Inecalcitol, a Novel Adjuvant Therapy Inhibiting Chronic Myeloid Leukemia (CML) Stem Cells, Activates a Macrophage Differentiation Pathway in Leukemic Progenitors

Despite the major success obtained with their use in chronic myeloid leukemia (CML), recent data obtained from treatment discontinuation trials suggest that tyrosine kinase inhibitors (TKI) alone are not sufficient to eradicate the most primitive CML stem cells in the majority of the patients. The mechanisms of this inefficiency might involve cell autonomous (activation of alternate signaling, reduced BCR-ABL expression) or non-cell autonomous (niche-related) pathways.  Several strategies of targeting the primitive stem cell compartment, in association with TKI, are currently being studied. Inecalcitol  (ICC) is a vitamin D3 analog exerting antiproliferative effects in several types of cancer cells. ICC  was tested in CD34+ cells isolated from CML patients at diagnosis (n= 18) in clonogenic assays as well as in the more primitive LTC-IC-derived progenitors. ICC alone inhibits the clonogenic growth in the majority of the CML patients at diagnosis (15/ 18 patients). The combination of ICC with either, Imatinib (IM), Dasatinib (DA) or Nilotinib (NIL) in clonogenic assays showed a synergistic effect for the inhibition of CFC growth (10 - 25% CFC survival) with no toxicity on normal progenitors. Synergistic effects of ICC and TKI was also demonstrated in LTC-IC-derived progenitors with IM, NIL and DA. To determine possible mechanism of action of ICC in CML stem cells, we have performed a gene profiling analysis of CD34+ cells obtained at diagnosis from 4 CML patients.  CD34+ cells were cultured for 7 days in the presence of growth factors with or without ICC. In phenotypic analyses, CD34+ cells treated in the presence of ICC showed an increase of  monocyte /macrophage differentiation features with increased expression of CD13/CD14 as well as CD11b expression. Day 0 and Day7 RNA with or without ICC treatment were then studied by transcriptome hybridation on human-v2 (8*60k) Agilent technologies microarrays. Data were treated with Feature Extraction 11.5.11, Genespring GX12, Mev 4.9, R software and GSEA 2.2.20.  Gene set enrichment analysis performed at day 7 of culture between ICC condition and control untreated cells showed an increase of cell differentiation markers under ICC (Normalized enrichment score = +1.94, p-value < 0.001)  One way ANNOVA with False Discovery Rate (FDR) correction allowed to discover 7176 modulated probes between the 3 experimental conditions (Day 0, Day7 without ICC, Day 7 with ICC). CYP24A1  was the gene with the greatest induction by the treatment (Fold Change = +150). CYP24A1 is responsible of the degradation of 1.25(OH)2D3 by the monocyte/macrophage cells. Unsupervised classification with 372 macrophage connected genes (ANOVA p<0.01, FDR with 1000 permutations) allowed to separate samples by their experimental classes. This macrophage expression profile allows to discriminate samples from Day7 control to those of treated cells at the same time on hierarchical classification. This was confirmed by first factorial map of principal component analysis which explained 72% (PCA1 axis) hematopoietic differentiation during 7 days and 10% (PCA2 axis) effect of the treatment at the end of the differentiation. 23 macrophagic genes were indeed found to be specifically induced by the treatment with a fold change greater than 2 as compared to the untreated control: 11 of them participate to integrin-interleukins-chemokines signalizations pathways (p-value adjust FDR = 4.04e-10). Some macrophagic ligands and receptors are over-expressed in CML cells treated with ICC, including CSF2, OSM, TNFSF11, CXCL12 as well as FAS and CXCR2. In summary, these results suggest that one of the major mechanisms of action of ICC in the leukemic progenitors involve differentiation and activation of macrophagic expression profile. This profile could be used to design further therapeutic actions and to predict response to ICC, which is now tested in combination with IM in a clinical trial in France.