Chronic myelogeneous leukemia (CML) is a two stage disease associated with expression of the BCR/ABL tyrosine kinase protein. Experiments in animals and humans have demonstrated that BCR/ABL is clearly responsible for the hyper-proliferation associated with chronic stage CML. However whether BCR/ABL expression directly causes blast crisis, and if so by what mechanism, is unknown. We hypothesize that BCR/ABL modulates the response to DNA damage, thus leading to a state of genomic instability. In support of this hypothesis, we have found that 1) BCR/ABL translocates from the cytoplasm to the nucleus after genotoxic stress; 2) BCR/ABL inhibits the repair of DNA double strand damage after etoposide and leads to a defect in an intra-S phase checkpoint causing a radioresistant DNA synthesis (RDS) phenotype; and 3) BCR/ABL disrupts ATR dependent signal transduction, characterized by the lack of phosphorylation of Chk1 and by the inability of BCR/ABL expressing cells to inactivate the cdc7/Dbf4 regulatory kinase after treatment with etoposide. Taken together, these exciting results show that BCR/ABL expression inhibits activation of ATR, leading to continued DNA synthesis during S phase. We hypothesize that such persistent DNA replication forks would collide with topoisomerase II/etoposide complexes, leading to increased DNA double strand breaks and the formation of chromosomal translocations. In order to confirm and extend this hypothesis, we propose to: 1) determine if BCR/ABL induces permanent chromosomal changes by studying cells using spectral karyotyping; 2) determine if BCR/ABL alters other functions of the ATR protein; and 3) determine the mechanism whereby BCR/ABL disrupts ATR function. Finally, we propose to develop animal models of CML blast crisis to confirm our hypotheses in vivo. Through these multiple approaches, we propose to document and elucidate this novel mechanism of CML progression induced by the BCR/ABL oncogene. More generally, we believe that these studies, based on the fundamental biology of the CML model, may provide insights into the molecular mechanisms of multi-stage carcinogenesis and establish paradigms for future studies of carcinogenesis in diverse systems and diseases.
