FLT3-ITD Mediated Growth Advantage of Human CD34+ PBPCs Is Dependent on Stromal Cells.

Activating mutations of the FLT3-receptor tyrosine kinase such as the short internal tandem duplication (ITD) represent the single most common genetic aberration in patients with acute myeloid leukemia (AML). FLT3-ITD mutations are present in about 25% of AML patients and are associated with high blast counts and a higher rate of relapse after conventional chemotherapy. In search for a valid model system for FLT3-ITD AML we greatly improved retroviral gene transfer of the mutated FLT3 receptor into human G-CSF mobilized CD34+ peripheral blood progenitor cells (PBPCs). In the past, retroviral vector production for FLT3 had been hampered by low virus titers related to transgene toxicity in the virus producing cells. Virus vector production in the presence of the FLT3 inhibitor GTP14562 strongly increased the virus vector titer. Subsequent ultracentrifugation and resuspension of the concentrated virus vector in fresh medium enabled us to consistently transduce CD34+ PBSCs to levels of greater than 50%. For individually tested CD34+ PBPC from 5 donors, co-cultivation of FLT3-ITD overexpressing PBPC on the irradiated mouse stromal cell line M210-B4 resulted in a 6 fold increased formation of early cobble stones and a dramatic proliferation advantage for suspended cells compared to naïve, GFP-transduced or FLT3-wildtype transduced PBPCs. The enhanced proliferation of the FLT3-ITD transduced cells was associated with decreased CD34 expression compared to control-transduced cells. Addition of the CXCR4 chemokine inhibitor AMD3100 to the co-culture decreased the cobble stone formation and growth advantage of FLT3-ITD transduced PBPCs. In contrast, cultivating the same FLT3-ITD overexpressing PBPCs in liquid culture (X-VIVO10, 1%HSA, TPO, SCF, FLT3 and IL-3) resulted in a moderate or no growth advantage or in an even reduced cell viability (dependent on the donor PBPCs used), which indirectly highlights the influence of stromal cells on FLT3-ITD overexpressing PBPCs. And in contrast to previously published data from murine cells, addition of the CXCR4 ligand stromal cell derived factor-1 (SDF-1) to liquid cultures did not confer an additional growth advantage for FLT3-ITD positive cells. Using an improved transduction protocol we demonstrate that the robust FLT3-ITD mediated growth advantage for human PBPCs is dependent on co-cultivation on a stromal cell layer and we show that the enhanced proliferation can be partly inhibited by the CXCR4 inhibitor AMD3100. Studies to delineate factors that confer FLT3-ITD specific cell growth on stromal cells are under way.