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Acute myeloid leukemia (AML) is an aggressive hematopoietic neoplasm with five-year overall survival rates To identify survival-critical AML genes irrespective of mutational status, we performed an shRNA screen on patient samples (N=12) using a barcoded lentiviral shRNA library. Transduced cells were cultured for 9 days on HS-5 stromal cells (to mimic the BM microenvironment) and subjected to NGS for barcode quantification. Sirtuin 5 (SIRT5), the only known enzyme with lysine desuccinylase, demalonylase, or deglutarylase activity, was amongst the top candidates. SIRT5 is implicated in regulating metabolic pathways, including energy metabolism. We first confirmed growth inhibition upon SIRT5 knockdown (KD) in cryopreserved AML cells from the original screen. Next, we transduced a panel of 25 AML cell lines with doxycycline (dox) - inducible shSIRT5 (dox-shSIRT5). SIRT5 KD strongly inhibited growth, reduced colony formation and increased apoptosis in 18/25 lines (SIRT5-dependent cell lines). Seven cell lines showed 80% reduction of SIRT5 protein and were considered SIRT5-independent. SIRT5 dependence was neither correlated with specific mutations nor basal SIRT5 expression. To control for off-target effects we tested three unique shSIRT5s targeting different sequences in three SIRT5-dependent and three SIRT5-independent cell lines, and confirmed the differential sensitivity for all lines and all constructs. We next transduced CD34+ cells from AML patients (n=15) or cord blood (CB, n=5) with dox-shSIRT5 and plated cells in colony assays. SIRT5 KD (~40% in AML and CB) reduced AML colonies by ~50%, with no effect on CB. Colony formation by SIRT5 null mouse bone marrow infected with FLT3-ITD or MLL-AF9 retrovirus was reduced by 50-60% compared to wild type controls. Metabolic profiling showed that SIRT5 KD induced a profound reduction of oxidative phosphorylation and glycolysis in SIRT5-dependent, but not SIRT5-independent cell lines. Mitochondrial reactive oxygen species (ROS) in SIRT5-dependent cell lines were strongly increased upon SIRT5 KD, and this increase preceded apoptosis. Ectopic expression of superoxide dismutase 2 (SOD2, mitochondrial) abrogated the increase in ROS and rescued cells from apoptosis. Metabolomics and RNAseq suggest that SIRT5-dependent cells exhibit profound metabolic disruption, including reduced TCA cycle activity, and recurrent transcriptional changes, while SIRT5 KD in SIRT5-independent cells is inconsequential. We next injected mice with CMK-1 cells (SIRT5-dependent) expressing dox-shSIRT5 and luciferase. Mice were randomized to dox-supplemented (dox-water) or control water 18 hours post injection, and monitored by luminescence imaging. Control mice showed abundant luminescence at week 3, and died before week 5, while mice receiving dox-water survived throughout the 13-week experiment without evidence of leukemia. When mice with established leukemia were switched to dox-water following week 3, luminescence rapidly decreased and the mice survived without evidence of leukemia until termination of the experiment (week 13). Dox-water had no effect on mice engrafted with OCI-AML3 cells (SIRT5-independent), all of whom died with extensive leukemic involvement despite downregulation of SIRT5 in leukemia cells. We are currently testing the requirement of SIRT5 in MLL-AF9-mediated AML using a retroviral BM transplant mouse model. Preliminary data suggest that absence of SIRT5 may prolong survival. Our data suggest that SIRT5 KD preferentially targets AML cells over normal cells. As SIRT5 null mice are viable with only minor metabolic abnormalities at steady state, these data implicate SIRT5 as a potential therapy target in AML and support the development of clinical SIRT5 inhibitors. Disclosures Deininger: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.

The mechanisms underlying tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML) patients lacking explanatory BCR-ABL1 kinase domain mutations are incompletely understood. To identify mechanisms of TKI resistance that are independent of BCR-ABL1 kinase activity, we introduced a lentiviral short hairpin RNA (shRNA) library targeting ∼5000 cell signaling genes into K562(R), a CML cell line with BCR-ABL1 kinase-independent TKI resistance expressing exclusively native BCR-ABL1. A customized algorithm identified genes whose shRNA-mediated knockdown markedly impaired growth of K562(R) cells compared with TKI-sensitive controls. Among the top candidates were 2 components of the nucleocytoplasmic transport complex, RAN and XPO1 (CRM1). shRNA-mediated RAN inhibition or treatment of cells with the XPO1 inhibitor, KPT-330 (Selinexor), increased the imatinib sensitivity of CML cell lines with kinase-independent TKI resistance. Inhibition of either RAN or XPO1 impaired colony formation of CD34(+) cells from newly diagnosed and TKI-resistant CML patients in the presence of imatinib, without effects on CD34(+) cells from normal cord blood or from a patient harboring the BCR-ABL1(T315I) mutant. These data implicate RAN in BCR-ABL1 kinase-independent imatinib resistance and show that shRNA library screens are useful to identify alternative pathways critical to drug resistance in CML.

Acute myeloid leukemia (AML) is an aggressive hematopoietic neoplasm that carries the worst prognosis among the hematologic malignancies. Up to 30% of AML patients exhibit activating mutations in FLT3 tyrosine kinase. FLT3 internal tandem duplications (ITDs) comprise ~70% of these mutations and are associated with a poor prognosis. Most patients treated with a single-agent FLT3 tyrosine kinase inhibitor (TKI) relapse within months due to secondary mutations in the FLT3 tyrosine kinase domain (TKD). Results from trials of FLT3 TKIs in AML reveal that leukemic blasts are more easily cleared from peripheral blood than from bone marrow (BM), suggesting that the BM microenvironment promotes survival of AML cells, including leukemia initiating cells, despite inhibition of FLT3. In this conceptual framework, extrinsic factors allow AML cells to survive TKI exposure until AML cell-intrinsic resistance is conferred by FLT3 TKD mutations, leading to clinical relapse. Here, we investigated the role of the BM microenvironment in protection of FLT3+AML cells from treatment with AC220 (quizartinib), a clinically available FLT3 TKI. To investigate the potential of the BM microenvironment to mediate TKI resistance in AML, we cultured FLT3-ITD+ AML cell lines, including MOLM-13, MOLM-14 and MV411, and the CML cell line, K562 (control; FLT3 wild-type), with graded concentrations of AC220 under the following conditions: (i) in regular medium (RM), (ii) in direct contact (DC) with human HS-5 BM stromal cells, or (iii) in HS-5 conditioned medium (CM). Cell proliferation and apoptosis assays revealed that, in RM,AC220 reduced proliferation and increased apoptosis of MOLM-13, MOLM-14 and MV411 cells, but had no effect on K562 cells. DC greatly reduced the effects of AC220 in all three FLT3-ITD+ AML cell lines, with comparable results observed between DC and CM. To confirm these data using primary cells, CD34+ blasts from a patient with newly diagnosed FLT3-ITD+ AML were similarly cultured in RM versus CM ± AC220. Consistent with results in cell lines, CM rescued primary AML cells from AC220-mediated cell death. These data indicate that soluble factors from the BM environment protect FLT3-ITD+ cells from the effects of FLT3 inhibition. Our lab and others have demonstrated that HS-5 DC and CM activate STAT3 in chronic myeloid leukemia, which mediates resistance to BCR-ABL1 TKIs (Bewry et al. Mol Cancer Ther 2008, Traer et al. Leukemia 2012, Eiring et al. Leukemia 2015). To interrogate the role of STAT3 in BM-mediated protection of AML cells from FLT3 inhibition, all cell lines were assessed for pSTAT3Y705 and total STAT3 by immunoblot analysis under each culture condition. In FLT3-ITD+ AML cells grown in RM, pSTAT3Y705 was undetectable, irrespective of AC220 dose. In contrast, pSTAT5Y694 was readily detected at steady state and suppressed by AC220. AML cells cultured in HS-5 DC or in HS-5 CM exhibited strong upregulation of pSTAT3Y705 that was unaffected by AC220, suggesting that soluble factor(s) promote STAT3 activation in AML. pSTAT5Y694, on the other hand, was slightly elevated by HS-5 DC or CM, but remained under control of FLT3 kinase activity. In order to mechanistically implicate STAT3 activation in stroma-based protection, we used a retroviral shRNA construct to knockdown STAT3 (shSTAT3) compared to an empty vector control (LMP) in MOLM-14 cells. STAT3 knockdown (~70%) was confirmed by qRT-PCR and immunoblot analyses. Cells containing shSTAT3 and LMP were cultured for 72 hours in RM or CM ± AC220, followed by analysis using MTS assays. As expected, CM increased the IC50 of AC220 from 1.37 nM to 6.24 nM in LMP-expressing cells (n=3). In contrast, shSTAT3 reduced the IC50 of AC220 from 6.24 nM to 2.87 nM (n=3) in CM, with minimal effects in RM. Similarly, pharmacologic inhibition of STAT3 using the novel STAT3 inhibitor, BP-5-087 (Eiring et al. Leukemia 2015), reduced the IC50 of AC220 from 10.07 nM to 5.91 nM in CM. Analogous experiments in additional FLT3-ITD+cell lines and primary AML cells, using shSTAT3, dominant-negative STAT3 constructs and BP5-087 are ongoing. Our data suggest that STAT3 is a critical signaling node in FLT3-independent TKI resistance mediated by the BM microenvironment. Therapeutic strategies designed to combine FLT3 and STAT3 inhibition may inhibit the survival of leukemic cells in the BM niche, thereby preventing subsequent clinical relapse conferred by TKD mutations. Disclosures Deininger: Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Nitric oxide (NO) induces apoptosis and differentiation in acute myeloid leukemia (AML) cells. The NO prodrug O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) is a lead agent of the arylated diazeniumdiolate class. JS-K is active in vitro and in vivo against AML, multiple myeloma, and several solid tumors. JS-K is directly cytotoxic to malignant cells and inhibits angiogenesis in vitro and in vivo. We have previously shown that JS-K is not toxic towards normal murine hematopoietic cells. Aiming at its clinical application to treat AML, we have developed a nanoscale micelle formulation for JS-K (P123/JS-K) using Pluronic®P123 polymers. The formulation (P123/DMSO) consists of 2.25% P123 and 2% dimethyl sulfoxide in phosphate buffered saline. P123/JS-K is currently at an advanced stage of pre-clinical development. Here, we compared the cytotoxicity of JS-K and its formulation between normal hematopoietic and AML cells. HL-60 AML cells or normal human CD34+ cells isolated from 2 different cord blood units were treated for 24 hours with P123/DMSO or P123/JS-K before plating in semi-solid media. Colonies were scored at 10 days. P123/DMSO alone had no effect on colony formation by HL-60 or normal CD34+ cells. P123/JS-K at concentrations of 0.1, 0.25, and 0.5 μM inhibited HL-60 colony growth by 27, 74, and 100%, respectively (P < 0.05 for the comparison between 0.25, 0.5 μM and controls). At the same concentrations, P123/JS-K inhibited the growth of erythroid colonies from the first cord blood sample, by 26, 43, and 61%, respectively. At a concentration of 0.1 μM, P123/JS-K did not inhibit the growth of erythroid colonies from the second cord blood sample but inhibited erythroid colony growth by 18, and 27% at concentrations of 0.25 and 0.5 μM, respectively. However, the differences with untreated controls were not statistically significant for either cord blood sample. At the same concentrations P123/JS-K inhibited the growth of myeloid colonies from the first cord blood sample, by 8, 3, and 37%, respectively. At a concentration of 0.1 μM, P123/JS-K did not inhibit the growth of myeloid colonies from the second cord blood sample but inhibited myeloid colony growth by 2, and 6% at concentrations of 0.25 and 0.5 μM, respectively. However, the differences with untreated controls were not statistically significant for either cord blood sample. With clinical development in mind, we also sought to determine whether the P123/DMSO formulation affects platelet function. P123/DMSO was added to normal donor blood obtained in either EDTA or citrate anticoagulant. With either anticoagulant, P123/DMSO did not affect the measured hemoglobin, white blood cell, or platelet counts. Inspection of peripheral blood smears (Wright stain) obtained with either anticoagulant revealed no platelet clumping. We then studied the effect of P123/DMSO on platelet aggregation in vitro in response to ADP, collagen, ristocetin, or arachidonic acid. Light transmission platelet aggregometry was performed using platelet-rich plasma from a healthy volunteer with normal platelet counts and function. In order to determine whether P123/DMSO could enhance or inhibit platelet aggregation, we chose a range of agonist concentrations from sub-threshold to concentrations expected to induce complete aggregation. P123/DMSO was added at a concentration expected to be equal to peak in vivolevels based on a prior dog toxicology study. Normal aggregation responses were observed in the saline control. There were no significant differences between this control and responses observed in the presence of P123/DMSO. Specifically, significantly decreased aggregation in P123/DMSO-treated platelets was not observed in response to agonists expected to elicit complete aggregation responses. Likewise, enhanced aggregation was not observed in response to sub-threshold agonist concentrations. Our results confirm the potent anti-leukemic activity and limited toxicity against normal human hematopoietic cells of JS-K formulated in P123 micelles. Furthermore, the formulation does not affect platelet aggregation. We therefore expect that P123/JS-K will have a favorable hematologic toxicity profile and as such, will constitute an important addition to our armamentarium for the treatment of AML. Disclosures Deininger: Gilead: Research Funding; BMS: Consultancy, Research Funding; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Shami:JSK Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

The majority of patients diagnosed with Philadelphia negative (Ph-) myeloproliferative neoplasms (MPNs) harbor somatic, gain-of-function mutations in JAK2, CALR or MPL. All of these mutations are associated with constitutive JAK/STAT signaling which confers a proliferative advantage to MPN cells and leads to malignant myeloid expansion at the expense of normal hematopoiesis. The bone marrow (BM) microenvironment in MPNs, particularly myelofibrosis (MF), is characterized by high concentrations of inflammatory cytokines, and we have previously shown that MF cells generate tumor necrosis factor alpha (TNF) in a JAK2-dependent manner. Elevated TNF promotes the survival of JAK2V617F mutant cells over their JAK2WT counterparts, creating a feedback loop in which the mutant cells enhance the inflammatory environment that supports their survival and expansion (Fleischman et al. Blood. 2011 Dec 8;118(24):6392-8). To determine which hematopoietic lineages contribute to increased levels of TNF in MPN, we measured intracellular TNF expression in immunophenotypically defined white blood or BM cells from MF patients and normal controls. TNF expression was relatively low in unstimulated cells. However, lipopolysaccharide (LPS) treatment induced a 16-fold greater increase of TNF expression in hematopoietic stem cells (HSCs) from MF patients relative to normal controls (p TNF signaling is mediated by two receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). To evaluate the contribution of each receptor to the TNF-mediated survival of MPN cells, we cultured CD34+ cells from MF patients or normal controls for 72 hours ± TNFR1 or TNFR2 blocking antibodies (BA), then plated in clonogenic assays. While TNFR1 BA had no effect, TNFR2 BA reduced colony numbers in MF specimens by 26% (p To identify TNF signaling networks that differentiate the TNF responsiveness of cells expressing JAK2V617F compared to JAK2WT, BM cells were harvested from JAK2V617F MPN mice and cultured for 16 hours ± TNFR1 or TNFR2 BA. Lin-Kit+ cells were sorted based on GFP (JAK2V617F) expression and 3 independent experiments were tested with Affymetrix Mouse Genome 430 2.0 Arrays. We focused our analysis on genes with differential regulation between JAK2V617F and JAK2WT, whoseexpression was equalized with TNFR2 BA. We then prioritized genes with reported involvement in TNF signaling. The top candidate was Mapk8 (Jnk1) whose expression was strongly downregulated in untreated JAK2V617F relative to JAK2WT expressing cells (3.4-fold; p Disclosures Deininger: Gilead: Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

To detect targeted antileukemia agents we have designed a novel, high-content in vivo screen using genetically engineered, T-cell reporting zebrafish. We exploited the developmental similarities between normal and malignant T lymphoblasts to screen a small molecule library for activity against immature T cells with a simple visual readout in zebrafish larvae. After screening 26 400 molecules, we identified Lenaldekar (LDK), a compound that eliminates immature T cells in developing zebrafish without affecting the cell cycle in other cell types. LDK is well tolerated in vertebrates and induces long-term remission in adult zebrafish with cMYC-induced T-cell acute lymphoblastic leukemia (T-ALL). LDK causes dephosphorylation of members of the PI3 kinase/AKT/mTOR pathway and delays sensitive cells in late mitosis. Among human cancers, LDK selectively affects survival of hematopoietic malignancy lines and primary leukemias, including therapy-refractory B-ALL and chronic myelogenous leukemia samples, and inhibits growth of human T-ALL xenografts. This work demonstrates the utility of our method using zebrafish for antineoplastic candidate drug identification and suggests a new approach for targeted leukemia therapy. Although our efforts focused on leukemia therapy, this screening approach has broad implications as it can be translated to other cancer types involving malignant degeneration of developmentally arrested cells.

The JAK2V617F mutation is a unifying feature in the majority of patients with myeloproliferative neoplasms (MPNs). The presence of JAK2V617 in a hematopoietic stem (HSC) or progenitor cell confers a proliferative advantage over native JAK2 counterparts. Several inflammatory cytokines are elevated in MPN patients and in murine models of JAK2V617F-driven MPN. In particular, expression of tumor necrosis factor alpha (TNF-α) is increased by constitutive JAK2 kinase activity and imparts a competitive advantage on JAK2V617Fexpressing cells. To identify cell types responsible for increased levels of TNF-α we performed intracellular cytokine staining in leukocytes from myelofibrosis (MF) patients and normal controls. Using a panel of markers to define cellular subsets we found expression of TNF-α was uniformly low in unstimulated cells. However, when cells were treated with lipopolysaccharide (LPS), TNF-α expression was 16-fold higher in HSCs of MF patients compared to normal controls (p To evaluate anti-TNF therapy in MPNs, we treated JAK2V617F mice with etanercept, a soluble TNF receptor fusion protein that binds and inactivates TNF-α. Etanercept did not significantly restrain JAK2V617F-associated WBC or hematocrit increases over a 10-week period, despite suppression of TNF-α activity in plasma. As TNF-α may activate pro-apoptotic (predominantly through TNF receptor 1, TNFR1) and pro-survival pathways (predominantly through TNF receptor 2, TNFR2), we reasoned that global blockade of TNF-α may not shift the balance in favor of normal hematopoiesis. To investigate this we sorted primitive (Lin-, cKit+) cells from mice with JAK2V617F-induced MPN by TNF receptor expression. TNFR2+ cells showed significantly increased colony formation compared to TNFR1+ cells, demonstrating that TNFR2 expression is associated with increased clonogenic potential. Additionally we treated these primitive cells with specific antibodies blocking TNFR1 or TNFR2. Colony assays performed after 3 days in liquid culture with TNFR-blocking antibodies confirmed that interrupting TNFR2-mediated survival signaling resulted in decreased colony formation (61% reduction) while blocking TNFR1 increased colony formation (5% increase). In addition we tested CD34+ cells from MF patient samples with TNFR-blocking antibodies. As seen with the murine MPN cells, colony formation was decreased with the TNFR2 blocking antibody (30% reduction) and increased with the TNFR1 blocking antibody (48% increase) consistent with a differential activation of survival signals by TNF-α in MPN cells. These studies were performed without addition of exogenous TNF-α, which confirms the ability and requirement for these primitive JAK2V617Fcells to utilize TNF-α production to actively support survival. Our data suggest that TNF-α generated by primitive MPN cells promotes their survival through activation of TNFR2, while TNFR1 activation is suppressive. If selective inhibition of TNFR2 shifts the equilibrium toward normal hematopoietic cells, this would support the use of TNFR2 blockade to treat MF and other myeloproliferative neoplasms. Genotyping of human MF and murine MPN colonies cultured with TNF receptor blocking antibodies and experiments in normal human CD34+ cells are ongoing and will be presented. Disclosures Deininger: BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMA, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy.

Polycythemia vera (PV) is a clonal disorder arising from a single stem cell while normal stem cells are present in the marrow but are suppressed by the PV clone by an unknown mechanism. Pegylated interferon alfa-2a (PegInfa), a better-tolerated form of Infa induces clinical remission, reduces JAK2V617F allelic burden, and may convert clonal to polyclonal hematopoiesis (EL Liu, Blood 2003). Tumor necrosis factor-α (TNFa) levels are increased in patients with myeloproliferative neoplasms, including PV (Fleischman, Blood 2011). We previously reported that transcripts of TNFα mRNA are higher in CD34+ cells compared to more differentiated cells. TNFa is markedly reduced in those PegInfa-treated patients with decreased JAK2V617F allelic burden and/or return of polyclonal hematopoiesis (detected in females by X-chromosome allelic usage ratio), while no such decrease was seen in PV with hydroxyurea-induced normalization of elevated hematocrit/platelets/leukocytes (Swierczek, ASH 2012). To directly interrogate the role of TNFα in inducing suppression of normal hematopoiesis, we used a TNFα blocking antibody (adalimumab) and examined its role on PV erythropoiesis using a 3-week liquid culture system characterized by synchronized differentiation of expanding erythroid progenitors (Bruchova, Exp Hemat, 2007). In vitro expanded PV erythroid progenitors were grown with or without adalimumab or PegInfa. We evaluated apoptosis, proliferation and differentiation at different stages of erythroid maturation and correlated these parameters with TNFα transcripts, JAK2V617F allelic burden and, in females, clonality. Although the initial mononuclear cells represented a heterogeneous population, the expansion process favors erythroid progenitors and results in their synchronized differentiation. The JAK2V617F allelic burden increased concomitant with erythroid expansion and reached its peak at day 11 (when the majority of cells are proerythroblasts and basophilic erythroblasts), indicating a preferential expansion of erythroid progenitors, and then declined progressively. The addition of adalimumab markedly reduced TNFα mRNA and JAK2V617F allelic burden compared to controls. We previously reported that in this in vitro liquid expansion system, PV erythroid progenitors exhibit accelerated differentiation at days 7-14 and increased proliferation at days 9-14, with a larger S-phase population (40%) than controls (20%) at day 11 (Bruchova Exp Hemat, 2007). Compared to controls, adalimumab increased proliferation and delayed differentiation at early stages of PV erythropoiesis, with the proportion of apoptotic cells consistently decreased compared to erythroid cells expanded without adalimumab. Furthermore, X-chromosome-based clonality assays revealed preferential expansion of normal progenitors in 1 informative female patient. We also measured the impact of TNFα inhibition with adalimumab on burst-forming units-erythroid (BFU-E) colonies from PV patients cultured ex vivo. As expected, JAK2WT, JAK2WT/V617F and JAK2V617F BFU-E colonies were detected in cultures performed in the absence of adalimumab, while the addition of adalimumab preferentially abrogated JAK2V617F homozygous BFU-Es. In analogous experiments, PegInfa markedly decreased TNFα mRNA and JAK2V617F allelic burden, and decreased differentiation, but unlike adalimumab, it decreased proliferation and had no demonstrable effect on apoptosis. Ongoing studies are being performed to correlate the TNFα mRNA expression changes seen with adalimumab treatment in erythroid progenitors with differences in TNFα protein levels using intracellular cytokine staining and flow cytometry. These data suggest that blocking TNFα can suppress the JAK2V617F clone, rescue normal dormant hematopoiesis, and provide a foundation for a combined PV therapy using TNFα blockers with either PegInfa or JAK2 inhibitors. Disclosures Deininger: BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMS, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy.

We have identified STAT3 as a convergence point for oncogenic signaling in tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia (CML) lacking BCR-ABL1 kinase domain mutations. In addition, we found that STAT3 activity contributes to disease in other myeloid disorders, including acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs). Utilizing TKI-resistant CML as a model system, we identified BP-5-087 as a small molecule inhibitor of STAT3 that reduces STAT3 phosphorylation and nuclear transactivation (Eiring et al. Leukemia, 2014). Binding of BP-5-087 to the STAT3 SH2 domain was initially assessed using fluorescence polarization (FP) assays and high-resolution computational docking simulations. To further validate the binding motif of BP-5-087, we conducted time-resolved electrospray ionization mass spectrometry/hydrogen-deuterium exchange experiments. Fold-change in deuterium uptake was analyzed for 68 STAT3 peptides representing 71% sequence coverage, and mapped onto the crystal structure of STAT3. This analysis precisely defined the binding epitope for BP-5-087 within the STAT3 SH2 domain. We next tested the effects of BP-5-087 in several myeloid malignancies using relevant disease models. (i) CML stem and progenitor cells from TKI-resistant patients without kinase domain mutations were treated with BP-5-087 ex vivo, using short-term liquid culture, clonogenic and LTC-IC assays. BP-5-087 treatment significantly reduced colony formation by CML stem and progenitor cells (p The in vivo activity of BP-5-087 was next evaluated in a murine model of JAK2 V617F-induced MPN. Briefly, Balb/c bone marrow was transduced with JAK2 V617F-GFP, followed by injection into lethally irradiated recipients. After disease induction, mice were treated with BP-5-087 (25 mg/kg) by once-daily oral gavage. No toxicities were observed after 40 days of treatment in BP-5-087-treated mice. While BP-5-087 did not significantly reduce the percentage of GFP+ cells, there was a 41% reduction of spleen weight in BP-5-087-treated mice compared to vehicle-treated controls (p To advance the lead optimization of our STAT3 inhibitor series, we instituted a comprehensive screening cascade. We first developed a computational model (quantitative structure-activity relationship, QSAR) to guide and prioritize selection of new inhibitor candidates for synthesis. Compounds are initially ranked using a methanethiosulfonate (MTS)-based cell viability assay in a TKI-resistant, STAT3-dependent CML cell line (AR230R). Inhibition of STAT3 is confirmed using a cell-based STAT3 reporter assay and an in vitro FP-based binding assay. Optimization of potency is balanced by the goals of reducing molecular weight (MW) and calculated LogP (cLogP) compared to BP-5-087 (MW: 694.8; cLogP: 7.3). Compounds with improvements in these categories are then subject to toxicity testing utilizing clonogenic assays with CD34+ CB cells. Non-toxic compounds are evaluated for their pharmacokinetic profile in Balb/c mice and tested for activity in primary samples from CML, AML and MPN patients. These activities have directed us to a lead compound, AM-1-124, which displays significant improvements in potency, MW, cLogP, and in vivo half-life compared to BP-5-087. AM-1-124 had minimal effects in the CB toxicity assay and induced apoptosis in primary AML patient samples at 2-fold lower concentrations than BP-5-087. With AM-1-124 as our current lead compound, we are continuing our iterative evaluation of novel STAT3 inhibitors utilizing our screening cascade. Design and testing of optimized, orally active inhibitors will enable further evaluation of STAT3 as a target in animal models of myeloid leukemia and will justify the clinical development of these compounds for patients in need of new targeted therapies. Disclosures Deininger: BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMA, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy.

Abstract 1519 INTRODUCTION: Receptor tyrosine kinases AXL and MER belong to the TYRO3 kinase family, first identified as a transforming gene in chronic myeloid leukemia and are found at high levels in various cancers, including hematopoietic malignancies like T cell acute lymphoblastic leukemia (T-ALL). Aurora kinases play important roles in chromosome alignment and cytokinesis during mitosis and are also aberrantly expressed in ALL. We describe here characterization and pre-clinical testing of Huntsman Cancer Institute-2084 (HCI-2084), a small molecule inhibitor of AXL and MER kinases that also has activity against Aurora Kinases A and B, as a novel therapeutic for T-ALL treatment, a leukemia with poor prognosis. METHODS: Quantitative RT-PCR and Western Blot confirmed elevated levels of AXL/MER expression in Jurkat, a human T-ALL, cell line. Our compound, HCI-2084, was developed using a computational structure-based approach against AXL kinase. HCI-2084 activity against AXL/MER, in cell-based assays was evaluated utilizing ATPlite. To test activity against Aurora kinase, Jurkat cells were treated with various concentrations of HCI-2084 and VX 680, a known aurora kinase inhibitor, for 24 hours. Cell lysates were evaluated for phospho-AKT (Ser473), phospho-Aurora (Thr288) and phospho-Histone H3 (Ser10) using the Meso Scale Discovery platform (MSD). Cell cycle analysis was performed on Jurkat cells incubated in the absence (negative control) or presence of 100nM HCI-2084 or VX-680 for 24 hours, stained with propidium iodide and analyzed by flow cytometry on FACS Calibur. Immunostaining was performed to evaluate the levels of phospho-Histone H3 (Ser10) and phospho-Aurora (Thr288) during mitosis in Jurkat cells incubated in the absence (negative control) or presence of 100nM HCI-2084 and VX-680 for 24 hours. In addition to the anti-phospho-Histone H3 and anti-phospho-Aurora antibodies, anti-alpha-tubulin and DAPI were used to determine the presence of mitotic chromosomes. To examine HCI-2084 as a therapeutic agent for T-ALL treatment in vivo, we tested it using transgenic Zebrafish (Danio rerio) with T-ALL driven by human MYC (hMYC). Over-expression of endogenous D. rerio axl /mertk transcripts in T-ALL from hMYC fish were verified by qRT-PCR. Fish were treated with HCI-2084, Dexamethasone (a known T-ALL therapeutic; positive control), and DMSO vehicle (HCI-2084 is reconstituted in DMSO; negative control). Trials were conducted by housing fish with fluorescently-labeled T-ALL (GFP-tagged) in water containing the agent being tested, for 14 days with monitoring of disease response by fluorescent microscopy. RESULTS: In in vitro studies, HCI-2084 showed potent activity in cell viability assays with an IC50 of 12 nM against AXL, 60 nM against MERTK and 15 nM against Aurora kinase. MSD assays demonstrated efficacy at 1 uM for reduction of phospho-AKT, phospho-Histone H3 and phospho-Aurora, as seen with VX 680. Cell cycle analyses performed on HCI-2084-treated Jurkat cells showed a significantly increased G2/M population and an accumulation of cells with ≥4N DNA, indicative of Aurora B inhibition and endo-reduplication (Figure A & B). Immunofluorescence analyzed using fluorescent microscopy demonstrated mitotic arrest with loss of phospho-Histone H3 and phospho-Aurora staining, demonstrating inhibition of Aurora kinase activity. HCI-2084 was also a potent therapeutic against Zebrafish T-ALL. Fish treated at 1uM HCI-2084 for 14 days, showed complete responses (CR), with efficacy comparable to our dexamethasone positive control (Figure C). Following treatment, fish remained disease-free for several days, and overall survival was prolonged significantly relative to untreated controls. CONCLUSION: HCI-2084 shows dual action in vitro against Jurkat cells with potent cytotoxicity via AXL/MER kinase inhibition and anti-proliferative activity via Aurora kinase inhibition. In vivo, HCI-2084 demonstrates activity against a MYC-driven vertebrate model of T-ALL, prolongs survival, and is well tolerated. We conclude that HCI-2084 is a potent dual AXL/MER kinase and Aurora kinase inhibitor which should be explored further as a potential novel therapeutic in the treatment of human T-ALL. Disclosures: Sharma: Millenium: Research Funding.