ABSTRACT
The preferential response to PARP inhibitors (PARPis) in BRCA-deficient and Schlafen 11 (SLFN11)-expressing ovarian cancers has been documented, yet the underlying molecular mechanisms remain unclear. As the accumulation of single-strand DNA (ssDNA) gaps behind replication forks is key for the lethality effect of PARPis, we investigated the combined effects of SLFN11 expression and BRCA deficiency on PARPi sensitivity and ssDNA gap formation in human cancer cells. PARPis increased chromatin-bound RPA2 and ssDNA gaps in SLFN11-expressing cells and even more in cells with BRCA1 or BRCA2 deficiency. SLFN11 was co-localized with chromatin-bound RPA2 under PARPis treatment, with enhanced recruitment in BRCA2-deficient cells. Notably, the chromatin-bound SLFN11 under PARPis did not block replication, contrary to its function under replication stress. SLFN11 recruitment was attenuated by the inactivation of MRE11. Hence, under PARPi treatment, MRE11 expression and BRCA deficiency lead to ssDNA gaps behind replication forks, where SLFN11 binds and increases their accumulation. As ovarian cancer patients who responded (progression-free survival >2 years) to olaparib maintenance therapy had a significantly higher SLFN11-positivity than short-responders (<6 months), our findings provide a mechanistic understanding of the favorable responses to PARPis in SLFN11-expressing and BRCA-deficient tumors. It highlight the clinical implications of SLFN11.
Subject(s)
BRCA1 Protein , BRCA2 Protein , DNA Replication , DNA, Single-Stranded , MRE11 Homologue Protein , Ovarian Neoplasms , Poly(ADP-ribose) Polymerase Inhibitors , Humans , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , DNA Replication/drug effects , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , Female , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Ovarian Neoplasms/metabolism , BRCA2 Protein/genetics , BRCA2 Protein/metabolism , MRE11 Homologue Protein/metabolism , MRE11 Homologue Protein/genetics , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , Cell Line, Tumor , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Replication Protein A/metabolism , Replication Protein A/genetics , Chromatin/metabolism , Phthalazines/pharmacologyABSTRACT
Schlafen (SLFN) 11 enhances cellular sensitivity to various DNA-damaging anticancer agents. Among the human SLFNs (SLFN5/11/12/13/14), SLFN11 is unique in its drug sensitivity and ability to block replication under DNA damage. In biochemical analysis, SLFN11 binds single-stranded DNA (ssDNA), and this binding is enhanced by the dephosphorylation of SLFN11. In this study, human cell-based assays demonstrated that a point mutation at the ssDNA-binding site of SLFN11 or a constitutive phosphorylation mutant abolished SLFN11-dependent drug sensitivity. Additionally, we discovered that nuclear SLFN13 with a point mutation mimicking the DNA-binding site of SLFN11 was recruited to chromatin, blocked replication, and enhanced drug sensitivity. Through generating multiple mutants and structure analyses of SLFN11 and SLFN13, we identified protein phosphatase 2A as a binding partner of SLFN11 and the putative binding motif in SLFN11. These findings provide crucial insights into the unique characteristics of SLFN11, contributing to a better understanding of its mechanisms.