Leukemic Stem Cells of Chronic Phase CML Patients Consistently Display Very High BCR-ABL Transcript Levels and Reduced Responsiveness to Imatinib Mesylate in Addition to Generating a Rare Subset That Produce Imatinib Mesylate-Resistant Differentiated Progeny.

Abstract

Imatinib mesylate (IM) is an inhibitor of the BCR-ABL oncoprotein associated with human chronic myeloid leukemia (CML). IM therapy has shown remarkable effects in initial clinical trials, but both clinical and laboratory studies increasingly suggest that, on its own, IM may have limited curative potential, due to a reduced IM sensitivity of the more primitive, slowly proliferating CD34+ CML cells thought to be responsible for sustaining the disease in vivo. To investigate the basis of this unresponsiveness, we compared the IM sensitivity and BCR-ABL expression of FACS-purified subsets of lin−CD34+ cells from 4 CML chronic phase patients. None of these had been treated with IM and their cells at all stages of differentiation were exclusively leukemic; i.e., >95% of the lin−CD34+CD38−, lin−CD34+CD38+ and lin+CD34− cells were BCR-ABL+ (by direct FISH) and all longterm culture-initiating cell (LTC-IC) -derived CFCs were Ph+. In the absence of IM, suspension cultures initiated with these lin−CD34+CD38− CML cells (0.5–5% of the lin−CD34+ cells) showed a net expansion of viable cells after 3 weeks; 100x with and 10x without added growth factors (GFs). Addition of 0.1–10 μM/ml IM reduced the yield of viable cells in a dose-dependent fashion, particularly when GFs were not added (100-fold decrease with 10 μM/ml IM). Parallel cultures of the corresponding lin−CD34+CD38+ CML cells showed these did not expanded as much (~8x +GFs, 2x -GFs) and were more sensitive to IM (1000-fold decrease after 3 weeks in 10 μM/ml IM -GFs). Quantitative real-time RT-PCR analysis revealed BCR-ABL transcripts to be present in the most primitive, freshly isolated lin−CD34+CD38− cells (n=12) at >300-fold higher levels than in the terminally differentiating lin+CD34− CML cells (n=21), at >10-fold higher levels than the normal BCR transcripts in the same lin−CD34+CD38− cells, and at 40-fold higher levels than in the less primitive lin−CD34+CD38+ cells (n=12), indicating a correlation between decreasing BCR-ABL transcripts and increasing IM sensitivity during CML stem cell differentiation in vivo. Interestingly, maintenance of the lin−CD34+CD38− CML cells for 3 weeks in vitro with 10 μM/ml IM (±GFs) consistently selected for a subset of leukemic cells (80–100% BCR-ABL+ by FISH) that showed complete resistance to 5 μM/ml IM in CFC assays, in marked contrast to the CFCs in the starting lin−CD34+CD38− cells that were inhibited 5–10-fold by 5 μM/ml IM. Moreover, although the Ph was the sole abnormality present in all direct metaphases, initial CFCs and LTC-IC-derived CFCs from all samples, a 17p+ abnormality was seen in 4/4 metaphases obtained from one colony generated from the cells present in one of the 3-week IM-containing cultures, suggesting the selective survival of differentiating progeny of rare, pre-existing, IM-resistant stem cells. Consistent with this possibility was the finding that BCR-ABL transcript levels in the cells present in the 3 week cultures were reduced 50-fold relative to the input lin−CD34+CD38− cells. Taken together, these findings suggest a previously undescribed epigenetic mechanism of IM unresponsiveness characteristic of chronic phase CML stem cells, in addition to the silent accumulation of genetically-determined IM-resistant members as the CML stem cell population expands during the development of the chronic phase of the disease.