Title: Investigating the effects of a de-regulated transcription machinery on replication stress in cancer
Funder: Cancer Research UK

Abstract: Background: There is growing interest in targeting oncogene-induced replication stress for cancer therapy. However, effective targeting will require mechanistic understanding of how oncogenes induce replication stress. It is appreciated that oncogenes can promote replication stress by de-regulating the cell cycle. However, we have strong evidence that deregulation of the basal transcription machinery, and overexpression of general transcription factors, is an alternative and important mechanism of oncogene-induced replication stress (Nat Commun 2016). Our follow-up work has uncovered new potential mechanisms for replication-transcription conflicts in cancer. New data suggest unexpected roles for the transcription machineries of RNA polymerases I and III, with their different modes of action from RNA polymerase II, as novel replication stress drivers. They also identify new candidate factors in the response to oncogene-induced replication stress, including HEXIM1 (Cell Reports 2018), and essential transcription regulator, and RNaseH2B, a RNaseH2 subunit that regulates the removal of RNA/DNA hybrids. Aims: We aim to discover new mechanisms and investigate new candidate factors in transcription-replication conflicts. We will use our expertise in oncogene-induced replication stress to generate essential new knowledge in cancer biology and add the transcription machinery to current models of replication stress. We will address the following questions: 1. How do RNA polymerases I and III contribute to oncogene-induced replication stress? 2. What is the role of RNaseH2B in the response to oncogene-induced replication stress? 3. How does HEXIM1 modulate transcription-replication conflicts, e.g. in response to BET inhibition? Methods: We will use non-cancer and cancer human cell lines to model oncogene activation or general transcription factor overexpression, and manipulate transcription with small molecule inhibitors. Replication-transcription conflicts will be measured by DNA fibre assay, EU click chemistry, gRT-PCR, S9.6 anti-R-loop antibody and DNA damage response assays. Molecular mechanisms will be investigated using HPLC, nucleolar imaging and -proteomics, ChIP-seq, DRIP-seq and RNA-seq, genome stability and survival assays, and protein biochemistry methods. We will follow up candidate pathways in cancer models where Birmingham has research strengths, colorectal cancer cell lines, colorectal cancer tissues and colorectal cancer organoid cultures. How the results of this research will be used: This programme will show new pathways that promote replication stress downstream of oncogenes and reveal mechanisms by which cancer cells deal with this stress. The new insights will help identify potential biomarkers to stratify patients for more effective therapy, and potential new therapeutic targets to be followed up in a future CRUK programme application.

Lay abstract: Background: There is growing interest in targeting oncogene-induced replication stress for cancer therapy. However, effective targeting will require mechanistic understanding of how oncogenes induce replication stress. It is appreciated that oncogenes can promote replication stress by de-regulating the cell cycle. However, we have strong evidence that deregulation of the basal transcription machinery, and overexpression of general transcription factors, is an alternative and important mechanism of oncogene-induced replication stress (Nat Commun 2016). Our follow-up work has uncovered new potential mechanisms for replication-transcription conflicts in cancer. New data suggest unexpected roles for the transcription machineries of RNA polymerases I and III, with their different modes of action from RNA polymerase II, as novel replication stress drivers. They also identify new candidate factors in the response to oncogene-induced replication stress, including HEXIM1 (Cell Reports 2018), and essential transcription regulator, and RNaseH2B, a RNaseH2 subunit that regulates the removal of RNA/DNA hybrids. Aims: We aim to discover new mechanisms and investigate new candidate factors in transcription-replication conflicts. We will use our expertise in oncogene-induced replication stress to generate essential new knowledge in cancer biology and add the transcription machinery to current models of replication stress. We will address the following questions: 1. How do RNA polymerases I and III contribute to oncogene-induced replication stress? 2. What is the role of RNaseH2B in the response to oncogene-induced replication stress? 3. How does HEXIM1 modulate transcription-replication conflicts, e.g. in response to BET inhibition? Methods: We will use non-cancer and cancer human cell lines to model oncogene activation or general transcription factor overexpression, and manipulate transcription with small molecule inhibitors. Replication-transcription conflicts will be measured by DNA fibre assay, EU click chemistry, gRT-PCR, S9.6 anti-R-loop antibody and DNA damage response assays. Molecular mechanisms will be investigated using HPLC, nucleolar imaging and -proteomics, ChIP-seq, DRIP-seq and RNA-seq, genome stability and survival assays, and protein biochemistry methods. We will follow up candidate pathways in cancer models where Birmingham has research strengths, colorectal cancer cell lines, colorectal cancer tissues and colorectal cancer organoid cultures. How the results of this research will be used: This programme will show new pathways that promote replication stress downstream of oncogenes and reveal mechanisms by which cancer cells deal with this stress. The new insights will help identify potential biomarkers to stratify patients for more effective therapy, and potential new therapeutic targets to be followed up in a future CRUK programme application.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS