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Juvenile myelomonocytic leukemia (JMML) is a pediatric myeloproliferative neoplasm that bears distinct characteristics associated with abnormal fetal development. JMML has been extensively modeled in mice expressing the oncogenic KrasG12D mutation. However, these models have struggled to recapitulate the defining features of JMML due to in utero lethality, nonhematopoietic expression, and the pervasive emergence of T cell acute lymphoblastic leukemia. Here, we have developed a model of JMML using mice that express KrasG12D in multipotent progenitor cells (Flt3Cre+ KrasG12D mice). These mice express KrasG12D in utero, are born at normal Mendelian ratios, develop hepatosplenomegaly, anemia, and thrombocytopenia, and succumb to a rapidly progressing and fully penetrant neonatal myeloid disease. Mutant mice have altered hematopoietic stem and progenitor cell populations in the BM and spleen that are hypersensitive to granulocyte macrophage-CSF due to hyperactive RAS/ERK signaling. Biased differentiation in these progenitors results in an expansion of neutrophils and DCs and a concomitant decrease in T lymphocytes. Flt3Cre+ KrasG12D fetal liver hematopoietic progenitors give rise to a myeloid disease upon transplantation. In summary, we describe a KrasG12D mouse model that reproducibly develops JMML-like disease. This model will prove useful for preclinical drug studies and for elucidating the developmental origins of pediatric neoplasms.

Hyperactivation of the mTOR pathway impairs hematopoietic stem cell (HSC) functions and promotes leukemogenesis. mTORC1 and mTORC2 differentially control normal and leukemic stem cell functions. mTORC1 regulates p70 ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding (eIF4E-binding) protein 1 (4E-BP1), and mTORC2 modulates AKT activation. Given the extensive crosstalk that occurs between mTORC1 and mTORC2 signaling pathways, we assessed the role of the mTORC1 substrate S6K1 in the regulation of both normal HSC functions and in leukemogenesis driven by the mixed lineage leukemia (MLL) fusion oncogene MLL-AF9. We demonstrated that S6K1 deficiency impairs self-renewal of murine HSCs by reducing p21 expression. Loss of S6K1 also improved survival in mice transplanted with MLL-AF9-positive leukemic stem cells by modulating AKT and 4E-BP1 phosphorylation. Taken together, these results suggest that S6K1 acts through multiple targets of the mTOR pathway to promote self-renewal and leukemia progression. Given the recent interest in S6K1 as a potential therapeutic target in cancer, our results further support targeting this molecule as a potential strategy for treatment of myeloid malignancies.

Purpose of Review: The protein tyrosine phosphatase Shp2 is encoded by PTPN11 and positively regulates physiologic hematopoiesis. Mutations of PTPN11 cause the congenital disorder Noonan syndrome and pathologically promote human leukemias. Given the high frequency of PTPN11 mutations in human disease, several animal models have been generated to investigate Shp2 in hematopoietic stem cell (HSC) function and leukemic transformation., Recent Findings: Two independent animal models bearing knockout of Shp2 in hematopoietic tissues clearly demonstrate the necessity of Shp2 in HSC repopulating capacity. Reduced HSC quiescence and increased apoptosis accounts for diminished HSC function in the absence of Shp2. The germline mutation Shp2D61G enhances HSC activity and induces myeloproliferative disease (MPD) in vivo by HSC transformation. The somatic mutation Shp2D61Y produces MPD in vivo but fails to induce acute leukemia, whereas somatic Shp2E76K produces MPD in vivo that transforms into full-blown leukemia. HSCs expressing Shp2D61Y do not generate MPD in recipient animals upon transplantation, whereas Shp2E76K-expressing HSCs yield MPD as well as acute leukemia in recipient animals. The mechanisms underlying the unique functions of Shp2D61Y and Shp2E76K in HSC transformation and leukemogenesis continue to be under investigation., Summary: Further understanding of the physiologic and pathologic role of Shp2 in hematopoiesis and leukemogenesis, respectively, will yield information needed to develop therapeutic strategies targeted to Shp2 in human disease.

Activating PTPN11 mutants promote hematopoietic progenitor hyperactivation of Erk and hypersensitivity to GM-CSF. We hypothesized that Kinase Suppressor of Ras 1 (KSR1) contributes to activating PTPN11-induced GM-CSF hypersensitivity. Bone marrow progenitors from WT and KSR1-/- mice expressing WT Shp2, Shp2E76K, or Shp2D61Y were evaluated functionally and biochemically. KSR1 activation and interaction with phospho-Erk was enhanced in Shp2D61Y- and ShpE76K-expressing cells. Genetic disruption of KSR1 partially normalized Shp2E76K-induced GM-CSF hypersensitivity, but failed to correct Shp2D61Y-induced GM-CSF hypersensitivity. Collectively, these studies suggest that cells expressing Shp2E76K have a greater dependence on KSR1 for GM-CSF hypersensitivity than cells expressing Shp2D61Y., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: Mutations in PTPN11, which encodes the protein tyrosine phosphatase Shp2, are commonly found in juvenile myelomonocytic leukemia (JMML). We hypothesized that PTPN11 mutations promote cell-cycle progression and confer enhanced survival to hematopoietic progenitors., Materials and Methods: Murine bone marrow low-density mononuclear cells were transduced with pMIEG3, pMIEG3-WT Shp2, pMIEG3-Shp2D61Y, or pMIEG3-Shp2E76K followed by cell-cycle and survival functional analysis as well as biochemical analysis for key cell-cycle and programmed cell-death regulatory proteins., Results: A higher proportion of hematopoietic progenitors bearing the gain-of-function Shp2 mutants were residing in the S or G2 phase of the cell cycle in response to low doses of granulocyte-macrophage colony-stimulating factor compared to cells transduced with empty vector (MIEG3) or with WT Shp2. Likewise, Shp2D61Y- or Shp2E76K-expressing hematopoietic cells demonstrated reduced apoptosis based on Annexin-V staining and produced increased progenitor colonies after 48 hours in minimal media compared to cells transduced with empty vector or WT Shp2. To differentiate enhanced survival vs hyperproliferation, cells were stained with PKH26 to distinguish undivided cells from divided progeny. Shp2D61Y- or Shp2E76K-expressing PKH26+ cells similarly demonstrated reduced apoptosis. Upon biochemical analysis, expression of Akt- and Erk-responsive cell-cycle and programmed cell-death regulatory proteins were altered, including increased levels of cyclin D1, Bcl2, and BclXL and reduced levels of p27, p21, and Bim., Conclusion: Collectively, these data demonstrate that gain-of-function Shp2 mutants promote hematopoietic progenitor cell-cycle progression and survival and imply that agents targeting the cell cycle or promoting apoptosis may have therapeutic potential in JMML.

Objective: Improved understanding of hematopoietic stem cell (HSC) differentiation, proliferation, and self-renewal is sought to develop improved stem cell-based therapies as well as to define novel therapies for stem cell-based diseases such as leukemia. Shp-2 is a widely expressed nonreceptor protein tyrosine phosphatase that participates early in hematopoietic development. The following study was performed to examine the role of Shp-2 in HSC function., Methods: Bone marrow low-density mononuclear cells were isolated from WT and Shp-2(+/-) littermate controls and utilized in competitive repopulation studies, homing analysis, cell-cycle analysis, and serial transplantation studies., Results: Haploinsufficiency of Shp-2 causes a threefold reduction in HSC repopulating units following transplantation into lethally irradiated recipients. Homing of Shp-2(+/-) and WT cells to the bone marrow and spleen compartments was equal. Cell-cycle analysis studies revealed that the Shp-2(+/-) lin(-)Sca-1(+)c-kit(+) cells are less quiescent than WT cells, providing a potential etiology for the observed reduced engraftment of the Shp-2(+/-) cells. Consistently, in serial transplantation studies, we observed a significant reduction of Shp-2(+/-) self-renewal compared to that of WT cells., Conclusion: These data demonstrate that Shp-2 is required for the physiologic homeostasis of the HSC compartment and potentially provide insight into how oncogenic Shp-2 may contribute to the pathogenesis of myeloproliferative disorders and leukemias.

The orphan homeobox gene Hex has been shown to be critical for normal murine liver development using Hex-/- mice. Because of the early lethality of the Hex-/- mice, however, in vitro embryonic stem cell differentiation assays were employed to elucidate the role of Hex in murine hematopoiesis. These studies revealed that Hex is not required for hemangioblast development but is required in a dose-dependent fashion for hemangioblast differentiation to definitive hematopoietic progenitors. Contained here are the methods needed to conduct embryonic stem cell-derived in vitro hematopoietic assays.

Juvenile myelomonocytic leukemia (JMML) is a lethal disease of young children characterized by hypersensitivity of hematopoietic progenitors to granulocyte-macrophage colony-stimulating factor (GM-CSF). Mutations in PTPN11, which encodes the protein tyrosine phosphatase Shp-2, are common in JMML. We hypothesized that PTPN11 mutations induce hypersensitivity of hematopoietic progenitors to GM-CSF and confer increased GM-CSF-stimulated phospho-extracellular signal-regulated kinase (Erk) levels. To test this hypothesis, the wild-type (WT) and 3 mutant Ptpn11 cDNAs (E76K, D61V, and D61Y) were transduced into murine bone marrow cells to examine GM-CSF-stimulated granulocyte-macrophage colony-forming unit (CFU-GM) growth, macrophage progenitor proliferation, and activation of the Ras signaling pathway. Expression of the Shp-2 mutants induced progenitor cell hypersensitivity to GM-CSF compared with cells transduced with vector alone or WT Shp-2. Macrophage progenitors expressing the Shp-2 mutants displayed both basal and GM-CSF-stimulated hyperproliferation compared with cells transduced with vector alone or WT Shp-2. Consistently, macrophage progenitors transduced with the Shp-2 mutants demonstrated constitutively elevated phospho-Erk levels and sustained activation of phospho-Erk following GM-CSF stimulation compared with vector alone or WT Shp-2. These data support the hypothesis that PTPN11 mutations induce hematopoietic progenitor hypersensitivity to GM-CSF due to hyperactivation of the Ras signaling axis and provide a basis for the GM-CSF signaling pathway as a target for rational drug design in JMML.

Controversies abound concerning hematopoietic stem cell transduction by recombinant adeno-associated virus 2 (AAV) vectors. For human hematopoietic cells, we have shown that this problem is related to the extent of expression of the cellular receptor for AAV. At least a small subset of murine hematopoietic cells, on the other hand, does express both the AAV receptor and the coreceptor, yet is transduced poorly. In the present study, we have found that approximately 85% of AAV genomes were present in the cytoplasmic fraction of primary murine c-Kit(+)Lin- hematopoietic cells. However, when mice were injected intraperitoneally with hydroxyurea before isolation of these cells, the extent to which AAV genomes were detected in the cytoplasmic fraction was reduced to approximately 40%, with a corresponding increase to approximately 60% in the nuclear fraction, indicating that hydroxyurea facilitated nuclear transport of AAV. It was apparent, nonetheless, that a significant fraction of the AAV genomes present in the nuclear fraction from cells obtained from hydroxyurea-treated mice was single stranded. We next tested whether the single-stranded AAV genomes were derived from virions that failed to undergo uncoating in the nucleus. A substantial fraction of the signal in the nuclear fraction of hematopoietic cells obtained from hydroxyurea-treated mice was also resistant to DNase I. That AAV particles were intact and biologically active was determined by successful transduction of 293 cells by virions recovered from murine hematopoietic cells 48 hr postinfection. Although hydroxyurea facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second- strand DNA synthesis and transgene expression. A better understanding of the underlying mechanism of viral uncoating has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.

Hematopoietic stem cells (HSCs) have long been defined as a cell with the capacity to repopulate the hematopoietic system of a lethally irradiated host. In clinical medicine, this property has been employed to reconstitute an individual's diseased hematopoietic system following ablation with a healthy, normal-functioning hematopoietic system by performing autologous and allogeneic stem cell transplantations. However, despite the widespread utilization of these pragmatic procedures for multiple human bone marrow diseases, much about the basic biology of the HSC and related primitive cells, such as the ontogenic origin of the HSC, the identification of the putative hemangioblast, and the potential of the HSC to contribute to alternative tissues, remains elusive. Basic scientists continue to investigate actively the origin of HSCs during mammalian ontogeny, the stimuli that induce HSCs to divide and differentiate normally, the relationship of HSCs to hemangioblasts, and the potential capacity of HSCs to transdifferentiate to other tissues such as endoderm-derived liver cells and ectoderm-derived neurons. This article will summarize the historical salient studies that have characterized the HSC and will review the active research currently being conducted to understand and define further the biologic properties and potential faculties of HSCs. The application of these studies to improved therapies for human disease, from leukemia to myocardial infarction, will be discussed.

Noonan syndrome (NS) is an autosomal dominant congenital disorder characterized by multiple birth defects including heart defects and myeloproliferative disease (MPD). Approximately 50% of NS patients have germline gain-of-function mutations in PTPNJ1, which encodes the protein-tyrosine phosphatase, Shp2. We provide evidence that conditional ablation of Stat3 in hematopoietic cells and cardiac valvular tissues leads to myeloid progenitor hyperplasia and pulmonary stenosis due to the leaflet thickening, respectively. Consistently, STAT3 activation is significantly compromised in peripheral blood cells from NS patients bearing Shp2-activating mutations. Biochemical and functional analyses demonstrate that activated Shp2 is able to down-regulate Tyr(P)-Stat3 and that constitutively active Stat3 rescues activating mutant Shp2-induced granulocyte-macrophage colony-stimulating factor hypersensitivity in bone marrow cells. Collectively, our work demonstrates that Stat3 is an essential signaling component potentially contributing to the pathogenesis of NS and juvenile myelomonocytic leukemia caused by PTPN11 gain-of-function mutations. [ABSTRACT FROM AUTHOR]

Objective: Monocytic zinc finger (MOZ) maintains hematopoietic stem cells and, upon fusion to the coactivator CREB-binding protein (CBP), induces acute myeloid leukemia (AML). Leukemic stem cells in AML often exhibit excessive signal-dependent activity of the transcription factor nuclear factor (NF)-κB. Because aberrant interaction between NF-κB and coactivators represents an alternative mechanism for enhancing NF-κB activity, we evaluated whether MOZ and MOZ-CBP cooperate with NF-κB to activate transcription from NF-κB–dependent promoters. Methods: The ability of MOZ, MOZ mutants, and MOZ-CBP to enhance expression of NF-κB–dependent promoters was tested in reporter studies. The interaction between MOZ and NF-κB was evaluated by both coimmunoprecipitation and glutathione S-transferase pulldown assays. Results: MOZ activates transcription from the NF-κB–dependent interleukin-8 promoter; interestingly, this effect is markedly enhanced by CBP. Although MOZ has less potent transcriptional activity than MOZ-CBP, both proteins cooperate with steroid receptor coactivator-1 to activate transcription. MOZ also induces multiple NF-κB–dependent viral promoters. Importantly, MOZ associates in a protein complex with the p65 subunit of NF-κB and interacts directly with p65 in vitro. Transcriptional activity of MOZ requires its C-terminal domain, which is absent from MOZ-CBP, indicating that the transcriptional activity of MOZ-CBP derives from its CBP sequence. Conclusions: MOZ interacts with the p65 subunit of NF-κB and enhances expression of NF-κB–dependent promoters. The more potent transcriptional activity of MOZ-CBP derives from its CBP sequence. Thus, interaction between NF-κB and MOZ-CBP may play an important role in the pathogenesis of certain acute myeloid leukemias. [Copyright &y& Elsevier]

Although hyperactivation of the Ras-Erk signaling pathway is known to underlie the pathogenesis of juvenile myelomonocytic leukemia (JMML), a fatal childhood disease, the PI3K-Akt signaling pathway is also dysregulated in this disease. Using genetic models, we demonstrate that inactivation of phosphatidylinositol-3-kinase (PI3K) catalytic subunit p1105, but not PI3K p110a, corrects gain-of-function (GOF) Shp2-induced granulocyte macrophage-colony-stimuiating factor (GM-CSF) hypersensitivity, Akt and Erk hyperactivation, and skewed hematopoietic progenitor distribution. Likewise, potent p1108-specific inhibitors curtail the proliferation of GOF Shp2-expressing hematopoietic cells and cooperate with mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK) inhibition to reduce proliferation further and maximally block Erk and Akt activation. Furthermore, the PI3K p1108-specific inhibitor, idelalisib, also demonstrates activity against primary leukemia cells from individuals with JMML. These findings suggest that selective inhibition of the PI3K catalytic subunit p1105 could provide an innovative approach for treatment of JMML, with the potential for limiting toxicity resulting from the hematopoietic-restricted expression of p1105. (Blood. 2014;123(18):2838-2842) [ABSTRACT FROM AUTHOR]