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Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 are remarkably effective therapies in chronic myeloid leukemia (CML). Despite success, TKIs do not target the CML leukemic stem cell (LSC), meaning patients must be treated for life at a high economic burden and often with significant side effects. Our previous work demonstrated a tumor suppressor role for G0/G1 switch gene 2 (G0S2) in CML, which is profoundly downregulated in TKI resistance (>3-fold, p To address this hypothesis, we first assessed ATGL protein expression in CML cell lines (K562 and KU812) in the presence and absence of the BCR-ABL TKI, imatinib. Importantly, ATGL protein expression remained unchanged in graded doses of imatinib, suggesting that ATGL expression occurs in a BCR-ABL1-independent manner. To assess the functional role of ATGL in CML and TKI resistance, we performed shRNA-mediated ATGL knockdown (shATGL) in both cell lines. Although ectopic G0S2 impaired survival of CML cell lines and patient samples, we observed a different phenotype upon ATGL knockdown. Rather, shATGL alone increased colony formation by ~30% in both cell lines in the absence but not presence of imatinib (K562, p=0.04; KU812, p=0.0024). Knockdown of ATGL had no effect on colony formation of normal cord blood CD34 + progenitors (p=0.742). Interestingly, when we ectopically expressed G0S2 into K562 cells with simultaneous ATGL knockdown, the phenotype in colony formation assays mimicked ectopic G0S2 expression, reducing survival by ~50% (p=0.05). These data suggest that the tumor suppressor role of G0S2 in CML is independent of ATGL. However, while ectopic G0S2 expression alone had no effect on apoptosis of CML cell lines, when combined with ATGL knockdown, imatinib-mediated apoptosis was markedly increased. This was similar to data observed in blast phase CML patient samples upon ectopic G0S2 expression. Consistently, RNA sequencing (RNAseq) data for CML patients revealed that ATGL mRNA is highly downregulated in blast phase (n=14) compared with patients in the chronic (n=52) or accelerated (n=11) phases of the disease (p The effect of G0S2 on lipid metabolism was further confirmed with RNAseq and metabolomics/lipidomics analyses. RNAseq revealed no overlapping pathways between K562 cells expressing ectopic G0S2 versus shATGL. Lipidomics analyses revealed that G0S2 knockdown reduced expression of tri- and di-glycerides, whereas ectopic G0S2 promoted triglyceride accumulation in K562 cells. G0S2 knockdown also resulted in substantial changes of phosphatidylethanolamine and phosphatidylcholine expression, implicating G0S2 in the production of lipid bilayer components. Finally, metabolomics data implicated a role for G0S2 as a negative regulator of the mitochondrial electron transport chain. Altogether, these findings suggest that G0S2 and lipid metabolism play a role in regulating leukemic stem and progenitor cell survival as well as TKI response in vitro. Therefore, restoring G0S2 expression and inhibiting FAO, combined with BCR-ABL1 inhibition, may be a novel clinical strategy to induce treatment-free remission in CML patients and eradicate the CML LSC. Disclosures No relevant conflicts of interest to declare.

Background Chronic phase chronic myeloid leukemia (CP-CML) is characterized by overproduction of differentiating myeloid cells, while blast phase CML (BP-CML) cells exhibit a differentiation block. Tyrosine kinase inhibitors (TKIs) are effective in CP-CML, but resistance is common in BP-CML and can occur without explanatory BCR-ABL1 kinase mutations (BCR-ABL1-independent resistance). Similarly, CML stem/progenitor cells (LSPCs) are insensitive to TKIs, and residual leukemia persists in the majority of CML patients on TKI therapy. We previously reported overlap between the transcriptomes of CD34 + cells from BP-CML and TKI-naïve CP-CML patients with primary TKI resistance, pointing to commonalities between LSPC quiescence, BCR-ABL1-independent TKI resistance, and BP-CML. Results To identify common mechanisms, we performed a meta-analysis of published CML transcriptomes. We identified a small set of genes with consistently low expression in LSPC quiescence, BCR-ABL1-independent TKI resistance, and BP-CML, including Membrane Spanning 4-Domains A3 (MS4A3), a signaling protein previously reported to inhibit hematopoietic cell cycle progression. Low MS4A3 in CD34 + cells from TKI-naïve CP-CML patients was associated with shorter survival on subsequent TKI therapy, suggesting that MS4A3 governs TKI response. To understand the function of MS4A3, we lentivirally introduced MS4A3 shRNA or an MS4A3 expression vector into CML CD34 + LSPCs. MS4A3 knockdown increased clonogenicity and imatinib resistance, while ectopic MS4A3 expression had opposite effects. MS4A3 KD also increased LSPC persistence ex vivo in LTC-IC assays, and in vivo in NSG mice xenografts, while modulating MS4A3 expression had no effect on normal CD34 + cells. We next generated Ms4a3+/+│-/-; Scl-tTA+; TRE-BCR-ABL1+ compound transgenic mice. Upon BCR-ABL1 induction, Ms4a3-/-; Scl-tTA+; TRE-BCR-ABL1+ mice developed leukocytosis comparable to Ms4a3+/+ controls. However, BM of Ms4a3-/-; Scl-tTA+; TRE-BCR-ABL1+ mice showed increased short-term HSCs and multipotent progenitor cells, and reduced granulocyte-macrophage progenitors. When Lin - BM cells from leukemic mice were transplanted into irradiated recipients, Ms4a3-/-; Scl-tTA+; TRE-BCR-ABL1+ cells showed increased engraftment and myeloid leukocytosis, validating our observations in human cells. To determine how MS4A3 is downregulated in CML, we expressed BCR-ABL1in 32D-cl3 cells. p210 BCR-ABL1 drastically reduced Ms4a3 expression, while kinase-inactive p210 BCR-ABL1-K271R had no effect. Moreover, we found that suppression of C/EBPε by MECOM reduces MS4A3, consistent with previous reports of MECOM as a driver of TKI resistance and progression to BP. Treatment of CML CD34 + cells with a library of epigenetic pathway inhibitors revealed that MS4A3 is suppressed by both DNA methylation and PRC2/EZH2-mediated H3K27 trimethylation, which was confirmed by patch-PCR and ChIPseq. These data indicate that multi-levelled mechanisms cooperate in the suppression of MS4A3 in CML. To determine how MS4A3 regulates clonogenicity and TKI response, we expressed MS4A3-EGFP fusion protein in LAMA-84 CML cells. We found that MS4A3 resides on the plasma membrane and in endosomes. Surface protein biotin labelling and tandem mass spectrometry ± MS4A3 KD showed that MS4A3 controls endocytosis of membrane proteins, including common β chain (βc) cytokine receptors. Specifically, MS4A3 promotes endocytosis of βc cytokine receptors upon GM-CSF/IL-3 stimulation of primary LSPCs and enhances downstream signaling and differentiation, suggesting that restoring MS4A3 expression has therapeutic efficacy. To test this, we manufactured a prototype MS4A3 protein-loaded liposomal nanoparticle (NP) using coating with the CD34 CD62L for targeted delivery to CD34 + cells. Compared to MS4A3-free NPs, MS4A3 NPs increased CD34 +CD38 + and CD34 -CD38 + at the expense of CD34 +CD38 - cells, reduced clonogenicity, and increased sensitivity to TKIs, mimicking ectopic MS4A3 expression. Conclusion MS4A3 governs response to differentiating myeloid cytokines, providing a unifying mechanism for the differentiation block characteristic of primitive LSPCs and BP-CML cells. We posit that LSPCs downregulate MS4A3 to evade βc cytokine-induced differentiation to maintain a primitive, TKI-insensitive state. MS4A3 re-expression or delivery of ectopic MS4A3 may eliminate LSPCs. Figure 1 Figure 1. Disclosures Druker: Aptose Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; EnLiven: Consultancy, Research Funding; Blueprint Medicines: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Aileron: Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; GRAIL: Current equity holder in publicly-traded company; Iterion Therapeutics: Membership on an entity's Board of Directors or advisory committees; Merck & Co: Patents & Royalties; Nemucore Medical Innovations, Inc.: Consultancy; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Pfizer: Research Funding; Recludix Pharma, Inc.: Consultancy; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharma: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees. Tyner: Agios: Research Funding; Astrazeneca: Research Funding; Array: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Petra: Research Funding; Seattle Genetics: Research Funding; Constellation: Research Funding; Schrodinger: Research Funding. Oehler: BMS: Consultancy; OncLive: Honoraria; Pfizer: Research Funding; Takeda: Consultancy; Blueprint Medicines: Consultancy. Radich: BMS: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Deininger: Sangamo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Fusion Pharma, Medscape, DisperSol: Consultancy; Novartis: Consultancy, Research Funding; SPARC, DisperSol, Leukemia & Lymphoma Society: Research Funding; Blueprint Medicines Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding.

FLT3-ITD+ acute myeloid leukemia (AML) accounts for ∼25% of all AML cases and is a subtype that carries a poor prognosis. microRNA-155 (miR-155) is specifically overexpressed in FLT3-ITD+ AML compared with FLT3 wild-type (FLT3-WT) AML and is critical for the growth of FLT3-ITD+ AML cells in vitro. However, miR-155's role in regulating FLT3-ITD-mediated disease in vivo remains unclear. In this study, we used a genetic mouse model to determine whether miR-155 influences the development of FLT3-ITD-induced myeloproliferative disease. Results indicate that miR-155 promotes FLT3-ITD-induced myeloid expansion in the bone marrow, spleen, and peripheral blood. Mechanistically, miR-155 increases proliferation of the hematopoietic stem and progenitor cell compartments by reducing the growth-inhibitory effects of the interferon (IFN) response, and this involves targeting of Cebpb. Consistent with our observations in mice, primary FLT3-ITD+ AML clinical samples have significantly higher miR-155 levels and a lower IFN response compared with FLT3-WT AML samples. Further, inhibition of miR-155 in FLT3-ITD+ AML cell lines using CRISPR/Cas9, or primary FLT3-ITD+ AML samples using locked nucleic acid antisense inhibitors, results in an elevated IFN response and reduces colony formation. Altogether, our data reveal that miR-155 collaborates with FLT3-ITD to promote myeloid cell expansion in vivo and that this involves a multitarget mechanism that includes repression of IFN signaling.

Introduction: Hispanics represent the largest minority group in the United States (U.S.), with 57.5 million individuals (18% of the population). Most U.S. Hispanics are of Mexican origin (63.2%), followed by Puerto Rican (9.5%), Cuban (3.9%), Salvadoran (3.8%), and Dominican (3.3%), but distribution varies by state. The majority of Hispanics in the U.S. reside in the Southwest region, and >11 million live in the state of Texas. Cancer is the leading cause of death in the Hispanic population, accounting for 21% of deaths in people of all ages. Health disparities for Hispanic cancer patients have previously been linked to disproportionate poverty and other barriers to optimal healthcare, and in the case of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), Hispanics were found to be diagnosed younger and to have worse overall survival (OS) than Non-Hispanic whites (NHWs) (ACS. Cancer Facts & Figures for Hispanics/Latinos 2018-2020). However, little is known about incidence and survival for Hispanic blood cancer patients residing at the U.S./Mexico border. To understand the impact of Hispanic ethnicity on outcomes for blood cancer patients diagnosed in this area, we examined OS in adult patients with hematologic malignancies throughout the state of Texas compared to Texas Health Service Region (HSR) 10, encompassing El Paso County. Methods: We retrospectively reviewed data available from the Texas Cancer Registry for hematologic malignancies diagnosed in the state of Texas between 1995 and 2016, focusing our analysis on chronic and acute leukemias (both myeloid and lymphoid), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPNs). Survival for Hispanic and NHW groups was compared using the log-rank test, and Cox regression analyses adjusting for age and diagnosis. Differences in age at diagnosis were evaluated using t-tests and generalized linear models. Similar analyses compared Hispanic patients from HSR 10 versus Hispanic patients from the rest of Texas. Research was conducted according to a local Institutional Review Board-approved protocol in accordance with the Declaration of Helsinki. Results: Of the 69,941 cases of hematologic malignancies with available information throughout the state of Texas, 18.29% self-identified as Hispanic. Surprisingly, in unadjusted analyses, Hispanic patients had significantly better OS than NHWs diagnosed with AML (p Conclusions: While Hispanic patients with AML, MDS, and CML had significantly better OS compared to NHWs in Texas as a whole, this could be explained by a significant reduction in the age of diagnosis for Hispanics. However, when comparing across Texas, El Paso Hispanics with ALL, AML, and CML have a worse prognosis than in the rest of the state. There appears to be evidence that disparities in outcome by ethnicity may be different in El Paso compared with the rest of Texas. Further study is required to identify factors responsible for the disparity in OS. Disclosures No relevant conflicts of interest to declare.

Recently, we showed that increased miR-181a expression was associated with improved outcomes in cytogenetically normal acute myeloid leukemia (CN-AML). Interestingly, miR-181a expression was increased in CN-AML patients harboring CEBPA mutations, which are usually biallelic and associate with better prognosis. CEBPA encodes the C/EBPα transcription factor. We demonstrate here that the presence of N-terminal CEBPA mutations and miR-181a expression are linked. Indeed, the truncated C/EBPα-p30 isoform, which is produced from the N-terminal mutant CEBPA gene or from the differential translation of wild-type CEBPA mRNA and is commonly believed to have no transactivation activity, binds to the miR-181a-1 promoter and up-regulates the microRNA expression. Furthermore, we show that lenalidomide, a drug approved for myelodysplastic syndromes and multiple myeloma, enhances translation of the C/EBPα-p30 isoform, resulting in higher miR-181a levels. In xenograft mouse models, ectopic miR-181a expression inhibits tumor growth. Similarly, lenalidomide exhibits antitumorigenic activity paralleled by increased miR-181a expression. This regulatory pathway may explain an increased sensitivity to apoptosis-inducing chemotherapy in subsets of AML patients. Altogether, our data provide a potential explanation for the improved clinical outcomes observed in CEBPA-mutated CN-AML patients, and suggest that lenalidomide treatment enhancing the C/EBPα-p30 protein levels and in turn miR-181a may sensitize AML blasts to chemotherapy.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a poor prognosis. FLT3 internal tandem duplications (ITDs) are found in ~30% of AML patients and are associated with inferior survival. Addition of FLT3 tyrosine kinase inhibitors (TKIs) such as midostaurin improve survival of AML treated with cytotoxic chemotherapy. However, relapse rates remain unacceptably high. Protection by stroma-derived survival signals is thought to enable survival of leukemia initiating cells (LICs) in the presence of FLT3 TKIs, providing a reservoir for subsequent overt relapse. We previously reported that HS-5 conditioned medium (CM) rescued FLT3-ITD positive AML cell lines and primary cells from the effects of the 2nd generation FLT3 TKI AC220 (quizartinib) as compared to cells cultured in regular medium (RM). Earlier work from our lab and others has demonstrated that HS-5 CM increases pSTAT3Y705 in chronic myeloid leukemia cells treated with BCR-ABL1 TKIs, leading to TKI resistance, while pSTAT5Y694 levels remain under the control of the BCR-ABL1 kinase (Eiring et al, Leukemia, 2015). Similar to BCR-ABL1, FLT3 signaling results in potent pSTAT5Y694 activation. We hypothesized that STAT3 and/or STAT5 activation contributes to stroma-mediated protection of FLT3-ITD+ AML cells upon FLT3 inhibition with AC220. We found that FLT3-ITD+ cell lines and primary cells grown in HS-5 CM exhibited strong upregulation of pSTAT3Y705 and pSTAT5Y694 that was unaffected by treatment with 10nM AC220. In RM, pSTAT3Y705 was undetectable irrespective of AC220 dose and pSTAT5Y694 was abolished by 10nM AC220. shRNA knockdown experiments of STAT3 and STAT5 in FLT3-mutated cell lines grown in CM and treated with AC220 showed that STAT5 knockdown alone consistently resulted in greater inhibition of cell proliferation than STAT3 knockdown alone. This suggests that in FLT3-ITD+ AML cells cultured in CM, upregulation of pSTAT5Y694 by stroma-derived soluble factors is the main contributor to AC220 resistance. We decided to use a candidate approach to identify the upstream kinases leading to pSTAT5Y694, focusing on inhibitors of pathways previously implicated in STAT5 activation in AML. We used phosphoflow to quantify pSTAT5Y694 in MOLM-13 cells grown for 24 hours in CM in the presence of 10nM AC220 combined with ruxolitinib, dasatinib, ibrutinib, PD173074 (FGFR1i) or PRT062607 (SYKi). Results from this screen indicated that the combination of dasatinib and AC220 most effectively decreased pSTAT5Y694 in MOLM-13 cells cultured in CM (minimum dasatinib dose =100nM). To further validate dasatinib as a candidate for combination drug treatment, we performed cell proliferation experiments in MOLM-13 cells cultured in CM for 72h with graded concentrations of AC220 and the candidate inhibitors at a minimum candidate drug dose of 100nM. Of the four inhibitors tested, the combination of dasatinib and AC220 most significantly decreased the IC50 of AC220 in MOLM-13 cells grown in CM (IC50 AC220 alone: 11.09nM; IC50 AC220 + 100nM dasatinib: 0.41nM; p It has recently been reported that FLT3-ITD AML cells harbor a highly glycolytic phenotype that can be partially suppressed by FLT3 TKIs (Huntly et al, Blood, 2018). Our preliminary Seahorse experiments show that the combination of dasatinib and AC220 more effectively inhibits glycolysis in MOLM-13 cells than AC220 alone, with differential modulation in CM versus RM. These results suggest that dasatinib inhibits a metabolic pathway critical to maintaining glycolysis in MOLM-13 cells challenged by AC220-based FLT3 inhibition. Moreover, these data indicate that extrinsic signals contribute to metabolic reprogramming of FLT3+ AML cells, enabling survival despite FLT3 inhibition. Experiments are underway to delineate how CM contributes to metabolic resistance and how dasatinib intercepts this survival signal. Our results support the concept of combining AC220 with dasatinib to reduce residual leukemia in FLT3+ AML and prolong survival. Further investigation is ongoing and will be reported at the time of abstract presentation. Disclosures Deininger: Blueprint: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Several RNA binding proteins (RBPs) have been implicated in the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase to the aggressively fatal blast crisis. In the latter phase, expression and function of specific RBPs are aberrantly regulated at transcriptional or posttranslational levels by the constitutive kinase activity of the BCR/ABL oncoprotein. As a result, altered expression/function of RBPs leads to increased resistance to apoptotic stimuli, enhanced survival, growth advantage, and differentiation arrest of CD34+ progenitors from patients in CML blast crisis. Here, we identify the mRNAs bound to the hnRNP-A1, hnRNP-E2, hnRNP-K, and La/SSB RBPs in BCR/ABL-transformed myeloid cells. Interestingly, we found that the mRNA encoding the transcription factor E2F3 associates to hnRNP-A1 through a conserved binding site located in the E2F3 3′ untranslated region (UTR). E2F3 levels were upregulated in CML-BCCD34+ in a BCR/ABL kinase- and hnRNP-A1 shuttling-dependent manner. Moreover, by using shRNA-mediated E2F3 knock-down and BCR/ABL-transduced lineage-negative bone marrow cells from E2F3+/+ and E2F3-/- mice, we show that E2F3 expression is important for BCR/ABL clonogenic activity and in vivo leukemogenic potential. Thus, the complexity of the mRNA/RBP network, together with the discovery of E2F3 as an hnRNP-A1-regulated factor, outlines the relevant role played by RBPs in posttranscriptional regulation of CML development and progression. © 2008 by The American Society of Hematology.

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.

Myelofibrosis (MF) is a hematopoietic stem cell neoplasm characterized by constitutive activation of JAK/STAT signaling due to mutations in JAK2, calreticulin or MPL. Many MF patients suffer from severe constitutional symptoms and have reduced life expectancy due to cytopenias, progression to acute myeloid leukemia and thromboembolic events. JAK kinase inhibitors such as ruxolitinib (RUX) reduce MF symptoms, but like all other drugs used in MF, are not curative, with persistence of mutant cells and prompt symptom rebound upon discontinuation. This defines a clinical need to identify strategies capable of inducing more profound and durable responses in MF. To identify previously unrecognized molecular vulnerabilities in MF, we infected HEL cells (homozygous for JAK2V617F) with a barcoded lentiviral shRNA library targeting ~5,000 human signal transduction genes, with 5-6 shRNAs/gene (Cellecta Human Module 1). Conditions were optimized to achieve a multitude of infection (MOI) of ~1. Barcode abundance was compared between days 0 and 9 after infection by next generation sequencing. Candidates were selected based on ≥ 15-fold reduction of abundance by ≥ 2 shRNAs targeting the same gene, similar to Khorashad et al. [Blood. 2015;125(11):1772-81]. Amongst the genes meeting these criteria, nuclear cytoplasmic transport (NCT) was significantly enriched, with RAN and RANBP2 amongst the top genes, suggesting that HEL cells may be highly dependent on NCT. For confirmation, HEL cells were stably transduced with doxycycline (DOX)-inducible shRAN. After 72 hours DOX-induced knockdown of RAN reduced viable cells by 77.3±5.5% and colony formation by 82.8±1.3% and dramatically increased apoptosis (uninduced: ~10% vs. induced: ~50%). Similar results were observed in SET-2 cells (heterozygous for JAK2V617F). We next cultured HEL and SET-2 cells with graded concentrations of the KPT-330 (selinexor, Karyopharm), an inhibitor of CRM-1, the core component of NCT, or RUX as a comparison. Selinexor was five-fold more potent than RUX against HEL cells (IC50: 98nM for KPT vs. 536 nM for RUX) and as potent as RUX in SET-2 cells (IC50:~100 nM). Importantly, RUX-resistant HEL cells (IC50:24µM) were highly sensitive to inhibition of NCT by knockdown of RAN or selinexor (IC50:160nM). Selinexor also selectively inhibited colony formation by primary MF vs. cord blood (CB) CD34+ cells (IC50:93nM for MF vs. 203nM for CB). Lastly, selinexor enhanced RUX-induced growth inhibition and apoptosis in primary MF CD34+cells cultured ex vivo for 72h (including both JAK2 mutation positive and negative MF samples, n=3 for each, and RUX resistant patient samples, n=6). Nuclear:cytoplasmic fractionation of HEL cells revealed that the expression and nuclear localization of the tumor suppressors FoxO3A and APC, but not of PP2A and nucleophosmin (NPM) were significantly increased upon knockdown of RAN, which may contribute to the increased apoptosis following NCT inhibition. To determine the in vivo effects of selinexor in MF, we induced MPN in Balb/c mice by transplanting donor marrow infected with JAK2V617F for three weeks, and then treated mice (n=13/group) with vehicle, selinexor (initial dose 20 mg/kg, 3x weekly, orally) or RUX (initial dose 50 mg/kg twice daily, orally) or the combination of RUX plus selinexor for up to 4 weeks. Combination treatment significantly reduced white blood cell counts and normalized spleen size. Compared to vehicle, selinexor alone significantly reduced GFP+cells in the spleen, and this effect was further enhanced with the combination treatment. Histopathology revealed that combination treatment restored splenic architecture, while bone marrow fibrosis was not significantly altered by selinexor or the combination. Mice in all groups, including the combined vehicle controls, experienced considerable weight loss, suggesting that toxicity may be partially due to high dose and frequent drug administration. Experiments with the next generation NCT inhibitor KPT-8602 [Etchin et al., Leukemia, 2016 Jun 24] are underway. In summary, our results suggest that MF cells are exquisitely dependent on NCT, and that NCT inhibition alone or in combination with RUX may reduce JAK2V617F allelic burden. This identifies NCT as a prime therapeutic target in MF. A phase I clinical trial of selinexor in refractory MF is in preparation. Disclosures Baloglu: Karyopharm Therapeutics: Employment, Equity Ownership. Deininger: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; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; 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.

Background: We have previously demonstrated that the transcriptional profile of diagnostic CD34+ cells from chronic phase chronic myeloid leukemia (CP-CML) patients exhibiting primary cytogenetic resistance to imatinib overlaps with that of patients with myeloid blast phase CML (BP-CML) (McWeeney et al. Blood 2010). These data suggest that primary resistance to tyrosine kinase inhibitors (TKIs) and advanced disease are biologically related. The hematopoietic cell cycle regulator, MS4A3, was identified as a principal component of the gene expression classifier predicting response to imatinib. Low MS4A3 correlated not only with primary TKI resistance, but also with shorter overall survival in CP-CML (n=35). Consistently, microarray (n=19 CP-CML; n=16 BP-CML), qRT-PCR (n=22 CP-CML; n=17 BP-CML), and immunoblot (n=3 CP-CML; n=3 BP-CML) analyses demonstrated that MS4A3 mRNA and protein levels are reduced in CD34+ progenitor cells from BP-CML versus CP-CML patients, with no difference between CP-CML and normal CD34+progenitors (n=3) (Eiring et al. ASH 2015 #14). These data suggest that MS4A3 may play a role in both primary TKI resistance and blastic transformation of CML. Results: To assess the functional role of MS4A3 in CML and TKI response, we used ectopic MS4A3 expression and shRNA-mediated MS4A3 knockdown in CD34+ cells from BP-CML and CP-CML patients, respectively. Ectopic expression of MS4A3 in BP-CML CD34+ progenitors (n=5) markedly reduced colony formation in the presence and absence of imatinib, consistent with a tumor suppressor role for MS4A3 in CML. While re-expression of MS4A3 alone did not increase apoptosis compared to empty vector-expressing controls, imatinib-induced apoptosis in BP-CML CD34+ cells was increased by 62%, with no effect on normal CD34+ cord blood cells (n=2). Conversely, shRNA-mediated MS4A3 knockdown (shMS4A3) in CP-CML CD34+ cells (n=7) reduced the effects of imatinib in colony formation and apoptosis assays, with no effect on normal CD34+ progenitors (n=4). In contrast to a previous report (Donato JL, et al. J Clin Invest 2002), we detected no change in cell cycle status of CML or normal CD34+ cells upon MS4A3 ectopic expression or knockdown (n=3). Altogether, these data suggest that MS4A3 positively regulates patient survival and imatinib response in CML progenitor cells. To evaluate MS4A3 in the leukemic stem cell compartment, we performed qRT-PCR on primary CP-CML cells (n=5) and observed that MS4A3 mRNA levels are 22-fold higher in committed CD34+38+ progenitors compared to more primitive CD34+38- stem cells, suggesting a role for MS4A3 in differentiation. Consistently, qRT-PCR, immunoblot, and flow cytometry demonstrated that MS4A3 mRNA and protein were upregulated in CP-CML CD34+ cells upon G-CSF treatment (n=3). Flow cytometry also revealed that shMS4A3 in CP-CML CD34+ cells resulted in a reduction of CD11b+ cells by ~45% in the presence of G-CSF (n=3). To assess the function of MS4A3 in CML stem cells, we performed long-term culture-initiating cell (LTC-IC) assays and xenografts into NSG mice upon MS4A3 knockdown in CP-CML (n=3). shMS4A3 increased Ph+ LTC-IC colony formation in the absence, and even more so in the presence, of imatinib, with no effects on Ph- LTC-ICs. Consistent with these data, shMS4A3 enhanced engraftment of CD34+CD45+GFP+ cells into the bone marrow of NSG recipient mice. Preliminary data in primary TKI-resistant and BP-CML CD34+ cells suggests regulation of this gene by promoter hypermethylation. Conclusions: Altogether, these data suggest that MS4A3 plays a key role in imatinib response of 1) patients with primary TKI resistance, 2) patients with BP-CML, and 3) the CML stem cell compartment. Since the effects of MS4A3 in CML do not involve changes to the cell cycle, experiments are underway to identify the mechanism by which MS4A3 improves imatinib response and survival in CML. Disclosures Druker: Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy. Deininger:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; 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; 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.

Introduction. Development of abnormal Philadelphia (Ph) negative clones following treatment of chronic myeloid leukemia (CML) patients with imatinib has been observed in 3 to 9% of patients. Here we report on a 77 year old male diagnosed with CML that responded to imatinib treatment and subsequently developed chronic myelomonocytic leukemia (CMML). He achieved major cytogenetic response within 3 months but this response coincided with the emergence of monocytosis diagnosed as CMML. Five months after starting imatinib treatment the patient succumbed to CMML. We analyzed five sequential samples to determine whether a chronological order of mutations defined the emergence of CMML and to characterize the clonal evolution of the CMML population. Materials and Method. Five samples (diagnostic and four follow up samples) were available for analysis. CMML mutations were identified by whole exome sequencing (WES) in CD14+ cells following the onset of CMML, using CD3+ cells as constitutional control. Mutations were validated by Sequenom MassARRAY and Sanger sequencing and quantified by pyrosequencing. Deep WES was performed on the diagnostic sample to determine whether the mutations were present at CML diagnosis. To determine the clonal architecture of the emerging CMML, colony formation assays were performed on the diagnostic and the next two follow-up samples (Samples 1-3). More than 100 colonies per sample were plucked for DNA and RNA isolation. The DNA from these colonies were tested for the presence of the confirmed CMML mutations and the RNA was used for detection of BCR-ABL1 transcript using a Taqman real time assay. Results. Four mutations were identified by Sequenom and WES throughout the patient's time course [KRASG12R, MSLNP462H, NTRK3V443I and EZH2I669M ]. Sequenom did not identify these at diagnosis while deep WES did. Clones derived from colony formation assay revealed three distinct clones present in all samples analysed. Clone 1 had only KRASG12R, clone 2 had KRASG12R, MSLNP462H, and NTRK3V443I, and clone 3 had all four mutations. All clones containing any of these four mutations were BCR/ABL1 negative. Analysis of clonal architecture indicated that KRASG12R was acquired first and EZH2I669M last, while MSLNP462H and NTRK3V443I were acquired in between. These CMML clones increased proportionately as clinical CML metamorphosed into clinical CMML after initiation of imatinib therapy. Consistent with the colony data, pyrosequencing revealed that the ratio between the mutants remained largely stable throughout the follow up period. Conclusion. This case illustrates how targeted therapy impacts clonal competition in a heterogeneous MPN. While the CML clone was dominant in the absence of imatinib, it was quickly outcompeted by the CMML clones upon initiation of imatinib therapy. The clonal architecture analysis, in combination with in vivo kinetics data, suggest that the KRASG12R mutation alone was able to produce a CMML phenotype as clones with just KRASG12R remained at a relatively stable ratio during follow up. Unexpectedly, acquisition of additional mutations, including EZH2I669M as the last mutational event identified in this patient, did not increase clonal competitiveness, at least in the peripheral blood. These data show that clonal evolution may not invariably increase clonal fitness, suggesting that factors other than Darwinian pressures contribute to clonal diversity in myeloproliferative neoplasms. Disclosures Deininger: Gilead: Research Funding; Bristol-Myers Squibb: 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; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Background: Mutations in the BCR-ABL1 kinase domain are a well-documented mechanism of resistance to tyrosine kinase inhibitors (TKIs), but less is known about primary resistance independent of BCR-ABL1 kinase activity. We reported a gene expression classifier of TKI-naïve CD34+ cells from chronic phase chronic myeloid leukemia (CP-CML) patients that predicts cytogenetic response to imatinib (McWeeney et al. Blood 2010). The expression signature associated with primary cytogenetic failure showed overlap with previously reported signatures of blast phase CML (BP-CML), suggesting that primary TKI resistance and advanced disease are biologically similar. Results: To identify critical genes involved in primary TKI resistance, we performed principal component analysis on the expression signature and identified the hematopoietic cell cycle regulator, MS4A3, as a key factor within this classifier. Importantly, low MS4A3 expression not only correlated with primary TKI resistance, but also with shorter overall survival (p92%) in CD34+ cells from BP-CML patients (n=17; p MS4A3 expression is also low in BP-CML cell lines, including K562, KYO-1, BV-173, KCL-22, and KU-812, with the notable exception of LAMA-84 cells. Thus, to understand the functional role of MS4A3 for TKI resistance, we introduced a doxycycline-inducible shRNA targeting MS4A3 (shMS4A3) into LAMA-84 cells. qRT-PCR confirmed 50-90% MS4A3 knockdown in the presence of doxycycline (0.1 µg/mL). Consistent with its role as a tumor suppressor, MTS assays revealed that MS4A3 knockdown increased the imatinib IC50 (n=3; p2-fold upregulation of MS4A3. As expected, ectopic MS4A3 reduced colony formation by 55% in AP-CML (n=2; p Conclusion: Our results suggest that MS4A3 is a tumor suppressor protein in CML that governs TKI responsiveness and is regulated in a BCR-ABL1 kinase-independent manner. MS4A3 loss confers TKI resistance to CP-CML patients destined to exhibit primary cytogenetic failure, and in BP-CML patients with refractory resistance. MS4A3 may also contribute to the innate resistance of primitive CML stem cells. Studies to identify the mechanism of MS4A3 downregulation in TKI resistance and how its loss biochemically impairs TKI response is currently underway and will be reported. Disclosures Agarwal: CTI BioPharma: Research Funding. Deininger:BMS: Other: Consulting & Advisory Role, Research Funding; Novartis: Other: Consulting or Advisory Role, Research Funding; Celgene: Research Funding; Genzyme: Research Funding; Gilead: Research Funding; ARIAD Pharmaceutical Inc.: Other: Consulting or Advisory Role; Incyte: Other: Consulting or Advisory Role; Pfizer: Other: Consulting or Advisory Role.

Purpose: Chronic myelomonocytic leukemia (CMML) is a clinically heterogeneous myelodysplastic/myeloproliferative neoplasm with short overall survival. Emerging data based on sequencing of candidate genes with a known role in myeloid leukemia have identified recurrent CMML mutations, some of which have been associated with poor prognosis. A comprehensive evaluation of the mutational landscape of CMML and its prognostic significance is lacking. Patients and Methods. We comprehensively characterized the mutational landscape of CMML by a 2-step design. We initially performed whole exome sequencing (WES) of paired leukemia and germline DNA from 21 patients with a confirmed CMML diagnosis (discovery cohort) to identify genes with somatic mutations. From this discovery cohort, 215 genes showing potential mutations as well as 61 genes selected from the literature were examined by targeted resequencing in a second cohort of 69 clinically annotated CMML patients, using two independent platforms for orthogonal confirmation Blood or marrow samples from 22 young and 17 old controls were included as controls. Results. We identified 22 genes with mutations in >3% of CMML patients, and 67/69 patients (97%) had one or more mutations in at least one of these genes. SRSF2, ASXL1 and TET2 were the most frequently mutated genes. Several novel CMML genes were identified, including FAT4 with a mutation prevalence of 10% and BCR, CBFA2T3, and TRPM1 with a prevalence of 3 – 9% (see Table). Total deleterious mutations per patient ranged from 0 – 11, with significant exclusion or association of various combinations of mutations in SETBP1, ASXL1, KRAS, TET2, and EZH2 observed. In univariate analysis hemoglobin < 9g/dL, white blood cell count > 15x109/L, no treatment with hypomethylating agents, mutations in ASXL1, EZH2, or NRAS and mutations in growth factor signaling genes were associated with shorter overall survival. In multivariate analysis, mutations in NRAS (P Conclusion. (i) The mutational landscape of CMML is complex and involves mutations in multiple genes, many affected with relatively low prevalence. (ii) Mutations in FAT4, a putative tumor suppressor and key regulator of the Hippo pathway, occur in approximately 10% of patients. (iii) Absence of therapy with hypomethylating agents and mutations in NRAS or the functional group of growth factor signaling genes are predictive of poor survival. Table. Count and prevalence of N=69 CMML patients having a deleterious mutation in one of the genes observed mutated at a prevalence ≥ 10%. Gene # CMML Patients with Mutation (%) SRSF2 34 (49%) TET2 28 (41%) ASXL1 25 (36%) RUNX1 14 (20%) SETBP1 11 (16%) KRAS 10 (14%) EZH2 8 (12%) FAT4 7 (10%) CBL 7 (10%) NRAS 7 (10%) Figure. Overall survival of N=48 CMML patients by HMA use and presence of deleterious mutation in NRAS. Figure. Overall survival of N=48 CMML patients by HMA use and presence of deleterious mutation in NRAS. Disclosures Mason: Agilent, Inc.: Research Funding. Deininger:Celgene: Research Funding; Agilent, Inc.: Research Funding.

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.

Background: The treatment and survival of chronic myeloid leukemia (CML) patients has greatly improved after the discovery of imatinib; however, disease persistence and drug resistance remain as clinical problems. McWeeney et al. (Blood 2010;115:315-325) identified a gene expression signature predictive of primary cytogenetic resistance to imatinib in treatment-naïve CML chronic phase (CML-CP) patients lacking BCR-ABL1 kinase domain mutations. Comparison of this gene classifier with other studies revealed extensive overlap of resistance genes with genes associated with CML blastic transformation, suggesting that CML-CP patients destined to fail imatinib may exhibit a gene profile reminiscent of advanced CML. Based on rank predictive score from the microarray, the top transcripts found to be dysregulated in newly diagnosed patients who subsequently emerged as imatinib non-responders were: PLCXD2, EGF16, GAS2, RXFP1, ITGA2, MS4A3, FCN1, EMCN, EMCN, CLIP4, ZNF44 and G0S2. Among these, MS4A3 and G0S2 were differentially downregulated in non-responders compared to responders. Conversely, high levels of MS4A3 (p=0.059) and G0S2 (p=0.036) correlated with higher likelihood of major cytogenetic response and longer overall survival. MS4A3 (HTM4) is a hematopoietic cell cycle regulator that inhibits G1/S phase cell cycle transition, whereas G0S2 is proapoptotic mitochondrial protein that interacts with and antagonizes BCL-2. In this study, we investigated the potential role of MS4A3 and G0S2 as tumor suppressors in CML and their influence on TKI resistance and blastic transformation. MS4A3 and CML: Expression of p210BCR-ABL1 in 32Dcl3 or Mo7e myeloid progenitor cells resulted in an 80% reduction of MS4A3 mRNA relative to parental cells by qRT-PCR analysis. Imatinib treatment slightly restored MS4A3 levels in 32D-p210 or Mo7e-p210 cells, but did not return levels to those of normal controls growing with cytokine support. Consistent with a role for MS4A3 in CML blastic transformation, qRT-PCR revealed low levels of MS4A3 in cell line models of blastic phase CML (CML-BP), including K562, KYO-1, and KBM, that were unaffected by treatment with imatinib. Furthermore, qRT-PCR confirmed that MS4A3 is downregulated (~20-fold) in CML CD34+ progenitor cells from CML-BP (n=3) compared to CML-CP (n=5) patients and normal controls (n=3), and that these levels were unaffected by imatinib. We then used tetracycline-inducible shRNA directed against MS4A3 (shMS4A3) to knockdown MS4A3 in primary CML CD34+ cells from newly diagnosed CML-CP patients subsequently responding to TKIs. Western blot and qRT-PCR analyses confirmed MS4A3 downregulation upon exposure to doxycyline (0.1 ug/mL). shMS4A3 upregulated colony formation by 37.6% (p G0S2 and CML: Consistent with a role for G0S2 in CML blastic transformation, qRT-PCR revealed that G0S2 mRNA is highly downregulated (~24-fold) in CML CD34+ progenitor cells from CML-BP (n=3) compared to CML-CP (n=5) patients and normal controls (n=3). G0S2 is also downregulated in TKI-resistant K562R and AR230R cells compared to parental TKI-sensitive counterparts. K562R and AR230R cells are resistant to all clinically approved TKIs, but lack BCR-ABL1 kinase domain mutations, implicating BCR-ABL1 kinase-independent TKI resistance. Ectopic expression of a Flag-tagged G0S2 (G0S2-Flag) significantly reduced colony formation in both parental K562 and AR230 cells, but had an even greater effect in TKI-resistant K562R and AR230R cells in the presence of imatinib. G0S2-Flag also impaired colony formation of CML-CP CD34+cells in both the presence (p Conclusions:These findings suggest a role for loss of MS4A3 or G0S2 tumor suppressor function in both TKI resistance in the absence of explanatory BCR-ABL1 kinase domain mutations and in CML blastic transformation. Studies to test the effects of restored MS4A3 or G0S2 expression in CML-BP and TKI-resistant patient samples are currently underway. 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.

Activation of JAK2, frequently as a result of the JAK2V617F mutation, is a characteristic feature of the classical myeloproliferative neoplasms (MPN) polycythemia vera, essential thrombocythemia and myelofibrosis and thought to be responsible for the constitutional symptoms associated with these diseases. BMS-911543 is a JAK2 selective inhibitor that induces apoptosis in JAK2-dependent cell lines and inhibits the growth of CD34+ progenitor cells from patients with V617F+ MPN. To explore the clinical potential of this inhibitor, we tested BMS-911543 in a murine retroviral transduction – transplantation model of JAK2V617F MPN. Treatment was initiated at two dose levels (3 mg/kg and 10 mg/kg) when the hematocrit exceeded 70%. Following the first week, white blood cell counts were reduced to normal in the high dose group and were maintained well below the vehicle-treated mice throughout the study. However, BMS-911543 had no effect on red cell parameters. After 42 days of treatment, the proportion of JAK2V617F - positive cells in hematopoietic tissues was identical to controls or slightly increased compared to controls. Plasma concentrations of IL-6, IL-15, and TNFα were elevated in MPN mice and reduced in the high dose treatment group, while other cytokines were unchanged. Collectively, these results show that BMS-911543 has limited activity in this murine model of JAK2V617F – driven MPN and suggest that targeting JAK2 alone may be insufficient to achieve effective disease control. 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.

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.

A β-catenin expression signature is associated with primary resistance to tyrosine kinase inhibitors (TKIs) (McWeeney et al. 2010), but its role in TKI resistance is not completely understood. To assess the role of β-catenin in TKI resistance, we used shRNA targeting β-catenin (shβcat) in in vitro models of BCR-ABL1 kinase-independent resistance. To model resistance in the absence of bone marrow (BM)-derived factors, we used TKI-resistant K562R cells that are adapted for growth in the presence of imatinib, as well as primary CD34+ progenitors from CML patients who had failed treatment with two or more TKIs. These cells are cultured in regular medium (RM), proliferate in 1.0-2.5 μM imatinib, and exhibit β-catenin expression that is independent of BCR-ABL1 kinase activity. To model resistance mediated by the BM microenvironment, we cultured TKI-sensitive parental K562 cells and CD34+ progenitors from newly diagnosed CML patients in direct contact (DC) with HS-5 BM stromal cells. HS-5 co-culture increases β-catenin protein levels and clonogenic potential by >3-fold despite continued suppression of BCR-ABL1 kinase activity. All cells lack BCR-ABL1 kinase domain mutations and undergo TKI-mediated kinase inhibition as detected by immunoblot analyses. CML cell lines and primary cells with BCR-ABL1-independent resistance were lentivirally transduced with shβcat or a scrambled control (shSCR), and knockdown was confirmed by immunoblot and/or qRT-PCR. In RM, shβcat reduced the clonogenicity of TKI-sensitive K562 cells and CD34+ cells from newly diagnosed CML patients by 49% and 39%, respectively, compared to shSCR controls. In TKI-resistant K562R cells and CD34+ cells from TKI-resistant CML patients, shβcat reduced clonogenicity by 60% and 50%, respectively, in the presence or absence of imatinib (0-10 μM), suggesting a role for β-catenin in the development or maintenance of TKI resistance. In contrast to cells grown in RM, clonogenicity of cell lines and patient samples cultured in HS-5 DC was unaffected by shβcat compared to imatinib alone. Immunoblot analyses revealed that β-catenin protein levels were fully restored in HS-5 DC, despite the continued presence of shβcat. qRT-PCR revealed that while cells in HS-5 DC have high amounts of β-catenin protein, the mRNA levels remained similar to shβcat-expressing cells cultured in RM, consistent with post-translational stabilization of β-catenin. Importantly, increased β-catenin was not observed when cells were cultured in HS-5 conditioned medium, indicating that stabilization requires DC with the bone marrow stroma. These data are consistent with a role for β-catenin in TKI resistance mediated by DC with the BM microenvironment, similar to a recent report (Zhang et al., 2013). To understand nuclear versus cytoplasmic distribution following HS-5 DC, CD34+ cells from newly diagnosed CML patients were cultured in RM or HS-5 DC for 36 hours and analyzed for β-catenin localization by immunfluorescence. As expected, cells cultured in RM had low levels of β-catenin in the nucleus and cytoplasm that decreased upon treatment with imatinib (2.5 μM). In contrast, cells cultured in HS-5 DC had a marked increase of β-catenin that was unaffected by treatment with imatinib. While detectable in the nucleus, the majority of β-catenin protein was localized in the cytoplasm and at the cell membrane, consistent with its role in cell-cell junctions. Accordingly, in CD34+ cells from newly diagnosed CML patients, an N-cadherin blocking antibody impaired the clonogenic potential of cells cultured in HS-5 DC, with no significant effect on cells grown in RM. Affymetrix Human Gene 1.0 ST arrays revealed high levels of genes encoding the CDH2 and CDH13 cadherins, which may be involved in N-cadherin-mediated β-catenin stabilization, even in the presence of shβcat. Preliminary data also suggests that HS-5 DC reduces luciferase reporter activity from a construct harboring sequential β-catenin binding elements (pGF1-Lef/Tcf-eFGP-luc), further supporting a role for cytoplasmic β-catenin in TKI resistance. These data demonstrate a critical role for β-catenin in BCR-ABL1 kinase-independent TKI resistance, and suggest new strategies for targeting TKI resistance in the absence of BCR-ABL1 kinase domain mutations. Disclosures: Deininger: Bristol-Myers Squibb: Advisory Boards Other, Consultancy, Research Funding; Ariad Pharmaceuticals: Advisory Boards, Advisory Boards Other, Consultancy; Novartis: Advisory Boards, Advisory Boards Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding.

Mutations in the BCR-ABL1 kinase domain are a well-established mechanism of tyrosine kinase inhibitor (TKI) resistance, but fail to explain many cases of clinical TKI failure. In the remaining patients, resistance occurs via activation of alternative signaling pathways that maintain survival despite BCR-ABL1 inhibition (BCR-ABL1-independent resistance). STAT3 mediates TKI resistance in chronic myeloid leukemia (CML) cells cultured in the presence of bone marrow-derived factors (Bewry et al., 2008; Traer et al., 2012; Nair et al., 2012), and also plays a critical role in survival of CML cells with BCR-ABL1-independent resistance (Eiring et al. #31, ASH 2012). While targeting transcription factors is notoriously difficult, our combination of synthetic chemistry, in vitro reporter assays, and computational modeling has led to a low micromolar mechanism-based STAT3 inhibitor, which, in combination with TKIs, shows promise as a treatment for CML patients with BCR-ABL1-independent resistance. The original compound of the series, SF1-066 (10 µM; Fletcher et al., 2009), combines with TKIs to reduce survival of CML CD34+ cells exhibiting BCR-ABL1-independent resistance (Eiring et al. #31, ASH 2012). To improve the potency and selectivity of SF1-066, we synthesized successive STAT3 inhibitor libraries and ranked candidates by structure-activity relationship using a luciferase-based reporter screen (Kraft et al. #2445, ASH 2012). This reporter assay quantifies STAT3 transcriptional activity in TKI-resistant AR230R cells, which grow in the continuous presence of imatinib (1.0 µM), lack BCR-ABL1 kinase domain mutations, and exhibit high levels of pSTAT3Y705, thereby enabling convenient, high-throughput screening for potency and selectivity in the context of endogenous STAT3 activation. Among three sequential STAT3 inhibitor libraries, BP5-087 emerged as the new lead compound. Fluorescence polarization assays verified that BP5-087 was 5-fold more effective than SF1-066 in outcompeting an SH2 peptide probe, and computational simulations predicted better overall binding of BP5-087 (-9.6 kcal/mol) versus SF1-066 (-7.6 kcal/mol) to the STAT3 SH2 interface. In AR230R cell growth assays, BP5-087 was effective at a 5-fold lower dose compared to SF1-066, with minimal effects on TKI-sensitive parental controls. Therefore, we tested BP5-087 in the context of primary TKI resistance. BP5-087 (1 µM) in combination with imatinib (2.5 µM) reduced colony formation and increased apoptosis of CD34+ cells from CML patients with BCR-ABL1-independent resistance. These cells have no BCR-ABL1 kinase domain mutations and undergo BCR-ABL1 kinase inhibition as detected by immunoblot analyses. In contrast, BP5-087 had no effect on CD34+ cells from newly diagnosed CML patients or normal individuals. Immunofluorescence demonstrated that dual treatment of TKI-resistant CML CD34+ cells resulted in reduced levels of nuclear pSTAT3Y705, consistent with an inhibitor of STAT3 dimerization. In more primitive CML stem cells, long term culture-initiating cell (LTC-IC) assays revealed that neither inhibitor alone had any effect on colony formation of primitive LTC-IC progenitors, whereas imatinib (2.5 µM) in combination with BP5-087 (1.0 µM) reduced LTC-IC colony formation by 66%. Consistent with this observation, immunofluorescence showed high levels of pSTAT3Y705 in primitive TKI-resistant CD34+CD38- cells when cultured in the presence but not absence of TKIs. To test the feasibility of BP5-087 for in vivo use, we treated mice orally with BP5-087 (25 mg/kg/day) for 4 weeks and observed no changes in body weight, peripheral blood cellularity, or bone marrow colony forming ability. Mass spectrometry confirmed that BP5-087 is orally bioavailable. In summary, BP5-087 is a systematically-derived, direct inhibitor of STAT3 that, in combination with TKIs, reduces survival of CML cells with BCR-ABL1-independent resistance. Further rounds of structure-activity optimization may reveal an inhibitor with a clinically-relevant effective concentration. Disclosures: Deininger: Bristol Myers Squibb: Advisory Boards Other, Consultancy, Research Funding; Ariad Pharmaceuticals: Advisory Boards, Advisory Boards Other, Consultancy; Novartis: Advisory Boards, Advisory Boards Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding.

Introduction BCR-ABL1 kinase domain mutations are detected in 30-60% of patients who develop resistance to tyrosine kinase inhibitors (TKIs) such as imatinib. However, the underlying mechanism(s) of resistance in the remaining patients are not known. To identify BCR-ABL1-independent mechanisms of resistance to TKIs, we used K562 cells that were adapted for long-term growth in 1 µM imatinib (K562-R). These cells lack BCR-ABL1 kinase domain mutations and survive despite continued suppression of BCR-ABL1 kinase activity. To screen for novel genes associated with BCR-ABL1-independent resistance, parental K562 and K562-R cells were lentivirally infected with a pooled shRNA library containing 27,000 shRNAs targeting ∼5,000 genes with known roles in cell signaling (Cellecta, Human Module 1). Using an unbiased, customized algorithm, we identified 18 genes with multiple shRNA hits that were depleted more than three-fold in K562-R cells compared to parental controls. Among the top five genes were RAN and XPO1 (CRM1), both of which encode components of a nucleocytoplasmic transport complex. Parental K562 cells also showed depletion for RAN and XPO1 shRNAs, suggesting a potential role in pathogenesis of CML and magnified activity in TKI resistance. Recently, enhanced expression of XPO1 was shown in Philadelphia chromosome positive leukemia and investigated as a potential therapeutic target in blast crisis CML (Walker et al. ASH 2012). Methods To validate the data from our shRNA library screen, we cloned a RAN shRNA into a tet-inducible lentiviral vector (Cellecta). Transduced parental and K562-R cells were treated with/out imatinib (2.5 mM) in the presence or absence of doxycycline. Parental and K562-R cells were treated with various concentrations of the oral XPO1 inhibitor Selinexor (KPT-330), which is now being evaluated in Phase 1 clinical trials in patients with solid and hematological malignancies. Following 72 hours of treatment, proliferation was measured by MTS assay (Promega). Additionally, CD34+ cells from newly diagnosed CML patients were lentivirally transduced with RAN shRNA and treated with/out imatinib (2.5 mM) in the presence or absence of doxycycline (n=3) or KPT-330 (n=1). Survival was assessed by colony formation assays. Normal cord blood CD34+cells were used as controls. Results Transduction of parental or K562-R cells with the inducible RAN shRNA and treatment with doxycycline reduced RAN mRNA levels by 57 and 76% respectively compared to the untreated control. This level of RAN suppression was sufficient to reduce the proliferation of parental K562 by 54%. It also reduced the IC50 of imatinib by > 3-fold in K562-R cells. In CD34+ cells from newly diagnosed CML patients, RAN shRNA alone had no significant effect on colony formation, whereas RAN shRNA in combination with imatinib (2.5 µM) reduced colony formation by 46% compared to imatinib alone. The IC50 of KPT-330 was 30-35% less in K562-R cells compared to parental controls suggesting a higher sensitivity of K562-R cells to XPO1 inhibition. We observed that 25 nM KPT-330 reduced colony formation by 52% in primary CML cells without reducing cord blood CD34+colonies. Combination of 2.5 µM imatinib and 25 nM KPT-330 reduced the colony formation significantly compared to imatinib alone in primary CML cells with no change in the control. Conclusion An unbiased, shRNA library screen and subsequent validations with primary CML cells suggested that the activity of nucleocytoplasmic transport is crucial for the survival of CML cells. Genetic and pharmacologic disruption revealed that nucleocytoplasmic transport is a target in both TKI sensitive and resistant CML cells. A deeper understanding of this pathway in CML will be important for understanding BCR-ABL1 independent resistance to TKI treatment Disclosures: Kauffman: Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Schacham:Karyopharm: Membership on an entity’s Board of Directors or advisory committees. Chenchik:Cellecta: Membership on an entity’s Board of Directors or advisory committees. Bonneau:Cellecta: Membership on an entity’s Board of Directors or advisory committees. Deininger:Bristol-Myers Squibb: Advisory Boards Other, Consultancy, Research Funding; Ariad Pharmaceuticals: Advisory Boards, Advisory Boards Other, Consultancy; Novartis: Advisory Boards, Advisory Boards Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding.

Abstract 31 In CML, TKI resistance in the absence of BCR-ABL1 kinase mutations is mechanistically unclear. Since extrinsic signals from the bone marrow (BM) microenvironment protect CML cells from TKIs in a STAT3-dependent manner (Bewry et al. 2008; Traer et al. 2011), we hypothesized that overt resistance may occur when CML cells maintain intrinsic STAT3 activation in the absence of extrinsic signals. We also asked whether combined targeting of STAT3 and BCR-ABL1 could produce synthetic lethality to overcome TKI resistance. To model extrinsic BCR-ABL1 kinase-independent resistance, we grew CML cell lines and CMLCD34+ progenitor cells from newly diagnosed patients in the presence of conditioned medium (CM) derived from HS-5 BM stromal cells. To model intrinsic resistance, we used the imatinib-resistant K562R and AR230R cell lines. Our models of TKI resistance proliferate in 1.0–2.5 μM imatinib and exhibit pSTAT3Y705 activation despite suppression of BCR-ABL1 kinase activity. To investigate the role of pSTAT3Y705 in TKI resistance, we used shRNA-mediated knockdown (shSTAT3), a dominant-negative mutant (dnSTAT3), and pharmacologic STAT3 inhibitors developed in our laboratories to block STAT3 activity. Compared to scrambled controls in the presence of HS-5 CM, shSTAT3 reduced colony formation (33.8% reduction, p Preliminary immunofluorescence data indicates that pSTAT3Y705 levels are higher in drug-resistant CML CD34+CD38− stem cells than in CD34+CD38+ progenitor cells, suggesting a potential role for pSTAT3Y705 in TKI resistance of primitive CML stem cells. To investigate whether pharmacologic STAT3 inhibition in combination with TKIs could overcome resistance, we tested a library of compounds that interfere with STAT3 dimerization. The most selective of these inhibitors, S3I-201.1066 (SF1-066), was biochemically shown to disrupt STAT3 dimerization and transcriptional activation. Compared to cells treated with 1.0 μM imatinib alone, addition of 10 μM SF1-066 impaired the clonogenicity of K562R cells by 68.6% (p We next developed a series of 14 second-generation STAT3 inhibitors and tested them using a luciferase reporter harboring sequential STAT3 cis-inducible elements to measure endogenous STAT3 activity in AR230R cells. Two lead compounds, BP2-047 and BP3-163, reduced luciferase activity by 69.2% (p Altogether, our data identifies STAT3 as a universal target in TKI-resistant CML cells, and suggests that simultaneous pharmacologic inhibition of STAT3 and BCR-ABL1 may be a suitable therapeutic strategy for treatment of patients with TKI resistance despite inhibition of BCR-ABL1 kinase activity. Disclosures: No relevant conflicts of interest to declare.

Abstract 2445 Constitutive activation of signal transducer and activator of transcription 3 (STAT3) correlates with drug resistance and a poor prognosis in many cancers. STAT3 signaling is mediated by phosphorylation at tyrosine-705 (p STAT3Y705), dimerization, and nuclear transactivation. In chronic myeloid leukemia (CML), pSTAT3Y705 is demonstrable under two distinct resistance scenarios: (1) extrinsic resistance, in which BCR-ABL1 kinase-independent survival signals originating from the bone marrow (BM) microenvironment activate pSTAT3Y705 in a JAK2- or TYK2-dependent manner, and (2) intrinsic resistance, in which BCR-ABL1 kinase-independent signals activate pSTAT3Y705 in response to kinase inhibition. Based on these observations, we identified TKI-resistant CML as an excellent model for developing and optimizing pharmacologic STAT3 inhibitors. Using K562 and AR230 CML cells that are resistant to 1 μM imatinib (K562R and AR230R; intrinsic resistance) and primary CML CD34+ progenitor cells exposed to BM stromal-derived conditioned medium (CM; extrinsic resistance), we examined the effects of direct pharmacologic inhibition of STAT3 in TKI-resistant CML. Here, we report the design and validation of next-generation STAT3 inhibitors identified through computational modeling and screening in AR230R CML cells expressing high levels of pSTAT3Y705. We initially examined the effects of an established STAT3 inhibitor, S3I-201.1066 (SF1–066). K562R or AR230R cells were treated with 1 μM imatinib and/or 10 μM SF1–066, followed by culture in methylcellulose medium and scoring after 14–16 days. Combination treatment reduced the clonogenicity of K562R and AR230R cells to 31.4% (p0.05). Next, CD34+ cells from newly diagnosed CML patients (n=4) were cultured for 96 hours in the presence of CM and treated with 2.5 μM imatinib, 10 μM SF1–066 or both. Equal numbers of cells were then plated in colony forming assays. Imatinib combined with SF1–066 reduced colony formation to 54.8% (p Computational modeling informed the design and synthesis of a second-generation, SF1–066-based library. We evaluated compounds with shared molecular functionalities using AR230R cells expressing a luciferase reporter containing sequential STAT3 sis-inducible elements (AR230R-SIE). At 10 μM, we found two inhibitors, BP2–047 and BP3–163, that reduced luminescence by 69.2% (p To account for the reduced luminescence in AR230R-NEG cells and analyze for more potent inhibitors, we performed the luciferase assay with candidate inhibitors at 5 μM. We identified BP5–087 and BP5–088, each with increased potency and STAT3 selectivity compared to SF1–066. These compounds decreased luminescence in AR230R-SIE cells by 36.1% (p0.05). Confirmation of STAT3 binding was obtained using fluorescence polarization assays, in which the EC50 values of BP5–087 and BP5–088 measured 8.5 μM and 4.6 μM, respectively. Studies with mouse and human liver microsomes also revealed that BP5–087 and BP5–088 exhibit enhanced metabolic stability compared to SF1–066. Interestingly, treatment with BP5–087 or BP5–088 (both at 1 and 5 μM) in CML CD34+ progenitors grown in CM showed increased cytoplasmic accumulation of pSTAT3Y705 compared to controls. Thus, we identified BP5–087 and BP5–088 as two of the most potent small-molecule binders of STAT3 reported. These compounds are promising frontrunners toward new therapies for TKI-resistant CML and other diseases in which STAT3 activation drives malignant phenotypes. Disclosures: No relevant conflicts of interest to declare.

Abstract 2778 In chronic myeloid leukemia (CML), imatinib and other tyrosine kinase inhibitors (TKIs) inhibit BCR-ABL1 tyrosine kinase activity but also target additional kinases including KIT. The role of KIT inhibition in the therapeutic efficacy of TKIs is controversial. We used TKIs with selective activity against ABL (PPY-A) or KIT (BAW667) and genetic tools to assess the role of KIT signaling for growth of CML cell lines and primary CML progenitor and stem cells. In Mo7eBCR-ABL1 or newly diagnosed CML CD34+ progenitor cells, immunoblotting confirmed that PPY-A (1 μM) suppresses BCR-ABL1 phosphorylation but not KIT tyrosine phosphorylation. In contrast, treatment of cells with a KIT-blocking antibody (K44.2, 200ng/mL), shRNA targeting KIT (shKIT), or the KIT selective inhibitor BAW667 (1 μM), suppressed KIT activity without affecting BCR-ABL1 kinase activity. Therefore, these systems are suitable to isolate the role of BCR-ABL1 vs. KIT inhibition. Treatment of Mo7eBCR-ABL1 cells with PPY-A resulted in suppression of growth by 91.7% (p To assess BCR-ABL1 vs. KIT inhibition in primary cells, CD34+ cells from newly diagnosed CML patients (n=4) and normal controls (n=3) were cultured in semisolid medium supplied with IL-3 and GM-CSF (no SCF), in the presence of 1 μM PPY-A combined with shKIT or 1 μM BAW667. KIT inhibition by shKIT or 1 μM BAW667 reduced CFU-GM formation by 40% compared to controls (p To assess the role of KIT vs. BCR-ABL1 inhibition on primitive CML cells we performed long-term culture-initiating cell (LTC-IC) assays on M2–10B4 murine stromal cells, using Lin− cells from newly diagnosed patients (n=3). Cultures were performed with K44.2, PPY-A, K44.2 plus PPY-A or 2 mM imatinib, with colonies plated at 1, 3, and 6 weeks. At 1 week colonies were reduced by 30% with K44.2 and 70% with PPY-A, but by 90% with the PPY-A / K44.2 combination or with imatinib. In contrast, at 6 weeks colony formation was unaffected by K44.2 but reduced by >95% with PPY-A, the PPY-A / K44.2 combination or imatinib. Week 3 colony growth was intermediate. Consistent with the LTC-IC assay, KIT inhibition with BAW667 enhanced PPY-A suppression of colony formation in Lin−CD34+CD38+ progenitor cells from newly diagnosed patients (n=3) by 18.7% (p Our findings suggest KIT inhibition is much more critical for suppression of mature progenitors compared to primitive CML cells. Since AKT is active in CML progenitors but suppressed by TGFβ in stem cells (Nature, 2010;463(7281):676; JCI, 2011;121(1):396), we speculate that upon BCR-ABL1 inhibition CML progenitors but not stem cells switch to an SCF-dependent mode of AKT activation, which renders these cells uniquely sensitive to dual inhibition of BCR-ABL1 and KIT signaling. Disclosures: No relevant conflicts of interest to declare.

Abstract 2481 Ponatinib (AP24534) is a pan-BCR-ABL inhibitor developed for treatment-refractory chronic myeloid leukemia (CML) and has significant activity in patients who fail second-line dasatinib and/or nilotinib tyrosine kinase inhibitor (TKI) therapy. A pivotal phase II trial (clinicaltrials.gov NCT01207440) is underway. BCR-ABL kinase domain mutation-mediated ponatinib resistance has been investigated in vitro (Cancer Cell 16, 2009, 401). Here, we developed ponatinib-resistant, BCR-ABL+ cell lines lacking a kinase domain mutation and investigated mechanisms of resistance to ponatinib and other TKIs. Methods: Four BCR-ABL+ CML cell lines (K562, AR230, BV173, and 32D(BCR-ABL)) were maintained in liquid culture containing ponatinib (0.1 nM) for 10 days. The ponatinib concentration was increased in small increments for a minimum of 90 days, yielding corresponding ponatinib-resistant cell lines. BCR-ABL kinase domain sequencing of sensitive and resistant cells confirmed BCR-ABL to be unmutated. Real-time qPCR was used to compare the expression of BCR-ABL in ponatinib-sensitive and -resistant cell lines. Immunoblot analysis (total and tyrosine-phosphorylated BCR-ABL) was used to the compare levels of BCR-ABL protein and to determine whether resistance to ponatinib corresponded with reduced (partially BCR-ABL-independent) or complete inhibition of BCR-ABL tyrosine phosphorylation (fully BCR-ABL-independent). Cell proliferation assays were performed on resistant and sensitive cell lines in the presence of ponatinib, nilotinib, and dasatinib. A small-molecule inhibitor screen composed of >90 cell-permeable inhibitors that collectively target the majority of the tyrosine kinome as well as other kinases (Blood 116, 2010, abstract 2754) is currently being applied to the 32D(BCR-ABL)R cell line in the presence of 24 nM ponatinib to assess synthetic lethality, with results analyzed using a companion drug sensitivity algorithm. As a second strategy to generate resistant lines, N-ethyl-N-nitrosourea (ENU) mutagenesis was done to investigate BCR-ABL kinase domain-mediated resistance in myeloid K562, AR230, BV173, and 32D(BCR-ABL) cells. After ENU exposure, cells were washed and cultured in 96-well plates with escalating ponatinib. Results: The four BCR-ABL+ cell lines initially grew in the presence of 0.1 nM but not 0.5 nM ponatinib. Upon gradual exposure to escalating ponatinib, each of the cell lines exhibited a degree of adaptation to growth in the presence of the inhibitor (range: 10 to 240-fold). Real-time qPCR showed a modest two-fold increase in BCR-ABL expression level in K562R, AR230R and BV173R cell lines relative to the respective parental lines. Based on immunoblot analysis, cell lines segregated into two categories of ponatinib resistance: partially (K562R and AR230R) or fully BCR-ABL-independent (BV173R and 32D(BCR-ABL)R). Cell proliferation assays showed that ponatinib resistant cell lines also exhibited resistance to nilotinib and dasatinib. The 32D(BCR-ABL)R cell line exhibited a level of ponatinib resistance comparable to that of the Ba/F3 BCR-ABLE255V cell line, which carries the most ponatinib-resistant BCR-ABL mutation. BCR-ABL tyrosine phosphorylation was efficiently blocked by low concentrations of ponatinib ( Conclusions: The ponatinib resistant, BCR-ABL+ cell lines described here exhibit either a partially or fully BCR-ABL independent mechanism of resistance. The molecular details of both processes will be reported, with an emphasis on the striking level of resistance (240-fold over starting conditions) exhibited by the 32D(BCR-ABL)R cell line. Our in vitro results indicate that BCR-ABL independent mechanisms may contribute to ponatinib resistance in myeloid CML cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 3742 In Philadelphia chromosome positive leukemia, mutations in the BCR-ABL kinase domain are a well established mechanism of resistance to tyrosine kinase inhibitors (TKIs), but fail to explain many cases of primary and acquired resistance. Extrinsic survival signals from the bone marrow microenvironment can protect CML cells from the effects of TKIs in a STAT3-dependent manner. To define the role of pSTAT3 in extrinsic versus intrinsic TKI resistance, we used BCR-ABL+ cell lines exhibiting ‘extrinsic resistance' through culture in conditioned media (CM) from human HS-5 bone marrow stromal cells (Re), or derivative lines expressing native BCR-ABL with ‘intrinsic resistance' adapted for growth in the presence of 1.0–2.5 μM imatinib (Ri). Immunoblot analysis showed near-complete suppression of BCR-ABL tyrosine kinase activity upon exposure to imatinib in all Re and Ri cells tested, suggesting BCR-ABL-independent resistance in both cases. K562-Ri and AR230-Ri cells proliferating in 1.0 μM imatinib showed upregulation of pAKTS473, pJAK2Y1007/1008, pSTAT3, and pSTAT5 compared to sensitive cells under the same conditions. When imatinib was increased (2.5 μM), pJAK2Y1007/1008 and pSTAT5 were markedly reduced, suggesting regulation by BCR-ABL kinase activity. In contrast, levels of pSTAT3 and pAKTS473 were further increased. Similar to Ri cells, extrinsically resistant cells (K562-Re, LAMA84-Re, KBM5-Re) showed increased pSTAT3 when cultured in HS-5 CM; however, upregulation was not observed for pAKTS473. Of key importance, pSTAT3 levels were also increased by HS-5 CM in CD34+ cells from newly diagnosed CML patients. To further investigate the role of pSTAT3 in TKI resistance, we used lentiviral shRNA to knockdown STAT3 (shSTAT3) in sensitive and resistant BCR-ABL+ cells. Cells were kept in culture for 24 hours with and without 1.0 μM imatinib in regular medium or CM, followed by trypan blue exclusion and/or plating in semisolid medium. Compared to cells expressing a scrambled control (shSCR), shSTAT3 reduced the in vitro growth of intrinsically resistant K562-Ri and AR230-Ri cells by 54.6% and 33.3% in the presence of imatinib (1.0 μM), respectively. Consistent with these observations, shSTAT3 impaired the clonogenic potential of K562-Ri cells by 65.0% following culture in 1.0 μM imatinib, and similar results were obtained for AR230-Ri cells, indicating that STAT3 plays a functional role in mediating intrinsic TKI resistance in multiple BCR-ABL-expressing cell lines. In extrinsic resistance, 3–12 hours of culture with CM from HS-5 stromal cells enhanced the clonogenic potential of K562-Re and LAMA84-Re cells by 25–40% in the presence of imatinib compared to controls cultured in regular medium. However, the protective effects of CM were abrogated by introduction of shSTAT3. In contrast, shSTAT3 had no effect on colony formation following growth in regular medium, consistent with the low level of pSTAT3 in parental K562 and LAMA-84 cells. These data suggest that STAT3 activation is a prominent feature of both extrinsic and intrinsic BCR-ABL-independent imatinib resistance. Additionally, preliminary experiments implicate pSTAT3 in intrinsic BCR-ABL-independent resistance to the third-generation TKI, ponatinib (AP24534), suggesting that activated STAT3 may have a role in resistance to other TKIs. Altogether, our data suggest that intrinsic and extrinsic pathways converge on STAT3 as a critical mediator of BCR-ABL-independent TKI resistance, and implicate this pathway as a potential therapeutic target for the treatment of patients with TKI resistance despite BCR-ABL inactivation. We posit that microenvironmental cues activate pathways that initially support survival of leukemia cells despite BCR-ABL inhibition, and that overt resistance develops when these cells establish an intrinsic mechanism to maintain activation of the same pathways. Disclosures: No relevant conflicts of interest to declare.

Abstract 3744 Background: BCR-ABL kinase domain (KD) mutations are a common mechanism of chronic myeloid leukemia (CML) resistance to tyrosine kinase inhibitor (TKI) therapy. It is well known that some patients harbor more than one KD mutation in the same sample, but the frequency of true compound mutations (defined as two or more mutations in the same allele) and the clonal relationships between mutant clones have not been established. Methods: The first group of samples (Group 1) was selected based on evidence of more than one BCR-ABL KD mutation by Sanger sequencing. Samples from a second group of patients (Group 2) who had one mutation in BCR-ABL KD by Sanger sequencing were also analyzed. The BCR-ABL KD was amplified using nested RT-PCR, the final amplicon was shotgun-cloned and 10 clones from each sample were sequenced. Results: Thus far, 18 samples from group 1 have been analyzed. Sequencing of 180 colonies from this group revealed >80 different mutations. Compound mutations were confirmed in 13 samples (Table 1), while 5 samples revealed the two mutations were in separate clones. T315I, F359V, V299L and M351T were over-represented among compound mutations and the T315I/F359V and V299L/M351T compound mutations were each detected in samples from two patients. On average, each patient sample had 7 different mutations per 10 clones (range: 3–14), some were silent or not previously shown to be associated with TKI resistance in biochemical or cell-based assays. In the 13 samples from Group 1 with confirmed compound mutations (Table 1), all respective individual mutations had been shown to confer TKI resistance in biochemical and/or in vivo studies. In 4 patients, the compound mutant clone was non-dominant (≤40% of clones). In 3 of these 4 patients (11, 13, 16; Table 1), both mutations were also detected independently in co-existent clones, while 1 patient (18) showed 2 compound mutant clones (T315/F359V and V299L/M351T) at comparable levels (40% each). In the remaining 9 patients from Group 1, the compound mutant represented >70% of clones. More than 2 mutations were observed in 41/102 screened compound mutant clones from Group 1. The number of mutations in these clones ranged from 3 to 6. Except V299L/M351T/E329G, which was observed in 5/10 screened clones of one patient (1), no clone with more than 2 mutations was detected in more than 1 clone per sample (≤10%). Notably, none of these additional mutations has yet been associated with TKI resistance in biochemical or cell-based assays. So far, samples from 7 patients in Group 2 have been analyzed. Sequencing of the clones from these patients also revealed the presence of low-level compound mutants. On average 30% of the clones had compound mutations (range: 20%-40%) and an average of 5 different mutations were detected among the 10 sequenced clones per patient (range: 3–6), some of which were silent or not previously shown to be associated with TKI resistance in biochemical or cell-based assays. Conclusions: 1. True compound mutations are common in patients with evidence of multiple mutations by direct sequencing. 2. Mutant clones evolve sequentially as well as in parallel, suggesting complex clonal relationships, in which the identical phenotype (mutation) may be acquired independently by multiple clones. 3. Clones harboring compound mutations comprised of more than 2 mutations rarely achieve dominance, suggesting that the number of different mutations compatible with maintenance of BCR-ABL kinase activity may ultimately be limited. 4. Low-level compound mutations are also detected by cloning and sequencing in the samples from patients who have had one detected mutation in screening (Group 2). Disclosures: Lange: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 515 The success of tyrosine kinase inhibitors as first line therapy for t(9;22) Chronic Myelogenous Leukemia (CML) depends on the addiction that Philadelphia-positive (Ph+) hematopoietic progenitors, but not quiescent Ph+ stem cells, have for BCR-ABL1 tyrosine kinase activity. We reported that the activity of the tumor suppressor PP2A is inhibited in a SET-dependent manner in CML progenitors and CD34+/CD38- stem cells from chronic phase (CP) and, to a greater extent, blast crisis (BC) CML patients (Neviani P. et al.: Cancer Cell 2005, J. Clin. Invest 2007, and ASH 2008). Restoration of PP2A activity by the immunosuppressive sphingosine analogue FTY720 markedly decreases the number of Ph+ but not normal long-term culture-initiating cells (LTC-IC) and quiescent stem cells (CFSEMAX/CD34+) by suppressing the BCR/ABL kinase-independent enhancement of b-catenin expression/transcriptional activity (Oaks JJ., et al., ASH 2009). Here we report that FTY720 induces apoptosis of Ph+ CD34+/CD38- cells independent from its phosphorylation as treatment with a phosphorylated FTY720 did not alter the number of Ph+ CFSEMAX/CD34+ cells. By contrast, two non phosphorylatable and non immunosuppressive FTY720 derivatives did significantly affect survival of Ph+ stem/progenitor cells. Interestingly, we also noted that the activity but not expression of BCR-ABL1 is considerably lower in quiescent CFSEMAX/CD34+ than CFSE+/CD34+ cells that underwent at least one division (∼80% decrease; n=3). Conversely, BCR-ABL1 expression is significantly higher in quiescent than proliferating CFSE+/CD34+ cells, suggesting that BCR-ABL1 might serve as a scaffold for other kinase(s) able to sustain survival and quiescence of Ph+ stem cells. Indeed, we found that expression of the K1172R kinase-deficient BCR-ABL1 mutant enhances expression and activity of Jak2, a kinase that is not only associated with BCR-ABL1 but is also capable of inactivating and being inactivated by PP2A. Accordingly, lentiviral shRNA-mediated BCR-ABL1 downregulation in Ph+ CD34+/CD38- stem cells resulted in marked (≥70% inhibition, P Thus, expression but not activity of the oncogenic product of the t(9;22) translocation is important for recruiting and allowing SET-mediated inhibition of PP2A and activation of Jak2; two events important for Ph+ stem cell survival and self renewal. Moreover, the ability of FTY720 and of its non-immunosuppressive derivatives to induce apoptosis of Ph+ progenitors and Ph+ but not normal quiescent stem cells emphasizes the notion that FTY720 and its derivatives represent strong and non-toxic anti-leukemic agents potentially useful not only for the treatment but, perhaps, for eradicating Ph+ leukemias. Disclosures: Holyoake: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 855 MicroRNAs (miRs) and heterogeneous ribonucleoproteins (hnRNPs) are post-transcriptional gene regulators that bind mRNA in a sequence-specific manner. We have reported that a) hnRNP-E2 suppresses CEBPA mRNA translation and inhibits myeloid maturation of bone marrow (BM) progenitors from chronic myelogenous leukemia patients in myeloid blast crisis (CML-BCCD34+; Perrotti et al, Nat Genet 2002); and b) miR-328 expression is lost in myeloid CML-BCCD34+ progenitors (n=6) and its restored expression at physiological levels rescues granulocytic differentiation and impairs clonogenic potential of primary BCR/ABL+ blasts (Eiring et al, ASH 2007). Here we show by Northern blot, real-time PCR, and microarray analyses that miR-328 levels increase during granulocytic differentiation of normal human CD34+ and mouse Lin− BM progenitors, but not during differentiation towards erythroid, megakaryocytic or monocytic lineages. BCR/ABL uses the same MAPKERK1/2-hnRNP-E2 signaling pathway to suppress both C/EBPα and miR-328, as pharmacologic or shRNA-mediated inhibition of these molecules restored miR-328 expression in BCR/ABL+ cells. In fact, two functional C/EBPα binding sites are present in the miR-328 promoter region and C/EBPα interacts in vivo with these regulatory elements to enhance miR-328 transcription. Importantly, we also show that restored maturation of BCR/ABL+ blasts requires direct interaction of hnRNP-E2 with the C-rich regions of miR-328. Indeed, RNA-immunoprecipitation (RIP) assays demonstrated that miR-328 directly binds to hnRNP-E2 independent of the RNA-induced silencing complex (RISC). Furthermore, ectopic miR-328, but not miR-181b, resulted in decreased in vivo binding of hnRNP-E2 to the uORF/spacer region of CEBPA mRNA, thereby releasing CEBPA from hnRNP-E2 translation inhibition and rescuing C/EBPa-driven neutrophil maturation (decoy activity). Differentiation of miR-328-expressing CML-BCCD34+ blasts (88.8±2.4% post-mitotic cells) correlated with induction of C/EBPa protein expression, whereas CEBPA mRNA and hnRNP E2 protein levels remained unchanged. The existence of a direct miR-328/hnRNP-E2/CEBPA interplay was formally demonstrated in vitro using RRL-directed translation assays and in vivo using the 6.15 clone of 32D-BCR/ABL cells that do not express endogenous CEBPA mRNA and require ectopic C/EBPα (wt-uORF-CEBPA) for differentiation. Addition of miR-328, but not miR-330, to hnRNP-E2-containing RRL reactions increased newly synthesized 35S-C/EBPa levels by >100%. Likewise, forced miR-328 expression in vivo resulted in decreased hnRNP-E2 binding to CEBPA mRNA, induction of C/EBPa protein but not mRNA and rescued granulocytic differentiation of 6.15-wt-uORF-CEBPA but not vector-transduced 6.15 cells. While hnRNP-E2 was not found in complex with basic RISC components (Dicer, TRBP2 and Ago2), RIP assays detected miR-328 associated to Dicer and Ago2 in miR-328-expressing cells, suggesting that it also acts through canonical RISC-dependent base-pairing with mRNA targets. Indeed, we identified the BCR/ABL-regulated PIM1 serine-threonine kinase as a bona fide miR-328 target in BCR/ABL+ cells. Ectopic miR-328 suppressed PIM1 protein but not mRNA levels, and this effect required integrity of the miR-328 binding site present in the PIM1 3'UTR. Forced expression of a wild-type but not kinase-deficient PIM1 lacking the 3'UTR into miR-328-expressing cells fully rescued BCR/ABL clonogenicity, suggesting that miR-328-induced PIM1 suppression accounts for reduced survival of miR-328-infected BCR/ABL+ blasts. To show that miR-328 acts on PIM1 in a RISC-dependent manner, we mutated the miR-328 seed sequence (miR-328-Mut) while retaining its C-rich character. Similar to wild-type miR-328, miR-328-Mut efficiently interacted with hnRNP-E2, restored C/EBPa protein expression and rescued granulocytic differentiation, but was unable to silence PIM1 in 32D-BCR/ABL cells, indicating that the C-rich character of miR-328 is essential for its decoy activity, while its seed sequence integrity is necessary for RISC-dependent pairing to mRNA targets. Thus, the discovery of dual activities for miR-328 not only adds a new layer of complexity to the mechanisms regulating CML disease progression, but also highlights the ability of miRNAs to alter mRNA metabolism by acting as molecular decoys for RNA-binding proteins. Disclosures: Cortes: Novartis: Research Funding.

Several RNA binding proteins (RBPs) have been implicated in the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase to the aggressively fatal blast crisis. In the latter phase, expression and function of specific RBPs are altered at transcriptional or post-translational levels by the increased constitutive kinase activity of the BCR/ABL oncoprotein, resulting in enhanced resistance to apoptotic stimuli, growth advantage and differentiation arrest of CD34+ CML blast crisis (CML-BC) progenitors. In the current study, we identified by RIP (RNA immunoprecipitation)-mediated microarray analysis that mRNA encoding the E2F3 transcription factor associates to the BCR/ABL-regulated RBP hnRNP A1. Moreover, RNA electrophoretic mobility shift and UV-crosslinking assays revealed that hnRNP A1 interacts with E2F3 mRNA through a binding site located in the 3’UTR of both human and mouse E2F3 mRNA. Accordingly, E2F3 protein levels were upregulated in BCR/ABL-transformed myeloid precursor cell lines compared to parental cells in a BCR/ABL-kinase- and hnRNP A1 shuttling-dependent manner. In fact, treatment of BCR/ABL-expressing myeloid precursors with the kinase inhibitor Imatinib (2mM, 24 hr) or introduction of a dominant-negative shuttling-deficient hnRNP A1 protein (NLS-A1) markedly reduced E2F3 protein and mRNA levels. Similarly, upregulation of BCR/ABL expression/activity in the doxycycline inducible TonB2.10 cell line resulted in increased E2F3 protein expression. BCR/ABL kinase-dependent induction of E2F3 protein levels was also detected in CML-BCCD34+ compared to CML-CPCD34+ progenitors from paired patient samples and to normal CD34+ bone marrow samples. Importantly, the in vitro clonogenic potential of primary mouse BCR/ABL+ lineage negative (Lin−) progenitors was markedly impaired in BCR/ABL+ E2F3−/− compared to BCR/ABL-transduced E2F3+/+ myeloid progenitors and upon shRNA-mediated downregulation of E2F3 expression (90% inhibition, P80% reduction in tumor burden, P

Altered microRNA (miR) expression contributes to aberrant post-transcriptional regulation of gene expression in different type of cancers; however, their role in the pathogenesis and progression of chronic myelogenous leukemia (CML) from chronic phase (CML-CP) to blast crisis (CML-BC) is still largely unknown. Microarray analysis of miR expression reveals that a discrete number of miRs are significantly upregulated (∼ 6.7% of the total 505 miRs present on the chip; 34 miRs) or downregulated (∼2.8% of the miRs present on the chip; 14 miRs) in an imatinib-sensitive manner in CML-BCCD34+ compared to CML-CPCD34+ progenitors and in BCR/ABL-expressing hematopoietic cell lines compared to untransformed parental cells. Among them, we focused our attention on miR-223, miR-15a/16-1 and miR-328, a microRNA with no currently known function, because of their importance in myelopoiesis, potential role as tumor suppressors and sequence homology with the 5’UTR of CEBPA mRNA, respectively. In 32D-BCR/ABL and K562 cells, Northern blot and TaqMan RT-PCR analyses revealed that expression of miR-223, miR-328, miR-15a and miR-16-1 was markedly suppressed (50–75% inhibition) by p210-BCR/ABL kinase activity and that imatinib treatment (1mM; 24h) restored the expression of these miRs to levels similar to those detected in non-transformed 32Dcl3 cells. Interestingly, sequence analysis of both miR-223 and miR-328 revealed homology with the hnRNP E2-binding site contained in the CEBPA uORF/spacer mRNA, a known target of the negative regulator of myeloid differentiation hnRNP E2. Accordingly, REMSA and UV-crosslinking experiments showed that synthetic miR-223 and to a greater extent miR-328 bind efficiently to recombinant hnRNP E2 protein and compete for its binding to an oligoribonucleotide containing the CEBPA uORF/spacer region, which is required for hnRNP E2-mediated translational inhibition of CEBPA in CML-BCCD34+ progenitors. Furthermore, both miR-223 and miR-328 bind endogenous hnRNP E2 from lysates of BCR/ABL-expressing but not parental cells, and from lysates of parental 32Dcl3 myeloid precursors ectopically expressing a Flag-tagged hnRNP E2 protein, suggesting that miR-223 and miR-328 may act as decoy molecules that interfere with the translation-inhibitory activity of hnRNP E2. Indeed, ectopic expression of miR-223 restored G-CSF-driven granulocytic maturation of differentiation-arrested 32D-BCR/ABL cells and restored C/EBPα expression, whereas it did not have any effect on cytokine-independent growth and clonogenic potential. Consistent with its ability to bind hnRNP E2, miR-328 also rescued C/EBPα expression and differentiation of cytokine-independent BCR/ABL-expressing myeloid precursor 32Dcl3 cells. By contrast, BCR/ABL-dependent colony formation was markedly reduced by overexpression of miR-15a and miR-16-1 (65–75% inhibition, P

Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome positive (Ph1) acute lymphoblastic leukemia (ALL) are two fatal BCR/ABL-driven leukemias against which the current therapy with Abl kinase inhibitors fails to induce a long-term response, as the majority of patients are either refractory or relapse after a few months of treatment. We recently reported that functional loss of the PP2A tumor suppressor occurs during CML disease progression and that restoration of PP2A activity impairs in vitro and in vivo BCR/ABL leukemogenesis. Here we assessed the therapeutic potential of the PP2A activator FTY720 in CML-BC and Ph1 ALL patient cells and in in vitro and in vivo models of these BCR/ABL+ leukemias. FTY720 (500 nM-2.5 mM) induces caspase-dependent apoptosis (70–98% annexin V+) and impairs the clonogenic potential (70–95% inhibition) of imatinib/dasatinib-sensitive and -resistant (T315I) p210 and p190 BCR/ABL-expressing myeloid and lymphoid progenitor cell lines (Ph1 K562, 32D-p210BCR/ABL, 32D-p210(T315I)BCR/ABL and BaF3-p190BCR/ABL), respectively, and of primary bone marrow CML-BCCD34+ (n=11) and Ph1 ALLCD34+/CD19+ (n=12) patients cells. Interestingly the cytokine (IL-3 or IL-7)-dependent growth and differentiation of normal CD34+ myeloid and CD34+/CD19+ lymphoid progenitors (n=8) is not affected by FTY720 treatment. Furthermore, pharmacologic doses of FTY720 markedly suppress leukemogenesis in SCID mice (n=13 per group) transplanted with myeloid and lymphoid progenitor cells transformed with p210BCR/ABL and p190BCR/ABL, respectively. In fact, the median survival has not yet been reached in FTY720-treated (10 mg/kg/day) BCR/ABL+ cell-injected mice. Conversely, all of untreated 32D-p210BCR/ABL, 32D-p210BCR/ABL(T315I) and BaF3-p190BCR/ABL leukemic mice died of an overt acute leukemia-like process with a median survival of 4.3, 4.8 and 4.1 weeks, respectively (P

Impaired differentiation is a common feature of many hematological malignancies including blast crisis chronic myelogenous leukemia (CML-BC). We previously reported that the inability of CML-BC myeloid progenitors to undergo terminal neutrophil differentiation depends on suppression of C/EBPα expression through the translation inhibitory activity of the BCR/ABL-regulated RNA binding protein hnRNP E2. Here we show that p210-BCR/ABL oncoprotein post-translationally enhances hnRNP E2 expression and translation-regulatory function. In fact, hnRNP E2 protein but not mRNA expression directly correlates with BCR/ABL levels in myeloid progenitors expressing different BCR/ABL levels, in Ph1 K562 and EM3 and BCR/ABL-inducible TonB.210 cells. Likewise, graded BCR/ABL expression in primary Lin− mouse bone marrow cells results in increasing hnRNP E2 levels. This, in turn, inhibits the expression of C/EBPα and that of the C/EBPα-regulated G-CSF (granulocytic-colony stimulating factor) receptor. In these models of CML-BC, increased hnRNP E2 expression results from the BCR/ABL- and ERK1/2-dependent enhancement of hnRNP E2 protein stability, as hnRNP E2 levels are downregulated by treatment with imatinib or the MEK inhibitors PD098059 and U0126, and after expression of dominant negative ERK1/2 mutants. Accordingly, hnRNP E2 protein levels rapidly decrease and are barely detectable in parental 32Dcl3 cells treated for 30 min. with the protein synthesis inhibitor cycloheximide (CHX). By contrast, they remain unchanged in 32D-BCR/ABL cells CHX-treated for 4 hours. Accordingly, hnRNP E2 protein expression is markedly reduced in IL-3-deprived parental but not 32D-BCR/ABL cells through a proteasome-dependent mechanism. Mechanistically, we have evidence that the BCR/ABL-activated ERK1/2 kinases phosphorylate hnRNP E2 on four different sites (S173, S189, T213, and S272), and that serine to alanine substitution of these phosphorylation sites abolishes hnRNP E2 phosphorylation in living cells and in vitro kinase assay. Moreover, the non-phosphatable hnRNP E2S173A, S189A, T213A, S272A mutant is less stable than wild-type and phosphomimetic hnRNP E2. Functionally, suppression of hnRNP E2 phosphorylation/expression by chemical inhibition of ERK1/2 activity results in decreased hnRNP E2 binding to the C-rich element contained in the uORF-spacer region of C/EBPα mRNA. This, in turn, restores C/EBPα expression and allows G-CSF-driven neutrophilic maturation of differentiation-arrested 32D-BCR/ABL cells and Lin− mouse bone marrow cells expressing high levels of p210-BCR/ABL oncoprotein. Likewise, treatment of G-CSF-cultured (48 hours) CML-BCCD34+ bone marrow patient cells with the MEK1 inhibitor U0126 (25 mM, 32 h) destabilizes hnRNP E2, thus restoring C/EBPα expression. Altogether our data not only indicate that the BCR/ABL-induced ERK1/2 activity is essential for the hnRNP E2 stability and suppression of C/EBPα-dependent granulocytic differentiation of CML-BC myeloid progenitors, but they also suggest the potential use of clinically-relevant MAPK inhibitors in the therapy of CML-BC.