Rap1 is a Potential Therapeutic Target for Non-myeloablative Conditioning

poster abstractvarious side effects including gastrointestinal mucositis. Identification of therapeutic targets and determining their role in HSC development and function is important to determine a regimen for nonmyeloablative conditioning. Previous studies have shown that GTPases play a critical role in self-renewal, engraftment and retention of HSCs. Rap1, a GTPase, is necessary for migration, adhesion as well as function of mature hematopoietic cells. To study the role of Rap1 in hematopoietic stem and progenitor cells (HSC/Ps), we have generated a mouse model in which the Rap1a and Rap1b isoforms of Rap1 are conditionally deleted in HSC/Ps (Rap1a/b -/-). Deficiency of Rap1a/b results in increased peripheral blood count as well as increase in HSCs in bone marrow along with a decrease in bone marrow cellularity. Rap1a/b deficient bone marrow HSC/Ps also have reduced adhesion capability in vitro. The self-renewal property of HSCs, in conjunction with their ability of multi-lineage reconstitution is important to repopulate the hematopoietic system of irradiated recipients of bone marrow transplant. Rap1a/b -/- HSCs show a defect in engraftment as well as multi-lineage reconstitution when they are transplanted into lethally irradiated hosts. Rap1 deficient HSCs show decreased homing into bone marrow of lethally irradiated recipients. To determine whether Rap1 can be used as a potential target for nonmyeloablative conditioning, we performed bone marrow transplant into WT and Rap1a/b -/- mice without prior irradiation. Deficiency of Rap1a/b in HSCs resulted in availability of bone marrow niche for exogenously transplanted HSCs to engraft along with subsequent multi-lineage reconstitution. Overall, our study reveals that Rap1a/b are important for homing and retention of hematopoietic cells in bone marrow and deletion of Rap1a/b in HSCs result in engraftment of exogenous HSCs within the bone marrow of non-irradiated recipients

COOPERATION OF AML1-ETO AND ONCOGENIC KIT IN ACUTE MYELOGENOUS LEUKEMIA

poster abstractA significant portion of AML patients have the cytogenetic abnormality t(8;21) which generates the fusion protein AML1-ETO, leading to a disruption of the core binding factor complex that regulates transcription of hematological genes. Patients harboring the translocation alone usually have a good prognosis; however, a substantial portion of patients bearing an additional oncogenic receptor tyrosine kinase, KIT, mutation have significantly worse prognosis. A mutation of aspartic acid to valine (KITD814V) in the activation loop results in altered substrate recognition and utilization, constitutive tyrosine autophosphorylation, and promiscuous signaling. Little is known concerning possible mechanisms of cooperation between AML1-ETO and KITD814V. Using an IL3 dependent murine myeloid cell line, we show that growth of AML1-ETO bearing cells remain ligand dependent, while cells that express both AML1-ETO and KITD814V demonstrate ligand independent proliferation. Furthermore, functional assays show that expression of AML1-ETO and KITD814V leads to an increase in cell cycling and decrease in apoptosis that may contribute to the observed ligand independent proliferation. Using a syngenic murine transplantation model we demonstrate that mice transplanted with AML1-ETO and KITD814V bearing cells succumb to a fatal myeloproliferative disease (MPD)-like phenotype, while AML1-ETO expressing mice remain disease free. This suggests that AML1-ETO alone is not sufficient to induce ligand independent growth, nor MPD, but may cooperate with KITD814V to enhance proliferation. Continuing research aims to investigate mechanisms of cooperation between KITD814V and AML1-ETO that contribute to ligand independent growth in vitro, transformation in vivo, and poor overall prognosis in AML patients bearing the two mutations

SHP2 phosphatase promotes mast cell chemotaxis toward stem cell factor via enhancing activation of the Lyn/Vav/Rac signaling axis

SHP2 protein-tyrosine phosphatase (encoded by Ptpn11) positively regulates KIT (CD117) signaling in mast cells and is required for mast cell survival and homeostasis in mice. In this study, we uncover a role of SHP2 in promoting chemotaxis of mast cells toward stem cell factor (SCF), the ligand for KIT receptor. Using an inducible SHP2 knockout (KO) bone marrow-derived mast cell (BMMC) model, we observed defects in SCF-induced cell spreading, polarization, and chemotaxis. To address the mechanisms involved, we tested whether SHP2 promotes activation of Lyn kinase that was previously shown to promote mast cell chemotaxis. In SHP2 KO BMMCs, SCF-induced phosphorylation of the inhibitory C-terminal residue (pY507) was elevated compared with control cells, and phosphorylation of activation loop (pY396) was diminished. Because Lyn also was detected by substrate trapping assays, these results are consistent with SHP2 activating Lyn directly by dephosphorylation of pY507. Further analyses revealed a SHP2- and Lyn-dependent pathway leading to phosphorylation of Vav1, Rac activation, and F-actin polymerization in SCF-treated BMMCs. Treatment of BMMCs with a SHP2 inhibitor also led to impaired chemotaxis, consistent with SHP2 promoting SCF-induced chemotaxis of mast cells via a phosphatase-dependent mechanism. Thus, SHP2 inhibitors may be useful to limit SCF/KIT-induced mast cell recruitment to inflamed tissues or the tumor microenvironment

Role of intracellular tyrosines in activating KIT induced myeloproliferative disease

Gain-of-function mutations in KIT receptor in humans are associated with gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), and acute myelogenous leukemia (AML). The intracellular signals that contribute to oncogenic KIT induced myeloproliferative disease (MPD) are poorly understood. Here, we show that oncogenic KITD814V induced MPD occurs in the absence of ligand stimulation. The intracellular tyrosine residues are important for KITD814V induced MPD, albeit to varying degrees. Among the seven intracellular tyrosines examined, tyrosine 719 alone plays a unique role in regulating KITD814V induced proliferation and survival in vitro, and MPD in vivo. Importantly, the extent to which AKT, ERK and Stat5 signaling pathways are activated via the seven intracellular tyrosines in KITD814V impacts the latency of MPD and severity of the disease. Our results identify critical signaling molecules involved in regulating KITD814V induced MPD, which might be useful for developing novel therapeutic targets for hematologic malignancies involving this mutation

The mirn23a and mirn23b microrna clusters are necessary for proper hematopoietic progenitor cell production and differentiation

Mice deficient for microRNA (miRNA) cluster mirn23a exhibit increased B lymphopoiesis at the expense of myelopoiesis, whereas hematopoietic stem and progenitor cell (HSPC) populations are unchanged. Mammals possess a paralogous mirn23b gene that can give rise to three mature miRNAs (miR-23b, miR-24-1, and miR-27b) that have identical seed/mRNA-targeting sequences to their mirn23a counterparts. To assess whether compound deletion of mirn23a and mirn23b exacerbates the hematopoietic phenotype observed in mirn23a−/− mice, we generated a compound mirn23a−/−mirn23bfl/fl:Mx1-Cre conditional knockout mouse and assayed hematopoietic development after excision of mirn23b. Loss of both genes in adult bone marrow further skewed HSPC differentiation toward B cells at the expense of myeloid cells, demonstrating a dosage-dependent effect on regulating cell differentiation. Strikingly, double-knockout (DKO) mice had decreased bone marrow cellularity with significantly decreased hematopoietic stem cell and HSPC populations, a phenotype not observed in mice deficient for mirn23a alone. Competitive transplantation assays showed decreased contribution of mirn23a−/−mirn23b−/− HSPCs to hematopoietic lineages at 6 and 12 weeks after transplantation. Defects in the proliferation of mirn23a−/−b−/− HSPCs was not observed; however, DKO cells were more apoptotic compared with both wild-type and mirn23a−/− cells. Together, our data show that complete loss of mirn23a/mirn23b miRNAs results in decreased blood production and affects lineage output in a concentration-dependent manner

Deficiency of Src family kinases compromises the repopulating ability of hematopoietic stem cells

OBJECTIVE: Src family kinases (SFK) have been implicated in regulating growth factor and integrin-induced proliferation, migration, and gene expression in multiple cell types. However, little is known about the role of these kinases in the growth, homing, and engraftment potential of hematopoietic stem and progenitor cells. RESULTS: Here we show that loss of hematopoietic-specific SFKs Hck, Fgr, and Lyn results in increased number of Sca-1(+)Lin(-) cells in the bone marrow, which respond differentially to cytokine-induced growth in vitro and manifest a significant defect in the long-term repopulating potential in vivo. Interestingly, a significant increase in expression of adhesion molecules, known to coincide with the homing potential of wild-type bone marrow cells is also observed on the surface of SFK(-/-) cells, although, this increase did not affect the homing potential of more primitive Lin(-)Sca-1(+) SFK(-/-) cells. The stem cell-repopulating defect observed in mice transplanted with SFK(-/-) bone marrow cells is due to the loss of Lyn Src kinase, because deficiency of Lyn, but not Hck or Fgr, recapitulated the long-term stem cell defect observed in mice transplanted with SFK(-/-) bone marrow cells. CONCLUSIONS: Taken together, our results demonstrate an essential role for Lyn kinase in positively regulating the long-term and multilineage engraftment of stem cells, which is distinct from its role in mature B cells and myeloid cells

S6K1 regulates hematopoietic stem cell self-renewal and leukemia maintenance.

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 an

DPP4 Truncated GM-CSF & IL-3 Manifest Distinct Receptor Binding & Regulatory Functions Compared to their Full Length Forms

Dipeptidylpeptidase 4 (DPP4/CD26) enzymatically cleaves select penultimate amino acids of proteins, including colony stimulating factors (CSFs), and has been implicated in cellular regulation. To better understand the role of DPP4 regulation of hematopoiesis, we analyzed the activity of DPP4 on the surface of immature blood cells and then comparatively assessed the interactions and functional effects of full-length (FL) and DPP4 truncated factors [(T)-GM-CSF and- IL-3] on both in vitro and in vivo models of normal and leukemic cells. T-GM-CSF and T-IL-3 had enhanced receptor binding, but decreased CSF activity, compared to their FL forms. Importantly, T-GM-CSF and T-IL-3 significantly, and reciprocally, blunted receptor binding and myeloid progenitor cell proliferation activity of both FL-GM-CSF and FL-IL-3 in vitro and in vivo. Similar effects were apparent in vitro using cluster forming cells from patients with Acute Myeloid Leukemia (AML) regardless of cytogenetic or molecular alterations and in vivo utilizing animal models of leukemia. This suggests that DPP4 T-molecules have modified binding and functions compared to their FL counterparts and may serve regulatory roles in normal and malignant hematopoiesis

Driver Mutations in Leukemia Promote Disease Pathogenesis through a Combination of Cell-Autonomous and Niche Modulation

Studies of patients with acute myeloid leukemia (AML) have led to the identification of mutations that affect different cellular pathways. Some of these have been classified as preleukemic, and a stepwise evolution program whereby cells acquire additional mutations has been proposed in the development of AML. How the timing of acquisition of these mutations and their impact on transformation and the bone marrow (BM) microenvironment occurs has only recently begun to be investigated. We show that constitutive and early loss of the epigenetic regulator, TET2, when combined with constitutive activation of FLT3, results in transformation of chronic myelomonocytic leukemia-like or myeloproliferative neoplasm-like phenotype to AML, which is more pronounced in double-mutant mice relative to mice carrying mutations in single genes. Furthermore, we show that in preleukemic and leukemic mice there are alterations in the BM niche and secreted cytokines, which creates a permissive environment for the growth of mutation-bearing cells relative to normal cells