through (haplo-insufficiency of the Apcmin-mutant) led to competitive advantage of HSC; however, these cells were exhausted after the second round of transplantation (31). Pronounced activation of Wnt signaling (Apc inactivation) impairs HSC self-renewal and differentiation (29) through unlatched cell-cycle activity of HSCs followed by loss of their function and apoptosis (32). Likewise, significant activation of Ctnnb1 through inactivation of its inhibitor GSK3β leads to expansion of the HSC pool, followed by stem cell exhaustion and bone marrow failure (33, 34).

Wnt signaling is activated in both AML–LSC (35) and myeloid blast crisis of chronic myelogenous leukemia (CML; ref. 36). In primary AML patient samples, immunohistochemical expression and constitutive activation of canonical Wnt pathway member β-catenin were also detectable in the bulk AML population (37). High expression of Ctnnb1 has been reported to correlate with poor prognosis, and γ-catenin seems to stabilize the nuclear “active” version of β-catenin in AML cells (38, 39). Of note, abundance of Ctnnb1 in these subentities can be considered to be a direct result of either transcriptional or posttranslational activation through the respective genetic alteration itself. Constitutive activation of oncogenic tyrosine kinases can stabilize cytoplasmatic β-catenin and presence of MLL fusions seems to activate Wnt signaling in a cell-intrinsic manner.

Several models have shown a significant impact of canonical Wnt signaling on development and maintenance of myeloid LSC (Table 1; refs. 27, 40–42). Mouse models of AML induced by MLL rearrangements revealed that self-renewal capacity of LSC is mediated—at least in part—by Ctnnb1 (41, 42). Constitutively expressed Ctnnb1 enabled progenitor cells to form leukemia with a similar efficacy as the corresponding stem cell controls (41). On the other hand, genetic deletion of Ctnnb1 led to reduction of LSC and thus to decreased leukemia formation. Interestingly, these effects could be mimicked using pharmacologic treatment, confirming the importance of Ctnnb1 for maintenance of AML–LSC. Interference of prostaglandin signaling has been shown to target the Wnt/β-catenin axis in HSC (43, 44), and abrogation of Ctnnb1 by the COX inhibitor indomethacin led to a 100-fold decrease in AML–LSC in secondary recipients (41). Moreover, COX inhibition of fully developed MLL-AF9–induced leukemia led to reduction of Ctnnb1 and of LSC frequency. These data indicate that certain subtypes of AML–LSC retain dependency on canonical Wnt signaling and suggest that self-renewal pathways can be selective therapeutic targets for LSC (45). In contrast, activation or accumulation of Ctnnb1 has been shown to mediate oncogenic potential (31, 36) in human and murine leukemia models and modest activation creates a preleukemic state in vivo (31).

Wnt–Notch interaction has been described in regard to the bone marrow stroma. As outlined above, mutations of Ctnnb1 have been found in bone marrow osteoblasts, resulting in overexpression of Notch ligands and consecutive activation of Notch signaling in HSPCs (22).

Therapeutically, development of Ctnnb1-targeting drugs remains a challenge. Several relevant pathway members have been used as target structures with varying success. Compounds such as XAV939 have been discovered recently and stabilize Axin by inhibition of the enzymes Tankyrase 1 and 2, thus promoting degradation of Ctnnb1. Tankyrases may serve as a bona fide target in colon cancer (46); however, their role in HSC appears more complicated. Antibodies directed against LRP receptors may influence the niche–cell interaction rather than cell intrinsic activation of Wnt signaling in AML. Recently, inhibitors of Ctnnb1 itself have been presented in preclinical studies of leukemia cells lines. Inhibitors of canonical Wnt signaling have not yet reached clinical trials for AML.

Hedgehog

The Hedgehog (Hh) signaling pathway is highly conserved in vertebrates (47), and three Hedgehog isoforms have been described in hematopoiesis: Sonic- (Shh), Desert- (Dhh), and Indian-Hedgehog (Ihh). The receptor, Patched (Ptch), acts as a negative modulator. In absence of Hedgehog, the negative modulator Patched represses the signal-transducer Smoothened (Smo; Fig. 3A and B; refs. 47, 48), which enables activation of target gene transcription through Gli proteins.

Figure 3.

Hedgehog signaling pathway and polarity regulators interacting with ESCPs (simplified view). A, the integral part of the Hedgehog signaling pathway is the transmembrane proteins “patched” (Ptch) and “smoothened” (Smo). They are located on the plasma membrane. B, once Hedgehog binds to Patched, Smoothened inhibition is interrupted, allowing its translocation to the membrane and activation by phosphorylation (1). Downstream, Smoothened targets and stabilizes ubiquitination of zinc-finger transcription factors members of the Gli family, which translocate to the nucleus. Gli1 and Gli2, as activators, switch on the transcription of targeted genes important in proliferation and survival as well as Gli1 and Ptch1/2. On the other hand, Gli3 and Gli2 act as repressive transcription factors, especially when Patched is active and Smoothened is inactive, and therefore not able to repress their degradation. SuFu prevents the active form of Gli from transactivating Hedgehog-responsive genes. Bona fide Hedgehog target genes include Gli1, Gli2, Ptch, and regulators of cell proliferation and survival. C and D, several cell fate determinants and polarity regulators have been described to interact with ECSPs such as canonical Wnt signaling, Hedgehog, or Notch. However, few of these interactions have been confirmed so far in hematopoietic cells. Llgl (a member of the “Scribble complex”) interacts with the “Par complex” (Par3, Par6, and aPKCs), parts of which interact closely with canonical Wnt signaling (downstream of Fzd and Strab receptors). C, the Par complex is regulated downstream of G-protein–coupled receptors (GPCR) and CDC42. Moreover, interactions between Llgl and the cytoskeleton (microtubules) have been reported. D, Msi-2 is a known interaction partner of Numb, a major determinant of binary cell fates (69, 70). However, the role of the Msi-2–Numb axis in regulation of HSC polarity has been controversially discussed (56). Numb has been described to influence ECSPs (Notch and Hedgehog) through ubiquitinylation and proteasomal degradation (69, 71). The E3 ubiquitin–ligase Itch modifies ubiquitinylation of activated Notch and the Hedgehog-intermediate Gli downstream of Numb. Itch negatively regulates the development and function of HSCs (71). In a straight knockout mouse model, genetic inactivation of Itch resulted in enhanced numbers and competitive advantage of HSCs. This gain-of-function can be partially explained by accumulation of activated Notch1 in Itch-deficient HSC.

Figure 3.

Hedgehog signaling pathway and polarity regulators interacting with ESCPs (simplified view). A, the integral part of the Hedgehog signaling pathway is the transmembrane proteins “patched” (Ptch) and “smoothened” (Smo). They are located on the plasma membrane. B, once Hedgehog binds to Patched, Smoothened inhibition is interrupted, allowing its translocation to the membrane and activation by phosphorylation (1). Downstream, Smoothened targets and stabilizes ubiquitination of zinc-finger transcription factors members of the Gli family, which translocate to the nucleus. Gli1 and Gli2, as activators, switch on the transcription of targeted genes important in proliferation and survival as well as Gli1 and Ptch1/2. On the other hand, Gli3 and Gli2 act as repressive transcription factors, especially when Patched is active and Smoothened is inactive, and therefore not able to repress their degradation. SuFu prevents the active form of Gli from transactivating Hedgehog-responsive genes. Bona fide Hedgehog target genes include Gli1, Gli2, Ptch, and regulators of cell proliferation and survival. C and D, several cell fate determinants and polarity regulators have been described to interact with ECSPs such as canonical Wnt signaling, Hedgehog, or Notch. However, few of these interactions have been confirmed so far in hematopoietic cells. Llgl (a member of the “Scribble complex”) interacts with the “Par complex” (Par3, Par6, and aPKCs), parts of which interact closely with canonical Wnt signaling (downstream of Fzd and Strab receptors). C, the Par complex is regulated downstream of G-protein–coupled receptors (GPCR) and CDC42. Moreover, interactions between Llgl and the cytoskeleton (microtubules) have been reported. D, Msi-2 is a known interaction partner of Numb, a major determinant of binary cell fates (69, 70). However, the role of the Msi-2–Numb axis in regulation of HSC polarity has been controversially discussed (56). Numb has been described to influence ECSPs (Notch and Hedgehog) through ubiquitinylation and proteasomal degradation (69, 71). The E3 ubiquitin–ligase Itch modifies ubiquitinylation of activated Notch and the Hedgehog-intermediate Gli downstream of Numb. Itch negatively regulates the development and function of HSCs (71). In a straight knockout mouse model, genetic inactivation of Itch resulted in enhanced numbers and competitive advantage of HSCs. This gain-of-function can be partially explained by accumulation of activated Notch1 in Itch-deficient HSC.

Close modal

Smo-deficient fetal liver cells (FLC) revealed decreased replating capacity in vitro whereas activation of the Hedgehog pathway through heterozygosity of the Smoothened inhibitor Patched led to increased colony formation of FLCs (49). Conditional activation of Hedgehog signaling led to exhaustion of HSC through increased cell-cycle activity. Inactivation of Smoothened in early development did not affect steady-state hematopoiesis but impaired long-term function of HSC (50), somewhat similar to β-catenin loss of function. However, Hedgehog signaling has been shown to be dispensable for maintenance of normal adult HSCs in conditional knockout mouse models (Table 1; refs. 51, 52).

Aberrant activation of Hedgehog signaling has been found in LSC. However, no convincing data are published on gene-expression changes of the Hedgehog pathway and implications of expression levels on prognosis or disease biology in AML with the exception of a recent report on Hedgehog activity in acute promyelocytic leukemia (53). Experimental data on the relevance and influence of Hedgehog signaling on AML–LSC are more limited than for Notch or canonical Wnt signaling. In regard to its role in myeloid neoplasia, most data have been published on the role of Hedgehog signaling in CML (49, 50). Here, genetic inactivation of Smoothened led to decreased penetrance and increased latency of CML (50). Similar effects could be observed using the Hedgehog inhibitor cyclopamine. In contrast, Hedgehog was described to be dispensable in maintenance of AML–LSC. Genetic inactivation of Smoothened in MLL-AF9–transformed LSC did not affect AML development in primary recipient mice (52).

Several small-molecule inhibitors of Hedgehog signaling have been developed for solid cancer (Table 2). Although the preclinical experimental data are less convincing in comparison with other ESCPs, Hedgehog inhibitors are being tested in a variety of myeloid malignancies. Currently, clinical trials are under way to investigate Hedgehog signaling inhibitors in AML: LDE225 and PF04449913 are evaluated in international multicenter phase II trials either as monotherapy or combination with chemotherapy (23). Of note, the LDE225 trial stopped recruitment in early 2014 due to lack of efficacy, highlighting the limited activity of Smoothened inhibition on the rapidly proliferating leukemic bulk.

Polarity regulators and cell fate determinants

Loss of cell polarity can influence tumor development by altering cell–cell matrix interactions. Moreover, changes in cell polarity are essential for regulation of symmetric versus asymmetric cell division in HSC and LSC. Only few of these proteins have been investigated in detail. Evidence for a role of Musashi-2 (Msi-2) not only in regulation of HSC but also AML–LSC has been provided recently (Fig. 3D). Although data are still limited to a few reports, recent findings suggest involvement of Scribble polarity complex proteins (Llgl1 and 2) in regulation of HSC polarity and potential implications in AML biology (Fig. 3C).

Msi-2 is a known regulator of the HSC pool and of HSC activity (54–56). Loss of Msi-2 was associated with reduction in short-term-HSCs (ST-HSC), impaired proliferation capacity, and competitive disadvantage. RNAi-based knockdown of Msi-2 also impaired long-term HSC (LT-HSC) function. long-term-HSCs are maintained through (a)symmetric cell division and this process is regulated by Msi-2 (57). Using a conditional knockout mouse model, genetic inactivation of Msi-2 severely impaired LT-HSC function as Msi-2−/− HSC become insensitive to TGFβ-mediated LT-HSC expansion (57). In AML, protein expression of Msi-2 was associated with unfavorable prognosis in AML independently of other known risk factors (58). Consistently, gene expression of MSI2 is correlated with dismal overall survival in AML (59). Moreover, Msi-2 has been investigated recently in leukemia mouse models (56, 60). Msi-2 inactivation was shown to be associated with a reduction in both symmetric division (56) and progression of CML (60). Increased expression of Msi2 was associated with aggressive disease and an immature phenotype of human AML and CML (56, 60).

Recently, mutation of Lethal-giant-larvae 2 (Llgl2) has been described as an early mutational event in progression from severe congenital neutropenia to AML (61). Decreased expression of Llgl1, its close homolog in mammals, could be found in AML–LSC of different origin (L-MPP/L-GMP), when compared with their normal counterparts (MPP/GMP; refs. 62, 63). Reduced Llgl1 expression was associated with significantly decreased overall survival in two independent patient cohorts treated for AML (63). However, it cannot be ruled out that these changes may be secondary due to genetic alterations in AML. Genetic inactivation of Llgl1 led to expansion of the HSC pool, suggesting a potential role of decreased expression in disease development. Of note, mouse models investigating the role of Llgl1 in the background of B- and T-cell leukemia did not generate any evidence for tumor-suppressor activity in the genetic models applied (64). These data suggest that the role of Llgl1 in the hematopoietic lineage might be restricted to specific cooperating mutations and a limited number of cellular contexts such as the HSC compartment.

Most recently, a switch from canonical to noncanonical Wnt signaling has been reported in aging of HSC, resulting in loss of cell polarity and skewing of lineage commitment (65). This aging-related process may also influence susceptibility of HSC for malignant transformation.

Factors that influence AML LSCs include (i) genetic/epigenetic variability, (ii) contributions of the bone marrow niche, and (iii) ECSPs that contribute to LSC maintenance. ECSPs can be modulated by underlying genetic as well as the bone marrow microenvironment. Of note, this review focused on detailed investigation of these pathways in various mouse models. However, human HSC/LSC may display a distinct dependency on these pathways and not all findings described may be transferable to human disease. These pathways may offer a novel target for future therapeutic interventions, focusing on eradication of LSC. Therefore, a window needs to be established for therapeutic intervention. Although a distinct requirement for adult HSC compared with LSC may facilitate therapeutic use in regard to hematopoietic toxicity, the requirement of evolutionarily conserved pathways for other somatic tissues needs to be considered. In AML, clinical phase I/II trials targeting prominent ESPC-related molecules have been recently initiated and results are eagerly awaited.

Besides their relevance in maintenance of LSC, these pathways offer therapeutic potential in regard to influencing regenerative potential of normal adult HSC. This is of major interest in the setting of allogeneic stem cell transplantation, which is strongly dependent on efficient homing and engraftment of HSPC.

The authors apologize to those whose work was not cited due to space limitations.

This work was supported by research funding from the German Research Council (DFG) to F.H. Heidel and T. Fischer within the collaborative research cluster 854 (CRC854; A20).

1.
Ley
TJ
,
Ding
L
,
Walter
MJ
,
McLellan
MD
,
Lamprecht
T
,
Larson
DE
, et al
DNMT3A mutations in acute myeloid leukemia.
N Engl J Med
2010
;
363
:
2424
33
.
2.
Mardis
ER
,
Ding
L
,
Dooling
DJ
,
Larson
DE
,
McLellan
MD
,
Chen
K
, et al
Recurring mutations found by sequencing an acute myeloid leukemia genome.
N Engl J Med
2009
;
361
:
1058
66
.
3.
Cancer Genome Atlas Research Network
. 
Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia.
N Engl J Med
2013
;
368
:
2059
74
.
4.
Burrell
RA
,
McGranahan
N
,
Bartek
J
,
Swanton
C
. 
The causes and consequences of genetic heterogeneity in cancer evolution.
Nature
2013
;
501
:
338
45
.
5.
Greaves
M
,
Maley
CC
. 
Clonal evolution in cancer.
Nature
2012
;
481
:
306
13
.
6.
Kreso
A
,
Dick
JE
. 
Evolution of the cancer stem cell model.
Cell Stem Cell
2014
;
14
:
275
91
.
7.
Duncan
AW
,
Rattis
FM
,
DiMascio
LN
,
Congdon
KL
,
Pazianos
G
,
Zhao
C
, et al
Integration of Notch and Wnt signaling in hematopoietic stem cell maintenance.
Nat Immunol
2005
;
6
:
314
22
.
8.
Wu
L
,
Aster
JC
,
Blacklow
SC
,
Lake
R
,
Artavanis-Tsakonas
S
,
Griffin
JD
. 
MAML1, a human homologue of Drosophila mastermind, is a transcriptional co-activator for NOTCH receptors.
Nat Genet
2000
;
26
:
484
9
.
9.
Patel
JP
,
Gonen
M
,
Figueroa
ME
,
Fernandez
H
,
Sun
Z
,
Racevskis
J
, et al
Prognostic relevance of integrated genetic profiling in acute myeloid leukemia.
N Engl J Med
2012
;
366
:
1079
89
.
10.
Roozen
PP
,
Brugman
MH
,
Staal
FJ
. 
Differential requirements for Wnt and Notch signaling in hematopoietic versus thymic niches.
Ann N Y Acad Sci
2012
;
1266
:
78
93
.
11.
Weng
AP
,
Ferrando
AA
,
Lee
W
,
Morris
JP
 IV
,
Silverman
LB
,
Sanchez-Irizarry
C
, et al
Activating mutations of NOTCH1 in human T-cell acute lymphoblastic leukemia.
Science
2004
;
306
:
269
71
.
12.
Rosati
E
,
Sabatini
R
,
Rampino
G
,
Tabilio
A
,
Di Ianni
M
,
Fettucciari
K
, et al
Constitutively activated Notch signaling is involved in survival and apoptosis resistance of B-CLL cells.
Blood
2009
;
113
:
856
65
.
13.
Robert-Moreno
A
,
Guiu
J
,
Ruiz-Herguido
C
,
Lopez
ME
,
Ingles-Esteve
J
,
Riera
L
, et al
Impaired embryonic haematopoiesis yet normal arterial development in the absence of the Notch ligand Jagged1.
EMBO J
2008
;
27
:
1886
95
.
14.
Mancini
SJ
,
Mantei
N
,
Dumortier
A
,
Suter
U
,
MacDonald
HR
,
Radtke
F
. 
Jagged1-dependent Notch signaling is dispensable for hematopoietic stem cell self-renewal and differentiation.
Blood
2005
;
105
:
2340
2
.
15.
Radtke
F
,
Wilson
A
,
Stark
G
,
Bauer
M
,
van Meerwijk
J
,
MacDonald
HR
, et al
Deficient T-cell fate specification in mice with an induced inactivation of Notch1.
Immunity
1999
;
10
:
547
58
.
16.
Lobry
C
,
Ntziachristos
P
,
Ndiaye-Lobry
D
,
Oh
P
,
Cimmino
L
,
Zhu
N
, et al
Notch pathway activation targets AML-initiating cell homeostasis and differentiation.
J Exp Med
2013
;
210
:
301
19
.
17.
Kim
HA
,
Koo
BK
,
Cho
JH
,
Kim
YY
,
Seong
J
,
Chang
HJ
, et al
Notch1 counteracts WNT/beta-catenin signaling through chromatin modification in colorectal cancer.
J Clin Invest
2012
;
122
:
3248
59
.
18.
Chen
PM
,
Yen
CC
,
Wang
WS
,
Lin
YJ
,
Chu
CJ
,
Chiou
TJ
, et al
Down-regulation of Notch-1 expression decreases PU.1-mediated myeloid differentiation signaling in acute myeloid leukemia.
Int J Oncol
2008
;
32
:
1335
41
.
19.
Kannan
S
,
Sutphin
RM
,
Hall
MG
,
Golfman
LS
,
Fang
W
,
Nolo
RM
, et al
Notch activation inhibits AML growth and survival: a potential therapeutic approach.
J Exp Med
2013
;
210
:
321
37
.
20.
Klinakis
A
,
Lobry
C
,
Abdel-Wahab
O
,
Oh
P
,
Haeno
H
,
Buonamici
S
, et al
A novel tumour-suppressor function for the Notch pathway in myeloid leukaemia.
Nature
2011
;
473
:
230
3
.
21.
Kode
A
,
Manavalan
JS
,
Mosialou
I
,
Bhagat
G
,
Rathinam
CV
,
Luo
N
, et al
Leukaemogenesis induced by an activating beta-catenin mutation in osteoblasts.
Nature
2014
;
506
:
240
4
.
22.
ClinicalTrials.gov database on the Internet.
Bethesda MD)
:
NIH
. 
c1993
.
(cited 2014 Aug 21). Available from
: http://www.clinicaltrials.gov.
23.
Hoey
T
,
Yen
WC
,
Axelrod
F
,
Basi
J
,
Donigian
L
,
Dylla
S
, et al
DLL4 blockade inhibits tumor growth and reduces tumor-initiating cell frequency.
Cell Stem Cell
2009
;
5
:
168
77
.
24.
Conboy
IM
,
Conboy
MJ
,
Smythe
GM
,
Rando
TA
. 
Notch-mediated restoration of regenerative potential to aged muscle.
Science
2003
;
302
:
1575
7
.
25.
Ruiz-Herguido
C
,
Guiu
J
,
D'Altri
T
,
Ingles-Esteve
J
,
Dzierzak
E
,
Espinosa
L
, et al
Hematopoietic stem cell development requires transient Wnt/beta-catenin activity.
J Exp Med
2012
;
209
:
1457
68
.
26.
Zhao
C
,
Blum
J
,
Chen
A
,
Kwon
HY
,
Jung
SH
,
Cook
JM
, et al
Loss of beta-catenin impairs the renewal of normal and CML stem cells in vivo.
Cancer Cell
2007
;
12
:
528
41
.
27.
Koch
U
,
Wilson
A
,
Cobas
M
,
Kemler
R
,
Macdonald
HR
,
Radtke
F
. 
Simultaneous loss of beta- and gamma-catenin does not perturb hematopoiesis or lymphopoiesis.
Blood
2008
;
111
:
160
4
.
28.
Rossi
L
,
Lin
KK
,
Boles
NC
,
Yang
L
,
King
KY
,
Jeong
M
, et al
Less is more: unveiling the functional core of hematopoietic stem cells through knockout mice.
Cell Stem Cell
2012
;
11
:
302
17
.
29.
Luis
TC
,
Naber
BA
,
Roozen
PP
,
Brugman
MH
,
de Haas
EF
,
Ghazvini
M
, et al
Canonical wnt signaling regulates hematopoiesis in a dosage-dependent fashion.
Cell Stem Cell
2011
;
9
:
345
56
.
30.
Lane
SW
,
Sykes
SM
,
Al-Shahrour
F
,
Shterental
S
,
Paktinat
M
,
Lo Celso
C
, et al
The Apcmin mouse has altered hematopoietic stem cell function and provides a model for MPD/MDS.
Blood
2010
;
115
:
3489
97
.
31.
Qian
Z
,
Chen
L
,
Fernald
AA
,
Williams
BO
,
Le Beau
MM
. 
A critical role for Apc in hematopoietic stem and progenitor cell survival.
J Exp Med
2008
;
205
:
2163
75
.
32.
Kirstetter
P
,
Anderson
K
,
Porse
BT
,
Jacobsen
SE
,
Nerlov
C
. 
Activation of the canonical Wnt pathway leads to loss of hematopoietic stem cell repopulation and multilineage differentiation block.
Nat Immunol
2006
;
7
:
1048
56
.
33.
Scheller
M
,
Huelsken
J
,
Rosenbauer
F
,
Taketo
MM
,
Birchmeier
W
,
Tenen
DG
, et al
Hematopoietic stem cell and multilineage defects generated by constitutive beta-catenin activation.
Nat Immunol
2006
;
7
:
1037
47
.
34.
Majeti
R
,
Becker
MW
,
Tian
Q
,
Lee
TL
,
Yan
X
,
Liu
R
, et al
Dysregulated gene expression networks in human acute myelogenous leukemia stem cells.
Proc Natl Acad Sci U S A
2009
;
106
:
3396
401
.
35.
Jamieson
CH
,
Ailles
LE
,
Dylla
SJ
,
Muijtjens
M
,
Jones
C
,
Zehnder
JL
, et al
Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML.
N Engl J Med
2004
;
351
:
657
67
.
36.
Simon
M
,
Grandage
VL
,
Linch
DC
,
Khwaja
A
. 
Constitutive activation of the Wnt/beta-catenin signalling pathway in acute myeloid leukaemia.
Oncogene
2005
;
24
:
2410
20
.
37.
Ysebaert
L
,
Chicanne
G
,
Demur
C
,
De Toni
F
,
Prade-Houdellier
N
,
Ruidavets
JB
, et al
Expression of beta-catenin by acute myeloid leukemia cells predicts enhanced clonogenic capacities and poor prognosis.
Leukemia
2006
;
20
:
1211
6
.
38.
Morgan
RG
,
Pearn
L
,
Liddiard
K
,
Pumford
SL
,
Burnett
AK
,
Tonks
A
, et al
γ-Catenin is overexpressed in acute myeloid leukemia and promotes the stabilization and nuclear localization of β-catenin.
Leukemia
2013
;
27
:
336
43
.
39.
Heidel
FH
,
Bullinger
L
,
Feng
Z
,
Wang
Z
,
Neff
TA
,
Stein
L
, et al
Genetic and pharmacologic inhibition of beta-catenin targets imatinib-resistant leukemia stem cells in CML.
Cell Stem Cell
2012
;
10
:
412
24
.
40.
Wang
Y
,
Krivtsov
AV
,
Sinha
AU
,
North
TE
,
Goessling
W
,
Feng
Z
, et al
The Wnt/beta-catenin pathway is required for the development of leukemia stem cells in AML.
Science
2010
;
327
:
1650
3
.
41.
Yeung
J
,
Esposito
MT
,
Gandillet
A
,
Zeisig
BB
,
Griessinger
E
,
Bonnet
D
, et al
beta-Catenin mediates the establishment and drug resistance of MLL leukemic stem cells.
Cancer Cell
2010
;
18
:
606
18
.
42.
Goessling
W
,
North
TE
,
Loewer
S
,
Lord
AM
,
Lee
S
,
Stoick-Cooper
CL
, et al
Genetic interaction of PGE2 and Wnt signaling regulates developmental specification of stem cells and regeneration.
Cell
2009
;
136
:
1136
47
.
43.
North
TE
,
Goessling
W
,
Peeters
M
,
Li
P
,
Ceol
C
,
Lord
AM
, et al
Hematopoietic stem cell development is dependent on blood flow.
Cell
2009
;
137
:
736
48
.
44.
Eaves
CJ
,
Humphries
RK
. 
Acute myeloid leukemia and the Wnt pathway.
N Engl J Med
2010
;
362
:
2326
7
.
45.
Huang
SM
,
Mishina
YM
,
Liu
S
,
Cheung
A
,
Stegmeier
F
,
Michaud
GA
, et al
Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling.
Nature
2009
;
461
:
614
20
.
46.
Irvine
DA
,
Copland
M
. 
Targeting hedgehog in hematologic malignancy.
Blood
2012
;
119
:
2196
204
.
47.
Mar
BG
,
Amakye
D
,
Aifantis
I
,
Buonamici
S
. 
The controversial role of the Hedgehog pathway in normal and malignant hematopoiesis.
Leukemia
2011
;
25
:
1665
73
.
48.
Dierks
C
,
Beigi
R
,
Guo
GR
,
Zirlik
K
,
Stegert
MR
,
Manley
P
, et al
Expansion of Bcr-Abl-positive leukemic stem cells is dependent on Hedgehog pathway activation.
Cancer Cell
2008
;
14
:
238
49
.
49.
Zhao
C
,
Chen
A
,
Jamieson
CH
,
Fereshteh
M
,
Abrahamsson
A
,
Blum
J
, et al
Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia.
Nature
2009
;
458
:
776
9
.
50.
Gao
J
,
Graves
S
,
Koch
U
,
Liu
S
,
Jankovic
V
,
Buonamici
S
, et al
Hedgehog signaling is dispensable for adult hematopoietic stem cell function.
Cell Stem Cell
2009
;
4
:
548
58
.
51.
Hofmann
I
,
Stover
EH
,
Cullen
DE
,
Mao
J
,
Morgan
KJ
,
Lee
BH
, et al
Hedgehog signaling is dispensable for adult murine hematopoietic stem cell function and hematopoiesis.
Cell Stem Cell
2009
;
4
:
559
67
.
52.
Yang
D
,
Cao
F
,
Ye
X
,
Zhao
H
,
Liu
X
,
Li
Y
, et al
Arsenic trioxide inhibits the Hedgehog pathway which is aberrantly activated in acute promyelocytic leukemia.
Acta Haematol
2013
;
130
:
260
7
.
53.
de Andres-Aguayo
L
,
Varas
F
,
Kallin
EM
,
Infante
JF
,
Wurst
W
,
Floss
T
, et al
Musashi 2 is a regulator of the HSC compartment identified by a retroviral insertion screen and knockout mice.
Blood
2011
;
118
:
554
64
.
54.
Hope
KJ
,
Cellot
S
,
Ting
SB
,
MacRae
T
,
Mayotte
N
,
Iscove
NN
, et al
An RNAi screen identifies Msi2 and Prox1 as having opposite roles in the regulation of hematopoietic stem cell activity.
Cell Stem Cell
2010
;
7
:
101
13
.
55.
Kharas
MG
,
Lengner
CJ
,
Al-Shahrour
F
,
Bullinger
L
,
Ball
B
,
Zaidi
S
, et al
Musashi-2 regulates normal hematopoiesis and promotes aggressive myeloid leukemia.
Nat Med
2010
;
16
:
903
8
.
56.
Park
SM
,
Deering
RP
,
Lu
Y
,
Tivnan
P
,
Lianoglou
S
,
Al-Shahrour
F
, et al
Musashi-2 controls cell fate, lineage bias, and TGF-beta signaling in HSCs.
J Exp Med
2014
;
211
:
71
87
.
57.
Byers
RJ
,
Currie
T
,
Tholouli
E
,
Rodig
SJ
,
Kutok
JL
. 
MSI2 protein expression predicts unfavorable outcome in acute myeloid leukemia.
Blood
2011
;
118
:
2857
67
.
58.
Thol
F
,
Winschel
C
,
Sonntag
AK
,
Damm
F
,
Wagner
K
,
Chaturvedi
A
, et al
Prognostic significance of expression levels of stem cell regulators MSI2 and NUMB in acute myeloid leukemia.
Ann Hematol
2013
;
92
:
315
23
.
59.
Ito
T
,
Kwon
HY
,
Zimdahl
B
,
Congdon
KL
,
Blum
J
,
Lento
WE
, et al
Regulation of myeloid leukaemia by the cell-fate determinant Musashi.
Nature
2010
;
466
:
765
8
.
60.
Beekman
R
,
Valkhof
MG
,
Sanders
MA
,
van Strien
PM
,
Haanstra
JR
,
Broeders
L
, et al
Sequential gain of mutations in severe congenital neutropenia progressing to acute myeloid leukemia.
Blood
2012
;
119
:
5071
7
.
61.
Goardon
N
,
Marchi
E
,
Atzberger
A
,
Quek
L
,
Schuh
A
,
Soneji
S
, et al
Coexistence of LMPP-like and GMP-like leukemia stem cells in acute myeloid leukemia.
Cancer Cell
2011
;
19
:
138
52
.
62.
Heidel
FH
,
Bullinger
L
,
Arreba-Tutusaus
P
,
Wang
Z
,
Gaebel
J
,
Hirt
C
, et al
The cell fate determinant Llgl1 influences HSC fitness and prognosis in AML.
J Exp Med
2013
;
210
:
15
22
.
63.
Hawkins
ED
,
Oliaro
J
,
Ramsbottom
KM
,
Ting
SB
,
Sacirbegovic
F
,
Harvey
M
, et al
Lethal giant larvae 1 tumour suppressor activity is not conserved in models of mammalian T- and B-cell leukaemia.
PLoS ONE
2014
;
9
:
e87376
.
64.
Florian
MC
,
Nattamai
KJ
,
Dorr
K
,
Marka
G
,
Uberle
B
,
Vas
V
, et al
A canonical to noncanonical Wnt signalling switch in haematopoietic stem-cell ageing.
Nature
2013
;
503
:
392
6
.
65.
Rubinfeld
B
,
Albert
I
,
Porfiri
E
,
Fiol
C
,
Munemitsu
S
,
Polakis
P
. 
Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly.
Science
1996
;
272
:
1023
6
.
66.
Behrens
J
,
Jerchow
BA
,
Wurtele
M
,
Grimm
J
,
Asbrand
C
,
Wirtz
R
, et al
Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta.
Science
1998
;
280
:
596
9
.
67.
Anastas
JN
,
Moon
RT
. 
WNT signalling pathways as therapeutic targets in cancer.
Nat Rev Cancer
2013
;
13
:
11
26
.
68.
Li
VS
,
Ng
SS
,
Boersema
PJ
,
Low
TY
,
Karthaus
WR
,
Gerlach
JP
, et al
Wnt signaling through inhibition of beta-catenin degradation in an intact Axin1 complex.
Cell
2012
;
149
:
1245
56
.
69.
Di Marcotullio
L
,
Ferretti
E
,
Greco
A
,
De Smaele
E
,
Po
A
,
Sico
MA
, et al
Numb is a suppressor of Hedgehog signalling and targets Gli1 for Itch-dependent ubiquitination.
Nat Cell Biol
2006
;
8
:
1415
23
.
70.
Wu
M
,
Kwon
HY
,
Rattis
F
,
Blum
J
,
Zhao
C
,
Ashkenazi
R
, et al
Imaging hematopoietic precursor division in real time.
Cell Stem Cell
2007
;
1
:
541
54
.
71.
Rathinam
C
,
Matesic
LE
,
Flavell
RA
. 
The E3 ligase Itch is a negative regulator of the homeostasis and function of hematopoietic stem cells.
Nat Immunol
2011
;
12
:
399
407
.