Investigating novel roles of topoisomerase regulatory interactions in transcription

Sammanfattning: Drugs targeting DNA Topoisomerases (TOPs, TOP1 and TOP2) are mainstays of anti-cancer therapy. While they have proven effective, the toxicity of current TOP drugs caused by DNA damage-induced apoptosis of non-cancer cells, limits their use in the clinic. Novel therapeutic approaches targeting TOP activity without promoting DNA damage would be ideal candidates to address this issue, however, this requires a deeper knowledge of TOP biology in the nuclear context. TOPs promote transcription and replication by cleaving and resealing DNA strands, thus removing inhibitory torsional stress generated during polymerase elongation. Although commonly considered to be constitutively active enzymes that rapidly drain excess supercoiling from the stressed regions of the genome, previous work from our lab demonstrated a new mechanism through which the RNA polymerase II (RNAPII) directly stimulates TOP1 above its intrinsic activity to ensure proficient transcription in human cells. These results suggest that two functional states characterize the activity of TOPs in the nucleus: a “basal state” and a “stimulated state”. If TOP stimulation is an obligatory step in transcription, then targeting the interface between TOP and its regulatory partner may abrogate TOP stimulation, while leaving basal TOP activity unimpaired. Since cancer cells often have deregulated transcriptional programs, such a strategy would have antitumor properties but with reduced toxicity. By integrating genome-wide maps of TOPs (from DNA binding to DNA cleavage-complex formation), biochemical assays and drug screens, we have set the following goals for our research: 1) to decipher the factors regulating TOP activity; 2) to understand the mechanisms of TOP regulation; 3) to target the regulation of TOP, to selectively affect the viability of cancer cells. To address these research questions, we have developed a fast and robust technique to map TOP1 activity across the genome: the TOP1 CAD-seq approach. Combining this method with transcriptomic analysis, we interrogated whether the RNAPII-TOP1 regulation is essential for transcriptional elongation and reported its fundamental role during mitotic transcription when the RNAPII translocates along highly compacted chromatin (Paper I). We also hypothesized that, besides the RNAPII, factors promoting transcription elongation are likely to stimulate the activity of TOP to remove the resulting supercoiling. We found that the master transcription factor MYC assembles TOP1 and TOP2 in a “topoisome” complex and stimulates their activity to allow high transcription rates (Paper II). Finally, we developed a treatment strategy that blocks RNAPII-TOP1 stimulation during transcription and tested the drugs in preclinical models of pancreatic cancers. We show that the treatment is effective and specific for the cancer cells as negligible transcriptional defects are observed in non-cancer cells (Manuscript III).