Abstract

In previous experiments we have demonstrated that BCR-ABL activates specifically STAT3 in the context of murine ES cells and in leukemic CD34+ cells in patients with chronic myelogenous leukemia. This activation occurs essentially through Tyr705 and Ser 727 phosphorylation and implicates Jak2 and MEK pathways (Coppo et al, Brit J Haematol, 2006). However, it is not known if STAT3 activation plays a role in the self-renewal of primitive stem cells or if it is predominantly involved in BCR-ABL-associated leukemogenesis. To determine the role of STAT3 at the primitive stem cell level, we have inhibited specifically STAT3 expression by using a shRNA-GFP-STAT3 construct which was lentivirally transduced into purified CD34+ cells from patients with CML. Western blot experiments determined the specificity of the shRNA-STAT3 construct in hematopoietic cell lines with specific inhibition of STAT3 with no interference with STAT1, STAT5a or STAT5b expression. 8 patients with CML at diagnosis were included in the study. CD34+ cells purified from cord blood (CB) or peripheral blood stem cell (PBCS) collects were used as controls. Each sample has been transduced with high titer lentiviruses expressing either sh-STAT3 or sh-luciferase control. After transduction, GFP+ cells were purified by cell-sorting and assayed in clonogenic assays as well as in longterm- culture assays in the presence of MS-5 stromal layers with weekly half-medium changes. At week+5, clonogenic assays were performed to evaluate the numbers of LTC-IC- derived progeny. The inhibition of STAT3 expression did not alter significantly the clonogenic cell potentials in CB-CD34+ cells (n=2) or PBSC (n=1) samples. In LTC-IC assays, STAT3 inhibition resulted in 1.8-fold reduced clonogenic output in one CB-CD34+ sample and increased the same clonogenic output by 6.7-fold in the second CB sample, with no effect in LTC-IC output in CD34+ cells purified from PBSC. Amongst CML samples, the numbers of LTC-IC-derived progenitors were reduced 3-fold after shRNA-mediated STAT3 inhibition in one patient (UPN2). In all other 7 patients, inhibition of STAT3 by shRNA led to either stable ( n = 1, UPN 4) or increased ( n= 6 ) LTC-IC derived clonogenic activity, with major increase of 5-week clonogenic output in 3 patients (luciferase vs shSTAT3 clonogenic outputs 28 vs 353 for UPN5; 130 vs 270 for UPN6; 295 vs 806 for UPN8). Thus, our results suggest that STAT3 activation seen in primary CML leukemic cells does not play a role in stem cell self-renewal detectable by LTC-IC assays. On the contrary, STAT3 inhibition seems to lead to a stimulating effect of primitive stem cells in the majority of the patients analyzed. These findings do not rule out the potential role of STAT3 in BCR-ABL induced leukemogenesis but suggest that STAT3 inhibition is not a clinically useful target at the stem cell level in CML.

Disclosures: No relevant conflicts of interest to declare.

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