ALBERT

Description: Mast cell maturation is poorly understood. We show that enhanced PI3K activation results in accelerated maturation of mast cells by inducing the expression of microphthalmia transcription factor (Mitf). Conversely, loss of PI3K activation reduces the maturation of mast cells by inhibiting the activation of AKT, leading to reduced Mitf but enhanced Gata-2 expression and accumulation of Gr1+Mac1+ myeloid cells as opposed to mast cells. Consistently, overexpression of Mitf accelerates the maturation of mast cells, whereas Gata-2 overexpression mimics the loss of the PI3K phenotype. Expressing the full-length or the src homology 3– or BCR homology domain–deleted or shorter splice variant of the p85α regulatory subunit of PI3K or activated AKT or Mitf in p85α-deficient cells restores the maturation but not growth. Although deficiency of both SHIP and p85α rescues the maturation of SHIP−/− and p85α−/− mast cells and expression of Mitf; in vivo, mast cells are rescued in some, but not all tissues, due in part to defective KIT signaling, which is dependent on an intact src homology 3 and BCR homology domain of p85α. Thus, p85α-induced maturation, and growth and survival signals, in mast cells can be uncoupled.

Description: Abstract 810 Gain-of-function mutations in the KIT receptor tyrosine kinase have been associated with highly malignant human neoplasms. In particular, an acquired somatic mutation at codon 816 in KIT involving an aspartic acid to valine substitution is found in ∼90% of patients with systemic mastocytosis (SM) and in ∼40% of core binding factor acute myeloid leukemia (AML). The presence of this mutation in SM and AML is associated with poor prognosis and overall survival. In mice, presence of this mutation is sufficient to recapitulate many cardinal features of human SM. This mutation changes the conformation of KIT receptor resulting in altered substrate recognition and constitutive tyrosine autophosphorylation leading to constitutive ligand independent growth, which is resistant to imatinib and shows little therapeutic efficacy in response to Dasatinib in most SM patients. As there are currently no efficacious therapeutic agents against this mutation, we sought to define novel therapeutic targets that contribute to aberrant signaling downstream from KITD816V that promote transformation of primary hematopoietic stem/progenitor cells (HSC/Ps) in diseases such as AML and SM. Previously, we and others have demonstrated that the regulatory subunit of PI3K, p85α, is required for KITD814V (murine homolog) induced transformation. Although difficult to target, we hypothesized that perhaps the downstream effectors of the PI3K signaling pathway, in particular p21 activated kinase (PAK1) and its upstream effectors including guanine exchange factors (GEF) such as Tiam1, Trio and Vav as well as the Rho family of GTPases (Rac) contribute to gain-of-function mutant-mediated transformation. We show that KITD814V (mouse) and KITD816V (human) bearing leukemic cells exhibit constitutive activation of PAK, Rac GTPases, and GEF Vav. Importantly, treatment of KITD814V bearing murine cells or mastocytosis patient derived cells bearing the KITD816V mutation with an allosteric inhibitor of PAK1 (i.e. IPA-3) results in significant inhibition in growth due to enhanced apoptosis. Consistently, expression of a dominant negative form of PAK1 (K299R) in KITD814V bearing cells profoundly inhibited their growth but not the growth of normal cells. Upstream of PAK, we show that suppression of Rac GTPases by expression of a dominant negative form of Rac (RacN17) abrogates activating KIT-induced hyperproliferation, and activity of downstream effector, PAK1. Although both Rac1 and Rac2 are activated due to the presence of KITD814V in primary HSC/Ps; loss of Rac1 only modestly corrects the growth of KITD814V bearing cells and loss of Rac2 contributes to only 50% correction. In contrast, loss of both Rac1 and Rac2 in HSC/Ps resulted in 75% correction in KITD814V induced ligand independent growth in vitro. In vivo, Rac repression significantly delayed the onset of KITD814V induced myeloproliferative neoplasms (MPN). Although, all KITD814V bearing mice died around 20 days of transplantation due to splenomegaly, increased white cell counts and massive lung infiltration by leukemic cells; KITD814V bearing mice in which Rac was repressed showed prolonged survival, significantly reducted spleen size, white cell counts and myeloid cell infiltration in the lungs. Prior studies have shown that Rac GTPases can be activated by GEFs such as Tiam1, Trio and Vav. To assess the specific role of these GEFs in KITD814V induced transformation, we utilized small molecule inhibitors that uniquely target different GEFs. We synthesized and utilized a novel inhibitor of Rac, EHop-016, which is based on the structure of an existing GEF inhibitor, NSC23766. While NSC23766 targets Tiam1 and Trio, EHop-016 targets Vav. The IC50 of EHop-016 is ∼50 fold lower than that of NSC23766. Using these two drugs, we demonstrate that EHop-016 is 50-fold more potent in inhibiting the growth of both murine and human patient derived leukemic cells compared to NSC23766. These observations were confirmed utilizing mice and bone marrow cells deficient in the expression of Vav1 engineered to express the KITD814V mutation. Taken together, a series of experiments using knockout mouse models, mouse models of MPN, dominant negative approaches, and a novel allosteric inhibitor of PAK1 and a novel small molecule inhibitor of GEF Vav provide a mechanism of KITD816V induced transformation and provide potential novel therapeutic targets for treating oncogenic KIT bearing neoplasms. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 858 Multiple genetic checks and balances regulate the complex process of hematopoiesis. Despite these measures, mutations in crucial regulatory genes are still known to occur, which in some cases results in abnormal hematopoiesis, including leukemogenesis and/or myeloproliferative neoplasms (MPN). An example of a mutated gene that contributes to leukemogenesis is the FMS- like tyrosine kinase 3 (Flt3) that encodes a receptor tyrosine kinase, which plays an essential role in normal hematopoiesis. Interestingly, Flt3 is one of the most frequently mutated genes (∼30%) in acute myeloid leukemia (AML). Although various pathways downstream of Flt3 activation that lead to leukemic transformation have been extensively studied, effective treatment options for Flt3ITD mediated leukemogenesis is still warranted. In this study we used genetic, pharmacological and biochemical approaches to identify a novel role of Focal adhesion kinase (FAK) in Flt3ITD induced leukemogenesis. We observed hyperactivation of FAK in Flt3ITD expressing human and mouse cell. Treatment with FAK specific small molecule inhibitors F-14 and Y-11, inhibited proliferation and induced cell death of Flt3ITD expressing cells. Similarly, treatment of primary AML patient samples (n=9) expressing Flt3ITD mutations with F-14 inhibited their proliferation. Consistently expression of a dominant negative domain of FAK (FRNK) inhibited hyperproliferation and induced death of Flt3ITD bearing cells. Further, low-density bone marrow (LDBM) cells derived from FAK−/− mice transduced with Flt3ITD showed significantly reduced growth compared to wild-type (WT) LDBM cells transduced with Flt3ITD. We also observed hyperactivation of Rac1 in Flt3ITD expressing cells downstream of FAK, which was downregulated upon treatment with FAK inhibitor F-14 and Y11. Moreover, expression of dominant negative Rac1N17, or treatment with Rac1 inhibitor NSC23766 inhibited hyperproliferation of ITD bearing cells. We next wanted to ascertain the underlying mechanism of FAK mediated activation of Rac1 in Flt3ITD expressing cells. Toward this end, we found RacGEF Tiam1 to be hyperactive in Flt3ITD expressing cells, which was downregulated upon pharmacological inhibition of FAK. A Tiam1-Rac1 complex was also co-immunoprecipitated from Flt3ITD bearing cells, and this association was perturbed upon pharmacological inhibition of FAK. While, Stat5 a key molecule in Flt3ITD mediated leukemic progression, is activated and recruited to the nucleus to express Stat5 responsive genes; however the mechanism of Stat5 translocation to the nucleus is unknown. We observed a novel mechanism involving FAK and Rac1GTPase, in regulating the nuclear translocation of active Stat5. Pharmacological inhibition of FAK and Rac1 resulted in reduced Rac1 and STAT5 translocation into the nucleus, indicating a role of FAK-Rac-STAT5 signaling in Flt3ITD induced leukemogenesis. More importantly, expression of Flt3ITD in Rac1−/− or FAK−/− LDBM cells, showed inhibition of Stat5 activation and its failure to translocate into the nucleus when compared to Flt3ITD expression in WT-LDBM cells. We also observed association between active Rac1 and active Stat5 in the nucleus and in whole cell lysates of Flt3ITD bearing cells, and also in human AML patient samples (n=3), which was attenuated upon pharmacological inhibition of FAK. To determine the effect of FAK inhibition in vivo on Flt3ITD induced MPN, syngeneic transplantation was performed, and mice were treated with vehicle or with FAK inhibitor F-14. While vehicle treated mice developed MPN within 30 days, mice treated with F-14 showed significant overall survival (*p

Description: Abstract 1435 Poster Board I-458 Gain-of-function mutations of the receptor tyrosine kinase KIT have been associated with gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM) and acute myelogenous leukemia (AML). A mutation of aspartic acid to valine (KITD814V) in the activation loop of KIT results in altered substrate recognition and constitutive tyrosine autophosphorylation. However, the intracellular mechanisms that contribute to promiscuous signaling via this mutation are poorly understood. We have previously shown that KITD814V is sufficient to induce ligand independent growth in primary hematopoietic stem and progenitor cells (HSC/P) in vitro as well as transformation in vivo. However, it is not known whether ligand induced (i.e. KITL) signals in vivo contribute to transformation. To assess this, we generated a chimeric receptor (CHR) in which the extracellular domain of KIT was replaced with the human-macrophage colony stimulating factor receptor (h-MCSFR) to inhibit endogenous binding of murine SCF. In vitro thymidine incorporation assay confirmed that the WTCHR is only functionally responsive to human (h)-MCSF stimulation, but not to murine (m)SCF nor murine MCSF stimulation, and that CHRD814V receptor maintains ligand independent growth potential similar to the KITD814V receptor. To determine if the myeloproliferative disease (MPD) could occur in the absence of endogenous ligand binding, a murine transplantation model was utilized to compare CHRD814V and KITD814V-induced transformation. In vivo results demonstrated no significant difference in the onset of MPD in mice bearing KITD814V vs. CHRD814V (median survival 52 days vs. 49 days, n=10 to 20 mice), with similar disease manifestation in the two groups including splenomegaly, hepatomegaly, myeloid cell infiltration and elevated white blood cell counts (n=10 to 20). Transforming potential in the absence of KIT extracellular domain strongly suggested that self-association of KITD814V was sufficient to induce transformation rather than influences from endogenous ligand. To further assess the relative contribution of intracellular tyrosine residues in transformation, a CHRD814V mutant was generated in which seven critical tyrosine residues including the binding sites for Src family kinases, Grb2, p85α regulatory subunit of class IA PI3Kinase, PLC-g, Ras-GAP, and Grb7 were mutated to phenylalanines (i.e.CHRD814V-F7). Our results show that CHRD814V-F7 bearing primary bone marrow cells lost ligand independent growth potential in vitro, suggesting that seven tyrosine induced signaling may play a critical role in KITD814V-induced ligand independent transformation. Consistently, transplantation studies demonstrated that mice bearing bone marrow cells expressing CHRD814V-F7 exhibited a notable delay in MPD development (median survival= 95 days, n=4 to 8). These results suggest that intracellular tyrosines are crucial for ligand independent proliferation and transformation. Next, we individually restored each of the seven tyrosine residues back into CHRD814V-F7 to determine the importance of pathways essential for ligand independent growth and transformation. We found that among the single tyrosine add back receptors, only restoring p85α subunit binding site (Y719) alone was sufficient to induce promiscuous cellular growth, proliferation, and survival. Transplantation studies revealed that restoration of Y719 was sufficient to induce transformation in vivo (median survival= 55 days, n=5). Furthermore, restoration of other individual tyrosine residues demonstrated a notable delay in CHRD814V-induced transformation in vivo (median survival= 83∼120 days, n=5 to 12) and a minimal contribution to ligand independent growth in vitro. Taken together, our results demonstrate: 1) KITD814V induced transformation in vivo occurs in the absence of ligand stimulation; 2) intracellular tyrosine residues play an essential role in KITD814V induced transformation in vitro and in vivo and; 3) of all the critical tyrosine residues present in KIT, presence of tyrosine at position 719 is sufficient to completely restore ligand independent growth in vitro and transformation in vivo. Disclosures No relevant conflicts of interest to declare.

Description: Intracellular mechanism(s) that contribute to promiscuous signaling via oncogenic KIT in systemic mastocytosis and acute myelogenous leukemia are poorly understood. We show that SHP2 phosphatase is essential for oncogenic KIT-induced growth and survival in vitro and myeloproliferative disease (MPD) in vivo. Genetic disruption of SHP2 or treatment of oncogene-bearing cells with a novel SHP2 inhibitor alone or in combination with the PI3K inhibitor corrects MPD by disrupting a protein complex involving p85α, SHP2, and Gab2. Importantly, a single tyrosine at position 719 in oncogenic KIT is sufficient to develop MPD by recruiting p85α, SHP2, and Gab2 complex to oncogenic KIT. Our results demonstrate that SHP2 phosphatase is a druggable target that cooperates with lipid kinases in inducing MPD.

Description: Abstract 868 Gain-of-function mutations in KIT receptor in humans are associated with gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), and acute myelogenous leukemia (AML). An activating KIT receptor mutation of aspartic acid to valine at codon 814 in mice (KITD814V) or codon 816 in humans (KITD816V) results in altered substrate recognition and constitutive tyrosine autophosphorylation leading to promiscuous signaling. Consequently, cells bearing oncogenic form of KIT (KITD814V) demonstrate ligand independent proliferation in vitro and MPD in vivo. However, the intracellular signals that contribute to KITD814V induced MPD are not known. Here, we show the constitutive phosphorylation of SHP2 in cells bearing KITD814V, but not WT KIT, which was inhibited by treatment with a novel SHP2 inhibitor, II-B08 (*p 〈 0.05). In addition, treatment with II-B08 suppressed the growth of cells bearing KITD814V, but not WT KIT (*p 〈 0.05), Human mast cell line HMC1.2 (*p 〈 0.05), and Human CD34+ cells bearing KITD816V (*p 〈 0.05). Likewise, deficiency of SHP2 in primary bone marrow cells resulted in a significant repression in constitutive growth of cells bearing KITD814V (*p 〈 0.05). To determine the mechanism behind the repression in ligand independent growth of cells bearing KITD814V by II-B08, we examined the role of SHP2 in cell survival. We observed a dose dependent increase in apoptosis of cells bearing KITD814V compared to WT KIT in the presence of II-B08 (*p 〈 0.05). Similarly, deficiency of SHP2 resulted in increased apoptosis of cells bearing KITD814V (*p 〈 0.05). In an effort to identify the mechanism behind reduced growth and increased apoptosis of cells bearing KITD814V in the presence of II-B08, we examined whether SHP2 regulates the activation of AKT in KITD814V bearing cells. We found constitutive activation of AKT in cells bearing KITD814V, but not WT KIT, which was significantly inhibited upon II-B08 treatment suggesting that SHP2 regulates ligand independent growth and survival of KITD814V bearing cells in part by regulating the activation of AKT. To further determine the signaling molecules that co-operate with SHP2 in KITD814V induced MPD, we examined proteins that potentially interact with SHP2 in KITD814V bearing cells and whether inhibition of SHP2 activity by II-B08 suppresses these co-operating protein interactions. SHP2 constitutively bound to p85α and Gab2 in cells bearing KITD814V, but not in WT KIT bearing cells, which was inhibited upon II-B08 treatment. To further assess whether the binding of SHP2 to p85α, Gab2 and KITD814V is sufficient to induce MPD, we generated a KIT mutant receptor, KITD814V-F7, in which seven tyrosine residues in KITD814V (known to bind SH2 containing proteins at tyrosine 567, 569, 702, 719, 728, 745, and 934) were converted to phenylalanine, and KITD814V-Y719, in which only tyrosine residue 719 (binding site for p85α) was added back to the KITD814V-F7 receptor. We observed constitutive binding of SHP2 and Gab2 to p85α in cells bearing KITD814V and KITD814V-Y719, but not in WT KIT or KITD814V-F7 bearing cells. In addition, conversion of all the seven intracellular tyrosine residues in KITD814V to phenylalanine (KITD814V-F7) resulted in complete loss of ligand independent growth in vitro (*p 〈 0.05) and significantly delayed the progression of MPD in vivo (*p 〈 0.05). Importantly, restoration of tyrosine residue at position 719 (KITD814V-Y719) was sufficient to induce ligand independent growth in vitro and MPD in vivo to KITD814V levels. Furthermore, deficiency of Gab2 resulted in significant repression in constitutive growth of cells bearing KITD814V (*p 〈 0.05). These results demonstrate that p85α recruits SHP2 and Gab2 to KITD814V at Y719, which might contribute to KITD814V induced MPD. Moreover, II-B08 enhances the efficacy of PI3Kinase inhibitor LY294002 in suppressing KITD814V induced ligand independent growth in vitro (*p 〈 0.05) and MPD in vivo (*p 〈 0.05). Taken together our results demonstrate that SHP2 is a druggable target which cooperates with PI3Kinase in inducing MPD. Disclosures: No relevant conflicts of interest to declare.

Description: KIT receptor signaling plays an important role in mast cell development. Gain-of-function mutations in KIT receptor have been identified in human diseases including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM) and acute myeloid leukemia (AML). Although KIT mutations found in GIST are sensitive to imatinib, KIT mutation (KITD816V) found in 90% of SM patients is imatinib-resistant and currently no therapies are available to treat the human diseases associated with this mutation. Our recent studies have identified Ten-Eleven-Translocation 2 (TET2) mutations in ~23% of SM patients and are associated with poor prognosis and overall survival. TET2 is a methylcytosine dioxygenase that plays a vital role in active DNA demethylation. Recent studies suggest that patients with mutations in TET2 and KITD816V develop more aggressive form of mastocytosis with worse prognosis. Although it is known that TET2 and KITD816V cooperate in SM patients, it is not clear how they cooperate with each other and what is the physiologic role of TET2 in normal mast cell development. We show that loss of Tet2 results in impaired maturation of mast cells in vivo and in bone marrow-derived mast cells (BMMC) compared to WT controls, which is associated with reduction in 5-hmc levels compared to WT BMMCs. We also observed reduction in the expression of mast cell-specific genesincluding Mast cell proteinase-5 (MCP-5), Mast cell proteinase-6 (MCP-6) and Carboxypeptidase A (CPA). To determine the mechanism behind altered mast cell differentiation in Tet2-/- BMMCs, we performed RNA-seq analysis in WT and Tet2-/- mast cells and observed altered expression of various genes involved in development of mast cells including Kit, FcεR1, Mitf, Notch, and Myc. We further confirmed altered expression of Mitf, Gata-2, and PU.1 in Tet2-/- BMMCs compared to WT BMMCs by western blotting. Since Tet2 regulates DNA demethylation, we tested whether altered BMMC differentiation in Tet2-/- mice is due to enhanced DNA methylation. We treated WT or Tet2-/- BM cells for 3 weeks with vehicle or 5-azacytidine (hypomethylating agent) and analyzed mast cell differentiation. Treatment with 5-azacytidine completely corrected the defective mast cell differentiation in Tet2-/- cells to WT levels. These results suggest that Tet2 plays a significant role in mast cell differentiation by regulating the expression of critical transcription factors including Mitf, Gata-2 and PU.1. We next analyzed the growth of Tet2-/- BMMCs in response to cytokines. Tet2-deficient BMMCs show enhanced cytokine mediated growth compared to WT BMMCs. Hyper-proliferation of Tet2-/- BMMCs is associated with reduced expression of tumor suppressor, PTEN, whose promoter is hypermethylated and a concomitant increase in the activation of the PI3K/AKT pathway. Since loss of function TET2 mutations have been observed in SM patients in addition to KITD816V mutation, we tested whether loss of Tet2 cooperates with KIT mutation in vitro and in vivo. Tet2-deficiency or knockdown in conjunction with the expression of KIT mutation resulted in significantly enhanced growth compared to cells bearing KIT mutation alone or lacking Tet2 expression. Likewise in human mastocytosis xenograft model, significantly enlarged tumors were observed in NSG mice transplanted with human mastocytosis cell line bearing the KITD816V mutation (HMC1.2) and knockdown of TET2 compared to HMC1.2 cells bearing only the KITD816V mutation. The cooperation between loss of Tet2 and KIT mutation was associated with further increase in PI3K/AKT activation and pharmacologic inhibitor treatment with a PI3K inhibitor GDC-0941 (Pan PI3K), but not TGX221 (p110β-specific) or IC87114 (p110δ-specific), significantly reduced the hyper-proliferation of Tet2-/- BMMCs and cell lines as well as primary BM blasts derived from SM patients bearing the KITD816V mutation. Consistently, combined loss of p110α and p110δ subunits of PI3K resulted in the most profound growth repression in oncogenic KIT bearing BM cells, but did not correct altered differentiation in Tet2-/- BMMCs. Taken together our results suggest that combinational therapy involving 5-azacytidine (which corrects the impaired mast cell differentiation) and PI3K inhibitor (which corrects the excessive proliferation) is a better therapeutic option for treating human mastocytosis patients bearing TET2 and KIT mutations. Disclosures No relevant conflicts of interest to declare.