Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are the primary pathways for repairing DNA double-strand breaks (DSBs) during interphase, whereas microhomology-mediated end-joining (MMEJ) has been regarded as a backup mechanism. Through CRISPR-Cas9–based synthetic lethal screens in cancer cells, we identified subunits of the 9-1-1 complex (RAD9A-RAD1-HUS1) and its interacting partner, RHINO, as crucial MMEJ factors. We uncovered an unexpected function for RHINO in restricting MMEJ to mitosis. RHINO accumulates in M phase, undergoes Polo-like kinase 1 (PLK1) phosphorylation, and interacts with polymerase θ (Polθ), enabling its recruitment to DSBs for subsequent repair. Additionally, we provide evidence that MMEJ activity in mitosis repairs persistent DSBs that originate in S phase. Our findings offer insights into the synthetic lethal relationship between the genes POLQ and BRCA1 and BRAC2 and the synergistic effect of Polθ and poly(ADP-ribose) polymerase (PARP) inhibitors.

The 5′-3′ structure-specific endonuclease ERCC1/XPF (Excision Repair Cross-Complementation Group 1/Xeroderma Pigmentosum group F) plays critical roles in the repair of cisplatin-induced DNA damage. As such, it has been identified as a potential pharmacological target for enhancing clinical response to platinum-based chemotherapy. The goal of this study was to follow up on our previous identification of the compound NSC143099 as a potent inhibitor of ERCC1/XPF activity by performing an in silico screen to identify structural analogues that could inhibit ERCC1/XPF activity in vitro and in vivo. Using a fluorescence-based DNA-endonuclease incision assay, we identified the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) as a potent inhibitor of ERCC1/XPF activity with an IC₅₀ (half maximal inhibitory concentration) in the nanomolar range in biochemical assays. Using DNA repair assays and clonogenic survival assays, we show that EGCG can inhibit DNA repair and enhance cisplatin sensitivity in human cancer cells. Finally, we show that a prodrug of EGCG, Pro-EGCG (EGCG octaacetate), can enhance response to platinum-based chemotherapy in vivo. Together these data support a novel target of EGCG in cancer cells, namely ERCC1/XPF. Our studies also corroborate previous observations that EGCG enhances sensitivity to cisplatin in multiple cancer types. Thus, EGCG or its prodrug makes an ideal candidate for further pharmacological development with the goal of enhancing cisplatin response in human tumors.

Identification of small molecule inhibitors of ERCC1-XPF that inhibit DNA repair and potentiate cisplatin efficacy in cancer cells

The radiation-induced bystander effect (RIBE) can increase cellular toxicity in a gap junction dependent manner in unirradiated bystander cells. Recent reports have suggested that cisplatin toxicity can also be mediated by functional gap junction intercellular communication (GJIC). In this study using lung and ovarian cancer cell lines, we showed that cisplatin cytotoxicity is mediated by cellular density. This effect is ablated when GJA1 or Connexin 43 (Cx43) is targeted, a gap junction gene and protein, respectively, leading to cisplatin resistance but only at high or gap junction forming density. We also observed that the cisplatin-mediated bystander effect was elicited as DNA Double Strand Breaks (DSBs) with positive H2AX Ser139 phosphorylation (γH2AX) formation, an indicator of DNA DSBs. These DSBs are not observed when gap junction formation is prevented. We next showed that cisplatin is not the “death” signal traversing the gap junctions by utilizing the cisplatin-GG intrastrand adduct specific antibody. Finally, we also showed that cells deficient in the structure-specific DNA endonuclease ERCC1-ERCC4 (ERCC1-XPF), an important mediator of cisplatin resistance, further sensitized when treated with cisplatin in the presence of gap junction forming density. Taken together, these results demonstrate the positive effect of GJIC on increasing cisplatin cytotoxicity.

Repair of DNA double strand breaks (DSBs) is integral to preserving genomic integrity. Therefore, defining the mechanisms underlying DSB repair will enhance our understanding of how defects in these pathways contribute to human disease and could lead to the discovery of new approaches for therapeutic intervention. Here, we established a panel of HaloTagged DNA damage response factors in U2OS cells which enables concentration-dependent protein labeling by fluorescent HaloTag ligands. Genomic insertion of HaloTag at the endogenous loci of these repair factors preserves expression levels and proteins retain proper subcellular localization, foci-forming ability, and functionally support DSB repair. We systematically analyzed total cellular protein abundance, measured recruitment kinetics to laser-induced DNA damage sites, and defined the diffusion dynamics and chromatin binding characteristics by live-cell single-molecule imaging. Our work demonstrates that the Shieldin complex, a critical factor in end-joining, does not exist in a preassembled state and that relative accumulation of these factors at DSBs occurs with different kinetics. Additionally, live-cell single-molecule imaging revealed the constitutive interaction between MDC1 and chromatin mediated by its PST repeat domain. Altogether, our studies demonstrate the utility of single-molecule imaging to provide mechanistic insights into DNA repair, which will serve as a powerful resource for characterizing the biophysical properties of DNA repair factors in living cells.

Purpose:ERCC1/XPF is a DNA endonuclease with variable expression in primary tumor specimens, and has been investigated as a predictive biomarker for efficacy of platinum-based chemotherapy. The failure of clinical trials utilizing ERCC1 expression to predict response to platinum-based chemotherapy suggests additional mechanisms underlying the basic biology of ERCC1 in the response to interstrand crosslinks (ICLs) remain unknown. We aimed to characterize a panel of ERCC1 knockout (D) cell lines where we identified a synthetic viable phenotype in response to ICLs with ERCC1 deficiency. Experimental Design:We utilized the CRISPR-Cas9 system to create a panel of ERCC1D lung cancer cell lines which we characterized. Results:We observe that loss of ERCC1 hypersensitizes cells to cisplatin when wildtype (WT) p53 is retained, while there is only modest sensitivity in cell lines that are p53^mutant/null. Additionally, when p53 is disrupted by CRISPR-Cas9 (p53*) in ERCC1D/p53^WT cells, there is reduced apoptosis and increased viability after platinum treatment. These results were recapitulated in two patient data sets utilizing p53 mutation analysis and ERCC1 expression to assess Overall Survival. We also show that kinetics of ICL- repair (ICL-R) differ between ERCC1D/p53^WT and ERCC1D/p53* cells. Finally, we provide evidence that cisplatin tolerance in the context of ERCC1 deficiency relies on DNA-PKcs and BRCA1 function. Conclusions:Our findings implicate p53 as a potential confounding variable in clinical assessments of ERCC1 as a platinum biomarker via promoting an environment in which error-prone mechanisms of ICL- repair may be able to partially compensate for loss of ERCC1.