Mechanism of Transcriptional control of oncogenic transcription factor fusions in tumor development
Gene expression networks are perturbed in every human malignancy. One common way that this occurs is through chromosomal translocation, such as t(8;21) or t(2;13), which fuse parts of two transcription factors to create a new chimeric protein. In these tumors that are driven by transcription factor fusions, the new factor is the best therapeutic target. The t(2;13) encoding PAX3-FOXO1 was found in alveolar rhabdomyosarcoma (aRMS) over 30 years ago. However, because transcriptional control is a highly dynamic process that changes rapidly in response to various cellular and extracellular cues, it has been difficult to define the mechanism of PAX3-FOXO1 function using slow genetic methods. We used a chemical-genetic approach to rapidly degrade a canonical transcription activator, PAX3-FOXO1, to determine the mechanism by which it disrupts gene expression programs. By coupling rapid protein degradation with the analysis of nascent transcription over short time courses and integrating CUT&RUN, ATAC-seq and eRNA analysis with deep proteomic analysis, we defined PAX3-FOXO1 function at a small network of direct transcriptional targets. Interestingly, PAX3-FOXO1 degradation impaired RNA polymerase pausing release and transcription elongation at many regulated gene targets, suggesting that drugs that target elongation might be effective in this hard to treat sarcoma. Inhibitors of the bromodomain and extra-terminal domain family (BETi) provide a therapeutic approach to target transcription elongation, BETi shows efficacy in preclinical models of AML, but the mechanism is unclear. We also explored the mechanism of action of BETi in t(8;21) AML cells, which contain fusion transcription factor AML1-ETO, and showed that BETi reduced AML cell size, lowered metabolic activity, triggered a rapid but reversible G0/G1 arrest, and with time, caused cell death. In addition, BETi inhibited the transcription of MYC and BCL2, suggesting the combination of BETi and the BCL2 inhibitor venetoclax. In conclusion, this dissertation shows how defining the detailed mechanism of transcriptional regulation by transcription factor fusions can identify promising new therapeutic targets.