ALBERT

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: Acute Myeloid leukemia (AML) has a poor prognosis in part due to lack of elimination of leukemic cells and acquisition of drug resistance. In particular mutations in the activation loop of KIT and internal tandem duplication (ITD) in Flt3 receptor are associated with poor prognosis in AML. Interestingly, more than 90% patients with aggressive systemic mastocytosis (ASM) carry the KITD816V mutation which is highly resistant to conventional therapeutics and invariably associated with poor survival. Since the efforts to target the kinase activity of FLT3 and KIT have met with little success; the focus has shifted to targeting critical downstream signaling regulators. We have elucidated the role of a novel downstream regulator of FLT3-ITD and KITD816V mediated leukemic transformation - LIM kinase and evaluated the efficacy of a novel inhibitor of LIM kinase (LIMK), Liminib. LIMK, a serine/threonine kinase has 2 isoforms; LIMK1 and LIMK2. They are substrates of the Rho associated kinase (ROCK) which we have shown to play a critical role in leukemic transformation (Cancer Cell, 2011). They in turn act on cofilin and phosphorylation by LIMK inactivates cofilin and thereby modulates F-actin reorganization. Western blot analysis showed marked increase in phospho cofilin levels in cells expressing FLT3-ITD or KITD814V oncogenes as compared to their WT counterparts. Dependence of cofilin phosphorylation on activation of ROCK and LIMK was confirmed by treating the cells with an inhibitor of ROCK, H1152 and Liminib, inhibitor of LIMK. Both the inhibitors effectively reduced oncogene-induced phospho-cofilin levels. Liminib, the LIMK inhibitor also inhibited cytokine independent proliferation driven by FLT3-ITD or KITD814V (〉90% at 100 nM). It also showed concentration dependent inhibition of proliferation in mutant oncogene bearing patient derived human cell lines such as MV411, Kasumi and HMC. FLT3-ITD expressing leukemic cells become resistant to Flt3 kinase inhibitor AC220 by acquiring additional point mutations. Interestingly, the cells expressing AC220 resistant FLT3-ITDD835F or FLT3-ITDF691L mutation showed similar sensitivity to Liminib as cells with FLT3-ITD. The growth inhibitory effect of Liminib was also validated in primary blasts from AML and SM patients. To further elucidate the mechanism of growth inhibition by Liminib in leukemic cells, we assayed for apoptosis induction in the treated cells. Cells expressing FLT3-ITD were significantly more susceptible (~ 73%) to induction of apoptosis as compared the WT-FLT3 cells (~33%) on treatment with Liminib. The extent of apoptosis induction by Liminib was similar in FLT3-ITD and AC220 resistant FLT3-ITDD835F or FLT3-ITDF691L mutation bearing cells. In addition to the effects of Liminib on F-actin polymerization; experimental evidence indicates involvement of mitochondrial pathway in the induction of apoptosis by Liminib. There was increase in accumulation of cofilin in the mitochondrial fraction in Liminib treated oncogene expressing cells as compared to the untreated cells. Phosphorylation of cofilin on Ser3 suppresses its mitochondrial translocation and treatment with LIMK inhibitor would inhibit cofilin phosphorylation leading to increased mitochondrial transport. Mitochondrial transport of cofilin has been observed with other inducers of apoptosis such as staurosporine and it possibly contributes to mitochondrial dysfunction during induction of apoptosis. To validate our pharmacologic findings using Liminib genetically and to assess which of the 2 isoforms of LIMK contributes to leukemogenesis, we generated LIMK1 and LIMK2 single and double knockout mice. 3 H-thymidine incorporation assay showed that deletion of either of the LIMK significantly reduced KIT D814V induced growth factor independent proliferation by approximately 50%. The extent of inhibition was more pronounced in Limk1-/- Limk2+/- or Limk1+/- Limk2-/- bone marrow cells (~80%) as compared to cells with deletion of a single isoforms of LIMK. Thus, there is some functional redundancy between the 2 isoforms and inhibition of both is likely to have better therapeutic value. Taken together we have identified a novel signaling pathway involving LIMK and cofilin that appear to be selectively activated in regulating oncogenic signaling in AML and MPN but not in normal hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Description: Heterozygous mutations in FLT3ITD, TET2 and DNMT3A are associated with human neoplasms. Whole exome sequencing of patients demonstrate co-occurrence of several of these mutations in myeloid malignancies; however it is unclear if and how these mutations cooperate and how does this cooperation manifest in disease. We assessed the consequence of co-existence of TET2, DNMT3A and FLT3ITD/WT mutations on AML development and overall survival. Mice were bred to obtain eight distinct genotypes WT, Tet2+/-, Flt3ITD/WT, Dnmt3aFlox/- MxCre, double het Tet2+/-; Dnmt3aFlox/- MxCre (TD), double het Tet2+/-; Flt3ITD/WT (TF), double het Flt3ITD/WT: Dnmt3aFlox/- MxCre (FD) and triple het Tet2+/-; Flt3ITD/WT; Dnmt3aFlox/+ MxCre (TFD) mice. These mice were treated with poly:IC and monitored for survival. Of the 8 genotypes examined, only TFD mice succumbed by 150 days. Peripheral blood (PB) counts, BM cellularity and splenomegaly were significantly higher in TFD mice compared to FD and TF mice. The absolute number of LSK cells in the BM was highest in TFD mice, although TF and FD mice also showed an increase relative to controls. A similar increase in the frequency of GMPs was noted in both double and triple heterozygous mice relative to controls as well as single heterozygous groups. An increase in Gr-1/Mac-1 cells was observed in TF and FD mice as well as in TFD mice relative to controls. To assess if the observed AML phenotype in TFD mice is transplantable, we transplanted BM cells from the 8 genotypes into lethally irradiated hosts. Recipients with TFD BM succumbed to rapid AML development and died within 45 days of transplantation. In contrast, none of the other recipients died during the entire monitoring period. A significant increase in PB neutrophil and monocyte counts with a significant reduction in red blood cells, and platelets counts was noted in TFD mice compared to other groups. Furthermore, a significant increase in BM cellularity, Lin- Kit+ progenitors, frequency and absolute number of LSK cells was also noted in mice with TF and FD BM but most significantly in recipients that received TFD BM. Given that the disease manifestation was qualitatively similar between FD, TF and TFD mice, although the severity greatest in TFD mice, we asked if these differences were due to quantitative or qualitative differences in gene expression between the various groups. RNA-seq analysis revealed distinct differences between WT and FD, TF and TFD BM cells. We observed 2328, 2168 and 1787 up-regulated genes and 1861, 1770 and 1430 downregulated genes in FD, TF and TFD vs. WT, respectively. The variations among FD, TF and TFD groups was smaller compared to the differences between WT and FD, TF and TFD groups, in which TF and TFD were more similar to each other compared to DF. Most of the cytokines regulating the differentiation of myeloid cells were downregulated in FD, TF and TFD, suggesting that all the mutant groups lost their differentiation ability. Of note, cytokines specific to stem cells and those involved in the differentiation of GMPs were all upregulated. Given the RNA-seq profile, we assessed the impact of using a combination of drugs that target Flt3ITD, inflammation as well as methylation status of cells in TFD mice. A cohort of mice was treated with a combination of AC220 (10 mg/kg, orally; Flt3 inhibitor), E3330 (20 mg/kg, i.p.; APE1 inhibitor; regulates the redox function of multiple transcription factors including NFkB and Stat3) and 2-dexoy-aza-cytidine (0.5 mg/kg, i.p). A significant correction in all PB, spleen and BM parameters was observed including in the frequency of Lin- Kit+ and LSK cells, HSCs, HPC1 and peripheral Gr1/Mac1 double positive cells in TFD mice. Collectively, these results demonstrate that a combination of three drugs targeting three different aspects of disease manifestation in TFD mice can significantly impact the leukemia progression both at the level of very primitive stem and progenitor cells as well as more mature myeloid and lymphoid cells. Disclosures No relevant conflicts of interest to declare.

Description: Histone deacetylase inhibitors (HDACi) are being evaluated in several clinical trials for the treatment of hematological malignancies and 2 of them have been approved for the treatment of T cell lymphoma. We have investigated the effectiveness of using 2 HDACi - Vorinostat (SAHA) and Panobionstat for treatment of systemic mastocytosis (SM) for which no treatments are available. SAHA and Panobinostat showed concentration dependent inhibition of proliferation in mastocytosis patient derived cell lines, HMC1.1 and HMC1.2 bearing either single mutation in the juxtamembrane domain KITG560V or double mutation also including the activation loop KITG560V,D816V, respectively. Interestingly HMC1.2 cells with 2 mutations in KITG560V, D816V were more sensitive to treatment with both SAHA (IC50 ~ 260 nM) and Panobinostat (IC50 ~11 nM) as compared to HMC1.1 cells (IC50 ~ 600 nM for SAHA and IC50 ~ 23 nM with Panobionstat) that have a single mutation in KIT. The inhibition of proliferation by HDACi treatment appeared to be associated with modulation of cell cycle and differentiation rather than induction of apoptosis. We further tested the effect of combining HDACi treatment with tyrosine kinase inhibitor, Dasatinib. Combination of Dasatinib (5 - 20 nM) with either SAHA or Panbionostat was more effective than any of the single drugs in HMC1.1 cells. However such a combination effect was not seen in HMC1.2 cells. Over 90% patients with aggressive systemic mastocytosis carry KITD816V mutation. This particular mutation of KIT is highly resistant to therapeutics and is associated with poor survival. Thus patients carrying the KITD816V mutation are likely to benefit from treatment with HDACi while juxtamembrane mutation bearing patients would benefit more from a combination of HDACi with kinase inhibitors such as Dasatinib. To further understand the mechanism behind HDACi inhibition of KITD816V mediated oncogenic signals, we expressed KITD814V (murine equivalent of human D816V) in myeloid cells and treated them with SAHA and Panobinostat. Cells expressing KITD814V showed cytokine independent proliferation and interestingly, cytokine independent proliferation was more sensitive to inhibition by both SAHA and Panbinostat as compared to the cytokine dependent proliferation. In contrast to HMC cells, inhibition of cytokine independent proliferation by HDACi in KITD814V oncogene expressing cells was associated with significant increase in apoptosis. The percent cells negative for Annexin V stain decreased from 74% in control to ~46% in 24h upon treatment with 500 nM SAHA or 10 nM Panbinostat (p〈 0.001; n=3). Induction of apoptosis by HDACi in KITD814V oncogene expressing cells was significantly suppressed in presence of cytokine. Consistent with the in vitro data, treatment with SAHA prolonged the survival of mice transplanted with myeloid cells expressing KITD814V mutation as compared to the vehicle treated group (Median survival - 21d (veh), 34d (SAHA), p=0.0015, n=5). In addition to increased histone acetylation, treatment of cells with HDACi also leads to increase in acetylation of chaperone proteins such as HSP90 which can mediate proteasomal degradation of oncogenes and has been suggested as one of the mechanisms by which HDACi mediates growth inhibition. In contrast, we found no significant decrease in mutant KIT protein expression or AKT expression and phosphorylation upon treatment with either of the HDACi. Though expression of Pim1 a pro survival serine/threonine kinase was down-regulated by SAHA and Panobinostat. Treatment with HDACi was also associated with differential modulation of STAT3, STAT5 and ERK pathways. Treatment with both SAHA and Panobinostat down-regulated STAT5 phosphorylation and up-regulated ERK1/2 phosphorylation with more increase in ERK1/2 phosphorylation observed in cells with higher sensitivity to inhibition by HDACi. Though increased activation of the ERK pathway has been associated with leukemogenesis; there is some evidence to indicate that hyper activation of ERK pathway in absence of additional leukemogenic signals activates a pro senescence program. Differential expression of Pim1 and along with differential modulation of STAT and ERK pathways may contribute to differential sensitivity of the cells to HDACi and are likely to be of therapeutic value in aggressive systemic mastocytosis that has so far been associated with poor prognosis. 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: Juvenile myelomonocytic leukemia (JMML) is a fatal leukemia affecting children under the age of 4 years and is characterized by myelomonocytic cell overproduction and hypersensitivity to GM-CSF. The only curative therapy is allogeneic stem cell transplantation; however, half of children relapse after this aggressive therapy. Approximately 85% of JMML patients bear loss-of-function (LOF) mutations in NF1 or CBL or gain-of-function (GOF) mutations in KRAS, NRAS, or PTPN11. Typically, these mutations are non-overlapping, with the net effect being Ras hyperactivation. Children bearing somatic GOF mutations within PTPN11, which encodes the protein tyrosine phosphatase, Shp2, exhibit the poorest prognosis. GOF Shp2 (Shp2D61Y and Shp2E76K) induces hyperactivation of both the Ras-MEK- Erk and PI3K-Akt pathways. While the Ras-MEK-Erk pathway clearly contributes to the pathogenesis of JMML, we hypothesize that the PI3K-Akt pathway cooperates with the Ras-MEK-Erk pathway to promote JMML. Recently published work indicates that genetic disruption of the PI3K regulatory subunit, p85a, reduces GOF Shp2-induced hypersensitivity to GM-CSF. However, as PI3K regulatory subunits cannot be easily inhibited pharmacologically, we examined the contribution of class IA PI3K catalytic subunits in GOF Shp2-induced JMML. Shp2 D61Y/+ ;Mx1Cre+ mice were crossed with mice bearing conditional knockout of p110a (Pik3caflox/flox) or bearing a kinase dead mutant of p110d (Pik3cdD910A/D910A). Shp2D61Y/+;Mx1Cre-, Shp2D61Y/+;Mx1Cre+, Shp2D61Y/+;Mx1Cre+; Pik3caflox/flox, and Shp2D61Y/+;Mx1Cre+; Pik3cdD910A/D910A mice were treated with polyI;polyC, and 8 weeks post-treatment, animals were euthanized followed by evaluation of spleen size, hypersensitivity of bone marrow low density mononuclear cells (LDMNCs) to GM-CSF, frequency of bone marrow phenotypically-defined common myeloid, granulocyte-monocyte, and megakaryocyte-erythroid progenitors (CMPs, GMPs, and MEPs), and GM-CSF-stimulated Erk and Akt activation. Genetic disruption of p110a failed to normalize GOF Shp2-induced splenomegaly, GM-CSF hypersensitivity in proliferation assays and methylcellulose-based progenitor assays, or hyperphosphorylation of Erk or Akt. In contrast, genetic ablation of p110d kinase activity significantly reduced spleen size, normalized progenitor hypersensitivity to GM-CSF, and reduced both Akt and Erk hyperactivation. Additionally, genetic inhibition of p110d normalized the skewed hematopoietic progenitor distribution reported in the Shp2D61Y/+;Mx1Cre+ mice, while genetic disruption of p110a failed to do so. This unique function of p110d in the context of GOF Shp2-expressing mice is significant, as p110d expression is restricted to hematopoietic cells and p110d bears transforming properties independent of Ras. While previously published work indicates that the PI3K p110a and p110d inhibitor, GDC-0941, inhibits proliferation of GOF Shp2-expressing cells, we tested if the potent p110d-specific inhibitor, GS-9820, is similarly effective. GOF Shp2-expressing bone marrow LDMNCs treated with GS-9820 demonstrated significantly reduced proliferation in a dose-dependent fashion, while GS-9820 failed to inhibit the proliferation of WT Shp2-expressing cells. GS-9820 treatment decreased Akt phosphorylation (S473 and T308) as well as reduced Erk phosphorylation, indicating that p110d inhibition also reduces signaling within the Ras-MEK-Erk pathway. While PI3K activates the canonical Akt-mTORC1 pathway, it also positively feeds back to the Ras-MEK-Erk pathway via activation of Rac-Pak-MEK; therefore, we evaluated if p110d inhibition adds to or is redundant with MEK inhibition. Treatment of GOF Shp2-expressing hematopoietic cells with the MEK inhibitor, PD0325901, effectively reduced proliferation, and addition of GS-9820 further significantly reduced proliferation, indicating that p110d works cooperatively with MEK to promote GOF Shp2-induced disease. Collectively, our findings suggest that PI3K catalytic subunit p110d functions in a Ras-MEK-Erk pathway-independent manner to promote GOF Shp2-induced hypersensitivity to GM-CSF, and suggest that PI3K p110d inhibition in combination with MEK inhibition may be a novel, optimal approach for the treatment of JMML. Disclosures: No relevant conflicts of interest to declare.