Introduction Homologous recombination repair (HRR) is a critical pathway for the repair of DNA damage caused by cisplatin or PARP inhibitors. HRR may be impaired by multiple mechanisms in cancer, which complicates assessing the functional HRR status in cells. Here, we monitored the ability of non-small cell lung cancer (NSCLC) cells to form subnuclear foci of DNA repair proteins as a surrogate of HRR proficiency. Methods We assessed clonogenic survival of 16 NSCLC cell lines in response to cisplatin, mitomycin C (MMC), and the PARP inhibitor olaparib. Thirteen tumor explants from NSCLC patients were subjected to cisplatin ex-vivo. Cells were assayed for foci of repair-associated proteins such as BRCA1, FANCD2, RAD51, and γ-H2AX. Results Four cell lines (25%) showed an impaired RAD51 foci forming ability in response to cisplatin. Impaired foci formation correlated with cellular sensitivity to cisplatin and MMC as well as olaparib. Foci responses complemented or superseded genomic information suggesting alterations in the ATM/ATR and FA/BRCA pathways. Because baseline foci in untreated cells did not predict drug sensitivity we adapted an ex-vivo biomarker assay to monitor damage-induced RAD51 foci in NSCLC explants from patients. Ex-vivo cisplatin treatment of explants identified 2 tumors (15%) exhibiting compromised RAD51 foci induction. Conclusions A fraction of NSCLC harbors HRR defects that may sensitize the affected tumors to DNA damaging agents including PARP inhibitors. We propose that foci-based functional biomarker assays represent a powerful tool for prospective determination of treatment sensitivity, but will require ex-vivo techniques for induction of DNA damage to unmask the underlying HRR defect.

In lung cancer patients whose tumors harbor activating mutations in the epidermal growth factor receptor (EGFR), increased responses to platinum-based chemotherapies are seen compared to wild-type cancers. However, the mechanisms underlying this association have remained elusive. Here, we describe a cellular phenotype of crosslinker sensitivity in a subset of EGFR-mutant lung cancer cell lines that is reminiscent of the defects seen in cells impaired in the Fanconi Anemia pathway, including a pronounced G2/M cell-cycle arrest and chromosomal radial formation. We identified a defect downstream of FANCD2 at the level of recruitment of FAN1 nuclease and DNA interstrand crosslink (ICL) unhooking. The effect of EGFR mutation was epistatic with FANCD2. Consistent with the known role of FANCD2 in promoting RAD51 foci formation and homologous recombination repair (HRR), EGFR-mutant cells also exhibited an impaired RAD51 foci response to ICLs, but not to DNA double-strand breaks. EGFR kinase inhibition affected RAD51 foci formation neither in EGFR mutant nor wild-type cells. In contrast, EGFR depletion or overexpression of mutant EGFR in wild-type cells suppressed RAD51 foci, suggesting an EGFR kinase-independent regulation of DNA repair. Interestingly, EGFR-mutant cells treated with the PARP inhibitor olaparib also displayed decreased FAN1 foci induction, coupled with a putative block in a late HRR step. As a result, EGFR-mutant lung cancer cells exhibited olaparib sensitivity in-vitro and in-vivo. Our findings provide insight into the mechanisms of cisplatin and PARP inhibitor sensitivity of EGFR-mutant cells, yielding potential therapeutic opportunities for further treatment individualization in this genetically defined subset of lung cancer.