Primary Founder Mutations in the PRKDC Gene Increase Tumor Mutation Load in Colorectal Cancer

The clonal composition of a malignant tumor strongly depends on cellular dynamics influenced by the asynchronized loss of DNA repair mechanisms. Here, our aim was to identify founder mutations leading to subsequent boosts in mutation load. The overall mutation burden in 591 colorectal cancer tumors was analyzed, including the mutation status of DNA-repair genes. The number of mutations was first determined across all patients and the proportion of genes having mutation in each percentile was ranked. Early mutations in DNA repair genes preceding a mutational expansion were designated as founder mutations. Survival analysis for gene expression was performed using microarray data with available relapse-free survival. Of the 180 genes involved in DNA repair, the top five founder mutations were in PRKDC (n = 31), ATM (n = 26), POLE (n = 18), SRCAP (n = 18), and BRCA2 (n = 15). PRKDC expression was 6.4-fold higher in tumors compared to normal samples, and higher expression led to longer relapse-free survival in 1211 patients (HR = 0.72, p = 4.4 × 10−3). In an experimental setting, the mutational load resulting from UV radiation combined with inhibition of PRKDC was analyzed. Upon treatments, the mutational load exposed a significant two-fold increase. Our results suggest PRKDC as a new key gene driving tumor heterogeneity.

Attenuation of Polycyclic Aromatic Hydrocarbon (PAH)-Mediated Pulmonary DNA Adducts and Cytochrome P450 (CYP)1B1 by Dietary Antioxidants, Omega-3 Fatty Acids, in Mice

Numerous human and animal studies have reported positive correlation between carcinogen-DNA adduct levels and cancer occurrence. Therefore, attenuation of DNA adduct levels would be expected to suppress tumorigenesis. In this investigation, we report that the antioxidants omega 3-fatty acids, which are constituents of fish oil (FO), significantly decreased DNA adduct formation by polycyclic aromatic hydrocarbons (PAHs). B6C3F1 male mice were fed an FO or corn oil (CO) diet, or A/J male mice were pre-fed with omega-3 fatty acids eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA). While the B6C3F1 mice were administered two doses of a mixture of seven carcinogenic PAHs including benzo(a)pyrene (BP), the A/J mice were treated i.p. with pure benzo[a]pyrene (BP). Animals were euthanized after 1, 3, or 7 d after PAH treatment. DNA adduct levels were measured by the 32P-postlabeling assay. Our results showed that DNA adduct levels in the lungs of mice 7 d after treatment were significantly decreased in the FO or EPA/DHA groups compared with the CO group. Interestingly, both qPCR and Western blot analyses revealed that FO, DHA and EPA/DHA significantly decreased the expression of cytochrome P450 (CYP) 1B1. CYP1B1 plays a critical role in the metabolic activation of BP to DNA-reactive metabolites. qPCR also showed that the expression of some metabolic and DNA repair genes was induced by BP and inhibited by FO or omega-3 fatty acids in liver, but not lung. Our results suggest that a combination of mechanism entailing CYP1B1 inhibition and the modulation of DNA repair genes contribute to the attenuation of PAH-mediated carcinogenesis by omega 3 fatty acids.

GWAS and functional studies implicate a role for altered DNA repair in the evolution of drug resistance in Mycobacterium tuberculosis

The emergence of drug resistance in Mycobacterium tuberculosis (Mtb) is alarming and demands in-depth knowledge for timely diagnosis. We performed genome-wide association analysis (GWAS) using 2237 clinical strains of Mtb to identify novel genetic factors that evoke drug resistance. In addition to the known direct targets, for the first time, we identified a strong association between the mutations in the DNA repair genes and the multidrug-resistant phenotype. To evaluate the impact of variants identified in the clinical samples in the evolution of drug resistance, we utilized knockouts and complemented strains in Mycobacterium smegmatis (Msm) and Mtb. Results show that variant mutations abrogated the function of MutY and UvrB. MutY variant showed enhanced survival compared with wild-type (Rv) when the Mtb strains were subjected to multiple rounds of ex vivo antibiotic stress. Notably, in an in vivo Guinea pig infection model, the MutY variant outcompeted the wild-type strain. Collectively, we show that novel variant mutations in the DNA repair genes abrogate their function and contribute to better survival under antibiotic/host stress conditions.

DNA Repair Genes as Drug Candidates for Early Breast Cancer Onset in Latin America: A Systematic Review

The prevalence of breast cancer in young women (YWBC) has increased alarmingly. Significant efforts are being made to elucidate the biological mechanisms concerning the development, prognosis, and pathological response in early-onset breast cancer (BC) patients. Dysfunctional DNA repair proteins are implied in BC predisposition, progression, and therapy response, underscoring the need for further analyses on DNA repair genes. Public databases of large patient datasets such as METABRIC, TCGA, COSMIC, and cancer cell lines allow the identification of variants in DNA repair genes and possible precision drug candidates. This study aimed at identifying variants and drug candidates that may benefit Latin American (LA) YWBC. We analyzed pathogenic variants in 90 genes involved in DNA repair in public BC datasets from METABRIC, TCGA, COSMIC, CCLE, and COSMIC Cell Lines Project. Results showed that reported DNA repair germline variants in the LA dataset are underrepresented in large databases, in contrast to other populations. Additionally, only six gene repair variants in women under 50 years old from the study population were reported in BC cell lines. Therefore, there is a need for new approaches to study DNA repair variants reported in young women from LA.

Unraveling the genetic architecture of hepatoblastoma risk: birth defects and increased burden of germline damaging variants in gastrointestinal/renal cancer predisposition and DNA repair genes

The ultrarare hepatoblastoma is the most common pediatric liver cancer. HB risk is related to a few rare syndromes, and the molecular bases remain elusive for most cases. We investigated the burden of rare damaging germline variants in 30 Brazilian patients with HB. A high frequency of prematurity (20%) and birth defects (37%), especially craniofacial (17%, including craniosynostosis) and kidney (7%) anomalies, was observed. Pathogenic or likely pathogenic variants mapped to 10 cancer predisposition genes (APC, CHEK2, DROSHA, ERCC5, FAH, MSH2, MUTYH, RPS19, TGFBR2 and VHL,) were detected in 33% of the patients, only 40% of them with a family history of cancer. These findings showed a predominance of CPGs with a known link to gastrointestinal/colorectal and renal cancer risk. A remarkable feature was an enrichment of rare damaging variants affecting different classes of DNA repair genes, particularly those known as Fanconi anemia genes. Moreover, several damaging rare variants mapped to genes impacting liver functions were observed. To our knowledge, this is the first comprehensive assessment of rare germline variants in HB patients, contributing to elucidating the genetic architecture of HB risk.

927 Wilms tumor reveals DNA repair gene hyper-expression is linked to lack of immune infiltration

BackgroundA T cell-rich tumor microenvironment has been associated with improved clinical outcome and response to immune checkpoint blockade therapies in several adult cancers. Understanding the mechanisms for lack of immune cell infiltration is critical for expanding immunotherapy efficacy in the clinic. To gain new insights into the mechanisms of poor tumor immunogenicity, we turned to pediatric cancers, which are generally unresponsive to checkpoint blockade.MethodsRNAseq and clinical data were obtained for Wilms tumor, rhabdoid tumor, osteosarcoma, and neuroblastoma from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database, and adult cancers from TCGA. Using an 18-gene tumor inflammation signature (TIS) representing activated CD8+ T cells, we identified genes significantly anti-correlated with the signature. Immunofluorescence was performed on metastatic melanoma samples for CD8, MSH2, and the tumor cell marker SOX10, and analyzed for relationship to anti-PD-1 efficacy.ResultsAmong the four pediatric cancers, we observed the lowest TIS scores in Wilms tumor. Wilms tumors demonstrated significantly lower T cell inflammation signatures than matched normal kidney samples, other pediatric tumor samples, and adult kidney tumor samples. Pathway analysis identified multiple types of DNA repair were upregulated in Wilms tumor and a score generated from the top 50 DNA repair genes strongly anti-correlated with TIS. This striking negative association was also observed in most adult tumor types. The anti-correlation was found to be independent of tumor mutation burden, suggesting that high expression of DNA repair pathway machinery may restrict tumor immunogenicity by mechanisms beyond prevention neoantigen accumulation. MSH2 was one of the top DNA repair genes identified from the Wilms tumor analysis and was confirmed to have a strong anti-correlation with TIS in melanoma samples from TCGA. Immunofluorescence from an independent cohort of metastatic melanoma patients revealed a significant negative correlation between CD8+ T cell numbers and MSH2+ SOX10+ tumor cell numbers. Additionally, non-responders to anti-PD-1 immunotherapy had significantly higher numbers of MSH2+ SOX10+ tumor cells than responders.ConclusionsIncreased tumor expression of DNA repair genes is associated with a less robust immune response in Wilms tumor, and this was also observed in the majority of TCGA tumor types. Surprisingly, the negative relationship between DNA repair score and TIS remained strong across TCGA when correcting for mutation count, indicating a potential role for DNA repair genes outside of preventing the accumulation of mutations. Strategies targeting DNA repair pathways could be considered as new therapeutic interventions to transform non-T cell-inflamed tumors into immune-responsive tumors.Ethics ApprovalThe study obtained ethics approval under IRB protocol 15-0837.