EGFR-Activating Mutations Correlate with a Fanconi Anemia–like Cellular Phenotype That Includes PARP Inhibitor Sensitivity

Abstract

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.

Gene expression and mutation-guided synthetic lethality eradicates proliferating and quiescent leukemia cells

“…GEMA, with emphasis on gene mutation analysis, may also be applied to identify additional oncogenes that sensitize cells to PARP1 inhibitor treatment, for example TMPRSS2-ERG-positive prostate carcinomas (DNA-PKcs deficiency), EGFR mutant-positive lung carcinomas (Fanconi anemia deficiency), EWSR1-FLI1-positive Ewing's sarcomas, and KRAS mutant-positive leukemias (40)(41)(42)(43).…”

DNA Damage Response Assessments in Human Tumor Samples Provide Functional Biomarkers of Radiosensitivity

“…Our own approach has been driven by the desire to obtain rapid read outs of treatment sensitivity using needle biopsies that can ultimately inform treatment decisions in real time 13,90 (Figure 3). In brief, core biopsy materials are obtained, placed in chilled complete cell culture medium with 10% serum, and equilibrated in a humidified cell culture incubator at 37°C and 5% CO 2 .…”

“…However, conducting the assay with IR alone at a relatively short time point (2 hours) is likely to only capture severe HRR defects due to BRCA1/2 disruption but will miss a number of other HRR defects. For example, our own in-vitro data indicate that tumor cells with an S-phase specific HRR defect, for example due to an alteration in the FA pathway, may display normal RAD51 foci induction after IR exposure but defective induction after replication-fork blocking treatments, such as cisplatin 90 . To detect this, a 24-hour time point would be required.…”

“…In-vitro analysis, however, indicated that γ-H2AX foci were actually superior to RAD51 foci in terms of predicting cell kill by cisplatin, likely because mechanisms other than loss of RAD51 function can also cause cisplatin sensitivity. In addition, HRR defects can manifest themselves not only as a failure to induce RAD51 foci but also a failure to resolve foci, presumably due to defects in resolvase proteins such as SLX4 or FAN1 90,110 . Detection of this phenomenon would require more than one time point and extending tissue incubation to at least 48 hours, which may be difficult to carry out on limited amounts of biopsy materials.…”

Radiotherapy in the Era of Precision Medicine

“…EGFR mutant patients are typically non-smokers with cancer genomes that contain less mutation and copy number burden and are less likely to contain alterations shown to confer therapeutic resistance (ie BRAF, KRAS, NFE2L2, and KEAP1) (41). Supporting a causative link between EGFR gain-of-function mutations and therapeutic sensitization are studies that demonstrate a link between mutant EGFR and diminished DNA repair capacity (60,63). Additional mechanistic work is needed to fully explore this interaction and to probe the differences in the therapeutic response of EGFR activation.…”