Project 2: TMEJ Regulation in Cancer and Treatment Resistance

PROJECT 2 SUMMARY Cancer cells frequently experience increased levels of replication stress, leading to the formation of DNA double- strand breaks (DSBs) that require efficient repair mechanisms for survival. One such repair pathway, Theta- mediated end joining (TMEJ) facilitated by DNA polymerase theta (Pol θ), is an error-prone process increasingly relied upon by cancer cells relative to their normal cell counterparts. Pol θ has thus emerged as a promising therapeutic target. Despite its potential, the specific replication-associated DNA damage substrates and genomic contexts that necessitate Pol θ/TMEJ activity are not well understood. This project aims to fill these critical knowledge gaps, enhancing our ability to exploit Pol θ inhibitors in cancer therapy. Aim 1 will elucidate the molecular mechanisms of TMEJ recruitment to clustered interstrand crosslinks (c-ICLs), building upon observations that a regulated homologous recombination (HR)-to-TMEJ handoff involving Rad51 ubiquitylation is required. Aim 2 will identify modulators of Pol θ dependency in HR deficient cancer cells. Specifically, we will examine the role of RNA/DNA hybrids and innate immune pathways in Pol θ synthetic lethality using genetically engineered mouse models of BRCA-deficient breast cancer. Aim 3 will establish mechanisms of Pol θ-mediated resistance to topoisomerase 1 (Top1) inhibitor-based antibody drug conjugates in HR proficient and HR deficient patient-derived cancer models. Project 2 will synergize with mechanistic approaches and insights utilized across all Projects in the Program and will be supported by the Administrative and Protein Expression Cores to reveal new insights into how Pol θ can be most effectively targeted to improve cancer outcomes.