MS4A3 Improves Imatinib Response and Survival in BCR-ABL1 Primary TKI Resistance and in Blastic Transformation of Chronic Myeloid Leukemia

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-naive 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 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 + 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 (unpublished observations) and Zheng et al.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 + 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 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 IC 50 (n=3; p + 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 + 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 + 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.