Mll-Af4 And Flt3 Activation Synergize To Induce Multi Step Leukemogenesis.

Abstract Acute lymphoblastic leukemia (ALL) expressing mixed-lineage leukemia (MLL)- AF4, the fusion product of t(4;11)(q21;q23), respond poorly to chemotherapy and have poor prognosis. MLL was required in normal hematopoietic proliferation and differentiation through Hox gene regulation. AF4 is a serine/proline rich nuclear protein with transcriptional activation domain and plays an important role in B and T lymphopoiesis. The MLL-AF4 fusion protein preserves the AT-hook and methyltransferase domains of MLL and the GTP binding, and nuclear localization regions of AF4. It is still controversial whether the MLL fusion protein is sufficient to induce acute leukemia without additional genetic alterations, although carcinogenesis in general is known to result from more than 1 genetic disorder accumulating during a lifetime. The mutations of FMS-like receptor tyrosine kinase 3 (FLT3) with constitutive tyrosine kinase activity are classified into FLT3-ITD or mutations within the activation loop (FLT3-mut), such as FLT3D835V. Recently FLT3-muts are found frequently in infant acute lymphoid leukemia with MLL rearrangements. In the present study, we intended to demonstrate MLL-AF4 fusion protein need activated FLT3-muts for leukemogenetic mechanisms. We successfully established a cell line expressing MLL-AF4 from proB ALL patients with t(4;11)(q21;q23). This cell line expressed CD10− CD15+ CD19+ phenotype and overexpressed c-myc by duplication chromosome 8. We have also succeeded to clone cDNA of MLL-AF4 from this cell line, and we got full length FLT3 cDNA from ORIGENE. After making FLT3-ITD and FLT3D835V mutation (FLT3-mut) by site-directed mutagenesis, we used them to confirm leukemogenetic mechanisms. Murine IL3 dependent cell line 32Dc was transduced with lentiviral vector (pCL20c Mp) encoding human MLL-AF4 cDNA (pCL20c CMp+MLL/AF4sEF1a-GFP) and/or FLT3-ITD or -mut (pCL20c Mp+FLT3 EF1a-DsRedExp). After confirming both mRNA expressions by RT-PCR and protein expressions by Western blot, each clone was isolated by FACSVantageSE. First, we examined growth profile under IL3 deprivation in each transduced 32Dc cell lines. 32Dc with FLT3-mut, and MLL-AF4 temporally grew and tended to show anti-apoptotic effect (day 5), but finally did not grow and demonstrated apoptotic cell death (day 10) under IL3 deprivation. By contrast, 32Dc with FLT3-ITD, and MLL-AF4 and FLT3-mut (MLL-FLT3-mut) could permanently grow and tended to show anti-apoptotic effect. We also examined their ability to confer clonogenic growth on 32Dc in semisolid media with presence or absence of IL3. Only 32Dc with FLT3-ITD and MLL-FLT3-mut could form coloies in semisolid media without IL3. Next we examined myeloid differentiation of each transduced 32Dc cell lines in response to granulocyte colony stimulating factor (G-CSF). Stimulation by G-CSF couldn’t promote morphologic differentiation of 32Dc with MLL-FLT3-mut, but promoted 32Dc with MLL-AF4 or FLT3-mut to granulocytes. These results clarify that MLL-AF4 plays an important role in a multi step leukemogenesis. Especially MLL rearrangement plays anti- apoptotic effect and AF4 rearrangement inhibit myeloid differentiation. However FLT3-mut may be necessary and sufficient for secondary genotoxicity on leukemogenesis.