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

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.

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.

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.