Comparative Effects of Wild Type and T315I Mutated bcr-ABL in Murine Embryonic Stem-Cell Derived Hematopoiesis.

Recent data suggest that there is a major discrepancy between the expression of BCR-ABL in primitive hematopoietic stem cells as compared to their differentiated counterparts where the expression is much lower. This high expression level predisposes this compartment to a major genetic instability leading to a mutator phenotype. The “gatekeeper” mutation T315I is the most problematic of these mutations leading to a resistance to all three clinically available tyrosine kinase inhibitors (TKI). This mutation is suspected to generate a specific signalling distinct from non-mutated BCR-ABL with high transforming potential but less TK activity. In order to study the effects of both mutated and non-mutated BCR-ABL in a primitive stem cell context, we have transduced retrovirally (Bosc 23 packaging cell line) both BCR-ABL and BCR-ABL T315I as well as an empty GFP control retrovirus, into day+5 embryoid bodies (EB’s) derived from D3 murine embryonic stem (ES) cells. In several experiments, the transduction efficiency was found to vary between 10–45% as evaluated by GFP expression at day+ 3 post-transduction. EB’s were then dissociated and put in hematopoietic differentiation conditions using clonogenic assays and in liquid culture in the presence of OP-9 stroma and hematopoietic growth factors including SCF, IL6 and IL3. Cells were collected weekly and analyzed with regard to their hematopoietic commitment, their amplification potential, their phenotype and their morphology as well as expression of BCR-ABL. In clonogenic assays, BCR-ABL and T315I6transduced cellD induced GFP+ growth-factor-independent colonies. In hematopoietic differentiation conditions, empty vector- and BCR-ABL-transduced cells underwent approximately 8-population doublings in vitro. On the other hand, BCR-ABL T315I mutant-transduced cells underwent a major expansion during this time with 16-Population doublings in 4 weeks. The addition of imatinib mesylate (1 and 2 mM) to cell cultures stimulated cell growth in T315I-BCR-ABL- transduced cells as compared to controls. Interestingly, phenotypic analysis demonstrated the appearance of CD45+ CD34+ cells in BCR-ABL-transduced cells as early as week+2–3 whereas this hematopoietic differentiation appeared to be delayed in BCR-ABL-T315I-transduced cells. Thus, our data suggest that in the context of a primitive hematopoietic stem cell context, T315I BCR-ABL exhibits a higher transforming potential. Current experiments underway are testing the long-term repopulating ability of BCR-ABL and BCR-ABLT315I- expressing cells in NOD/SCID reconstitution assays as well as the occurrence of ABL-kinase mutations in the presence and in the absence of TKI. Thus, the murine ES-cell-derived hematopoiesis could be an important experimental tool to recapitulate the early stages of hematopoiesis and the role of T315I mutation in the rare, primitive stem cell populations.