Imatinib Mesylate Does Not Inhibit BCR-ABL Kinase Activity in CML Stem Cells In Vitro.

Imatinib mesylate (IM, Gleevec, Novartis), a specific inhibitor of the tyrosine kinase BCR-ABL, promotes an impressive complete cytogenetic remission rate in patients with chronic phase Chronic Myeloid Leukaemia (CML). In most cases however, molecular disease persists and may lead to eventual relapse. IM selectively interacts with BCR-ABL thereby inhibiting activation of downstream targets, in particular phosphorylation of CrkL. We have previously demonstrated the sensitivity of rare Ph+ CD34+ CML stem cells (<5% of total CD34+ cells) to the anti-proliferative but, not the pro-apoptotic effects of IM, hence their persistence in vitro. We hypothesised that these rare cells may also behave differently from total CD34+ cells in terms of their kinase response to IM. The effect of IM on the phosphorylation status of CrkL was therefore investigated, in total CD34+ and in primitive CD34+/38- cells from 6 untreated chronic phase CML patients. Ph+ CD34+ and CD34+/38- cells were cultured in serum free medium for up to 72h, in the presence and absence of IM (5mM). After 16 and 72h the phosphorylation status of CrkL was measured with an antibody specifically designed to recognise only the phosphorylated form of the protein. After 16h total CD34+ cells showed a significant dephosphorylation of CrkL in response to IM. However, after 72h the remaining viable BCR-ABL+ cells failed to show any de-phosphorylation of CrkL, consistent with an IM selected, resistant population of stem cells. Primitive CD34+/38- cells from the same patients exhibited no de-phosphorylation of CrkL after either 16 or 72h, confirming their innate/inherent resistance to the drug. These data show, for the first time, that very primitive CML cells exhibit BCR-ABL dependent resistance (i.e. BCR-ABL kinase remains active during drug exposure) and suggest that the drug is not reaching or binding its target at inhibitory concentrations. Novel approaches to eliminate these cells will be critical to the development of curative strategies for CML.