Oncogenic Kras Initiates Leukemia in Hematopoietic Stem Cells

How oncogenes modulate the self-renewal properties of cancer-initiating cells is incompletely understood. Activating KRAS and NRAS mutations are among the most common oncogenic lesions detected in human cancer, and occur in myeloproliferative disorders (MPDs) and leukemias. We investigated the effects of expressing oncogenic Kras^G12D from its endogenous locus on the proliferation and tumor-initiating properties of murine hematopoietic stem and progenitor cells. MPD could be initiated by Kras^G12D expression in a highly restricted population enriched for hematopoietic stem cells (HSCs), but not in common myeloid progenitors. Kras^G12D HSCs demonstrated a marked in vivo competitive advantage over wild-type cells. Kras^G12D expression also increased the fraction of proliferating HSCs and reduced the overall size of this compartment. Transplanted Kras^G12D HSCs efficiently initiated acute T-lineage leukemia/lymphoma, which was associated with secondary Notch1 mutations in thymocytes. We conclude that MPD-initiating activity is restricted to the HSC compartment in Kras^G12D mice, and that distinct self-renewing populations with cooperating mutations emerge during cancer progression.     

Temporal Changes in PTEN and mTORC2 Regulation of Hematopoietic Stem Cell Self-Renewal and Leukemia Suppression

Pten deletion from adult mouse hematopoietic cells activates the PI3-kinase pathway, inducing hematopoietic stem cell (HSC) proliferation, HSC depletion, and leukemogenesis. Pten is also mutated in human leukemias, but rarely in early childhood leukemias. We hypothesized that this reflects developmental changes in PI3-kinase pathway regulation. Here we show that Rictor deletion prevents leukemogenesis and HSC depletion after Pten deletion in adult mice, implicating mTORC2 activation in these processes. However, Rictor deletion had little effect on the function of normal HSCs. Moreover, Pten deletion from neonatal HSCs did not activate the PI3-kinase pathway or promote HSC proliferation, HSC depletion, or leukemogenesis. Pten is therefore required in adult, but not neonatal, HSCs to negatively regulate mTORC2 signaling. This demonstrates that some critical tumor suppressor mechanisms in adult cells are not required by neonatal cells. Developmental changes in key signaling pathways therefore confer temporal changes upon stem cell self-renewal and tumor suppressor mechanisms.     

Activation of Notch1 promotes development of human CD8 single positive T cells in humanized mice

Notch1 mutations are found in more than 50% of human T cell acute lymphoblastic leukemia (T-ALL) cells. However, the functions of Notch1 for human T cell development and leukemogenesis are not well understood. To examine the role of Notch1, human hematopoietic stem cells (HSCs), which had been transduced with a constitutively active form of Notch1 (ICN1), were transplanted into severely immunodeficient NOD/Shi-scid-IL2rγ^null (NOG) mice. We found that the great majority of the ICN1-expressing hematopoietic cells in the bone marrow expressed surface markers for T cells, such as CD3, CD4, and CD8, and that this T cell development was independent of the thymus. Accordingly, phenotypically mature CD8⁺ single positive (SP) T cells were observed in the spleen. Furthermore, T-ALL developed in one NOG recipient mouse out of 26 that had been secondary transferred with the T cells developed in the first NOG mice. These results indicate that Notch1 signaling in HSCs promotes CD8⁺ SP T cell development, and that T cell leukemogenesis may require additional oncogenic factors other than Notch1 activation.     

Luteinizing hormone signaling restricts hematopoietic stem cell expansion during puberty

The number and self‐renewal capacity of hematopoietic stem cells (HSCs) are tightly regulated at different developmental stages. Many pathways have been implicated in regulating HSC development in cell autonomous manners; however, it remains unclear how HSCs sense and integrate developmental cues. In this study, we identified an extrinsic mechanism by which HSC number and functions are regulated during mouse puberty. We found that the HSC number in postnatal bone marrow reached homeostasis at 4 weeks after birth. Luteinizing hormone, but not downstream sex hormones, was involved in regulating HSC homeostasis during this period. Expression of luteinizing hormone receptor (Lhcgr) is highly restricted in HSCs and multipotent progenitor cells in the hematopoietic hierarchy. When Lhcgr was deleted, HSCs continued to expand even after 4 weeks after birth, leading to abnormally elevated hematopoiesis and leukocytosis. In a murine acute myeloid leukemia model, leukemia development was significantly accelerated upon Lhcgr deletion. Together, our work reveals an extrinsic counting mechanism that restricts HSC expansion during development and is physiologically important for maintaining normal hematopoiesis and inhibiting leukemogenesis.     

Deficiency of β Common Receptor Moderately Attenuates the Progression of Myeloproliferative Neoplasm in NrasG12D/+ Mice

Activating Ras signaling is a major driver in juvenile and the myeloproliferative variant of chronic myelomonocytic leukemia (JMML/MP-CMML). Numerous studies suggest that GM-CSF signaling plays a central role in establishing and maintaining JMML/MP-CMML phenotypes in human and mouse. However, it remains elusive how GM-CSF signaling impacts on JMML/MP-CMML initiation and progression. Here, we investigate this issue in a well characterized MP-CMML model induced by endogenous Nras^G12D/+ mutation. In this model, Nras^G12D/+ hematopoietic stem cells (HSCs) are required to initiate and maintain CMML phenotypes and serve as CMML-initiating cells. We show that the common β chain of the GM-CSF receptor (βc) is dispensable for Nras^G12D/+ HSC function; loss of βc does not affect the expansion, increased self-renewal, or myeloid differentiation bias in Nras^G12D/+ HSCs. Therefore, βc^−/− does not abrogate CMML in Nras^G12D/+ mice. However, βc deficiency indeed significantly reduces Nras^G12D/+-induced splenomegaly and spontaneous colony formation and prolongs the survival of CMML-bearing mice, suggesting that GM-CSF signaling plays an important role in promoting CMML progression. Together, our results suggest that inhibiting GM-CSF signaling in JMML/MP-CMML patients might alleviate disease symptoms but would not eradicate the disease.     

Wnt signaling in the niche

There is much interest in understanding the signals in the bone marrow niche that keep hematopoietic stem cells (HSCs) in a quiescent state. In the current issue of Cell Stem Cell, Fleming et al. (2008) report that blocking Wnt signaling in the niche increases the number of proliferating HSCs and reduces their ability to reconstitute the hematopoietic system of irradiated recipient mice. These findings show that Wnt/beta-catenin activity is crucial for the maintenance of HSC quiescence in the bone marrow niche.     

Glycogen synthase kinase-3 is an in vivo regulator of hematopoietic stem cell repopulation

The in vivo regulation of hematopoietic stem cell (HSC) function is poorly understood. Here, we show that hematopoietic repopulation can be augmented by administration of a glycogen synthase kinase-3 (GSK-3) inhibitor to recipient mice transplanted with mouse or human HSCs. GSK-3 inhibitor treatment improved neutrophil and megakaryocyte recovery, recipient survival and resulted in enhanced sustained long-term repopulation. The output of primitive Lin(-)c-Kit(+)Sca-1(+) cells and progenitors from HSCs increased upon GSK-3 inhibitor treatment without altering secondary repopulating ability, suggesting that the HSC pool is maintained while overall hematopoietic reconstitution is increased. GSK-3 inhibitors were found to modulate gene targets of Wnt, Hedgehog and Notch pathways in cells comprising the primitive hematopoietic compartment without affecting mature cells. Our study establishes GSK-3 as a specific in vivo modulator of HSC activity, and suggests that administration of GSK-3 inhibitors may provide a clinical means to directly enhance the repopulating capacity of transplanted HSCs.     

mTOR activation induces tumor suppressors that inhibit leukemogenesis and deplete hematopoietic stem cells after Pten deletion

Pten deficiency depletes hematopoietic stem cells (HSCs) but expands leukemia-initiating cells, and the mTOR inhibitor, rapamycin, blocks these effects. Understanding the opposite effects of mTOR activation on HSCs versus leukemia-initiating cells could improve antileukemia therapies. We found that the depletion of Pten-deficient HSCs was not caused by oxidative stress and could not be blocked by N-acetyl-cysteine. Instead, Pten deletion induced, and rapamycin attenuated, the expression of p16(Ink4a) and p53 in HSCs, and p19(Arf) and p53 in other hematopoietic cells. p53 suppressed leukemogenesis and promoted HSC depletion after Pten deletion. p16(Ink4a) also promoted HSC depletion but had a limited role suppressing leukemogenesis. p19(Arf) strongly suppressed leukemogenesis but did not deplete HSCs. Secondary mutations attenuated this tumor suppressor response in some leukemias that arose after Pten deletion. mTOR activation therefore depletes HSCs by a tumor suppressor response that is attenuated by secondary mutations in leukemogenic clones.     

Wnt signaling in the niche enforces hematopoietic stem cell quiescence and is necessary to preserve self-renewal in vivo

Wingless (Wnt) is a potent morphogen demonstrated in multiple cell lineages to promote the expansion and maintenance of stem and progenitor cell populations. Wnt effects are highly context dependent, and varying effects of Wnt signaling on hematopoietic stem cells (HSCs) have been reported. We explored the impact of Wnt signaling in vivo, specifically in the context of the HSC niche by using an osteoblast-specific promoter driving expression of the paninhibitor of canonical Wnt signaling, Dickkopf1 (Dkk1). Here we report that Wnt signaling was markedly inhibited in HSCs and, unexpectedly given prior reports, reduction in HSC Wnt signaling resulted in reduced p21Cip1 expression, increased cell cycling, and a progressive decline in regenerative function after transplantation. This effect was microenvironment determined, but irreversible if the cells were transferred to a normal host. Wnt pathway activation in the niche is required to limit HSC proliferation and preserve the reconstituting function of endogenous hematopoietic stem cells.     

Quantification of self-renewal capacity in single hematopoietic stem cells from normal and Lnk-deficient mice

Despite being a hallmark of hematopoietic stem cells (HSCs), HSC self-renewal has never been quantitatively assessed. Establishment of a clonal and quantitative assay for HSC function permitted demonstration that adult mouse HSCs are significantly heterogeneous in degree of multilineage repopulation and that higher repopulating potential reflects higher self-renewal activity. An HSC with high repopulating potential could regenerate approximately 1000 HSCs, whereas the repopulating activity of regenerated HSCs on average was significantly reduced, indicating extensive but limited self-renewal capacity in HSCs. Comparisons of wild-type mice with mutant mice deficient in the signal adaptor molecule Lnk showed that not only HSC numbers but also the self-renewal capacity of some HSCs are markedly increased when Lnk function is lost. Lnk appears to control HSC numbers by negatively regulating HSC self-renewal signaling.     

mTOR Complex 1 Plays Critical Roles in Hematopoiesis and Pten-Loss-Evoked Leukemogenesis

The mechanistic target of rapamycin (mTOR) pathway serves as a key sensor of cellular-energetic state?and functions to maintain tissue homeostasis. Hyperactivation of the mTOR pathway impairs hematopoietic stem cell (HSC) function and is associated with leukemogenesis. However, the roles of the unique mTOR complexes (mTORCs) in hematopoiesis and leukemogenesis have not been adequately elucidated. We deleted the mTORC1 component, regulatory-associated protein of mTOR (Raptor), in mouse HSCs and its loss causes a nonlethal phenotype characterized by pancytopenia, splenomegaly, and the accumulation of monocytoid cells. Furthermore, Raptor is required for HSC regeneration, and plays largely nonredundant roles with rapamycin-insensitive companion of mTOR (Rictor) in these processes. Ablation of Raptor also significantly extends survival of mice in models of leukemogenesis evoked by Pten deficiency. These data delineate critical roles for mTORC1 in hematopoietic function and leukemogenesis and inform clinical strategies based on chronic mTORC1 inhibition.     

Lack of evidence that hematopoietic stem cells depend on N-cadherin-mediated adhesion to osteoblasts for their maintenance

Recent studies have proposed that bone marrow hematopoietic stem cells (HSCs) are maintained via N-cadherin-mediated homophilic adhesion with osteoblasts. However, there is not yet any evidence that N-cadherin-expressing cells have HSC activity or that osteoblasts are required for HSC maintenance. We were unable to detect N-cadherin expression in highly purified HSCs by polymerase chain reaction, by using commercial anti-N-cadherin antibodies, or by beta-galactosidase staining of N-cadherin gene trap mice. Only N-cadherin-negative bone marrow cells exhibited HSC activity in irradiated mice. Finally, biglycan-deficient mice had significant reductions in trabecular bone and osteoblasts but showed no defects in hematopoiesis, HSC frequency, or function. Thus, reductions in osteoblasts do not necessarily lead to reductions in HSCs. Most bone marrow HSCs in wild-type and biglycan-deficient mice localized to sinusoids, and few localized within five cell diameters of the endosteum. These results question whether significant numbers of HSCs depend on N-cadherin-mediated adhesion to osteoblasts.     

Gadd45a deletion aggravates hematopoietic stem cell dysfunction in ATM-deficient mice

Ataxia telangiectasia mutated (ATM) kinase plays an essential role in the maintenance of genomic stability. ATM-deficient (ATM^-/-) mice exhibit hematopoietic stem cell (HSC) dysfunction and a high incidence of lymphoma. Gadd45a controls cell cycle arrest, apoptosis and DNA repair, and is involved in the ATM-p53 mediated DNA damage response. However, the role of Gadd45a in regulating the functionality of ATM^-/- HSCs is unknown. Here we report that Gadd45a deletion did not rescue the defects of T-cells and B-cells development in ATM^-/- mice. Instead, ATM and Gadd45a double knockout (ATM^-/- Gadd45a^-/-) HSCs exhibited an aggravated defect in long-term self-renewal capacity compared to ATM^-/- HSCs in HSC transplantation experiments. Further experiments revealed that the aggravated defect of ATM^-/- Gadd45a^-/- HSCs was due to a reduction of cell proliferation, associated with an accumulation of DNA damage and subsequent activation of DNA damage response including an up-regulation of p53-p21 signaling pathway. Additionally, ATM^-/- Gadd45a^-/- mice showed an increased incidence of hematopoietic malignancies, as well as an increased rate of metastasis than ATM^-/- mice. In conclusion, Gadd45a deletion aggravated the DNA damage accumulation, which subsequently resulted in a further impaired self-renewal capacity and an increased malignant transformation in ATM^-/- HSCs.     

Mouse tissues that undergo neoplastic progression after K-Ras activation are distinguished by nuclear translocation of phospho-Erk1/2 and robust tumor suppressor responses

Mutation of K-Ras is a frequent oncogenic event in human cancers, particularly cancers of lungs, pancreas, and colon. It remains unclear why some tissues are more susceptible to Ras-induced transformation than others. Here, we globally activated a mutant oncogenic K-Ras allele (K-Ras(G12D)) in mice and examined the tissue-specific effects of this activation on cancer pathobiology, Ras signaling, tumor suppressor, DNA damage, and inflammatory responses. Within 5 to 6 weeks of oncogenic Ras activation, mice develop oral and gastric papillomas, lung adenomas, and hematopoietic hyperproliferation and turn moribund. The oral, gastric, and lung premalignant lesions display activated extracellular signal-regulated kinases (Erk)1/2 and NF-κB signaling as well as activated tumor suppressor and DNA damage responses. Other organs such as pancreas, liver, and small intestine do not exhibit neoplastic progression within 6 weeks following K-Ras(G12D) activation and do not show a potent tumor suppressor response. Even though robust Erk1/2 signaling is activated in all the tissues examined, the pErk1/2 distribution remains largely cytoplasmic in K-Ras(G12D)-refractory tissues (pancreas, liver, and intestines) as opposed to a predominantly nuclear localization in K-Ras(G12D)-induced neoplasms of lung, oral, and gastric mucosa. The downstream targets of Ras signaling, pElk-1 and c-Myc, are elevated in K-Ras(G12D)-induced neoplastic lesions but not in K-Ras(G12D)-refractory tissues. We propose that oncogenic K-Ras-refractory tissues delay oncogenic progression by spatially limiting the efficacy of Ras/Raf/Erk1/2 signaling, whereas K-Ras-responsive tissues exhibit activated Ras/Raf/Erk1/2 signaling, rapidly form premalignant tumors, and activate potent antitumor responses that effectively prevent further malignant progression.     

The role of Wnt signaling in hematopoietic stem cell development

Hematopoietic stem cells (HSCs) can self-renew and differentiate into all cell types of the blood. This is therapeutically important as HSC transplants can provide a curative effect for blood cancers and disorders. The process by which HSCs develop has been the subject of extensive research in a variety of model organisms; however, efforts to produce bonafide HSCs from pluripotent precursors capable of long-term multilineage reconstitution have fallen short. Studies in zebrafish, chicken, and mice have been instrumental in guiding efforts to derive HSCs from human pluripotent stem cells and have identified a complex set of molecular signals and cellular interactions mediated by such developmental regulators as fibroblast growth factor, Notch, transforming growth factor beta (TGFβ), and Wnt, which collectively promote the stepwise developmental progression toward mature HSCs. Tight temporal and spatial control of these signals is critical to generate the appropriate numbers of HSCs needed for the life of the organism. The role of the Wnt family of signaling proteins in hematopoietic development has been the subject of many studies owing in part to the complex nature of its signaling mechanisms. By integrating cell fate specification with cell polarity establishment, Wnt is uniquely capable of controlling complex biological processes, including at multiple stages of embryonic HSC development, from HSC specification to emergence from the hemogenic epithelium to subsequent expansion. This review highlights key signaling events where specific Wnt signals instruct and guide hematopoietic development in both zebrafish and mice and extend these findings to current efforts of generating HSCs in vitro.     

Fetal liver: an ideal niche for hematopoietic stem cell expansion

Fetal liver (FL) is an intricate and highly vascularized hematopoietic organ, which can support the extensive expansion of hematopoietic stem cells (HSCs) without loss of stemness, as well as of the downstream lineages of HSCs. This powerful function of FL largely benefits from the niche (or microenvironment), which provides a residence for HSC expansion. Numerous studies have demonstrated that the FL niche consists of heterogeneous cell populations that associate with HSCs spatially and regulate HSCs functionally. At the molecular level, a complex of cell extrinsic and intrinsic signaling network within the FL niche cells maintains HSC expansion. Here, we summarize recent studies on the analysis of the FL HSCs and their niche, and specifically on the molecular regulatory network for HSC expansion. Based on these studies, we hypothesize a strategy to obtain a large number of functional HSCs via 3D reconstruction of FL organoid ex vivo for clinical treatment in the future.     

Development of a multi-step leukemogenesis model of MLL-rearranged leukemia using humanized mice

Mixed-lineage-leukemia (MLL) fusion oncogenes are intimately involved in acute leukemia and secondary therapy-related acute leukemia. To understand MLL-rearranged leukemia, several murine models for this disease have been established. However, the mouse leukemia derived from mouse hematopoietic stem cells (HSCs) may not be fully comparable with human leukemia. Here we developed a humanized mouse model for human leukemia by transplanting human cord blood-derived HSCs transduced with an MLL-AF10 oncogene into a supra-immunodeficient mouse strain, NOD/Shi-scid, IL-2Rγ(-/-) (NOG) mice. Injection of the MLL-AF10-transduced HSCs into the liver of NOG mice enhanced multilineage hematopoiesis, but did not induce leukemia. Because active mutations in ras genes are often found in MLL-related leukemia, we next transduced the gene for a constitutively active form of K-ras along with the MLL-AF10 oncogene. Eight weeks after transplantation, all the recipient mice had developed acute monoblastic leukemia (the M5 phenotype in French-American-British classification). We thus successfully established a human MLL-rearranged leukemia that was derived in vivo from human HSCs. In addition, since the enforced expression of the mutant K-ras alone was insufficient to induce leukemia, the present model may also be a useful experimental platform for the multi-step leukemogenesis model of human leukemia.     

Interleukin-1β inhibits normal hematopoietic expansion and promotes acute myeloid leukemia progression via the bone marrow niche

Enhanced interleukin-1β (IL-1β) signaling is a common event in patients with acute myeloid leukemia (AML). It was previously demonstrated that chronic IL-1β exposure severely impaired hematopoietic stem cell (HSC) self-renewal capability in mice and promoted leukemia cell growth in primary AML cells. However, the role of IL-1β in the murine bone marrow (BM) niche remains unclear. Here, we explored the role of IL-1β in the BM niche in Il-1r1^−/− mice, chronic IL-1β exposure mice and mixed lineage leukemia-AF9 fusion gene (MLL-AF9)–induced AML mice models. We demonstrated that IL-1R1 deficiency did not affect the function of HSCs or niche cells under steady-state conditions or during transplantation. Chronic exposure to IL-1β decreased the expansion of Il-1r1^−/− hematopoietic cells in Il-1r1^+/+ recipient mice. These results indicated that IL-1β exposure impaired the ability of niche cells to support hematopoietic cells. Furthermore, we revealed that IL-1R1 deficiency in niche cells prolonged the survival of MLL-AF9–induced AML mice. The results of our study suggest that inhibition of the IL-1β/IL-1R1 signaling pathway in the niche might be a non–cell-autonomous therapy strategy for AML.