The Leukemic Fusion Gene NUP98-HOXD13 Suppresses Non-Homologous End Joining Factors and Impairs DNA Double Strand Break Repair

Abstract 521

Chromosomal translocations are hallmark features of hematologic malignancies that often require collaborating mutations for malignant transformation. These secondary mutations can occur spontaneously, or rather, be induced by the primary translocation. Mutations can occur as a result of DNA damage and misrepair with DNA double strand breaks being one of the most serious types of cell damage. Double strand breaks are classically repaired by the non-homologous end joining (NHEJ) mechanism and impaired NHEJ has been shown to promote mutagenesis. Transgenic mice expressing the myeloid leukemic fusion gene NUP98-HOXD13 (NHD13) develop Myelodysplastic syndrome and progress to acute leukemia after acquiring secondary mutations. Our studies have shown that B lymphocyte development and class switch recombination are impaired in stimulated B lymphocytes from NHD13 mice. Based on this, we used in vitro class switch recombination (CSR) to delineate the DNA break induction and repair mechanisms in NHD13 B lymphocytes. Naïve B lymphocytes were harvested from wild type (WT) and NHD13 spleens and cultured in the presence of E.coli Lipopolysaccharide (LPS) and IL-4 to induce CSR. The DNA break induction pattern was determined using phosphorylated H2AX labeling combined with confocal microscopy and flow cytometry. Our results showed that NHD13 B lymphocytes had a comparable break induction pattern, but significantly reduced DNA repair. Analysis of the cell cycle pattern of stimulated B cells at 24 hour intervals showed cell cycle arrest at the G2/M phase at 72 hours following stimulation—a hallmark feature of impaired DNA break repair. We analyzed the expression of classical NHEJ and alternative end joining factors including Ku70, Ku80, DNA Protein Kinase catalytic subunit (DNAPKcs), Xrcc4, DNA ligase 4, Ligase 1, and Ligase 3. Our results showed reduced expression of DNAPKcs, Ligase 4 and Xrcc4 in NHD13 B lymphocytes at 72 hours following stimulation, suggesting that cells failed to initiate NHEJ-mediated DNA repair. Our results suggest that a myeloid leukemic gene can impair the DNA repair mechanism and may indirectly promote mutations necessary for malignant transformation.