Pol\u03b8 inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance

Diana Zatreanu,Kimberley Wiggins,Theresia Schaedler,Joana Neves,Katherine Ewings,Vera Grinkevich,Robert A Heald,Cameron Bell,Peter Blencowe,Mathew Calder,Ellen Macdonald,Diego Grande,Geoff S Higgins,Remko Prevo,Luned Badder ,Rebecca Marlow,Simon N Willis ,Marie Boursier,Mark Charles,Omar Alkhatib,André Nussenzweig ,Eleanor Knight ,Martin L Stockley ,Graeme C.m Smith ,Claire Mcwhirter,Stephen J Pettitt,Jane Curry ,Jayesh B Majithiya ,Andrew Tutt,Harry Finch,Rachel Brough,Christopher Bot ,Hollie Mccarron,Simon J Boulton,Nan Shao,Chris Pedder,Lerin Geo,Susan Moore ,Ed Wheatley,Aditi Gulati,Daniela Novo,Laurent Rigoreau,Marco Ranzani,Sandhya Sridhar,Christina Alli,Eeson Rajendra,Leon Pang,Sophie Langdon,Tennyson Ekwuru,Martin E Swarbrick ,Syed Haider,Joost Schimmel,Graeme Hewitt,Marcel Tijsterman,Elsa Callén ,Niall M.b Martin ,Amanda Ferdinand,Helen M.r Robinson ,Wojciech Krajewski,Ai Ching Wong,Christopher J Lord

doi:10.1038/s41467-021-23463-8

Abstract

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.

Highlights

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
We found that ART558 exposure elicited a series of DNA damage related biomarkers to a greater degree in TMEJ-mediated repair of a NanoLuciferease-encoding TMEJ reporter substrate
The observations described above demonstrate the discovery of a potent and selective small molecule Polθ inhibitor that elicits BRCA-gene synthetic lethality, and targets cells with PARPi resistance caused by 53BP1/Shieldin defects

Summary

Introduction

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. We assessed the effect of ART558 on cellular TMEJ, using PCR and luminescence-based DNA reporter assays that were adapted from a previously described MMEJ assay[13] (Fig. 1h) These assays demonstrated that ART558, but not the control compound ART615, was able to inhibit Polθ-mediated DNA DSB repair with sub micromolar potency, but did not inhibit canonical NHEJ (Fig. 1i, j) further demonstrating excellent selectivity of the molecule. ART615, the inactive isomer of ART558, did not enhance the radiosensitivity of Polq wild type cells (Fig. 1l) Taken together these data suggested that ART558 is a potent and selective inhibitor of the polymerase activity of Polθ, increases retention of Polθ at sites of DNA damage and is active in modulating cellular TMEJ and radiosensitivity

Methods

Results

Conclusion