The mutational landscape of the sensitivity of cancer to ionizing radiation.

Abstract

3129 Background: The impact of common or rare gene mutations on the sensitivity of cancers to ionizing radiation remains largely unknown. We conducted a systematic, arrayed (single variant per well) profiling effort to identify gene mutations that alter cellular sensitivity to radiation and validated some of our findings using a clinical cohort of patients who received thoracic radiotherapy alone. Methods: Candidate mutations were prioritized on the basis of genotype-phenotype associations from our previously completed large-scale cancer cell line irradiation profiling study (doi: 10.1038/ncomms11428), location within conserved protein domains, and functional impact (MutationAssessor). We used site-directed mutagenesis to generate mutant clones (2 clones per variant) and transferred the ORFs into lentiviral vectors in SV40 lung primary immortalized cells (BEAS2B). For clinical validation, an IRB-approved study was used to identify patients treated with lung radiotherapy alone. 197 patients with primary (stage I–IV) or recurrent lung cancer and patients with other cancer types and solitary metastases or oligometastases to the lung were included. Death without evidence of local failure was treated as a competing event, and Fine and Gray regression modeling was used to examine potential predictors of local failure. Results: Over 600 cancer variants were tested in ̃1200 experimental replicates, comprising 91 genes. We identified known and new radioresistant and radiosensitive variants involved in several cellular functional categories including cellular signaling, cytoskeleton, cell cycle, apoptosis, DNA methylation, and DNA repair. Variants that conferred resistance in BEAS2B cells were significantly more likely to confer resistance in TERT-HU1 and NCI-H520 cells, suggesting that most functional variants are cellular context indifferent. Variants under somatic oncogenic selection (hotspot mutants) were significantly more likely to confer resistance to radiation. Several infrequent cancer variants ( < 1% prevalence in cancer), including those in ERBB3, SMAD4, TGFBR1, VHL, CTNNB1, and MAP2K1, conferred radiation resistance. Some genes (e.g. KEAP1) demonstrated significant intragenic allelic variation in the magnitude of conferred resistance and other genes (e.g. CTNNB1) displayed both resistance and sensitivity in a protein domain-dependent manner. KRAS (resistant; HR 2.23; P= 0.02) and CTNNB1 exon 3 (sensitive; HR 0.3; P = 0.04) mutants conferred resistance and sensitivity, respectively, to radiotherapy in our clinical cohort. Conclusions: We report on a large-scale profiling effort to identify mutant alleles that govern radiation survival. Our results reveal new insights into potentially actionable determinants of tumor sensitivity to radiotherapy and accelerate clinical validation of common and rare gene mutations that impact radiation sensitivity.