ALBERT

Description: Recent studies, including studies from our group have shown that somatic mutations in DNA methyltransferase type 3A (DNMT3A) are frequently associated with systemic mastocytosis (SM), myelodysplastic syndrome and AML. Importantly, the presence of this mutation is associated with poor prognosis and overall patient survival. We have shown that approximately 12% of patients with SM possess DNMT3A mutations, which are commonly co-expressed with an activating mutation of KIT (KITD816V in humans and KITD814V in mice). However, it is unclear how DNMT3A mutations contribute to myeloid lineage derived mast cell growth and differentiation either alone or in conjunction with KITD816V. Further, the mechanisms by which loss of DNMT3A impairs various mast cell functions either alone or in cooperation with KITD816V are poorly understood. Utilizing mice conditionally deficient in Dnmt3a in the hematopoietic compartment, we show that loss of Dnmt3a in vivo results in increased numbers of mast cells relative to controls. In vitro, loss of Dnmt3a results in accelerated and enhanced differentiation of mast cells from its bone marrow (BM) precursors. Previous studies have shown that the level of expression of the transcription factor, Microthalamia (MITF) (high MITF expression drives mast cell commitment) and CEBPα (high C/EBPα drives basophil commitment) in Basophil-Mast cell common progenitor (BMPs) drives the fate of these cells towards either basophilic lineage or mast cell lineage. To assess why loss of Dnmt3a results in increased mast cell lineage commitment, we performed RNAseq analysis and found elevated expression of MITF and a profound repression in the expression of C/EBPα in BM precursors. Importantly, restoring the expression of Dnmt3a in Dnmt3a deficient BM cells, completely restored enhanced differentiation, along with the expression of MITF and C/EBPα. To our surprise, no appreciable differences in the expression of Gata-1, Gata-2 or PU.1 were noted in the absence of Dnmt3a relative to controls. These results demonstrate that early loss of C/EBPα in Dnmt3a null cells contributes to enhanced maturation of mast cells from their precursors. We next assessed the growth and survival potential of Dnmt3a mast cells and found a significant increase in SCF-mediated growth of these cells compared to controls. Biochemical analysis revealed greater PI3Kinase activation in Dnmt3a null cells, and these findings were supported by genome wide transcriptome analysis involving Ingenuity pathway analysis (IPA) and Gene set enrichment analysis (GSEA). Importantly, mast cells derived from Dnmt3a null BM lacking the expression of p85α regulatory subunit of PI3Kinase (Dnmt3a-/-:p85α-/-) or pharmacologic inhibition of PI3Kinase completely corrected SCF induced hyper proliferation in these cells to near normal levels. The increase in PI3Kinase activity in Dnmt3a null cells was associated with the loss of PTEN expression. To understand how loss of Dnmt3a and expression of an oncogenic form of KIT cooperate to drive MPN, we generated mice in which the expression of oncogenic KIT was temporally and spatially regulated in a cell type and tissue specific manner using the Mx-Cre system along with the expression of Dnmt3a. Consistent with earlier studies, we observed that KitD814V/+ mutation alone only resulted in the development of lymphoid neoplasms. In contrast, Dnmt3a-/- :KitD814V/+ mice manifested robust signs of MPN development including splenomegaly with increased numbers of neutrophils and monocytes in the peripheral blood associated with a significant reduction in lymphoid cells including B cells. Importantly, we noted a 5-fold increase in the fraction of LSKs and GMPs in the BM of Dnmt3a-/- :KitD814V/+ mice compared to controls and consequently an increase in the presence of mature Gr-1 and Mac-1 positive myeloid cells in the BM, spleen and in the PB of these mice relative to controls. The observed MPN was more aggressive in Dnmt3a-/- :KitD814V/+ mice compared to Dnmt3a+/- :KitD814V/+ mice and of stem cell origin as transplantation of BM cells from these mice recapitulated the development of MPN in recipient mice, similar to that observed in the original animals. The impact of PI3Kinase inhibition on mitigating oncogenic KIT and loss of Dnmt3a induced MPN and overall survival will be discussed. Disclosures No relevant conflicts of interest to declare.

Description: Enhanced blast clearance/remission followed for relapsed/refractory AML patients, in whose blasts were annotated by mutant Flt3 in company with additional TET2 mutation, when a combination of Sorafenib/Vorinostat was supplemented by Bortezomib (Sayar, et al. Oncotarget, 2018). Pharmacodynamic analysis of day0/4 marrow blasts demonstrated early inhibition of HOXA9/10, and/or MEIS1, which occurred upon re-regulation of Wnt pathway participants whose activity had been affected by mTET2. We hypothesized Wnt effectors which drive HOXA overexpression enlist β-Catenin-dependent transactivation of HOXA (Bei, Eklund et al. JBC, 2012), and the role of Bortezomib extends beyond previously recognized AML-specific targets: Flt3ITD, p52NFκB. Therefore a series of primary AML blasts chosen across a spectrum of cytogenetic and molecular categories were analyzed herein to examine the hypothesis that proteasome inhibitors (PI) provide novel targeting action in combination with a new Flt3/Syk inhibitor (FSI) currently in clinical trial, TAK-659 (K Pratz, Blood(abstract) 2018) or, alternatively, when using a previously-reported compound with similar FSI selectivity, R406 (Puissant, Stegmaier, et al. Cancer Cell, 2014). Either of these agents alone or in combination with Bortezomib or Ixazomib were tested to learn respective mechanism of action, and to identify sensitive molecular phenotypes. In order to study β-Catenin as target, we incubated AML blasts in culture with the following treatments: control, FSI, PI, or PI+FSI, while using informative dosing in tandem cultures for proliferation, apoptosis, gene expression and proteomic endpoints. Because ubiquitination directs substrate proteolysis as well as subcellular localization for transcriptional regulators, we isolated within each test group both cytoplasmic and nuclear protein. Also, because activity of β-Catenin involves phosphorylation-initiated and ubiquitin-directed trafficking, we probed immunoblots for both nonphosphorylated [active] (S33,S37,T41)- and phosphorylated β-Catenin species. For Flt3/Syk inhibition by TAK-659, we used concentrations at, and below, a clinically-achievable optimal concentration of 250 nM (opcit). In all cases of Flt3mutant AML's studied, we observed synergy between Flt3/Syk inhibitor with PI, including at clinically suboptimal concentrations of individual agents. This cooperative activity involved loss of active, nonphosphorylated β-Catenin (80kD) from the nucleus, and the cytoplasmic accumulation of large ubiquitin-laden species of phospho-β-Catenin, ranging from 100-175kD, under conditions where apoptotic cell death occurred. A cytoplasmically-localized gatekeeper for stopping canonical Wnt pathway activation, Axin1, was not limiting for the loss of nuclear β-Catenin by the combination: FSI plus PI, but was modestly upregulated by PI alone. However, nuclear-localized FOXM1, a known nuclear chaperone for β-Catenin, was frequently significantly reduced by PI or the combination of FSI/PI. Both Flt3ITD (cytoplasmic) and p52NFκB (nuclear) were lost in additive fashion by combination treatments. At the transcriptional level (real-time RT-PCR), c-jun (a transactivator for FOXM1 and CTNNB1), Syk, as well as FOXM1, HOXA and MEIS1 were additively repressed by combination FSI plus PI in select AML blasts. Certain AML molecular phenotypes demonstrated heightened sensitivity to the combination of TAK-659 plus PIs. Within blasts bearing hierarchically-related mutant effectors: IDH2, TET2, or WT1-all recognized to dampen Wnt-inhibitory pathways, we observed that the combination of TAK-659 with PI was cooperatively active in both Flt3mutant and Flt3wild-type AMLs, while accompanied by an inhibitory effect on β-Catenin. In summary, proteasome inhibition with Ixazomib or Bortezomib cooperated with Flt3/Syk inhibition to cause apoptotic cell death in both Flt3mutant and wild-type AML's with Wnt pathway DNA/chromatin modifying enzyme mutations, a process which appeared to depend upon loss of nuclear-active β-Catenin and FOXM1, and accompanied by accumulation of large, ubiquitin-laden cytoplasmic β-Catenin species. Thus, proteasome inhibition in combination with inhibition of Flt3/Syk holds a central role in targeted therapy of certain poor-risk de novo AMLs, by interdiction of a leukemic stem cell (LSC) signature and causing apoptosis of AML blasts. Disclosures Roodman: Amgen Denosumab: Membership on an entity's Board of Directors or advisory committees.

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: Myeloid malignancies including AML, CMML, MPN and MDS are considered clonal blood disorders. Hematopoietic stem and progenitor cells (HSPCs) with mutation(s) in AML-related genes such as TET2 or DNMT3A represent what are commonly defined as preleukemic HSPCs. The selection and expansion of preleukemic-HSPC clones precedes the development of AML. Additionally, preleukemic-HSPCs can transform through serial acquisition of additional somatic mutations over time and contribute to the development of full-blown AML. What is unclear is the nature of environmental signals that might contribute to the "switch" from a preleukemic state to a leukemic state in these cells. In this context, inflammation has been hypothesized to play an essential role, but precisely how inflammatory signals influence the growth, survival, differentiation and the overall engraftment potential of preleukemic-HSPCs is yet to be determined. Mouse models carrying loss of function alleles in Tet2 or Dnmt3a manifest an expanded HSPC pool, including a hematopoietic stem cell (HSC)-enriched fraction defined by cell surface markers Lineage-/Sca-1+/c-Kit+ (LSK). Some of these genetically modified mice go on to develop CMML or MPN with modest penetration when aged. Majority of pre-leukemic mutations on their own are insufficient to cause AML in mice, suggesting that a single mutation among the above-described mutations define a pre-leukemic state and perhaps additional cooperating mutations are necessary to provide a more effective selection advantage for preleukemic-HSPC leading to the development of full-blown leukemia. Inflammation has been linked to tumor induction and transformation in solid tissues. Inflammation caused by environmental exposure, infection, autoimmunity, or ageing may result in mutations and genomic instability in somatic cells as well as in reprogramming of the tumor microenvironment (i.e. through regulating angiogenesis and expression of cytokines and chemokines). Considering that both innate and adaptive immune cells are generated from HSPCs and are involved in regulating local as well as whole-body inflammatory processes, the relationship between inflammation and hematopoietic malignancies is likely to be complex. While the influence of inflammatory stress on normal HSPCs has recently gained significant attention, little is known about how preleukemic HSPCs respond to inflammation. Because HSPCs reside in the bone marrow and are surrounded by mature immune cells, the inflammatory microenvironment is likely to influence the growth and self-renewal of these cells in part by producing pro-inflammatory cytokines and chemokines. In support of this hypothesis are epidemiologic findings demonstrating that infection may act as a trigger for AML development in humans. In the present study, we asked whether Tet2-deficient HSPCs maintain growth and survival advantage during pathological stress by examining how Tet2-KO preleukemic-HSPCs respond to acute and chronic inflammation. We show a rapid increase in the frequency and absolute number of Tet2-deficient mature myeloid cells and HSPCs in response to acute inflammatory stress, which results in enhanced production of inflammatory cytokines, including IL-6, and resistance to apoptosis. Functionally, Tet2-deficient preleukemic-HSPCs showed resistance to inflammation-induced damage and apoptosis relative to controls. IL-6 induces hyperactivation of the Shp2-Stat3 signaling axis, resulting in increased expression of a novel anti-apoptotic lncRNA Morrbid in Tet2-KO myeloid cells and HSPCs. Expression of activated Shp2 in HSPCs phenocopies Tet2 loss, with regard to hyperactivation of Stat3 and Morrbid. In vivo, pharmacologic inhibition of Shp2 or Stat3 or genetic loss of Morrbid in Tet2-deficient mice rescues inflammatory stress-induced abnormalities in HSPCs and mature myeloid cells including clonal hematopoiesis. Our results suggest that the anti-inflammatory drugs E3330 and SHP099, which suppress the hyper-activation of Morrbid, could be of clinical benefit for TET2 mutations-induced clonal hematopoiesis and leukemogenesis. Our results provide insight into the selection advantage that might render Tet2-deficient HSPCs susceptible to transformation and suggest that anti-inflammation therapy could be of clinical benefit for individuals carrying TET2 mutations. Disclosures Kelley: Apexian Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Mohseni:Novartis Institutes of Biomedical Research: Employment.

Description: Mutations in DNA methyltransferase type 3A (DNMT3A) are frequently associated with myeloid malignancies including myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), and myeloproliferative neoplasms (MPN) including systemic mastocytosis (SM). Presence of this mutation is associated with poor prognosis and reduced overall patient survival. Earlier studies have shown that hematopoietic specific loss of Dnmt3a in mice results in lethal, fully penetrant MPN with myelodysplasia (MDS/MPN) characterized by extramedullary hematopoiesis, peripheral cytopenias, splenomegaly and hepatomegaly. However, it's unclear how DNMT3A mutations contribute to myeloid skewing, and induce myeloid malignancies. Further, the mechanism(s) by which loss of DNMT3A impairs various mast cell functions leading to mastocytosis are also not defined. We show that loss of Dnmt3a in vitro results in accelerated and enhanced differentiation of mast cells from its bone marrow (BM) precursors and in vivo results in increased number of mast cells. Genome wide transcriptome analysis revealed elevated expression of the transcription factor MITF and a profound repression in the expression of C/EBPα in BM precursors derived from Dnmt3a-/- mice. Importantly, restoring the expression of Dnmt3a in Dnmt3a deficient BM cells, completely restored enhanced differentiation, along with correction in levels of MITF and C/EBPα, suggesting that early loss of C/EBPα in Dnmt3a-/- cells contributes to enhanced maturation of mast cells from its precursors. Furthermore, biochemical analysis revealed greater PI3Kinase activation in Dnmt3a-/- cells, and these findings were supported by genome wide transcriptome analysis. Importantly, mast cells derived from Dnmt3a-/- mice lacking the expression of p85α regulatory subunit of PI3Kinase or pharmacologic inhibition of PI3Kinase completely corrected cytokine induced hyperproliferation in these cells to near normal levels. Insights from mast cell studies led us to hypothesize that the aberrant hematopoietic regulation in Dnmt3a-/- mice described above may be due to hyperactivation of the PI3Kinase signaling in hematopoietic stem and progenitors (HSCPs). We investigated the role of PI3Kinase signaling in Dnmt3a loss induced myeloid malignancy using pharmacological inhibitor, GDC0941 (PI3Kα/δ inhibitor) and BAY1082439 (PI3Kα/β inhibitor). PI3Kα/β inhibitor treatment of Dnmt3a-/- mice markedly reduced monocytosis, neutrophilia, enhanced WBC counts, and improved RBCs, hematocrits and platelet numbers compared to control mice. In contrast, PI3Kα/δ inhibition moderately corrected the monocytosis and WBC aberrancy, however correction in erythroid dysregulation was not observed. Inhibiting PI3Kα/β signaling dramatically reduced splenomegaly and hepatomegaly in Dnmt3a-/- mice. In contrast, PI3Kα/δ inhibition moderately reduced the spleen size, however the correction in hepatomegaly was insignificant. Importantly, clonal hematopoiesis due to loss of Dnmt3a in a competitive transplant setting was rescued by inhibiting the PI3Kinase pathway. In the BM, PI3Kα/β inhibition moderately decreased LSK cells, however a marked decrease in GMP, and significant increase in MEP, and CLP was observed compared to controls. Flow cytometry analysis revealed that PI3Kα/β inhibition reduced Dnmt3a-/- BM cell migration to liver, and completely corrected the extramedullary hematopoiesis in the spleen compared to controls. Further, Dnmt3a loss induced alteration in erythroid development in the BM, spleen and PB was corrected upon PI3Kα/β blockade compared to controls. Genome wide transcriptome analysis revealed that PI3Kα/β inhibition markedly reduced the expression of GMP related genes, fetal liver hematopoiesis transcription program, and expression levels of inflammatory cytokines, growth factors, and chemokines in Dnmt3a depleted malignant mice. However PI3Kα/β inhibition increased the expression of genes involved in erythroid development in Dnmt3a-/- mice compared to controls. These results demonstrate that Dnmt3a ablation induces liver specific expansion of hematopoietic cells, and extramedullary hematopoiesis in spleen via aberrant activation of PI3Kinase signaling in HSCPs. Consistent with this notion, PI3K inhibitor treatment of malignant Dnmt3a-/- bearing mice showed significantly improved overall survival compared to controls. Disclosures No relevant conflicts of interest to declare.

Description: Recent attempts at single agent targeted therapy of AMLs described by mutation of Flt3 or nuclear epigenetic effectors, has led to the conclusion that combination targeted approaches will be required (CM McMahon et al. Cancer Discov 2019). The simplest combination therapy would involve inhibitors directed at mutant drivers at each level (receptor, nuclear). However, the number of those inhibitors is limited. A broader strategy would target common endpoints for converging pathways such as Wnt/beta-catenin activation elicited by mutation of IDH1/2, TET2, DNMT3A. We found marked cytoplasmic accumulation of ubiquitinated protein (especially inactive b-catenin excluded from the nucleus) by treatment with proteasome inhibitor(PI) to be an efficient, dose-dependent inducer of endoplasmic reticulum (ER) stress apoptosis in mutant Flt3/Wnt effector AML's, requiring concentrations =/〉100nM Ixazomib, or =/〉20nM Bortezomib, when used alone on cultured blasts. Indeed, a compensatory pathway to protective autophagic escape from PI in poor-risk AML is linked to the levels of NRF2, a major transcriptional activator of NADPH quinone reductase1 (NQO1) that buffers oxidative stress. However, the Flt3/Syk inhibitor (FSI) in clinical trial, TAK-659, at =/ HOXA in AML, as compared to the RAS and WT1 pathways, and have established a combination therapy (TAK-659 plus PI) that affects the inhibitory effectors elicited by these co-mutational states, which are responsible for negating activity for most Flt3 selective targeted agents, so as to allow antileukemic response. Disclosures Roodman: Amgen: Membership on an entity's Board of Directors or advisory committees. Konig:Agios: Consultancy; Amgen: Honoraria.