B. Yard

A genetic basis for the variation in the vulnerability of cancer to DNA damage

Radiotherapy is not currently informed by the genetic composition of an individual patients tumour. To identify genetic features regulating survival after DNA damage, here we conduct large-scale profiling of cellular survival after exposure to radiation in a diverse collection of 533 genetically annotated human tumour cell lines. We show that sensitivity to radiation is characterized by significant variation across and within lineages. We combine results from our platform with genomic features to identify parameters that predict radiation sensitivity. We identify somatic copy number alterations, gene mutations and the basal expression of individual genes and gene sets that correlate with the radiation survival, revealing new insights into the genetic basis of tumour cellular response to DNA damage. These results demonstrate the diversity of tumour cellular response to ionizing radiation and establish multiple lines of evidence that new genetic features regulating cellular response after DNA damage can be identified.

Radiotherapy in the Era of Precision Medicine

Current predictors of radiation response are largely limited to clinical and histopathologic parameters, and extensive systematic analyses of the correlation between radiation sensitivity and genomic parameters remain lacking. In the era of precision medicine, the lack of -omic determinants of radiation response has hindered the personalization of radiation delivery to the unique characteristics of each patient׳s cancer and impeded the discovery of new therapies that can be administered concurrently with radiation therapy. The cataloging of the -omic determinants of radiation sensitivity of cancer has great potential in enhancing efficacy and limiting toxicity in the context of a new approach to precision radiotherapy. Herein, we review concepts and data that contribute to the delineation of the radiogenomic landscape of cancer.

RNF138 interacts with RAD51D and is required for DNA interstrand crosslink repair and maintaining chromosome integrity.

The RAD51 family is integral for homologous recombination (HR) mediated DNA repair and maintaining chromosome integrity. RAD51D, the fourth member of the family, is a known ovarian cancer susceptibility gene and required for the repair of interstrand crosslink DNA damage and preserving chromosomal stability. In this report, we describe the RNF138 E3 ubiquitin ligase that interacts with and ubiquitinates the RAD51D HR protein. RNF138 is a member of an E3 ligase family that contains an amino-terminal RING finger domain and a putative carboxyl-terminal ubiquitin interaction motif. In mammalian cells, depletion of RNF138 increased the stability of the RAD51D protein, suggesting that RNF138 governs ubiquitin-proteasome-mediated degradation of RAD51D. However, RNF138 depletion conferred sensitivity to DNA damaging agents, reduced RAD51 focus formation, and increased chromosomal instability. Site-specific mutagenesis of the RNF138 RING finger domain demonstrated that it was necessary for RAD51D ubiquitination. Presence of RNF138 also enhanced the interaction between RAD51D and a known interacting RAD51 family member XRCC2 in a yeast three-hybrid assay. Therefore, RNF138 is a newly identified regulatory component of the HR mediated DNA repair pathway that has implications toward understanding how ubiquitination modifies the functions of the RAD51 paralog protein complex.

Abstract 3315: Functional genomic profiling of lung adenocarcinoma identifies BRAF mutations as novel therapeutic targets

Patients with non-small cell lung cancer (NSCLC) display a wide spectrum of oncologic outcomes, suggesting significant underlying biologic diversity. Despite two notable exceptions in the cases of EGFR mutations and ALK rearrangements, current therapeutic management is largely homogeneous for a given stage. To advance genotype-directed therapy in NSCLC, we sought to identify genetic determinants of therapeutic resistance by leveraging cancer genomic data with a recently developed high-throughput platform for measuring radiation survival (Cancer Res. 2013. 73(20): 6289-98). To adequately represent the biologic spectrum of lung cancer and maximize power to detect clinically relevant genotypes, we profiled 104 lung cancer cell lines, including 89 NSCLC and 15 small cell lung cancer (SCLC) lines. We used our recently validated high-throughput proliferation assay to measure survival. Genomic correlates of radiosensitivity were explored by accessing Oncomap data from the Cancer Cell Line Encyclopedia, the COSMIC database of the Cancer Genome Project, and The Cancer Genome Atlas. Radiation survival across lineages reflected clinical experience and the literature regarding differential response to radiation, inasmuch as lung squamous cell carcinoma and adenocarcinoma (ACA) had similar radiosensitivity, whereas SCLC was less radiosensitive. Importantly, radiosensitivity varied more within a lineage than across lineages, with a 6-fold difference in integral survival among ACA lines. Correlation with cancer genomic data revealed clustering of BRAF mutations within the most resistant ACA lines (p = 0.035). When radiation survival distributions were compared by mutation status, BRAF-mutant ACA lines were significantly more resistant than BRAF wild-type ACA lines (p = 0.023). Some of the mutations identified by our analysis have been previously annotated by The Cancer Genome Atlas lung adenocarcinoma dataset and others appear to be novel. The identified BRAF mutations located in the highly conserved kinase domain enhanced kinase activity in a fashion analogous to the well-known BRAF V600E mutation. Integration of high-throughput radiation survival profiling with large-scale cancer genomic data suggests BRAF mutations are associated with therapeutic resistance in lung ACA. Our analysis nominates BRAF pathway inhibitors, which are commercially available, as therapeutic sensitizers in select BRAF-mutant lung ACA. Further investigation has the potential to yield an additional genotype-directed therapy that could impact up to 7% of patients with lung ACA, a prevalence comparable to that of ALK rearrangements (4%) or EGFR mutations (10%). Citation Format: Mohamed Abazeed, Brian Yard, Drew Adams, Pablo Tamayo, Jason Hearn, Eui Kyu Chie, Stuart Schreiber, Matthew Meyerson, Craig Peacock, Peter Hammerman. Functional genomic profiling of lung adenocarcinoma identifies BRAF mutations as novel therapeutic targets. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3315. doi:10.1158/1538-7445.AM2015-3315