Reuben Kapur - US grants

protein isoforms; alleles; hematopoietic stem cells; hematopoiesis; hematopoietic growth factor; mitogen activated protein kinase; complementary DNA; genetic mapping; cell line; laboratory mouse; genetically modified animals; nucleic acid sequence;

DESCRIPTION (provided by applicant): Cellular interactions between hematopoietic stem and progenitor cells (SC/Ps) and the hematopoietic microenvironment (HM) play a critical role in regulating hematopoiesis. At least three classes of molecules contribute to the maintenance of hematopoiesis. Cytokines and chemokines stimulate the survival, growth, and migration of SC/Ps. Adhesion molecules (AMs) support the physical association of SC/Ps within the hematopoietic niches and regulate growth, homing and mobilization of SC/Ps. Nevertheless, little is known about the signaling mechanisms that control SC/P functions downstream from these molecules. Identification of key intracellular signaling molecules and pathways involved in regulating SC/P functions will facilitate the design of specific molecular therapies for the treatment of malignancies involving defects in SC/P function. Our long-range goal is to understand signaling mechanisms that control growth and actin-based functions in SC/Ps. Understanding these mechanisms is critical to the biology of SC/P amplification, homing and mobilization for translational applications of transplantation and gene therapy of blood disorders. The objective of this application is to determine how Src family of signaling molecules regulate growth and actin-based functions in c-Kit+Scal+ Lin- cells. The central hypothesis of the application is that hematopoietic specific Src family kinase (SFK) possess both unique as well as overlapping mechanisms to regulate growth and actin-based functions in c- Kit+Sca-l+Lin- cells. Among the known SFKs, three highly homologous SFKs Lyn, Hck, and Fgr are expressed only in SC/Ps. Amino acid sequence differences in these molecules are restricted predominantly to the amino terminus region. Despite their sequence similarities, we will present preliminary data to support our hypothesis that SFKs differentially (positively and negatively) regulate the magnitude and duration of cytokine-mediated growth in SC/Ps in part via regulation of phosphatase activation. The SC/P phenotypic changes we have identified associated with Lyn-deficiency are apparent in spite of normal Hck and Fgr expression. How these hematopoietic SFKs differentially (positively and negatively) modulate SC/P growth and survival given their limited amino acid differences will constitute a focus of this proposal. Our preliminary observations also lead us to hypothesize that SFKs regulate actin-based functions in SC/Ps, including defective chemotaxis in vitro and redistribution in vivo of c-Kit+Lin-Sca-l+ cells. Our proposed studies will provide unique insights into the physiologic significance of Lyn, Hck, and Fgr SFKs in the regulation of SC/P growth and actin-based functions.

DESCRIPTION (provided by applicant): Myeloproliferative neoplasms or MPNs are a heterogeneous group of complex hematologic diseases, which share the common characteristic of myeloid cell overproduction. Mastocytosis, especially the systemic form of the disease, also known as systemic mastocytosis (SM) is considered a particularly difficult form of MPN to treat. Activating mutations of KIT are found in over 90% patients with SM, characterized by clonal expansion and accumulation of myelomastocytic progenitors within various tissues leading to organ failure and poor overall survival. With the exception of chronic myelogenous leukemia (CML), there are no effective therapies for MPNs. In the case of CML, targeting the tyrosine kinase BCR-ABL with imatinib (gleevec) or second generation tyrosine kinase (TK) inhibitors such as nilotinib and desatinib appears to be sufficient for treating most patients; however, a significant number of these patients go on to develop drug resistance. In contrast, in other types of MPNs, including SM, targeting the activated version of the receptor tyrosine kinase receptor KIT alone has been ineffective; particularly in patients that harbor the activating mutation of KIT in the catalytic domain, KITD816V, which are completely resistant to imatinib or second generation tyrosine kinase inhibitors. Recent studies in patients with mastocytosis have shown the presence of Tet2 mutations in ~30% patients. In these patients, mutations in Tet2 are associated with higher leukocyte counts, monocyte counts, serum tryptase levels, mast cell burden, splenomegaly and the presence of activating KIT mutation, KITD816V. Thus, Tet2 mutations are frequent in SM; they segregate with KITD816V and significantly influence the phenotype including overall survival and may help explain why anti-KITD816V therapy alone may not be effective for treating these patients. Our long range goal is to elucidate the aberrant signaling mechanism(s) induced by activating KIT mutations and epigenetic regulators such as Tet2 that promote pathologic over production of myeloid/mast cells in SM, with the intent of defining novel therapeutic targets for this disease. The objective of this application is to define the role of Tet2 and activating mutation of KIT and downstream pathways in the initiation and progression of mast cell growth, development and transformation utilizing state of the art mouse genetic models of SM and primary patient samples. Our proposed studies will provide unique insights into the physiologic significance of the in vivo interactions between Tet2 and the oncogenic KIT in regulating normal as well as abnormal myeloid/mast cell biology.

ABSTRACT KIT is a unique receptor with important functional roles in melanocytes, germ cells, interstitial cells of Cajal, mast cells, and hematopoietic stem cells. Consistent with the importance of KIT in these defined tissues, activating mutations of KIT have been described in germ cell tumors, gastrointestinal stromal tumors (GISTs), sinonasal lymphomas, acute myeloid leukemia (AML), and systemic mastocytosis (SM). A significant proportion of these diseases commonly bear the KIT activation loop mutation KITD816V. Activation loop mutations of KIT have also been observed in core binding factor-acute myeloid leukemia (CBF-AML), leukemias that bear either the t(8;21) or inv(16) cytogenetic abnormality, generating the fusion genes AML1- ETO or CBF¿-MYH11, respectively. Studies examining both adult and pediatric AML have indicated that the presence of the KITD816V mutation in CBF-AML carrying t(8;21) worsens the prognosis based on several clinical indices. Although KIT mutations within the juxtamembrane region that are commonly found in GISTs are sensitive to inhibition by the tyrosine kinase inhibitor, imatinib mesylate (Gleevec); KIT mutations within the carboxy-terminal lobe of the tyrosine kinase domain, such as KITD816V, stabilizes the KIT activation loop conformation in its active form, which precludes sufficient imatinib binding for tyrosine kinase inhibition. Therefore, in contrast to successful use of Gleevec to treat GISTs, Gleevec is ineffective for the treatment of the hematologic diseases harboring the KIT activation loop mutants (i.e. KITD816V), including SM and CBF- AML. Importantly, nature of the receptor proximal and/or downstream signals by which activation loop mutations in KIT (KITD816V) induce transformation in primary hematopoietic cells are poorly defined. We have evidence to demonstrate that KITD816V (KITD814V in mice) induced transformation in primary hematopoietic stem and progenitor cells results in constitutive activation of GEF Vav/Rho GTPase Rac pathway and that genetic disruption of hematopoietic specific Vav1 and/or Rac2 in mice abrogates ligand independent growth via KITD814V, leading us to hypothesize that signals involved in KITD814V induced transformation may in part be mediated via the hyperactivation of this pathway. Furthermore, we have evidence demonstrating that mutating the tyrosine residues within the intracellular domain of KITD814V results in complete loss of KITD814V induced ligand independent growth, leading us to hypothesize that the intracellular tyrosines within the juxtamembrane and the kinase insert region of KITD814V are likely to contribute to KITD814V induced transformation. Based on these findings, the central hypothesis of this application is that hyperactivation of the Vav/Rac pathway contributes to the etiology of diseases associated with systemic mastocytosis, AML as well as other diseases involving the KITD814V mutation. Our proposed studies will provide mechanistic insight into the physiologic significance of the Vav/Rac pathway as well as the involvement of the juxtamembrane and the kinase insert sequences in regulating KITD814V induced transformation for which currently no drugs exist.

DESCRIPTION (provided by applicant): Acute myeloid leukemia (AML) is a lethal disease which dramatically increases in incidence in individuals >65 years of age, the fastest growing population in the United States. Regrettably, the 5 year survival rate drops dramatically in patients over 65 years (4.3%) compared to patients less than 65 years (34.45%). These dismal statistics have led scientists to investigate the molecular mechanism(s) underlying the transforming process leading to AML in an effort to develop novel, molecularly-targeted, effective, and less toxic therapies for use in this lethal disease. Internal tandem duplications (ITD), an in-frame mutation leading to insertion or duplication of several amino acids near the juxtamembrane domain, in fms-like tyrosine kinase receptor (FLT3), are seen in nearly 25% of all AML patients and confer a poor prognosis. Expression of the Shp2 protein tyrosine phosphatase is consistently elevated in primary leukemia cell specimens from multiple adult acute leukemias compared to Shp2 levels in bone marrow mononuclear cells from healthy controls. However, how Shp2 contributes to myeloid leukemogenesis is unknown. We present preliminary studies demonstrating that Shp2 is constitutively associated with FLT3 in mutant N51-FLT3- and N73-FLT3-bearing cells and that genetic disruption of Shp2 and pharmacologic inhibition of Shp2 preferentially reduces N51-FLT3-induced hyperproliferation compared to that observed in WT FLT3-bearing cells. As a corollary, since Shp2 has been shown to regulate the activation of the Rac subfamily of Rho-GTPases in hematopoietic cells, we predicted that Rac1 and/or Rac2 may also be relevant effectors of FLT3-ITDs. Consistently, we present preliminary findings demonstrating that pharmacologic Rac inhibition using NSC23766 or genetic disruption of Rac2 results in significantly reduced N51-FLT3-induced hyperproliferation. Based on our preliminary data, the central role of STAT5 hyperactivation in FLT3-ITD-induced leukemia, and the reported positive roles of Shp2 and Rac1/Rac2 promoting activated STAT5 nuclear accumulation, the central hypothesis of this application is that, mechanistically, Shp2 and Rac1/Rac2 contribute to FLT3-ITD-induced leukemia by facilitating STAT5 nuclear localization and the expression of STAT5-responsive pro-leukemogenic genes. The objectives of this application are to define the consequences of genetic disruption of Shp2 and Rac1/Rac2 on the activation and nuclear localization of STAT5 and on the development of FLT3-ITD-induced myeloproliferative disorder (MPD) in vivo, to examine the efficacy of a Shp2 inhibitor and a Rac1 inhibitor in an AML xenograft model in vivo, and to define the intracellular tyrosines within the juxtamembrane and the duplicated juxtamembrane of FLT3-ITD that contribute to ligand-independent proliferation.

DESCRIPTION (provided by applicant): Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by poor prognosis. While incremental improvements in chemotherapy regimens have been described for AML treatment in recent years; majority of AML patients relapse and a significant number of them die of this disease. Although the precise mechanism(s) leading to relapse are poorly understood, lack of elimination of leukemia stem cells (LSCs) and the acquisition of drug resistance mutations such as those found in patients with FLT3 receptor may be some of the significant contributors. Internal tandem duplications (ITD) in FLT3 are observed in nearly 30% of all AML patients and confer poor prognosis. Likewise, KIT activation loop mutations (e.g. KITD816V), that are resistant to Gleevec, are exclusively associated with ~ 50% of core binding factor (CBF)-AMLs and 95% of systemic mastocytosis (SM) patients (a form of myeloproliferative neoplasm (MPN)) and confer poor overall survival. While several clinical trials have been conducted utilizing FLT3 and KIT inhibitors for above indications, none have shown significant long-term clinical efficacy. Thus, fo the elderly, who demonstrate the highest incidence of and mortality from these diseases, continued investigation for novel molecularly-targeted and less toxic therapies are desperately needed. Recently, four independent clinical studies comprising of 157 AML patients demonstrated overexpression of focal adhesion kinase (FAK) in up to 50% of patient derived BM cells but not in normal cells. In a significant number of these patients, FAK was hyper-phosphorylated on Y397, a critical residue for its activation. FAK+ AML cells displayed significantly higher migration and resistance to daunorubicin compared with FAK- cells and FAK expression significantly correlated with high blast cell counts, early death and shorter survival rate. In addition to FAK, a recent study utilizing 112 AML patients also showed that expression of phosphorylated (p) pStat5 in newly diagnosed AML patients is associated with poor overall survival. Remarkably, constitutive activation of pStat5 was seen in 100% of mastocytosis patients bearing the KITD816V mutation. Importantly, a strong correlation between the presence of pStat5 and FLT3ITD mutations was observed in AML patients. These clinical findings suggest that FLT3ITD/KITD814V, FAK and Stat5 are likely to contribute to the development of AML and MPNs; however, the relationship between these signaling molecules in the development or progression of AML or MPNs is poorly understood. Importantly, although Stat5 has been implicated in several hematologic malignancies involving LSCs; how precisely activation of Stat5 is regulated in the cytoplasm or in the nucleus and what are the signaling molecules involved in its nuclear import in the context of AML or MPN remains an enigma. We will define the role of FAK and its downstream effectors including PAK1 and Stat5 in regulating FLT3ITD/KITD814V induced transformation. We hypothesize that hyper-activation of FAK via FLT3ITD/KITD814V and downstream phosphorylation and nuclear translocation of Stat5 in a PAK1 dependent manner contributes to FLT3ITD/KITD814V driven AML.

PROJECT SUMMARY/ABSTRACT Diabetes mellitus (DM) is a strong risk factor for cancer development. However, it is unclear if DM is also a risk factor for transforming pre-leukemic stem cells (pre-LSCs) into full-blown leukemia such as acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML) or severe form of myeloproliferative neoplasm (MPN). To this end, extensive whole exome sequencing on peripheral blood (PB) cells of more than 20 thousand persons who were unselected for hematologic phenotypes were examined for 160 genes that are recurrently mutated in leukemia. Two most mutated genes found were epigenetic regulators: Ten eleven translocation methylcytosine dioxygenase 2 (TET2) and DNA cytosine-methyltransferase 3A (DNMT3A). Notably, both these genes are mutated at very high frequency in patients with MPNs and AML. Importantly, epidemiological findings demonstrate that persons with DM are more likely to have these mutations than those without DM. However, there is a significant gap in knowledge regarding our understanding of DMs causative role in MPN and leukemogenesis. Studies using murine models of loss of Tet2 function (Tet2-/-) show that Tet2-deficient hematopoietic stem cells (HSCs) have abnormal global 5-hydroxymethylcytosine (5-hmC) levels and local 5- hmC levels in genes responsible for self-renewal of HSCs. In addition, Tet2-/- HSCs show a competitive advantage (i.e. clonal hematopoiesis) over normal HSCs but do not progress to AML development. Moreover, mice with haplo-insufficiency of Tet2 (Tet2+/-) manifest milder form of these phenotypes only when aged, indicating that Tet2 functions as a putative tumor suppressor. We discovered that Tet2-deficient mice manifest a sustained response to acute inflammation and aged naïve Tet2-/- mice show elevated expression of a series of genes encoding the innate immune/pro-inflammatory pathway components including S100A8/9, TLR4, NF?B1 and IL-6. As patients with DM develop chronic inflammation and have increased expression of S100A8/A9, the central hypothesis of this proposal is that diabetic individuals that carry a single genetic lesion in the form of TET2 in their HSCs (in absence of any hematologic malignancies), will be more susceptible to developing severe MPN and/or myeloid leukemia, in part to an overactive pro-inflammatory cytokine signaling cascade or a forward feeding positive signaling loop and in part to hyperglycemia (HG) induced further global reduction in 5-hmC levels in pre-LSCs bearing loss of Tet2, thus mimicking pre-LSCs lacking all forms of TET. Indeed, by introducing Tet2 mutation into a murine model of diabetes (Ins2Akita/+), our preliminary data demonstrate that the diabetic mice haploinsufficient for Tet2 (Ins2Akita/+;Tet2+/-) progressively develop lethal AML/severe MPN. Importantly, treatment of Tet2-/- mice exposed to an acute inflammatory challenge with an anti-inflammatory molecule, APX3330, reverses this phenotype. Based on these preliminary results, we hypothesize that progressive DM acts as a significant risk factor for transforming pre-LSCs and/or clonal hematopoiesis into heme malignancies in an age-dependent manner.

PROJECT SUMMARY/ABSTRACT Although majority of patients with acute myeloid leukemia (AML) do respond transiently to frontline chemotherapy, relapse occurs frequently and is the most common cause of death in older AML patients who have an overall cure rate of only 15% and make up ~90% of the AML patient population. Recent whole genome and exome sequencing studies suggest that accumulation of stepwise genetic and epigenetic changes in hematopoietic stem cells (HSCs) results in the formation of pre-leukemia stem cells (pre-LSC) that play a crucial role not only in disease origination but also in leukemia relapse. While the presence of pre-LSCs has been fairly well documented in both humans and mouse models of AML, mechanisms responsible for the growth/survival of pre-LSCs and signals leading to the progression of these pre-LSCs into full-blown LSCs and AML blasts are poorly understood. Mutations in epigenetic-modifying genes, such as TET2 and DNMT3A, are frequently found in pre-LSCs and when paired with genetic mutations such as FLT3-ITD, result in full-blown AML. Based on studies performed in animal models, Tet2 or Dnmt3A mutations alone do not result in AML, and thus single mutations in these genes recapitulate a pre-LSC state. However, combinations of these mutations with FLT3- ITD lead to full-blown AML and portend a poor prognosis in humans. These AML murine models are characterized by definable, functionally altered pre-LSCs and LSCs, closely resembling human disease with regard to key molecular, cellular and phenotypic features, and allow for prospective identification and functional study of mechanisms driving the formation of pre-LSCs and progression to LSCs in AML. Given that FLT3-ITD often occurs in the presence of other cooperating epigenetic mutations such as TET2 and DNMT3A, in this competitive renewal, we have focused on how Shp2 integrates signals from these distinct genes (an epigenetic regulator vs. a receptor tyrosine kinase) to regulate the growth and survival of both pre-LSCs and LSCs. To this end, we have novel preliminary data to suggest that Shp2 regulates loss of Tet2 mediated clonal hematopoiesis in pre-LSCs by forming a feed-forward loop involving the production of inflammatory cytokines including IL-6 as well as by inducing the expression of a novel lncRNA, Morrbid. MORRBID is significantly upregulated in AML patient derived cells including in AML patients with FLT3-ITD as well as bearing TET2 mutations, where it is associated with poor overall survival. We show that loss of Morrbid in pre-LSCs lacking Tet2 or in LSCs lacking Tet2 and expressing FLT3-ITD, renders these cells susceptible to apoptosis, in part by upregulation of a pro- apoptotic protein Bim. We further demonstrate, using a novel allosteric SHP2 inhibitor, currently in clinical trials, to potently inhibit the growth of mouse and human leukemic AML cells. Importantly, SHP2 inhibitor, shows no toxicity against normal cells but uniquely impacts the growth of leukemic cells alone and in combination with 5- Azacytidine. We hypothesize that targeting SHP2, targets both Tet2 loss mediated signals, as well as FLT3-ITD induced signals, that converge on a novel lncRNA, Morrbid in pre-LSCs and LSCs, respectively.

PROJECT SUMMARY/ABSTRACT Juvenile myelomonocytic leukemia (JMML) is a common myeloproliferative neoplasm (MPN) in childhood. JMML is characterized as being Ras-driven due to mutations in NF1, CBL, KRAS, NRAS, or PTPN11, and cells from JMML patients show hypersensitivity to GM-CSF. Chemotherapeutic agents are mostly ineffective in JMML, and the only curative treatment is allogeneic hematopoietic stem cell transplantation (HSCT). A common clinical picture in JMML is that it presents as a hyperinflammatory syndrome, and is often difficult to distinguish from viral infections. Thus, a component of JMML is associated with hyperinflammatory state and hyperactive innate immune cells. Further, unlike other MPNs, JMML rarely progresses to blast crisis; rather, mortality is due to extramedullary myeloid cell expansion leading to organ failure. Importantly, following allogeneic HSCT, 50% of patients succumb to leukemia relapse, implicating a role for bone marrow microenvironment (BME) in JMML development and progression. The hyperinflammatory nature of JMML may damage the BME, altering the expansion of normal donor cells following transplant, permitting residual leukemia cells to outcompete the normal graft, and leading to relapse. Utilizing mouse models of JMML, we demonstrate relapse in mice bearing PTPN11 mutations, we show altered composition of the BME in PTPN11 bearing mice and provide evidence that JMML patients that have a higher neutrophil count at the time of HSCT are more likely to relapse. These data combined with previous studies demonstrating hyperactive and inflamed neutrophils due to PTPN11 mutations suggests that these cells may contribute to relapse. We will examine this in detail. We have been analyzing multiple RNA sequencing datasets for lncRNAs that are differentially expressed in JMML. In doing so, we identified several novel lncRNAs whose expression is differentially regulated. We will examine how one of these lncRNAs contributes to JMML pathogenesis. We have recently shown that PI3K catalytic subunit p110? contributes to both Akt and Erk hyperactivation, and promotes PTPN11-induced GM-CSF hypersensitivity and hyperproliferation, thus partially contributing to the progression of JMML. Given the lack of complete rescue by loss of p110? in PTPN11-induced JMML, we sought out putative tyrosine kinases that signal together with p110? in the PI3K-Akt signaling pathway that must be targeted for optimal JMML therapy. We present preliminary data demonstrating that Bruton's Tyrosine Kinase (BTK) inhibition collaborates with PI3K p110? inhibition to reduce the activation of Akt and Erk in PTPN11-expressing cells. We will study the mechanism behind this cooperation. Overall, the proposed Aims will shed novel insight into JMML development and pathogenesis as well as identification of novel therapeutic targets.

PROJECT SUMMARY/ABSTRACT The incidence of acute myeloid leukemia (AML) has been on the rise. Activating mutations in the fms like tyrosine kinase 3 (FLT3) are present in 25-30% of AML, ~10% of myelodysplastic (MDS) and 5-6% of acute lymphoblastic leukemia (ALL) patients. The common mutations include missense point mutations in the kinase domain, in frame deletions and internal tandem duplications (ITD) in the juxta membrane domain leading to constitutive activation of the receptor tyrosine kinase (RTK) activity. FLT3-ITD is present in ~25% AML patients with normal karyotype and is considered an independent prognostic marker. Patients with FLT3-ITD mutation are at a higher risk of disease relapse and reduced overall 5-year survival. Activating mutations of FLT3 contribute to deregulated proliferation of hematopoietic progenitor cells leading to myeloproliferative neoplasm (MPN). We and others have shown that co-occurrence of mutations that enhance the self-renewal of hematopoietic stem cells (HSC) can transform these cells into AML.However, despite the high prevalence rate and the clinical significance of FLT3 mutations in the pathogenesis of AML, there are limited options for targeted therapy. In 2017, Midostaurin (Rydapt), a multi-kinase inhibitor became the first targeted therapy to be approved by food and drug administration (FDA) for the treatment of AML, followed by Gilteritinib (Xospata), a FLT3 and AXL1 specific inhibitor in 2018. Additional experimental drugs specific for mutant FLT3 in various stages of clinical trials including Quizartinib and Crenolanib have also been described, although they are known to develop both intrinsic and acquired resistance in response to FLT3 targeted therapy the intrinsic resistance in AML to therapy with FLT3 directed inhibitors depends on the presence of co-occurring mutations acquired resistance is due to activation of parallel survival pathways and/or acquisition of secondary mutations in FLT3-ITD. More recently, emergence of RAS mutations has been reported in AML patients treated with Gilteritinib. Thus, there is a critical unmet need to identify and develop potent and selective inhibitor(s) for mutant FLT3 to provide additional therapeutic options for treating AML patients with these mutations. To this end, we have recently identified a novel class of naphthyridine based FLT3 inhibitors that not only selectively target FLT3-ITD at sub-nanomolar concentrations but are also effective against the drug resistance conferring secondary mutations acquired in response to targeted therapy. Based on our preliminary data, we hypothesize that in comparison to recently FDA approved FLT3 inhibitors, including Gilteritinib, we have identified novel and potent drugs with inhibitory activity against FLT3-ITD as well as gatekeeper mutations of FLT3 for a more robust and durable AML treatment. We will utilize two of these inhibitors (KRX-101 & KRX-107; also defined as HSN608 & HSN748, respectively) to further characterize its biological impact on primary de novo AMLs, drug resistant AMLs as well as relapsed/refractory AMLs bearing FLT3 mutations along with other co-occurring mutations.