Abstract 6179: Comparative study of Polθ helicase and polymerase inhibitor biochemical and cellular potency
Authors: Ahlskog Johanna K. , Pohjakallio Antti , Paul Ralf , Augustin Martin , Schneider Elisabeth , Johri Sakshi , Tervahauta Tuomas , Beisel Hans-Georg , Neumann Lars , Lindqvist Julia , Moilanen Anu 
Published: April 2023

Background: DNA Polymerase θ (Polθ) is active in Theta-mediated end joining (TMEJ), a repair pathway that utilizes resected 3’ ends and micro-homology sequences to repair double strand breaks. Polθ contains an N-terminal helicase domain and a C-terminal polymerase domain. The helicase domain facilitates stripping of RPA/RAD51 from single stranded DNA and is involved in DNA-end recognition, and micro-homology search and pairing. The polymerase domain performs DNA synthesis following micro-homology pairing. Lack of Polθ is synthetic lethal in homologous recombination deficient (HRD) cells. In this study, we utilized existing small molecule inhibitors targeting either helicase or polymerase domain of Polθ, to assess their translational potential in HRD in vitro cancer cell models.

Methods: Small molecules inhibiting either the helicase or polymerase activity of Polθ were studied with biochemical and cellular assays. For the biochemical studies, recombinant helicase or polymerase domains were purified. The helicase activity of Polθ was analyzed using ADP-Glo and the polymerase activity by primer extension assay. Binding assays confirmed inhibitor binding to recombinant proteins. To study inhibitor efficacy in cellular models, we probed cell viability of BRCA2-deficient cell lines.

Results: Both helicase and polymerase inhibitors showed good potency in inhibiting Polθ enzymatic activity in ATPase or primer extension assays, respectively, with IC50 values in low-nanomolar range. However, the viability of BRCA2-deficient cell lines was not significantly affected after treatment with either helicase or polymerase inhibitors. The measured compound properties (stability, efflux, solubility) did not explain the lack of efficacy in cellular viability assays. Combining Polθ inhibitor treatment with ionizing radiation led to a modest enhancement of radiation-induced decrease in cell viability.

Conclusions: Recently, significant advances have been made in Polθ inhibitor development prompted by the synthetic lethal interaction between Polθ and HRD discovered in several screening efforts. Both helicase and polymerase inhibitors are highly potent in biochemical assays. However, without exogenous genotoxic stress, the potential of Polθ inhibitors as therapeutic target may not be reached in vitro. Additional studies are required to understand the translational gap between biochemical and biological read-outs.

Citation Format: Johanna K. Ahlskog, Antti Pohjakallio, Ralf Paul, Martin Augustin, Elisabeth Schneider, Sakshi Johri, Tuomas Tervahauta, Hans-Georg Beisel, Lars Neumann, Julia Lindqvist, Anu Moilanen. Comparative study of Polθ helicase and polymerase inhibitor biochemical and cellular potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6179.