Targeting the CML Stem/Progenitor Cell with JAK2 (BMS-911543)/ABL Inhibitor Combination Therapy

Abstract Abstract 2743 The hallmark of chronic myeloid leukemia (CML) is the BCR-ABL fusion gene that originates in hematopoietic stem cells. The BCR-ABL oncoprotein has constitutively elevated tyrosine kinase (TK) activity and drives CML pathogenesis. The first specific BCR-ABL inhibitor, Imatinib Mesylate (IM), has had a major impact on the treatment of chronic phase CML. However, early relapse and molecular evidence of residual disease remain a significant issue in some IM-treated patients. We and others have recently demonstrated that CML stem/progenitor cells are insensitive to IM, genetically unstable, and characterized by elevated BCR-ABL expression and TK activity. The prevention of the development of resistant subclones through the specific targeting of CML stem/progenitor cells consequently represents a key unmet medical need in CML therapy. We recently identified the presence of a physical complex between oncoproteins encoded by AHI-1 (Abelson helper integration site 1), BCR-ABL and JAK2. This molecular complex contributes to the transforming activity of BCR-ABL and plays a key role in the IM responsivity/resistance of primary CML stem/progenitor cells. We therefore hypothesized that a combination treatment strategy designed to destabilize the complex might provide a rational approach to the elimination of CML stem/progenitor cells. Several JAK2 inhibitors are currently in various stages of clinical development, but myelosuppressive effects of these inhibitors on normal hematopoietic stem/progenitor cells remains a concern. We have examined a new and selective JAK2 inhibitor (BMS-911543) in combination with a number of different tyrosine kinase inhibitors (TKIs), including IM, dasatinib (DA), and nilotinib (NL) in CML cells. First, Western blot analysis showed that combination treatment with IM and BMS-911543 was more effective at reducing pSTAT5 levels in K562 cells and IM-resistant K562 cells (K562R) than in cells treated with BMS-911543 or IM alone. BMS-911543 treatment alone was not though effective in reducing pSTAT5 levels. Consistent with the Western blot analysis, in colony-forming cell (CFC) assays CML cell lines derived from myeloid or B-lymphoid blast crisis patient samples that expressed high levels of BCR-ABL (K562, K562R, BV173, and UT7 transduced with BCR-ABL) demonstrated reduced colony numbers, especially in medium-sized colonies (50–500 cells per colony) following combination treatment (BMS-911543 and IM) as compared with cells treated with BMS-911543 or IM alone (∼2–3 fold for all cell lines, p<0.05). To determine whether the results obtained following the simultaneous targeting of BCR-ABL and JAK2 in CML cell lines would extend to primary primitive CML cells and whether this combined BCR-ABL-JAK2 targeting approach could also be therapeutically effective for CML patients with inadequate responses to treatment with TKI monotherapy, we investigated CML cells obtained at the time of diagnosis from four CML patients who were classified retrospectively after the initiation of IM therapy as non-responders. To assess the effects on CFCs, CD34+ cells isolated from these samples were plated directly in methylcellose medium containing growth factors and TKIs (5μM IM and NL and 150nM DA) and BMS-911543 (100 and 300nM) alone, or in combination. The number of colonies obtained in cultures containing the respective TKIs and BMS-911543 alone was reduced from control values by about 50%. However, when TKI and BMS-911543 was present, a more significant reduction in colony formation was observed (77–86% inhibition of colony growth, p<0.05). Interestingly, combination treatment significantly inhibits BFU-E colony formation as compared with treatment with TKI alone (92–100% inhibition for the combinations vs 63–66% inhibition for the single agents, p<0.05). Most significantly, our data indicate far less toxicity on normal bone marrow CD34+ cells (n=4) as compared with CML samples treated with either single agent or combination therapy (2–3 fold from 100 to 600nM, p<0.05). The combination approach appears to be effective against treatment-naïve CML stem/progenitor cells derived from patients who were subsequently shown to be clinically resistant to IM therapy, suggesting a rational strategy for improving the clinical outcome of CML patients destined to develop IM resistance that involves the simultaneous targeting of both BCR-ABL and JAK2 in CML stem/progenitor cells. Disclosures: No relevant conflicts of interest to declare.

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