Combined DNA-PK and PARP Inhibition as a Therapeutic Strategy in BRCA-Mutated Prostate Cancer: An in Vitro Pilot Study.

IntroductionDNA double-strand breaks (DSBs) are repaired via homologous recombination (HR) or the more error-prone non-homologous end joining (NHEJ). breast cancer gene 1 (BRCA1) and breast cancer gene 2 (BRCA2) are key genes in HR, and their mutations are associated with aggressive prostate cancer (PCa). While PARP inhibitors (PARPi) improve survival in BRCA-mutated PCa, their efficacy in late-stage disease is limited and often accompanied by serious side effects. This study aims to develop an in vitro model of BRCA-mutated PCa and evaluate the therapeutic potential of DNA-dependent protein kinase (DNA-PK) inhibitors that target the NHEJ pathway.MethodsThe genes BRCA1 and BRCA2 were targeted for knockout (KO) in lymphnode cancer of the prostate (cell line) [LNCaP] using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. The KO were assessed via Western blot analysis. Scramble LNCaP, BRCA1 KO, and BRCA2 KO cells were treated with the PARPi talazoparib, the DNA-PK inhibitor nedisertib and their combination. The impact on cell proliferation was evaluated using the CellTiter-Glo assay and synergy of the treatments was analyzed with SynergyFinder. Cytotoxic effects were measured by flow cytometry using an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit. The presence of DSB was quantified using immunofluorescence.ResultsBRCA2 and BRCA1 protein expression were successfully downregulated in the knockout (KO) cell lines. After two days of treatment with talazoparib and/or nedisertib, a significant decrease in cell proliferation was observed. Additive effects of the combination treatment were detected exclusively in the BRCA KO cells. These cells also exhibited significantly higher rates of necrosis after treatment compared to scramble cells and, DNA DSB were significantly more prevalent in the BRCA KO cells. Additionally, BRCA1/2 loss is inversely correlated with DNA-PK, with knockout leading to increased DNA-PK expression to support NHEJ.ConclusionBRCA knockout LNCaP models were established, exhibiting increased DNA-PK activity and indicating greater susceptibility to DNA-PK inhibition. Therapeutically targeting NHEJ presents a promising approach in treating BRCA-mutated PCa. Further in vivo investigations are required to assess the tolerability of this drug combination.