ALBERT

Description: Juvenile Myelomonocytic Leukemia is the most common pediatric myeloproliferative neoplasm (MPN). JMML is characterized by myeloid populations with mutually-exclusive mutations in Ras-Erk signaling genes, most commonly PTPN11, which confer growth hypersensitivity to GM-CSF. JMML is notable among pediatric MPNs as being refractory to chemotherapy and having a 50% relapse rate following allogeneic hematopoietic stem cell (HSC) transplantation. As such, there is an urgent need for novel JMML therapies. The recent discovery of yolk sac myeloid lineages that persist into adulthood independently of bone marrow HSC contributions suggests a mechanism for JMML relapse following HSC transplantation. In this study, we sought to determine whether yolk sac HSC-independent myeloid progenitors bear hallmarks of MPN in a mouse model of JMML. Using the Vav1 promoter-directed Cre recombinase, we generated a mouse model of JMML that expresses the PTPN11D61Y gain of function mutation in all waves of embryonic and adult hematopoiesis, including yolk sac myeloid progenitors that emerge prior to and independently from HSCs. PTPN11D61Y/+; VavCre+ mice are viable, born at expected Mendelian ratios, and develop peripheral blood monocytosis as early as 4 weeks of age. Given this early onset, we hypothesized MPN may develop in these mice during embryonic development. E14.5 fetal liver progenitors from PTPN11D61Y/+; VavCre+ embryos displayed marked GM-CSF hypersensitivity in methylcellulose colony forming assays (Figure-1A), possessed hyperactive Ras-Erk pathway signaling (Figure-2), and had a skewed progenitor distribution with a greater proportion of megakaryocyte-erythroid progenitors (63.5% vs. 50.1%, p

Description: Key Points Compared with ubiquitously expressed PI3K p110α, genetic inhibition of PI3K p110δ uniquely normalizes mutant Shp2-induced GM-CSF hypersensitivity. Potent pharmacologic inhibitors of PI3K p110δ cooperate with MEK inhibition to reduce mutant Shp2-induced hyperproliferation.

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 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: Shp2 is a non-receptor protein-tyrosine phosphatase encoded by PTPN11 and implicated in the Ras, JAK-STAT and PI3K pathways. Activating mutations in Shp2 are found in patients with developmental disorders such as Noonan and LEOPARD syndrome, as well as, hematologic malignancies. Although rare in most other solid tumors, Shp2 mutations are common in juvenile myelomonocytic leukemia (JMML) accounting for ~35% of cases. To understand its role as a cooperating mutation in AML we sequenced PTPN11 in human samples. Here we report that Shp2 mutations are present in human AML at a rate of 6.6% (6/91) in the ECOG E1900 dataset. To investigate the biological function of Shp2 mutations we asked how this functions in a cooperative model of leukemogenesis with the MLL-AF9 fusion protein. Despite showing increased genetic stability compared to other leukemias, MLL leukemias commonly contain type I mutations that can functionally cooperate resulting in more aggressive leukemias. These mutations often occur in genes encoding components of the Ras pathway including mutually exclusive mutations of NRAS, KRAS, PTPN11 and NF1 and account for ~37% of MLL rearranged leukemias. However, the mechanisms of cooperation with MLL fusion proteins are poorly understood. We found that the Shp2E76K activating mutation commonly found in humans significantly accelerates MLL-AF9 mediated leukemogenesis. The E76K mutation results in structural changes that confer increased phosphatase activity to the Shp2 protein and increased Ras signaling. We attribute the MLL-AF9/Shp2E76K cooperation to a more rapid leukemic initiation as evidenced in colony formation assays using mouse bone marrow HSPCs. Cells transduced with MLL-AF9/Shp2E76K expanded faster than MLL-AF9 cells at early stages following transduction, indicating more efficient transformation of myeloid progenitors than MLL-AF9 alone. Cytokine independent growth is achieved in MLL-AF9 cells following expression of Shp2E76K through the constitutive activation of the IL3 signaling pathway and ERK phosphorylation. Importantly, addition of Shp2E76K significantly accelerated MLL-AF9 mediated acute myeloid leukemia in mice, indicating activated Shp2 cooperates with MLL-AF9 in vivo. In addition, leukemic stem cell frequency was increased by greater than 4 fold due to Shp2E76K expression. As Shp2 is reported to regulate an anti-apoptotic gene program, we investigated these in the context of MLL-AF9 leukemic cells with and without Shp2E76K. While Bcl2, BclXL and Mcl-1, were upregulated in Shp2E76K cells, Mcl-1 showed the highest upregulation in response to Shp2E76K. Further, expression of Mcl-1 with MLL-AF9 in colony assays phenocopies expression of Shp2E76K suggesting that, mechanistically, Shp2 mutations may cooperate through activation of an anti-apoptotic gene program, primarily through Mcl-1. Finally, we asked how leukemic cells bearing Shp2E76K would respond to small molecule inhibition of Mcl-1. MLL-AF9 leukemic cells expressing Shp2E76K were desensitized to small molecule mediated Mcl-1 inhibition consistent with increased Mcl-1 protein. These data were confirmed in human cells where U937 cells, which contain an activating Shp2 mutation, exhibited resistance to Mcl-1 inhibition compared to ML2 or K562 cell which both bear wild type Shp2. Together, these data suggest patients with hyperactive Shp2 signaling may respond poorly to drugs targeting Mcl-1 due to an overabundance of the protein. 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