Imatinib mesylate (IM) is a tyrosine kinase inhibitor (TKI) that induces clinical responses in most chronic myeloid leukemia (CML) patients. Nevertheless, early relapses and later emergence of IM-resistant disease pose serious concerns for many. The inadequacies of IM therapy are due, at least in part, to the unique properties of CML stem/progenitor cells that make them generally less responsive to IM and, indeed, other TKIs, and also confer on them a genetic instability that leads to a high rate of formation of BCR-ABL mutants. Improved treatment approaches to prevent the development of resistant subclones by targeting other key molecular elements active in CML stem/progenitor cells are thus clearly needed. One candidate is a complex that forms in CML stem/progenitor cells between the oncoproteins encoded by AHI-1 (Abelson helper integration site 1), BCR-ABL and the JAK2 kinase. This complex contributes to the transforming activity of BCR-ABL both in vitro and in vivo and also plays a role in the IM response/resistance of primary CML stem/progenitor cells. We now describe the results of experiments designed to test the ability of ABL and JAK2 inhibitors to block the activity of this protein complex in CML cells. K562 cells engineered to stably overexpress AHI-1 showed a significantly reduced sensitivity to both IM (at 1 and 5 μM) and TG101209, a JAK2 inhibitor, (at 0.5 and 1 μM), as determined by assays for cell viability, apopotosis, and colony-forming activity. K562 cells engineered to suppression AHI-1 showed an opposite effect, with a heightened sensitivity to IM at concentrations as low as 1 μM. In addition, IM together with TG101209 was more effective at killing AHI-1-overexpressing K562 cells, IM-resistant K562 cells and IM-resistant T315I-mutant cells than either treatment alone. Western blot and co-IP experiments demonstrated a significant reduction of p-BCR-ABL, p-JAK2 and p-STAT5 in cells treated with IM plus TG101209 compared to cells treated with IM or TG101209 alone. Importantly, treatment with 5 μM IM, 150 nM dasatinib (DA) or 5 μM nilotinib (NL) in combination with 100 nM TG101209 caused a significantly greater reduction in the viability of primary CD34+CD38− and CD34+CD38+ CML cells when these responses were compared to any of the TKIs or TG101209 alone (~2-4 fold, n=3). Apoptotic cells at 72 hours were also significantly increased for all drug combinations compared to single agent treatments (40%-52% for the combinations vs 15%-18% for the single agents). CFSE tracking analysis of cell division in these cells further demonstrated additive anti-proliferative activity from the TKI plus TG101219 combinations, although some rare undivided cells were not eliminated. Nevertheless, exposure of CD34+ CML cells from IM-nonresponders (n=4) to TG101209 plus IM or DA did cause a greater inhibition (81% and 85%) of patients' colony-forming cells as compared to the same cells treated with the combination of IM plus DA only, or IM or DA only (60%, 41% and 50% inhibition, p<0.05). Long-term culture-initiating cell assays were undertaken to compare the effect of these combination treatments versus the effects of TKIs or TG101209 alone on very primitive CML cells. The results again showed a more significant reduction of these cells treated with the combination (n=3). Intracellular staining revealed a greater reduction in the levels of p-CrKL and p-STAT5 in CD34+ CML cells treated for 24 hours with the combination of TKIs plus TG101219 as compared to single TKI-treated cells (~44% vs 65% for p-CrKL and 36% vs 57% for p-STAT5, n=3). Strikingly, the combination treatment produced an even greater inhibition of both p-CrKL and p-STAT5 after 72 hours while p-CrKL was almost fully reactivated with TKIs alone (~29% vs 89% for p-CrKL and 23% vs 50% for p-STAT5). These results point to the possibility of achieving improved therapeutic outcomes in CML patients by simultaneously targeting both BCR-ABL and JAK2 activities in the critical TKI-insensitive CML stem/progenitor reservoir.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.