ALBERT

Description: AML is thought to arise from leukemia stem cells (LSCs); however, recent evidence suggests that the transforming events may initially give rise to pre-leukemic hematopoietic stem cells (pre-leukemic HSCs), preceding the formation of fully transformed LSCs. Pre-leukemic HSCs have been shown to contribute to normal blood development and harbor a selective growth advantage compared to normal HSCs. Pre-leukemic HSCs can acquire subsequent mutations, and once differentiation capacity is impaired, leukemia emerges. Recently, acquired somatic TP53 mutations, including p53R248W and p53R273H, were identified in healthy individuals as well as AML patients, suggesting that TP53 mutations may be early events in the pathogenesis of AML. We found that p53R248W HSCs showed a multi-lineage repopulation advantage over WT HSCs in transplantation experiments, demonstrating that mutant p53 confers a pre-leukemic phenotype in murine HSCs. Although TP53 mutations are limited in AML, TP53 mutations do co-exist with mutations of epigenetic regulator, ASXL-1, or receptor tyrosine kinase, FLT3, in AML. Mutations in Asxl-1 are present in ~10-30% of patients with myeloid malignancies and confer poor prognosis. Loss of Asxl-1 in the hematopoietic compartment leads to a myelodysplastic-like syndrome in mice and reduced stem cell self-renewal. Internal tandem duplications in Flt3 (Flt3-ITD) occur in ~30% of AML patients and are associated with adverse clinical outcome. Flt3-ITD-positive mice develop a myeloproliferative neoplasm (MPN) and HSCs expressing Flt3-ITD have decreased self-renewal capabilities. We hypothesize that mutant p53 drives the development of pre-leukemic HSCs with enhanced self-renewal capability, allowing clonal expansion and subsequent acquisition of Asxl-1 or Flt3 mutations leading to the formation of fully transformed leukemia stem cells. To define the role of mutant p53 in Asxl-1+/- HSCs, we generated p53R248W/+ Asxl-1+/- mice and performed in vitro serial replating assays as well as in vivo competitivebone marrow transplantation experiments. We found that p53R248W significantly enhanced the serial replating ability of Asxl-1-deficient bone marrow cells. Interestingly, while bone marrow from Asxl-1+/- mice had very poor engraftment compared to wild type bone marrow cells 16 weeks post-transplantation, the expression of p53R248W in Asxl-1+/- bone marrow rescued the defect. To examine the role of mutant p53 in Flt3-ITD-positive HSCs, we generated p53R248W/+ Flt3ITD/+ mice. We found that p53R248W enhanced the replating ability of Flt3ITD/+ bone marrow cells. Despite the fact that Flt3ITD/+ bone marrow cells displayed decreased repopulating ability compared to wild type cells 16 weeks post-transplant, expression of p53R248W in Flt3ITD/+ cells rescued the defect. We are monitoring leukemia development in primary and secondary transplant recipients as well as in de novo p53R248W/+ Asxl-1+/- and p53R248W/+ Flt3ITD/+ animals and predict that mutant p53 may cooperate with Asxl-1 deficiency or Flt3-ITD in the formation of LSCs to accelerate leukemia development in Asxl-1 deficient or Flt-ITD-positive neoplasms. Mechanistically, dysregulated epigenetic control underlies the pathogenesis of AML and we discovered that mutant p53 regulates epigenetic regulators, including Ezh1, Ezh2, Kdm2a, and Setd2, in HSCs. H3K27me3 is catalyzed by EZH1 or EZH2 of the Polycomb repressing complex 2 (PRC2). Both Ezh1 and Ezh2 are important for HSC self-renewal. SETD2 is a histone H3K36 methyltransferase and mutations in SETD2 have been identified in 6% of patients with AML. SETD2 deficiency resulted in a global loss of H3K36me3 and increased self-renewal capability of leukemia stem cells. We found that there were increased levels of H3K27me3 and decreased levels of H3K36me3 in p53R248W/+ HSCs compared to that of the WT HSCs. In ChIP experiments, we found that p53R248W, but not WT p53, was associated with the promoter region of Ezh2 in mouse myeloid progenitor cells, suggesting that p53R248W may directly activate Ezh2 expression in hematopoietic cells. Given that Asxl-1 has been shown to regulate H3K27me3 in HSCs, the synergy between mutant p53 and Asxl-1 deficiency on LSC self-renewal could be due to changes in histone modifications. Overall, we demonstrate that mutant p53 promotes the development of pre-leukemic HSCs by a novel mechanism involving dysregulation of the epigenetic pathways. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2420 Internal tandem duplications in the fms-like tyrosine kinase receptor (FLT3-ITDs) confer a poor prognosis in individuals with acute myeloid leukemia (AML). Based on the finding that the protein tyrosine phosphatase, Shp2, interacts with WT FLT3 tyrosine (Y) 599, which is commonly duplicated in FLT3-ITDs, we hypothesized that increased recruitment of Shp2 to FLT3-ITDs contributes to FLT3 ligand (FL)-independent hyperproliferation and aberrant STAT5 activation. Co-immunoprecipitation studies demonstrated constitutive association of Shp2 with the FLT3-ITD, N51-FLT3, as well as with STAT5. Additionally, we found that genetic disruption of Ptpn11, the gene encoding Shp2, significantly reduced N51-FLT3-induced hematopoietic cell hyperproliferation and STAT5 hyperphosphorylation in vitro. To investigate these findings further, Lin- bone marrow cells from Shp2flox/flox;Mx1Cre+ animals were retrovirally transduced with N51-FLT3, sorted to homogeneity, and transplanted into lethally irradiated congenic recipients. Transplanted animals were treated with polyI:polyC to delete Shp2 or with phosphate buffered saline (PBS control) 4 – 6 weeks following transplantation, and animals were followed temporally. The majority of PBS-treated animals (16/18) died of hematologic malignancy. In contrast, animals with Shp2 deletion (polyI:polyC-treated, n=16) succumbed to malignant disease less frequently (10/16), demonstrated a significantly prolonged survival (p=0.024 by log-rank test), and had smaller spleen sizes compared to the PBS-treated animals. Notably, Y599 has been shown to recruit Shp2 to WT FLT3 and mutation of Y599 to phenylalanine (F) within WT FLT3 causes a reduction in FL-stimulated cell proliferation. Thus, we generated point mutants including N51-Y599F1 bearing the Y to F mutation at the first Y599 and N51-Y599F1/2 bearing Y to F mutation at both the first and duplicated Y599. Murine bone marrow low density mononuclear cells were transduced with each construct and subjected to 3H-thymidine incorporation and immunoblot for proliferation and STAT5 activation, respectively. While mutation of the first Y599 alone failed to reduce proliferation or STAT5 phosphorylation, mutation of both the first and duplicated Y599 significantly reduced cellular proliferation and phospho-STAT5 levels. To investigate molecular mechanisms underlying how constitutive association of Shp2 with STAT5 may promote FLT3-ITD-induced leukemogenesis, we utilized the human FLT3-ITD positive AML-derived cell line, MV411. While previous studies have demonstrated nuclear localization of Shp2 in AML samples, the role of nuclear Shp2 in leukemia has never been investigated. We utilized in situ immunofluorescence to examine nuclear distribution of Shp2 and potential co-localization with phospho-STAT5. Strong nuclear expression of Shp2 was observed in MV411 cells, and upon merging of images, nuclear Shp2 co-localized strongly with nuclear phospho-STAT5, suggesting that Shp2 may work with STAT5 within the nucleus to enhance gene expression promoting leukemogenesis. We chose to examine the BCL2L1 promoter, a STAT5-responsive promoter which regulates expression of the prosurvival protein, Bcl-XL. Using chromatin immunoprecipitation assays, we found Shp2 is present at functional interferon-g activation sites (GAS) within the BCL2L1 promoter. Furthermore, knockdown of Shp2 in MV411 cells resulted in reduced phospho-STAT5 levels and reduced BCL2L1 promoter-directed luciferase expression. Moreover, using a novel small molecule Shp2 inhibitor, the proliferation of N51-FLT3-expressing bone marrow progenitors and primary AML samples was significantly reduced in a dose-dependent manner. Our findings suggest that constitutive association of Shp2 with N51-FLT3 promotes hyperproliferation and that either genetic disruption of Shp2 expression or mutation of the Shp2 binding sites on N51-FLT3 significantly abrogates N51-FLT3-induced hyperproliferation, STAT5 hyperactivation, and N51-FLT3-induced hematologic malignancy in vivo. Furthermore, Shp2 and STAT5 appear to work functionally in the nucleus to promote STAT5-responsive, pro-leukemogenic gene expression. Collectively, these studies demonstrate that Shp2 positively contributes to FLT3-ITD-induced leukemia and suggest that Shp2 inhibition may provide a novel therapeutic approach to AML. Disclosures: No relevant conflicts of interest to declare.

Description: Macrophages are professional phagocytic cells, and express pattern recognition receptors such as C-type lectins and integrins for the detection of invading pathogens. Both Dectin-1 (a C-type lectin) and complement receptor 3 (CR3, a β2-integrin) are expressed on innate immune cells including macrophages, neutrophils, and dendritic cells. Dectin-1 stimulation by b-glucan-containing particles (zymosan) and CR3 stimulation by serum opsonized zymosan (SOZ) activate Erk- and Akt-dependent signaling resulting in phagocytosis and production of an oxidative burst. Shp2, a protein tyrosine phosphatase encoded by Ptpn11, promotes activation of Ras-Erk and PI3K-Akt signaling, supports hematopoietic development, and is commonly mutated in juvenile myelomonocytic leukemia (JMML). However, no studies have examined the role of Shp2 in Dectin-1- or CR3-stimulated NADPH oxidase activation or ROS production. As activation of Erk and Akt stimulates NADPH oxidase by phosphorylating p47phox, we hypothesized that Shp2 positively regulates ROS production in response to Dectin-1 or CR3 stimulation. Using murine peritoneal exudate macrophages (PEMs), both zymosan and SOZ exposure induced maximal ROS production 10 minutes post-stimulation, which corresponded to maximal induction of Shp2 phosphorylation (Y580, proposed to promote Shp2 phosphatase activity) and Erk phosphorylation. Using bone marrow derived macrophages (BMMs) from mice bearing a conditionally deleted allele of Ptpn11 (Shp2flox/flox;Mx1Cre+), ROS production was significantly reduced in response to zymosan and SOZ in Shp2flox/flox;Mx1Cre+ BMMs compared to control Shp2flox/flox;Mx1Cre- BMMs. Notably, the phagocytic index of the Shp2flox/flox;Mx1Cre+ and Shp2flox/flox;Mx1Cre- BMMs was similar, and protein components of the NADPH oxidase complex (p40phox, p67phox, and p47phox) were expressed at similar levels. To define the biochemical role of Shp2 in ROS production, we generated yellow fluorescent protein (YFP)-tagged Shp2 constructs bearing mutation of the N-SH2 (R32K) or phosphatase (C463A) domain and retrovirally expressed these constructs in murine BMMs. When subjected to zymosan or SOZ stimulation, mutation of either the N-SH2 or phosphatase domain resulted in reduced ROS production. Using time-lapse confocal videomicroscopy, we found that Shp2-R32K-YFP failed to translocate to the phagosome in SOZ-stimulated BMMs; however, phosphatase dead Shp2-C463A-YFP strongly translocated to the phagosome despite producing lower ROS levels. These findings specifically pointed to Shp2 phosphatase function as crucial in positively regulating NADPH oxidase and ROS production. Accordingly, we reasoned that macrophages expressing JMML-associated gain-of-function (GOF) Shp2 mutants, characterized to have increased phosphatase activity, would produce elevated ROS levels. As anticipated, BMMs retrovirally expressing GOF Shp2-D61Y or GOF Shp2-E76K and PEMs from mice bearing a conditionally induced gain-of-function allele of Ptpn11 (Shp2D61Y/+;Mx1Cre+) similarly produced significantly elevated levels of zymosan- and SOZ-stimulated ROS compared to WT Shp2-expressing BMMs or PEMs, respectively. Given the positive role of Shp2 phosphatase in promoting zymosan- and SOZ-stimulated ROS production, we investigated putative Shp2 substrates in response to zymosan stimulation. SHPS-1 (SH2 domain-containing protein tyrosine phosphatase substrate 1) is a myeloid inhibitory immunoreceptor expressed on macrophages, requires tyrosine phosphorylation to exert its inhibitory effect, and has been shown to be de-phosphorylated by Shp2. Consistent with its potential function in regulation ROS production, SHPS-1 is strongly associated with phagosomes in zymosan-stimulated PEMs. In immunoblot analysis, reduced phospho-SHPS-1 levels kinetically correlated with maximal zymosan-stimulated Shp2 phosphorylation and ROS production, and increased levels of phospho-SHPS-1 were found in BMMs expressing phosphatase dead Shp2-C463A compared to cells expressing WT Shp2. Collectively, these findings indicate that Shp2 phosphatase function positively regulates Dectin-1- and CR3-stimulated NADPH oxidase activation and ROS production in macrophages, and that mechanistically, Shp2 may exert its positive effect by de-phosphorylating and thus negatively regulating the inhibitory function of SHPS-1. Disclosures No relevant conflicts of interest to declare.

Description: Acute myeloid leukemia (AML) is a heterogeneous disease with multiple signaling pathways contributing to its pathogenesis. Mutations in receptor tyrosine kinase KIT and FLT3 are found in approximately 40% of AML patients and targeted therapies for inhibiting KIT and FLT3 have failed, thus new targets for therapeutic intervention need to be identified. The phosphatase of regenerating liver (PRL) family of phosphatases, consisting of PRL1, PRL2, and PRL3, represents an intriguing group of proteins being validated as biomarkers and therapeutic targets in human cancer. While PRL2 is highly expressed in some subtypes of human AML, including AML1-ETO+ AML and AML with mixed lineage leukemia (MLL) translocations, its role in AML is largely unknown. To determine the role of PRL2 in the pathogenesis of AML, we utilized two murine models of human AML induced by transducing mouse HSCs with AML1-ETO or MLL-AF9. We found that PRL2 is important for the progression and maintenance of leukemia induced by AML1-ETO or MLL-AF9 through enhancing leukemia stem cell (LSC) self-renewal. To elucidate the mechanisms by which PRL2 promotes LSC maintenance, we performed genome wide RNA-seq analysis of MLL-AF9+ LSCs. Gene Set Enrichment Analysis (GESA) indicates that PRL2 deficiency alters the MLL-AF9 signature essential for LSC self-renewal. We have recently identified PRL2 to be important for the proliferation and self-renewal of hematopoietic stem cells (HSCs) through the regulation of KIT signaling. Notably, PRL2 null hematopoietic progenitor cells showed decreased KIT phosphorylation as well as ERK phosphorylation following SCF stimulation, suggesting that PRL2 is important for KIT activation. Given that KIT inactivation could be mediated by removal from the cell surface and intracellular degradation, we reasoned that PRL2 may regulate KIT receptor internalization and stability. That was indeed the case. We found that the KIT protein half-life in PRL2 null hematopoietic progenitor cells (Kit+) was significantly decreased compared to WT cells. Furthermore, PRL2 null progenitor cells showed enhanced KIT ubiquitination compared to WT cells and less KIT was found on the surface of PRL2 null progenitor cells compared to WT cells following SCF stimulation. We also found that loss of PRL2 in human AML cells resulted in enhanced internalization of KIT. These observations demonstrate that PRL2 deficiency results in less KIT on the cell surface and a lower global KIT level in the cell. Upon SCF stimulation, KIT binds to and induces the phosphorylation of CBL proteins, which in turn act as E3 ligases, mediating the ubiquitination and degradation of KIT. To understand how PRL2 modulates the turnover of KIT in hematopoietic cells, we performed GST-pulldown assays and found that the substrate-trapping mutant PRL2/CS-DA showed an increased association with KIT and CBL compared to wild-type PRL2 in Kasumi-1 cells, suggesting that KIT and CBL may be PRL2 substrates. Furthermore, we found that PRL2/CS-DA mutant showed enhanced association with FLT3 and CBL compared to wild-type PRL2 in MV4-11 cells. Our data suggest that PRL2 dephosphorylates CBL and inhibits CBL activity toward KIT and FLT3, leading to sustained activation of downstream signaling pathways. To determine the functional significance of PRL2 in human AML with KIT and FLT3 mutations, we utilized two well-established murine model of myeloproliferative neoplasms (MPN) induced by KITD814V or FLT3-ITD. We found that loss of Prl2 decreased the ability of oncogenic KITD814V and FLT3-ITD to promote mouse hematopoietic stem and progenitor cell (HSPC) proliferation in vitro andthe development of MPN in vivo. Furthermore, we found that genetic and pharmacological inhibition of PRL2 decreased the proliferation and survival of human AML cells bearing KIT or FLT3 mutations. Taken together, we demonstrate that PRL2 promotes leukemia stem cell (LSC) self-renewal and maintenance through sustaining the activity of oncogenic KIT and FLT3 signals. Our findings suggest that pharmacological inhibition of PRL2 holds potential as a novel therapy for acute myeloid leukemia, and might also be applicable to the treatment of other human cancers. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 827 Internal tandem duplications in the FMS-like receptor tyrosine kinase receptor (FLT3-ITDs) are present in approximately 25% of patients with acute myeloid leukemia (AML) and confer a poor prognosis. FLT3-ITDs induce FLT3 ligand (FL)-independent hyperproliferative cellular growth and promiscuous activation of STAT5; however, the molecular mechanism underlying aberrant activation of these signaling molecules is largely unknown. Upon FLT3 ligand (FL) stimulation, tyrosine 599 (Y599) of WT-FLT3 recruits the protein tyrosine phosphatase, Shp2 and induces cell proliferation and Erk activation. In several FLT3-ITDs, including N51-FLT3 and N73-FLT3, Y599 is duplicated. These findings led us to hypothesize that increased recruitment of Shp2 to N51-FLT3 or N73-FLT3, via Y599, contributes to N51-FLT3- and N73-FLT3-induced cellular hyperproliferation and promiscuous STAT5 activation. Using Baf3 cells stably expressing WT-FLT3, N51-FLT3, or N73-FLT3, co-immunoprecipitation assays demonstrated that Shp2 associates with WT-FLT3 in a FL-dependent manner, while Shp2 is constitutively associated with N51-FLT3 and N73-FLT3 independent of FL-stimulation. To investigate the functional role of Shp2 in FLT3-ITD-induced leukemogenesis, Baf3 cells expressing WT-FLT3, N51-FLT3, or N73-FLT3 were transfected with a mammalian expression vector encoding a U6 polymerase III–directed Shp2-specific short-hairpin RNA (shRNA) or a scrambled shRNA and selected in puromycin. Immunoblot analysis revealed significant reduction of Shp2 expression by the Shp2-specific shRNA and no change in Shp2 expression by the scrambled shRNA in all cell lines. Upon knock-down of Shp2 in Baf3/WT-FLT3 cells, proliferation was minimally reduced based on thymidine incorporation assays; however, knock-down of Shp2 in Baf3/N51-FLT3 and Baf3/N73-FLT3 cells significantly reduced proliferation, both at baseline and in response to FL stimulation. To further test the role of Shp2 in FLT3-ITD-induced transformation, Shp2flox/flox mice were crossed with Mx1Cre transgenic mice to produce Shp2flox/flox Mx1Cre+ mice. Shp2flox/floxMx1Cre+ and Shp2flox/floxMx1Cre- pairs were treated with 3 intraperitoneal injections of polyI/polyC to induce recombination of the Shp2 allele and generate a null allele in the Mx1Cre+ animals. PCR and immunoblot analysis revealed ablation of the flox allele and reduction of Shp2 protein, respectively, in Shp2flox/floxMx1Cre+ bone marrow as soon as 2 weeks post injection. Low density mononuclear cells (LDMNCs) isolated from polyI/polyC-treated Shp2flox/floxMx1Cre- and Shp2flox/floxMx1Cre+ mice were transduced with either pMSCV-WT-FLT3 or pMSCV-N51-FLT3. Transduced cells were sorted for EGFP (enhanced green fluorescent protein) expression and subjected to thymidine incorporation assays. Shp2flox/floxMx1Cre+ cells transduced with N51-FLT3 demonstrated significantly reduced proliferation at baseline and in response to FL stimulation compared to Shp2flox/floxMx1Cre- cells expressing N51-FLT3. To pharmacologically examine the role of Shp2 in FLT3-ITD-induced-hyperproliferation and constitutive activation of Stat5, we utilized a Shp2 inhibitor, II-B08, identified from a library of indole salicylic acid derivatives. II-B08 exhibits an IC50 of 5.5 μM for Shp2 and demonstrates excellent cellular activity. Bone marrow LDMNCs retrovirally transduced with WT-FLT3 or N51-FLT3 were incubated in the absence of or in the presence of increasing concentrations of II-B08 and plated into thymidine incorporation assays. A minimal and non-significant reduction in proliferation was observed in WT-FLT3 expressing cells in the presence of II-B08; however N51-FLT3 expressing cells demonstrated a highly significant reduction in cellular proliferation in a concentration-dependent fashion. In addition to these functional analyses, biochemical studies revealed a reduction in Stat5 activation in N51-FLT3 expressing cells treated with II-B08 compared N51-FLT3 expressing cells treated with DMSO in two independent experiments. Collectively, these data suggest that constitutive recruitment of Shp2 to N51-FLT3 and N73-FLT3 contributes to the FLT3-ITD-induced hyperproliferative phenotype and imply that inhibition of Shp2 function may provide a novel therapeutic approach to FLT3-ITD-bearing leukemias. 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: Internal tandem duplications of the FMS-like receptor tyrosine kinase (FLT3-ITDs), an in-fame insertion of several amino acids within the juxtamembrane domain, are present in 25% of acute myeloid leukemia (AML) patients and confer a poor prognosis. FLT3-ITDs induce FLT3 ligand (FL)-independent hyperactivation of Erk and promiscuous activation of STAT5; however, the molecular mechanisms underlying aberrant activation of these signaling molecules is largely unknown. Tyrosine 599 (Y599) of WT FLT3 recruits the protein tyrosine phosphatase, Shp2, upon stimulation with FL, resulting Erk activation. In several FLT3-ITDs, including N51-FLT3 and N73-FLT3, Y599 is duplicated. These findings led us to hypothesize that increased recruitment of Shp2 to N51-FLT3 or N73- FLT3, via Y599, results in enhanced Shp2 activation and contributes to N51-FLT3- and N73-FLT3-induced cellular hyperproliferation, Erk hyperactivation, and promiscuous STAT5 activation. Using Baf3 cells stably expressing WT FLT3, N51-FLT3, or N73- FLT3, co-immunoprecipitation assays demonstrated that Shp2 is phosphorylated and associates with WT FLT3 in a FL-dependent manner. However, in contrast, Shp2 is constitutively hyperphosphorylated and associated with FLT3-N51 and FLT3-N73 independent of FL stimulation. To investigate the functional role of Shp2 in Flt3-ITD-induced leukemogenesis, Baf3 cells expressing WT FLT3, N51-FLT3, or N73-FLT3 were transfected with a mammalian expression vector encoding a U6 polymerase III– directed Shp2-specific short-hairpin RNA (shRNA) or a scrambled shRNA and selected in puromycin. Western blot analysis revealed significant reduction of Shp2 expression by the Shp2-specific shRNA and no change in Shp2 expression by the scrambled shRNA in all cell lines. Upon knock-down of Shp2 in Baf3/WT-FLT3 cells, proliferation was minimally reduced based on thymidine incorporation assays; however, knock-down of Shp2 in Baf3/N51-FLT3 and Baf3/N73-FLT3 cells significantly reduced proliferation, both at baseline and in response to FL stimulation. Collectively, these data suggest that constitutive recruitment of Shp2 to N51-FLT3 and N73-FLT3 contributes to the FLT3- ITD-induced hyperproliferative phenotype and imply that inhibition of Shp2 function may provide a novel therapeutic approach to FLT3-ITD-bearing leukemias.

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.