Abstract: Myelofibrosis is a hematopoietic stem cell neoplasm characterized by bone marrow reticulin fibrosis, extramedullary hematopoiesis, and frequent transformation to acute myeloid leukemia. Constitutive activation of JAK/STAT signaling through mutations in JAK2, CALR, or MPL is central to myelofibrosis pathogenesis. JAK inhibitors such as ruxolitinib reduce symptoms and improve quality of life, but are not curative and do not prevent leukemic transformation, defining a need to identify better therapeutic targets in myelofibrosis. Experimental Design: A short hairpin RNA library screening was performed on JAK2V617F-mutant HEL cells. Nuclear–cytoplasmic transport (NCT) genes including RAN and RANBP2 were among top candidates. JAK2V617F-mutant cell lines, human primary myelofibrosis CD34+ cells, and a retroviral JAK2V617F-driven myeloproliferative neoplasms mouse model were used to determine the effects of inhibiting NCT with selective inhibitors of nuclear export compounds KPT-330 (selinexor) or KPT-8602 (eltanexor). Results: JAK2V617F-mutant HEL, SET-2, and HEL cells resistant to JAK inhibition are exquisitely sensitive to RAN knockdown or pharmacologic inhibition by KPT-330 or KPT-8602. Inhibition of NCT selectively decreased viable cells and colony formation by myelofibrosis compared with cord blood CD34+ cells and enhanced ruxolitinib-mediated growth inhibition and apoptosis, both in newly diagnosed and ruxolitinib-exposed myelofibrosis cells. Inhibition of NCT in myelofibrosis CD34+ cells led to nuclear accumulation of p53. KPT-330 in combination with ruxolitinib-normalized white blood cells, hematocrit, spleen size, and architecture, and selectively reduced JAK2V617F-mutant cells in vivo. Conclusions: Our data implicate NCT as a potential therapeutic target in myelofibrosis and provide a rationale for clinical evaluation in ruxolitinib-exposed patients with myelofibrosis.

Abstract: 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.

Abstract: The chronic phase of chronic myeloid leukemia (CP-CML) is characterized by the excessive production of maturating myeloid cells. As CML stem/progenitor cells (LSPCs) are poised to cycle and differentiate, LSPCs must balance conservation and differentiation to avoid exhaustion, similar to normal hematopoiesis under stress. Since BCR-ABL1 tyrosine kinase inhibitors (TKIs) eliminate differentiating cells but spare BCR-ABL1-independent LSPCs, understanding the mechanisms that regulate LSPC differentiation may inform strategies to eliminate LSPCs. Upon performing a meta-analysis of published CML transcriptomes, we discovered that low expression of the MS4A3 transmembrane protein is a universal characteristic of LSPC quiescence, BCR-ABL1 independence, and transformation to blast phase (BP). Several mechanisms are involved in suppressing MS4A3, including aberrant methylation and a MECOM-C/EBPε axis. Contrary to previous reports, we find that MS4A3 does not function as a G1/S phase inhibitor but promotes endocytosis of common β-chain (βc) cytokine receptors upon GM-CSF/IL-3 stimulation, enhancing downstream signaling and cellular differentiation. This suggests that LSPCs downregulate MS4A3 to evade βc cytokine-induced differentiation and maintain a more primitive, TKI-insensitive state. Accordingly, knockdown (KD) or deletion of MS4A3/Ms4a3 promotes TKI resistance and survival of CML cells ex vivo and enhances leukemogenesis in vivo, while targeted delivery of exogenous MS4A3 protein promotes differentiation. These data support a model in which MS4A3 governs response to differentiating myeloid cytokines, providing a unifying mechanism for the differentiation block characteristic of CML quiescence and BP-CML. Promoting MS4A3 reexpression or delivery of ectopic MS4A3 may help eliminate LSPCs in vivo.

Abstract: 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.

Abstract: 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.

Abstract: Normal human bone marrow cells are critical for studies of hematopoiesis and as controls to assess toxicity. As cells from commercial vendors are expensive, many laboratories resort to cancer-free bone marrow specimens obtained during staging or to umbilical cord blood cells, which may be abnormal or reflect a much younger age group compared to the disease samples under study. We piloted the use of femoral heads as an alternative and inexpensive source of normal bone marrow. Femoral heads were obtained from 21 successive patients undergoing elective hip arthroplasty. Mononuclear cells (MNCs) were purified with Ficoll, and CD3 〈 sup 〉 + 〈 /sup 〉 , CD14 〈 sup 〉 + 〈 /sup 〉 , and CD34 〈 sup 〉 + 〈 /sup 〉 cells were purified with antibody-coated microbeads. The median yield of MNCs was 8.95 × 10 〈 sup 〉 7 〈 /sup 〉 (range, 1.62 × 10 〈 sup 〉 5 〈 /sup 〉 –2.52 × 10 〈 sup 〉 8 〈 /sup 〉 ), and the median yield of CD34 〈 sup 〉 + 〈 /sup 〉 cells was 1.40 × 10 〈 sup 〉 6 〈 /sup 〉 (range, 3.60 × 10 〈 sup 〉 5 〈 /sup 〉 –9.90 × 10 〈 sup 〉 6 〈 /sup 〉 ). Results of downstream applications including qRT-PCR, colony-forming assays, and ex vivo proliferation analysis were of high quality and comparable to those obtained with standard bone marrow aspirates. We conclude that femoral heads currently discarded as medical waste are a cost-efficient source of bone marrow cells for research use.

Abstract: 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 (p 〈 0.01), prompting us to study the role of MS4A3 in more detail. Since the signatures for primary cytogenetic failure and BP-CML overlapped, we next assessed whether MS4A3 expression was downregulated in BP-CML versus CP-CML. qRT-PCR confirmed that MS4A3 mRNA levels are markedly reduced (by 〉 92%) in CD34+ cells from BP-CML patients (n=17; p 〈 0.01) versus CP-CML patients (n=23) and normal controls (n=3). There was no significant difference between normal and chronic phase CD34+ cells, suggesting that MS4A3 is specifically downregulated upon CML blastic transformation. Microarray data from Oehler et al. (unpublished observations) and Zheng et al. (Leukemia 2006) also showed that MS4A3 mRNA levels are reduced in CD34+ cells from BP-CML patients (n=16; p 〈 0.001) compared to CP-CML patients (n=19). Immunoblot analyses confirmed that MS4A3 protein was detectable in CD34+ cells from newly diagnosed CP-CML patients, but not in samples from patients with primary resistance who failed multiple TKIs but lack clinically relevant BCR-ABL1 mutations. Finally, in CP-CML samples from newly diagnosed patients, MS4A3 mRNA levels are 12-fold reduced in primitive CD34+38- stem cells compared to more committed CD34+38+ progenitor cells (n=3; p 〈 0.05), which are innately resistant to BCR-ABL1 inhibition. Consistent with BCR-ABL1 kinase independence, MS4A3 mRNA and protein levels were unaffected by ex vivo TKI treatment. 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; p 〈 0.05) and abolished the effects of imatinib in colony formation assays (n=2; p 〈 0.05). We next assessed the effects of shMS4A3 in CD34+ cells from newly diagnosed CP-CML patients. qRT-PCR confirmed ~50% MS4A3 knockdown in primary cells. Despite the incomplete knockdown, shMS4A3 enhanced colony formation in the presence of imatinib in a dose-dependent manner (n=4; p 〈 0.00001), and abolished imatinib-induced apoptosis (n=3; p 〈 0.001). We also assessed the effects of ectopic MS4A3 expression in CD34+ cells from advanced phase CML (AP-CML) patients. qRT-PCR confirmed 〉 2-fold upregulation of MS4A3. As expected, ectopic MS4A3 reduced colony formation by 55% in AP-CML (n=2; p 〈 0.01), and enhanced sensitivity to imatinib-induced apoptosis by 35% (n=2; p 〈 0.01). Neither shMS4A3 nor ectopic MS4A3 had any effect on survival of normal cord blood CD34+ cells (n=2). 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.