Loss of the p12 subunit of DNA polymerase delta leads to a defect in HR and sensitization to PARP inhibitors.

Highlights • p12-knockout generates a HR deficiency phenotype. • Pol δ 4 is required for full expression of cellular HR activity. • p12 knockout cells are sensitized to PARP inhibitors. • p12 expression is a potential biomarker for the use of PARP inhibitors. • p12 deficiency may have a role in SCLC tumorigenesis. Abstract Human DNA polymerase δ is normally present in unstressed, non-dividing cells as a heterotetramer (Pol δ 4). Its smallest subunit, p12, is transiently degraded in response to UV damage, as well as during the entry into S-phase, resulting in the conversion of Pol δ 4 to a trimer (Pol δ 3). In order to further understand the specific cellular roles of these two forms of Pol δ, the gene (POLD4) encoding p12 was disrupted by CRISPR/Cas9 to produce p12 knockout (p12KO) cells. Thus, Pol δ 4 is absent in p12KO cells, leaving Pol δ 3 as the sole source of Pol δ activity. GFP reporter assays revealed that the p12KO cells exhibited a defect in homologous recombination (HR) repair, indicating that Pol δ4, but not Pol δ 3, is required for HR. Expression of Flag-tagged p12 in p12KO cells to restore Pol δ 4 alleviated the HR defect. These results establish a specific requirement for Pol δ 4 in HR repair. This leads to the prediction that p12KO cells should be more sensitive to chemotherapeutic agents, and should exhibit synthetic lethal killing by PARP inhibitors. These predictions were confirmed by clonogenic cell survival assays of p12KO cells treated with cisplatin and mitomycin C, and with the PARP inhibitors Olaparib, Talazoparib, Rucaparib, and Niraparib. The sensitivity to PARP inhibitors in H1299-p12KO cells was alleviated by expression of Flag-p12. These findings have clinical significance, as the expression levels of p12 could be a predictive biomarker of tumor response to PARP inhibitors. In addition, small cell lung cancers (SCLC) are known to exhibit a defect in p12 expression. Analysis of several SCLC cell lines showed that they exhibit hypersensitivity to PARP inhibitors, providing evidence that loss of p12 expression could represent a novel molecular basis for HR deficiency. [ABSTRACT FROM AUTHOR]