DNA damage inducers, i.e., genotoxins, are some of the most effective agents in cancer therapy. Persistent damage to the genomic DNA can activate programmed cell death. Therefore, fundamental understanding of genotoxin-induced cell death mechanisms holds the promise of enhancing the efficacy of cancer therapeutics. The varied responses of tumors to genotoxins are a reflection of the variable death responses to DNA damage among different normal cell types, which suggest developmental programs may set different thresholds for damage signals to trigger death. Studies from our laboratory have identified the nuclear Abl tyrosine kinase as a developmental context-dependent activator of cell death to DNA damage. Through tyrosine phosphorylation of the C-terminal repeated domain (CTD) of RNA polymerase II, we have found that Abl regulates RNA splicing in genotoxin-treated cells. The human genome contains approximately twenty thousand genes but encodes many more proteins through the alternative usage of variable exons. Alternative splicing has been shown to generate pro-death and anti-death protein isoforms from a single gene, underscoring the importance of splicing regulation in programmed cell death. There has not been a systematic study of the effects of genotoxins on alternative splicing. The proposed study will fill this gap by pursuing the splicing regulatory function of nuclear Abl. Specifically, we will (1) delineate the role of Abl in the alternative splicing of the CD44 gene, because we have found that activation of Abl leads to the exclusion of CD44 variable exons 4 and 5 in doxorubicin-treated cells;(2) conduct a large-scale survey of DNA damage-induced and Abl-dependent alternative splicing of several thousand genes using a fiber-optic bead array-based technology;(3) identify DNA damage-induced genomic binding sites for Abl using the human genome tiling arrays, because we have found that doxorubicin causes an enhanced association of Abl with the CD44 variable exon 5;(4) investigate the role of alternative splicing in cell death response to DNA damage by testing the hypothesis that doxorubicin specifically reduces the levels of CD44 variants to enhance death receptor-induced cell killing;and (5) construct mouse tumor models to investigate the role of nuclear Abl in tumor responses to chemotherapy. The proposed research will investigate a previously unappreciated effect of DNA damage on RNA splicing, through a previously unknown mechanism of splicing regulation by CTD tyrosine phosphorylation. Because Abl is not mutated in sporadic human cancers and because Abl can activate p53-independent cell death, results from the proposed research will shed light on how to exploit the pro-apoptotic function of nuclear Abl to kill tumor cells.
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