The Leukemia Oncogene E2a-Hlf Impairs Checkpoint Activation.

The E2a-Hlf oncogene results from the translocation of chromosomes 17 and 19. This is a rare translocation found in pro-B cell leukemia cells of older children and adolescents. However, the resulting leukemia has been fatal in all reported cases despite intensive therapy. Evidence suggests that E2a-Hlf contributes to leukemogenesis by impairing apoptosis, but the precise molecular mechanisms of malignant transformation are not well understood. We have observed that E2a-Hlf confers a survival disadvantage relative to un-transduced cells and hypothesize that impaired checkpoints play a major role in genetic instability, malignant transformation, and resistance to therapy. In retrovirally transduced murine pro-B lymphocytes (FL5.12) we have observed that when co-cultured with un-transduced cells, the percentage of E2a-Hlf expressing cells diminishes over time. Furthermore, after retroviral transduction of wild-type primary hematopoietic progenitor cells and transplantation into lethally irradiated recipients, E2a-Hlf expressing cells are undetectable after 2 weeks. In contrast, after transduction and transplantation of replication impaired hematopoietic progenitors (E2F1 and E2F2 mutated), E2a-Hlf expressing cells persist for up to 6 months. We have also observed a survival disadvantage in fibroblasts, indicating that the effect is not tissue specific. When exposed to genotoxic agents such as bleomycin, E2a-Hlf expressing cells have substantially decreased activation of the checkpoint proteins p53, Chk1, and Chk2 as determined by Western blot analysis using phospho-specific antibodies. This effect appears to be downstream of DNA damage focus formation, as levels of ATR and phosphorylated ATM (Western blotting) and gamma-H2AX foci formation (immunofluorescence) are not affected by E2a-Hlf expression. Future studies will be directed at determining how checkpoint activation is inhibited by E2a-Hlf. A better understanding of the molecular mechanisms of E2a-Hlf mediated malignant transformation may lead to targeted and more effective therapy.