Vitamin D3 Analog Inecalcitol Synergizes Vith Imatinib To Inhibit Selectively The Growth Of Chronic Myeloid Leukemia (CML) Progenitors and Stem Cells

Tyrosine kinase inhibitor (TKI) therapies which are the first line drugs in chronic myeloid leukemia (CML) have profoundly changed the prognosis of the disease and prolonged survival. However, it is clear that TKI are not able to eradicate efficiently the most primitive leukemic cells, due to their quiescence or oncogene-independence. It would therefore be of major interest to determine if compounds targeting CML progenitors / stem cells can be used in combination with TKI. Inecalcitol (19, nor 14 epi 23-yne-1,25 (OH)2D3) (ICC) is a vitamin D3 analog which has been shown to exert antiproliferative effects in several types of experimental tumors. As compared to calcitriol, the active for of natural vitamin D3, ICC has been shown to induce less hypercalcemia in vivo in mice and a higher differentiation inducing effect. The effects of ICC in primary CML progenitors and stem cells has not been tested so far. We first studied the effects of ICC on the U937 cell line as this cell line is a model responsive to the differentiation-inducing effects of vitamin D3. We have previously generated U937 cell lines expressing BCR–ABL (Ahmed et al, Leukemia Lymphoma, 2001). The effects of ICC were tested in these cells at day +2, +4 and +7 of culture using ICC at the dose of 5 nM and by the assessment of differentiation based on the morphology and the appearance CD11b and CD14 markers in flow cytometry. In dose-response experiments, we have cultured U937 cells in the presence of 1, 2 and 5 nM of ICC and we have established that the concentration of 5 nM was the most efficient in terms of differentiation induction. In cell kinetics experiments, culture of U937Neo cells as compared to U937-BCR-ABL cells, ICC alone ( 5nM) induced a similar differentiation effect with 45% of control cells and 30% of BCR-ABL cells expression CD11b at day 2, respectively and 30 % of both expressing CD11b at day 5. ICC induced the expression of CD14 at day 2 and day 4 with similar efficiencies, suggesting that BCR-ABL did not interfere with the differentiation inducing effect of ICC. In parallel with the differentiation effect, the cell mortality increased as compared to non-treated cells by 15% at day 5. To determine the effects of ICC in CML progenitors, we have used ICC alone at 1 nM and 5 nM final concentrations and established that the latter had the most inhibitory effect on CD34+ derived colony-forming-cell (CFC) growth (25% and 50% reduction as compared to untreated control n= 2 exp). In further experiments, CD34+ cells isolated from CML patients at diagnosis (n= 5) were tested in clonogenic assays (500 CD34+ cells / dish in triplicates) using a combination of ICC (5 nM) and Imatinib Mesylate (IM) which was used either at a concentration of 0.2 or 0.5 mM. In all CML samples (n= 5) there was a synergistic effect of the combination of IM and ICC, either at IM 0.2 mM and ICC 5nM ( 17% CFC survival n= 2) or IM 0.5 mM and ICC 5nM concentrations (10% CFC survival, n=3). On the other hand, this combination did not inhibit clonogenic cell growth from normal control CD34+ cells tested in the same conditions either with ICC alone at 5 nM ( 111% mean CFC survival as compared to untreated controls n= 3) or in combination of ICC 5 nM and IM 0.5 mM ( 100 % CFC survival n= 3). We have then used CD34+ cells from CML (n=2) and control ( n= 2) CD34+ cells to test the effects of ICC and IM in long-term culture initiating cell (LTC-IC) assays. Long-term cultures were started using 4.10⁴ cells on MS5 cell stroma in three conditions by adding IM 0.5 mM, ICC 5nM or the combination of both in cultures, with weekly half-medium changes during which drugs were added to cultures in replaced medium. At week+5 cultures were terminated and LTC-IC progeny was evaluated. The numbers of non-adherent cells collected from long-term cultures were reduced significantly in the presence of IM and ICC in CML samples. A similar pattern of inhibition was also observed in control CD34+ cultures but in terms of clonogenic activity at week 5, there was a slightly higher inhibition of LTC-IC-derived CFC numbers (n=2) in CML. Thus, these results establish that ICC, a clinically used derivative of vitamin D3 has a clear activity in CML progenitors by itself and a major synergistic effect with Imatinib. The combination is not toxic to normal progenitors. This would suggest that ICC could be used in clinical setting, especially if in future experiments, a synergistic effect could be observed with more potent second generation TKI such as Nilotinib and Dasatinib.