Genetic disruption of DNA polymerase theta (Pol¿) activity has been shown to effectively target BRCA1 mutated
cells, while leaving BRCA1 wild-type (WT) cells intact. Pol¿ facilitates theta-mediated DNA end joining (TMEJ)
repair by promoting DNA synapsis and repair synthesis at break sites containing 3' single stranded (ss)DNA
overhangs. Although small molecule inhibitors of Pol¿ activity are currently under development for the treatment
of BRCA1 mutant cancers, very little is known regarding the mechanisms that activate TMEJ and result in Pol¿-
dependency in BRCA1 mutant cells. Moreover, the current paradigm assumes that homologous recombination
(HR)-deficiency confers Pol¿i sensitivity, therefore PARPi responsiveness is expected to be a biomarker for Pol¿
inhibitor (Pol¿i) sensitivity. In our preliminary data, we unexpectedly identified commonly used Brca1 mutant
cells that grow relatively unperturbed with genetic Polq (Pol¿) knockout (KO), indicating that Pol¿i and PARPi
sensitivity may not necessarily correlate. In this proposal, we will elucidate the molecular requirements for TMEJ
activation and identify biological factors that distinguish PARPi and Pol¿i sensitivity. We will address the following
Specific Aims: 1) reveal the molecular basis of TMEJ activation in Brca1 mutant cells; 2) uncover genetic Pol¿-
dependencies in Brca1 mutant cells; and 3) examine pharmacologic Pol¿ inhibition in Brca1 mutant cells.
Collectively, these studies will be informative for future clinical studies employing Pol¿i.