Hematopoietic Cells Undergo Homotypic Cell Fusion: A Novel Platform to Study Genomic Instability and Leukemogenesis.

Abstract 1196

Gene copy number variation (CNV) and translocation-derived gene fusion products are commonly observed in hematological malignancies. Mechanisms precipitating CNV include failed cytokinesis or spontaneous cell fusion. Heterotypic fusion of hematopoietic cells with non-hematopoeitic cells has been reported following injury in diverse tissues. Cell fusion involves blending membranes, intracellular material, and nuclear material from two parental cells to form a genetically and immunophenotypically distinct (often hyperdiploid) single daughter cell. We therefore hypothesized that if we could generate genetically chimeric, hematopoietic cells via cell-cell fusion, they might provide an instructive model for determining the role CNV plays in leukemogenesis. To generate genetically abnormal hematopoietic cells in vivo, a transplantation model with radiation induction was employed in which donor and host mice possess numerous unique cell surface and genomic markers. Fusion was determined by co-expression of donor and host markers among hematopoietic cells isolated from recipient BM, spleen, and thymus by serial fluorescence activated cell sorting. The presence of ‘bona fide’ fused cells was confirmed by performing rigorous immunophenotypic and genotypic analyses of individual cells, including immunofluoresence staining and z-stack analysis, single nucleotide polymorphism (SNP) PCR, and fluorescent in situ hybridization (FISH). Fused cell populations were detected in both mature myeloid and lymphoid lineages. Consistent with the proposed model of studying early, initiating leukemogenic events, cell fusion was also detected in the progenitor cell populations including colony forming unit (CFU-C) myeloid progenitors, and c-kit+, sca-1+, lin- (KSL) cells. Moreover, transplanted fused hematopoietic cells could reconstitute lethally irradiated secondary hosts >16 weeks after transplant. Thus, hematopoietic progenitor cells are capable of homotypic cell fusion, and subsequent to cell fusion, these cells are further able to divide, differentiate, and are competent to contribute to functional hematopoiesis. Additional assays are underway to more definitively address the functional significance of hematopoietic cell fusion as a repair mechanism, or during homeostasis. Importantly, we detected evidence of genomic instability in fused hematopoietic cells using a variety of assays. Fused cells with loss of specific donor and/or host marker genes were identified by immunofluorescence and genomic DNA analysis. DNA strand breakage, evaluated by single cell gel electrophoresis (comet assay) showed the presence of a significantly larger tail moment in fused cells suggesting a higher degree of DNA damage in these cells. Studies for evaluation of p53 status and histone H2AX activation are underway. In summary, our data not only provide novel evidence that homotypic cell fusion occurs between hematopoietic cells following injury, but also that fused cells retain their replicative potential in vitro and in vivo and exhibit greater genomic instability compared to non-fused counterparts. Because most existing murine models of leukemogenesis focus on events in the promotion of disease, we believe fusion and reductive division of hematopoietic cells shown here will serve as a novel platform to study the role of CNV as a leukemia-initiating event.