Cancer cells deficient in homologous recombination (HRD) are dependent on alternative DNA repair pathways for double strand break (DSB) repair, such as theta-mediated end joining (TMEJ). Our previous research demonstrated that the presence of HRD enhances the efficacy of CP-506, a novel hypoxia-activated DNA crosslinking agent. Therefore, we hypothesized that inhibiting polymerase theta (POLQ) in HR-deficient cells would further enhance the efficacy of CP-506.

Genetic analyses were performed using TCGA Pan-cancer datasets obtained from UCSC Xena and cBioportal. Isogenic cancer cell lines proficient or deficient in homologous recombination (BRCA1 and BRCA2), non-homologous end joining (NHEJ; DNA-PKcs), or the Fanconi anemia pathway (FA; FANCA and FANCD2) were cultured as 2D monolayers or 3D spheroids. Cell viability, clonogenic survival, and spheroid growth inhibition (SGI) were assessed following CP-506 and/or POLQ inhibitor (ART558) exposure.

Analysis of TCGA pan-cancer datasets revealed a positive correlation between POLQ expression and HRD (r = 0.46) or hypoxia (r = 0.85) scores across pan-cancer datasets. ART558 treatment decreased cell viability in a dose-dependent manner, which was most pronounced in BRCA2-deficient cells (parental IC50: 53.8 ± 9.5 µM vs BRCA2-/- IC50: 14.7 ± 21.7 µM; p < 0.01). FA-deficient cells showed increased sensitivity under normoxic but not anoxic conditions (≤ 0.02% O2). In contrast, BRCA1- or NHEJ-deficiency did not alter sensitivity to ART558. Combination treatment with CP-506 and ART558 further decreased cell viability specifically in BRCA2-deficient cells, but not in parental or NHEJ-deficient cells. Furthermore, CP-506 (25 µM) or ART558 (5 µM) alone modestly reduced clonogenic survival in BRCA2-deficient cells (surviving fraction: 0.57 and 0.37, respectively), while the combination resulted in an enhanced reduction in survival (surviving fraction: 0.01). Whereas combination treatment of parental cells showed only a modest reduction in survival (surviving fraction: 0.55). Similarly, the SGI of BRCA2-deficient spheroids was more pronounced upon combination therapy (81.3 ± 0.5%) as compared to CP-506 (1.6 ± 18.0%; P < 0.0001) or ART558 (47.3 ± 6.6; P < 0.0001) alone. In parental spheroids, a marginal increase in SGI was observed upon combination treatment with the highest ART558 concentration (10 µM; 28.9 ± 8.2%) compared to CP-506 (9.0 ± 7.9%; P < 0.05) but not compared to ART558 (22.5± 10.6%; P = 0.84) alone.

These findings suggest that TMEJ may act as a compensatory DNA repair pathway in BRCA2-deficient cancer cells following CP-506 treatment. Inhibition of POLQ could be a promising strategy to enhance the therapeutic efficacy of CP-506. Further in vivo studies are warranted to validate these findings.

We gratefully acknowledge Lerin Geo, Helen Robinson, and Graeme Smith from Artios Pharma for kindly providing ART558 for this study.