BMS-214662 Targets Quiescent Chronic Myeloid Leukaemia Stem Cells and Enhances the Activity of Both Imatinib and Dasatinib (BMS-354825).

Chronic myeloid leukaemia (CML) is a clonal disease of stem cell origin associated with expression of the Philadelphia chromosome and its oncogenic fusion protein product Bcr-Abl. Despite an impressive rate of complete cytogenetic response in chronic phase CML, the majority of patients treated with imatinib mesylate (IM) show persistent molecular disease. Recent work by our group shows that this molecular persistence results from a population of quiescent CML stem cells which are not effectively targeted by IM, the novel, oral, multi-targeted kinase inhibitor dasatinib (BMS-354825; which targets Bcr-Abl and Src kinases), or several rationally designed drug combinations¹. Further in vitro studies by our group have demonstrated that the only combination to have an improved response in the quiescent stem cell sub-population was IM with the farnesyl transferase inhibitor (FTI) lonafarnib. BMS-214662 is an atypical non-peptidomimetic cytotoxic FTI, which has been shown to preferentially kill non-dividing cells² and has anti-leukaemic activity in acute myeloid leukaemia. We assessed the efficacy of this compound alone and in combination with IM and dasatinib in primary CD34⁺ CML cells in vitro using a CFSE-based flow cytometry method to track cell division, caspase-3 activity to measure apoptosis and dephosphorylation of Crkl to determine Bcr-Abl kinase activity. Primary CD34⁺ CML cells were cultured for 6 days in serum free medium supplemented with 5 growth factors (IL-3, IL-6, Flt-3 ligand, G-CSF and SCF). Conditions studied were: (1) no drug control, (2) IM (5μM; ~IC₉₀ dose), (3) dasatinib (150nM; ~IC₉₀ dose) (4) BMS-214662 (250nM; ~IC₅₀ dose), (5) IM plus BMS-214662, (6) dasatinib plus BMS-214662. After 6 days culture, there was a significant reduction in total viable cells in all treatment arms relative to the no drug control (P=0.001). The combinations of IM plus BMS-214662 and dasatinib plus BMS-214662 showed increased cytotoxic effect over either IM or dasatinib alone (P=0.024 and P=0.034, respectively). While the IM and dasatinib arms showed significant accumulation of undivided CFSE^max CD34⁺ CML cells over the no drug control (P=0.04 and P=0.023, respectively), the arms containing BMS-214662 either alone or in combination showed a reduction in these primitive cells to <50% of the no drug control. This reduction was highly statistically significant when either IM or dasatinib alone was compared to the combination with BMS-214662 (P=0.01 and P=0.043, respectively). There were no significant differences in undivided CFSE^max CD34⁺ CML cells between the BMS-214662 containing arms. At 72 hours, caspase-3 activity was increased in the BMS-214662-containing arms with increased apoptosis in the undivided CFSE^max CD34⁺ CML cells. BMS-214662 induced dephosphorylation of Crkl in remaining viable cells at 72 hours and 6 days, suggesting inhibition of Bcr-Abl kinase activity. In conclusion, BMS-214662 is highly effective against CML cells, including, for the first time, the primitive quiescent stem cell fraction, overcoming the accumulation of this population seen with IM or dasatinib in vitro.