The Mutational Landscape of Cancer's Vulnerability to Ionizing Radiation.

PURPOSE: Large-scale sequencing efforts have established that cancer-associated genetic alterations are highly diverse, posing a challenge to the identification of variants that regulate complex phenotypes like radiation sensitivity. The impact of the vast majority of rare or common genetic variants on the sensitivity of cancers to radiotherapy remains largely unknown. EXPERIMENTAL DESIGN: We developed a scalable gene editing and irradiation platform to assess the role of categories of variants in cells. Variants were prioritized on the basis of genotype-phenotype associations from a previously completed large-scale cancer cell line radiation profiling study. Altogether, 488 alleles (396 unique single-nucleotide variants) from 92 genes were generated and profiled in an immortalized lung cell line, BEAS-2B. We validated our results in other cell lines (TRT-HU1 and NCI-H520), in vivo via the use of both cell line and patient-derived murine xenografts, and in clinical cohorts. RESULTS: We show that resistance to radiation is characterized by substantial inter- and intra-gene allelic variation. 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. We combined results from our platform with gene expression and metabolite features and identified the upregulation of amino acid transporters that facilitate oxidative reductive capacity and cell-cycle deregulation as key regulators of radiation sensitivity. CONCLUSIONS: Our results reveal new insights into the genetic determinants of tumor sensitivity to radiotherapy and nominate a multitude of cancer mutations that are predicted to impact treatment efficacy.

Functional characterization and identification of mouse Rad51d splice variants.

BACKGROUND: The homologous recombination (HR) pathway is vital for maintaining genomic integrity through the restoration of double-stranded breaks and interstrand crosslinks. The RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) are essential for this process in vertebrates, and the RAD51D paralog is unique in that it participates in both HR repair and telomere maintenance. RAD51D is also known to directly interact with the RAD51C and XRCC2 proteins. Rad51d splice variants have been reported in mouse and human tissues, supportive of a role for alternative splicing in HR regulation. The present study evaluated the interaction of the Rad51d splice isoform products with RAD51C and XRCC2 and their expression patterns. RESULTS: Yeast-2-hybrid analysis was used to determine that the Mus musculus Rad51d splice variant product RAD51DDelta7b (deleted for residues 219 through 223) was capable of interacting with both RAD51C and XRCC2 and that RAD51D+int3 interacted with XRCC2. In addition, the linker region (residues 54 through 77) of RAD51D was identified as a region that potentially mediates binding with XRCC2. Cellular localization, detected by EGFP fusion proteins, demonstrated that each of the splice variant products tested was distributed throughout the cell similar to the full-length protein. However, none of the splice variants were capable of restoring resistance of Rad51d-deficient cell lines to mitomycin C. RT-PCR expression analysis revealed that Rad51dDelta3 (deleted for exon 3) and Rad51dDelta5 (deleted for exon 5)transcripts display tissue specific expression patterns with Rad51dDelta3 being detected in each tissue except ovary and Rad51dDelta5 not detected in mammary gland and testis. These expression studies also led to the identification of two additional Rad51d ubiquitously expressed transcripts, one deleted for both exon 9 and 10 and one deleted for only exon 10. CONCLUSION: These results suggest Rad51d alternative splice variants potentially modulate mechanisms of HR by sequestering either RAD51C or XRCC2.

Homologous Recombination-Mediated DNA Repair and Implications for Clinical Treatment of Repair Defective Cancers.

Double-strand DNA breaks (DSBs) are generated by ionizing radiation and as intermediates during the processing of DNA, such as repair of interstrand cross-links and collapsed replication forks. These potentially deleterious DSBs are repaired primarily by the homologous recombination (HR) and nonhomologous end joining (NHEJ) DNA repair pathways. HR utilizes a homologous template to accurately restore damaged DNA, whereas NHEJ utilizes microhomology to join breaks in close proximity. The pathway available for DSB repair is dependent upon the cell cycle stage; for example, HR primarily functions during the S/G2 stages while NHEJ can repair DSBs at any cell cycle stage. Posttranslational modifications (PTMs) promote activity of specific pathways and subpathways through enzyme activation and precisely timed protein recruitment and degradation. This chapter provides an overview of PTMs occurring during DSB repair. In addition, clinical phenotypes associated with HR-defective cancers, such as mutational signatures used to predict response to poly(ADP-ribose) polymerase inhibitors, are discussed. Understanding these processes will provide insight into mechanisms of genome maintenance and likely identify targets and new avenues for therapeutic interventions.

Cellular and Genetic Determinants of the Sensitivity of Cancer to α-Particle Irradiation.

Targeted α-particle-emitting radionuclides have great potential for the treatment of a broad range of cancers at different stages of progression. A platform that accurately measures cancer cellular sensitivity to α-particle irradiation could guide and accelerate clinical translation. Here, we performed high-content profiling of cellular survival following exposure to α-particles emitted from radium-223 (223Ra) using 28 genetically diverse human tumor cell lines. Significant variation in cellular sensitivity across tumor cells was observed. 223Ra was significantly more potent than sparsely ionizing irradiation, with a median relative biological effectiveness of 10.4 (IQR: 8.4-14.3). Cells that are the most resistant to γ radiation, such as Nrf2 gain-of-function mutant cells, were sensitive to α-particles. Combining these profiling results with genetic features, we identified several somatic copy-number alterations, gene mutations, and the basal expression of gene sets that correlated with radiation survival. Activating mutations in PIK3CA, a frequent event in cancer, decreased sensitivity to 223Ra. The identification of cellular and genetic determinants of sensitivity to 223Ra may guide the clinical incorporation of targeted α-particle emitters in the treatment of several cancer types. SIGNIFICANCE: These findings address limitations in the preclinical guidance and prediction of radionuclide tumor sensitivity by identifying intrinsic cellular and genetic determinants of cancer cell survival following exposure to α-particle irradiation.See related commentary by Sgouros, p. 5479.

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.

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 patient's 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.