Cancer genome sequencing efforts have revealed tumor-associated gene variants that can direct therapy, functionally significant variants that do not currently direct therapy or variants that are of unknown functional significance. The impact of all of these categories of variants on the efficacy of radiotherapy remains largely unknown. To gain insight into the genetic landscape of radioresistance, we previously established a high-throughput platform that measures cell line survival to radiation. We then used this technology to analyze the radiosensitivity of more than 500 cancer cell lines annotated by the Cancer Cell Line Encyclopedia and discovered gene sets and pathways that regulate radiation survival. Here, we used the results from this study to identify gene variants (missense mutations, short inserts/deletions, and premature stop codons) that are associated with either radiosensitivity or radioresistance and functionally annotate a multitude of these variants.
Candidate variants from more than 100 ORFs were prioritized on the basis of their location within conserved protein domains (UniProt), predicted functional impact (MutationAssessor), and variant allele frequency. We used site-directed mutagenesis to generate mutant clones and transferred the ORFs into lentiviral vectors for stable expression under a PGK promoter in both SV40 and hTERT immortalized cell lines. For a subset of variants, expression of the endogenous gene was repressed by CRISPRi to model loss of heterozygosity. The effect of individual variants on radiation response was assessed using our high-throughput platform and benchmarked against the canonical, cyto- and radio-protective NFE2L2 E79K gain-of-function mutation and validated by colony forming assay.
We successfully adapted our high-throughput platform to measure the radiosensitivity of individual variants expressed in immortalized cell lines. Integral survival was significantly correlated between our high-throughput platform and clonogenic survival measurements. We discovered radioresistant conferring variants in genes involved in cell cycle transition, apoptosis (intrinsic), and MEK/ERK signaling. In contrast, variants associated with radiation sensitivity were identified in genes with established roles in DNA damage response pathways as well as in putative DNA repair factors.
Determining the impact of gene variants remains a major obstacle in the implementation of personalized radiotherapy. Here, we report on a large-scale profiling effort to identify and classify mutant alleles that govern radiation survival across multiple tumor lineages. Our results reveal new insights into the mechanisms of cellular survival to radiation and genome maintenance.
Citation Format: Brian D. Yard, Aaron P. Petty, Mohamed E. Abazeed. Systematic annotation of genetic variants that determine sensitivity to radiation: A pan-cancer encyclopedia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 984.