To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.

Human DNA polymerase theta (Polθ), which is essential for microhomology-mediated DNA double strand break repair, has been proposed as an attractive target for the treatment of BRCA deficient and other DNA repair pathway defective cancers. As previously reported, we recently identified the first selective small molecule Polθ in vitro probe, 22 (ART558), which recapitulates the phenotype of Polθ loss, and in vivo probe, 43 (ART812), which is efficacious in a model of PARP inhibitor resistant TNBC in vivo. Here we describe the discovery, biochemical and biophysical characterization of these probes including small molecule ligand co-crystal structures with Polθ. The crystallographic data provides a basis for understanding the unique mechanism of inhibition of these compounds which is dependent on stabilization of a “closed” enzyme conformation. Additionally, the structural biology platform provided a basis for rational optimization based primarily on reduced ligand conformational flexibility.

Chk1 plays a crucial role in the DNA damage and replication checkpoints in vertebrates and may therefore be an important determinant of tumour cell responses to genotoxic anticancer drugs. To evaluate this concept we compared the effects of the nucleoside analogue 5-fluorouracil (5FU) on cell cycle progression and clonogenic survival in DT40 B-lymphoma cells with an isogenic mutant derivative in which Chk1 function was ablated by gene targeting. We show that 5FU activates Chk1 in wild-type DT40 cells and that 5FU-treated cells accumulate in the S phase of the cell cycle due to slowing of the overall rate of DNA replication. In marked contrast, Chk1-deficient DT40 cells fail to slow DNA replication upon initial exposure to 5FU, despite equivalent inhibition of the target enzyme thymidylate synthase, and instead accumulate progressively in the G1 phase of the following cell cycle. This G1 accumulation cannot be reversed rapidly by exogenous thymidine or removal of 5FU, and is associated with increased incorporation of 5FU into genomic DNA and severely diminished clonogenic survival. Taken together, these results demonstrate that a Chk1-dependent replication checkpoint which slows S phase progression can protect tumour cells against the cytotoxic effects of 5FU.

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