Growth Factor Deprivation Combined with Prolonged Inhibition of BCR-ABL Does Not Eradicate Functional CML Stem Cells

Chronic myeloid leukaemia (CML) is a myeloproliferative disorder that originates in a haemopoietic stem cell (HSC) and is characterised by the BCR-ABL oncogene. We have previously shown that cancer stem cells exist in all patients with newly diagnosed CML. These cells are primitive, quiescent, transplantable in NOD/SCID mice and insensitive to the tyrosine kinase inhibitor (TKI) imatinib. This intrinsic resistance may in part be explained by high expression of BCR-ABL in the most primitive cells. Our overall aim was to achieve complete and prolonged inhibition of BCR-ABL within CML CD34+ cells in vitro, using the highest clinically achievable concentration of a novel dual Src/ABL TKI, dasatinib, in serum free medium in the absence of growth factors. The drug was replenished every 3 days and cells remaining at 12 days were analysed. Despite growth factor deprivation combined with 12 days continuous treatment with dasatinib 150nM, 10% of the starting CD34+ cells were recovered. CFSE stained CML cells showed that treatment with dasatinib resulted in a “backing up” of cells within the undivided cell population and earlier cell divisions, demonstrating that dasatinib, like imatinib, is anti-proliferative. Inhibition of P-CrkL and hence BCR-ABL was maximal at the earliest time-point (day 4) and in the cells that were able to proliferate through several divisions, likely the most mature cells. Cells which remained undivided/quiescent and maintained expression of stem cell markers, CD34 and CD133, demonstrated significantly elevated levels of P-CrkL, as compared to the mature cycling populations. Following drug washout, the remaining viable cells achieved 5-fold expansion when cultured in growth factors for 7 days. Moreover, these cells were able to form colonies following culture in LTCIC assays, which were BCR-ABL positive by FISH and did not show gene mutation. Retrospective studies which assessed the efficacy of TKI treatment times and concentrations also showed that transient dasatinib exposure for 1 hour at 150nM, resulted in a ~80% reduction of viable CML cells when assessed 72 hours later and was as effective as 72 hours continuous exposure at concentrations as high as 1000nM. Our next approach will be to combine BCR-ABL knock-down using shRNA with dasatinib to fully inhibit BCR-ABL in the stem cell population in an effort to determine whether these cells are dependent on BCR-ABL for survival and proliferation.