RAD51 Gene Research Articles

Regulation of yeast <i>RAD9</i> gene in energy charge, intracellular ROS, and cell cycle arrest in response to DNA damage

In various environmental conditions, eukaryotic cells are exposed to many kinds of exogenous toxic agents as well as to endogenous agents like reactive oxygen species (ROS) generated from oxidative metabolism that can all result in damage to DNA. To cope with these types of damage, yeast cells have evolved a number of mechanisms and specific response systems regulated by key control genes. One of which is RAD9 gene that regulates DNA damage and repair checkpoints, and cell cycle arrest. Thus, a series of methods, e.g. oxygen consumption monitoring, physicochemical analysis, and flow cytometry, were used in the present study to investigate the role of the RAD9 gene by using the BY4742 (wild type) and specific knock-out yeast strains (∆rad9) and elucidate the function of this gene in cellular defense mechanism and metabolic response to DNA damage triggered by methyl methanesulfonate (MMS) treatment. The results indicated that fully functional DNA damage repair and cell cycle checkpoint (RAD9, wild type) significantly enhanced mitochondrial activity and oxygen consumption, reduced intracellular ROS accumulation. Fully functional mitochondria attenuated ROS accumulation, enabled efficient mitochondrial electron transport chain (mtETC) and ATP synthesis, and stabilized cellular energy status. Also, high mitochondrial activity acted as a protective mechanism against oxidative stress. In contrast, deletion of the RAD9 (∆rad9) resulted in high ROS accumulation and damaged to mitochondrial DNA, leading to strong inhibition of mitochondrial activity and oxygen consumption. Furthermore, low mitochondrial activity in cells lacking RAD9 (∆rad9) led to the development of oxidative stress. Subsequently, high ROS accumulation in ∆rad9 cells caused a block of the mtETC, repression of ATP synthesis, fluctuation of cellular energy status, and induction of cell cycle arrest at S and G2/M phases.

The Impact of Oleuropein on Cisplatin-Induced Toxicity in Cochlear Cells in Relation to the Expression of Deoxyribonucleic Acid Damage-Associated Genes.

Different organs respond differently to cisplatin (CDDP)-induced toxicity. Oleuropein (OLE) is a natural phenolic antioxidant. The purpose of this study was to determine the potential protective effect of OLE against CDDP-induced ototoxicity by evaluating expression of genes associated with deoxyribonucleic acid (DNA) damage and repair in cochlear cells. House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were treated using CDDP, OLE, and OLE-CDDP. The water-soluble tetrazolium salt assay was used for monitoring cell viability. Deoxyribonucleic acid damage in cells due to the CDDP, OLE, and combination treatments was determined using a flow-cytometric kit. The change in the expression of 84 genes associated with CCDP, OLE, and OLE-CDDP treatments that induced DNA damage was tested using the reverse transcription polymerase chain reaction array. Changes ≥3-fold were considered significant. House Ear Institute-Organ of Corti 1 cell viability was significantly reduced by CDDP. The OLE-CDDP combination restored the cell viability. Cisplatin increased the H2AX ratio, while OLE-CDDP combination decreased it. Some of the DNA damage-associated genes whose expression was upregulated with CDDP were downregulated with OLE-CDDP, while the expression of genes such as Gadd45g and Rev1 was further downregulated. The expression of DNA repair-related Abl1, Dbd2, Rad52, and Trp53 genes was downregulated with CDDP, whereas their expression was upregulated with OLE-CDDP treatment. In cochlear cells, the OLE-CDDP combination downregulated DNA damage-associated gene expression relative to that upregulated mainly by CDDP. The results revealed that OLE has a potential protective effect on CDDP-induced ototoxicity in cochlear cells by altering the expression of DNA damage-related genes.

RAD51 High-Expressed Hepatocellular Carcinomas Are Associated With High Cell Proliferation

IntroductionRAD51 is a pivotal DNA repair gene managing double-stranded DNA break recognition and repair. RAD51 high expression was associated with adverse outcomes in other cancer types. This study aims to investigate the tumor microenvironment and immune landscape in the RAD51 high-expressed Hepatocellular Carcinoma (HCCs). MethodsA total of 467 patients from two large independent cohorts with clinical and transcriptomic data were obtained. The cohort was dichotomized based on the median RAD51 gene expression. xCell and Gene Set Enrichment Analysis (GSEA) were used. ResultsRAD51 high-expressed HCCs were associated with worse recurrence-free, progression-free, disease-specific, and overall survival (all P < 0.05). While RAD51 high-expressed HCCs were associated with intratumoral heterogeneity, homologous recombination deficiency, and fraction altered scores, mutation or neoantigens were not increased in this group. xCell analysis demonstrated inconsistent immune cell infiltration between two cohorts. Cytolytic activity as well as GSEA with immune-related gene sets also demonstrated inconsistent results between two cohorts as well. On the other hand, RAD51 expression was significantly increased in higher-grade tumors, larger tumors, and higher clinical stages. RAD51 high-expressed HCCs were found to have elevated proliferation score. Furthermore, GSEA exhibited significant enrichment of all the cell proliferation–related gene sets in the Hallmark collection, including E2F targets, G2M checkpoint, Mitotic spindle, MYC targets, and MTORC1 signaling consistently in both cohorts (all false discovery rate < 0.25). ConclusionsRAD51 high-expressed HCCs were associated with worse survival and with increased cell proliferation and were not necessarily associated with immune infiltration or inflammation.

Single Nucleotide Polymorphisms in APE1, hOGG1, RAD51 Genes and their Association with Radiotherapy Induced Toxicity among Head and Neck Cancer Patients.

Radiotherapy (RT) is a crucial treatment for head and neck cancer however, it causes adverse reactions to the normal tissue and organs adjacent to target tumor. The present study was carried out to investigate possible association of single nucleotide polymorphism in DNA repair genes with toxicity effects of radiotherapy on normal tissue. Three hundred and fifty head and neck cancer patients receiving radiotherapy treatment were enrolled in this study. The adverse after effects of radiotherapy on the normal tissue in the form of skin reactions were recorded. Single nucleotide polymorphisms of APE1 (rs1130409), hOGG1 (rs1052133) and Rad51 (rs1801320, rs1801321) genes were studied by polymerase chain reaction-Restriction fragment length polymorphism (PCR-RFLP) and direct DNA sequencing methods and their association with development of severe radio-toxicity effects was evaluated logistic regression analysis. The 172G/T polymorphism of Rad51 was 2.85 times higher and significantly associated with skin reactions (OR=2.85, 95% CI: 1.50-5.41; p=0.001) and severe oral mucositis (OR=4.96, 95% CI: 2.40-10.25; p<0.0001). These results suggested that the polymorphic nature of Rad51 is responsible for risk of radiotherapy adverse effects in HNC patients. The variant 326Cys and heterozygous 326Ser/Cys genotype of hOGG1 was significantly associated with high tumor grade (OR=3.16 95% CI: 1.66-5.99; p=0.0004, and OR=3.97 95% CI: 2.15-7.34; p=<0.0001 respectively). The homozygous variant 172TT genotype of Rad51 showed positive association with poor response of both tumor and nodes towards radiotherapy treatment (p=0.007 and p=0.022). Interpretation of our results revealed significant association of rs1801321 SNP of Rad51 with development of adverse toxicity reactions in normal tissue of head and neck cancer patients treated with radiotherapy.

Identification of DNA Repair-related Genes as Biomarkers Reflecting MSI, TMB, and TIL in Colorectal Cancer.

There are no established biomarkers for immune checkpoint inhibitors (ICI) in colorectal cancer (CRC) with microsatellite stability (MSS) or proficient mismatch repair (pMMR). Therefore, this study aimed to identify biomarkers for ICI benefit in patients with pMMR by analyzing the down-regulated DNA repair-related genes involved in highly immunogenic and immune responses, and comparing their expression levels and clinical features. Mismatch repair (MMR), tumor-infiltrating lymphocytes (TIL), and tumor mutation burden (TMB) were evaluated in 13 CRC cases and mRNA expression levels of 95 DNA repair-related genes were measured. DNA repair-related genes with reduced mRNA expression in the high immunogenicity and high immune response groups were identified. Then, the mRNA expression levels of the identified DNA repair-related genes were measured in 135 patients with CRC. Hierarchical cluster analysis was performed using the mRNA expression levels to compare the clinicopathological characteristics of each cluster. ATR, LIG4, and RAD52 mRNA levels were significantly down-regulated in the high immunogenicity group. GADD45B, SMUG1, and XRCC6 mRNA levels were significantly down-regulated in the high immune response group. Cases in the cluster with reduced mRNA expression of the six genes were pMMR cases. CD8 mRNA expression level was higher in this cluster than in the other clusters. Decreased mRNA expression levels of ATR, LIG4, RAD52, GADD45B, SMUG1, and XRCC6 genes were associated with high cytotoxic T cell and TMB levels, suggesting that these genes could serve as biomarkers for ICI efficacy in pMMR cases.

SingleNucleotide Polymorphisms as Biomarkers of Mepolizumab and Benralizumab Treatment Response in Severe Eosinophilic Asthma.

The most promising treatment options for severe uncontrolled asthma (SUA) have emerged in recent years with the development of monoclonal antibodies for blocking selective targets responsible for the underlying inflammation, such as mepolizumab and benralizumab. However, there is variability in treatment response that is not fully controlled. The variability of the response to mepolizumab and benralizumab could be influenced by single-nucleotide polymorphisms (SNPs), and it would be useful to detect these and use them as predictive biomarkers of response. We conducted a retrospective observational cohort study of 72 Caucasian patients recruited from a tertiary hospital with severe uncontrolled eosinophilic asthma treated with mepolizumab and benralizumab. Polymorphisms in the IL5 (rs4143832, rs17690122), RAD50 (rs11739623, rs4705959), IL1RL1 (rs1420101, rs17026974, rs1921622), GATA2 (rs4857855), IKZF2 (rs12619285), FCGR2A (rs1801274), FCGR2B (rs3219018, rs1050501), FCGR3A (rs10127939, rs396991), FCER1A (rs2251746, rs2427837), FCER1B (rs1441586, rs573790, rs569108), and ZNF415 (rs1054485) genes were analyzed by real-time polymerase chain reaction (PCR) using Taqman probes. The response was analyzed after 12 months of treatment. In patients under mepolizumab treatment, a treatment response defined as a reduction in exacerbations was associated with ZNF415 rs1054485-T (p = 0.042; OR = 5.33; 95% CI = 1.06-30.02), treatment response defined as a reduction in oral corticosteroids use was associated with the number of exacerbations in the previous year (p = 0.029; OR = 3.89; 95% CI = 1.24-14.92), and treatment response defined as improvement in lung function was associated with the age at the beginning of biological therapy (p = 0.002; OR = 1.10; 95% CI = 1.04-1.18), FCER1B rs569108-AA (p < 0.001; OR = 171.06; 95% CI = 12.94-6264.11), and FCER1A rs2427837-A (p = 0.021; OR = 8.61; 95% CI = 1.71-76.62). On the other hand, in patients under benralizumab treatment, treatment response, defined as a reduction in exacerbations, was associated with ZNF415 rs1054485-T (p = 0.073; OR = 1.3 × 108; 95% CI = 1.8 × 10-19-NA), FCER1B rs569108-AA (p = 0.050; OR = 11.51; 95% CI = 1.19-269.78), allergies (p = 0.045; OR = 4.02; 95% CI = 1.05-16.74), and sex (p = 0.028; OR = 4.78; 95% CI = 1.22-20.63); and treatment response defined as improvement in lung function was associated with polyposis (p = 0.027; OR = 9.16; 95% CI = 1.58-91.4), IKZF2 rs12619285-AA (p = 0.019; OR = 9.1; 95% CI = 1.7-75.78), IL5 rs4143832-T (p = 0.017; OR = 11.1; 95% CI = 1.9-112.17), and FCER1B rs1441586-C (p = 0.045; OR = 7.81; 95% CI = 1.16-73.45). The results of this study show the potential influence of the studied polymorphisms on the response to mepolizumab and benralizumab and the clinical benefit that could be obtained by defining predictive biomarkers of treatment response.

GENETIC POLYMORPHISMS IN RAD51, RAD52 AND RAD54 GENES OF HOMOLOGOUS RECOMBINATION REPAIR PATHWAY IN BREAST CANCER: A CASE-CONTROL STUDY FROM PUNJAB

A case-control study was conducted on 600 subjects (300 cases and 300 controls) to understand the association of genetic polymorphisms of RAD51 (rs1801320, rs1801321, rs121917739, rs2619681), RAD52 (rs4987207, rs4987208) and RAD54 (rs2295466) genes in breast cancer patients and controls in population of Punjab. Genotyping of the selected SNPs of RAD51 and RAD52 gene was done by PCR-RFLP, whereas ARMS-PCR was used for RAD54 gene polymorphism. Our study identified significant associations between specific SNPs in the RAD51 and RAD52 genes and breast cancer risk. Notably, three RAD51 SNPs (rs1801320, rs1801321, and rs121917739) and one RAD52 SNP (rs4987208) showed significant genotype frequency differences between cases and controls. Genetic model analysis revealed that minor alleles of four RAD51 SNPs (rs1801320, rs1801321, rs121917739, and rs2619681) and two RAD52 SNPs (rs4987207 and rs4987208) were linked to increased breast cancer risk. Haplotype analysis further supported these findings, with 10 RAD51 haplotypes and two RAD52 haplotypes (G-G and T-G) significantly associated with higher breast cancer risk. Additionally, the GT + TT genotype of RAD52 rs4987207 was associated with lower odds of metastasis, while the TG + GG genotype of rs4987208 was linked to lymph node involvement and higher tumor grade.

Germline pathogenic variants in the MRE11, RAD50, and NBN (MRN) genes in cancer predisposition: A systematic review and meta-analysis.

The MRE11, RAD50, and NBN genes encode the MRN complex sensing DNA breaks and directing their repair. While carriers of biallelic germline pathogenic variants (gPV) develop rare chromosomal instability syndromes, the cancer risk in heterozygotes remains controversial. We performed a systematic review and meta-analysis of 53 studies in patients with different cancer diagnoses to better understand the cancer risk. We found an increased risk (odds ratio, 95% confidence interval) for gPV carriers in NBN for melanoma (7.14; 3.30-15.43), pancreatic cancer (4.03; 2.14-7.58), hematological tumors (3.42; 1.14-10.22), and prostate cancer (2.44, 1.84-3.24), but a low risk for breast cancer (1.29; 1.00-1.66) and an insignificant risk for ovarian cancer (1.53; 0.76-3.09). We found no increased breast cancer risk in carriers of gPV in RAD50 (0.93; 0.74-1.16; except of c.687del carriers) and MRE11 (0.87; 0.66-1.13). The secondary burden analysis compared the frequencies of gPV in MRN genes in patients from 150 studies with those in the gnomAD database. In NBN gPV carriers, this analysis additionally showed a high risk for brain tumors (5.06; 2.39-9.52), a low risk for colorectal (1.64; 1.26-2.10) and hepatobiliary (2.16; 1.02-4.06) cancers, and no risk for endometrial, and gastric cancer. The secondary burden analysis showed also a moderate risk for ovarian cancer (3.00; 1.27-6.08) in MRE11 gPV carriers, and no risk for ovarian and hepatobiliary cancers in RAD50 gPV carriers. These findings provide a robust clinical evidence of cancer risks to guide personalized clinical management in heterozygous carriers of gPV in the MRE11, RAD50, and NBN genes.

Reduced levels of MRE11 cause disease phenotypes distinct from ataxia telangiectasia-like disorder.

The MRE11/RAD50/NBS1 (MRN) complex plays critical roles in cellular responses to DNA double-strand breaks. MRN is involved in end binding and processing, and it also induces cell cycle checkpoints by activating the ataxia-telangiectasia mutated (ATM) protein kinase. Hypomorphic pathogenic variants in the MRE11, RAD50, or NBS1 genes cause autosomal recessive genome instability syndromes featuring variable degrees of dwarfism, neurological defects, anemia, and cancer predisposition. Disease-associated MRN alleles include missense and nonsense variants, and many cause reduced protein levels of the entire MRN complex. However, the dramatic variability in the disease manifestation of MRN pathogenic variants is not understood. We sought to determine if low protein levels are a significant contributor to disease sequelae and therefore generated a transgenic murine model expressing MRE11 at low levels. These mice display dramatic phenotypes including small body size, severe anemia, and impaired DNA repair. We demonstrate that, distinct from ataxia telangiectasia-like disorder caused by MRE11 pathogenic missense or nonsense variants, mice and cultured cells expressing low MRE11 levels do not display the anticipated defects in ATM activation. Our findings indicate that ATM signaling can be supported by very low levels of the MRN complex and imply that defective ATM activation results from perturbation of MRN function caused by specific hypomorphic disease mutations. These distinct phenotypic outcomes underline the importance of understanding the impact of specific pathogenic MRE11 variants, which may help direct appropriate early surveillance for patients with these complicated disorders in a clinical setting.

Synthetic Lethality between Cohesin and WNT Signaling Pathways in Diverse Cancer Contexts.

Cohesin is a highly conserved ring-shaped complex involved in topologically embracing chromatids, gene expression regulation, genome compartmentalization, and genome stability maintenance. Genomic analyses have detected mutations in the cohesin complex in a wide array of human tumors. These findings have led to increased interest in cohesin as a potential target in cancer therapy. Synthetic lethality has been suggested as an approach to exploit genetic differences in cancer cells to influence their selective killing. In this study, we show that mutations in ESCO1, NIPBL, PDS5B, RAD21, SMC1A, SMC3, STAG2, and WAPL genes are synthetically lethal with stimulation of WNT signaling obtained following LY2090314 treatment, a GSK3 inhibitor, in several cancer cell lines. Moreover, treatment led to the stabilization of β-catenin and affected the expression of c-MYC, probably due to the occupancy decrease in cohesin at the c-MYC promoter. Finally, LY2090314 caused gene expression dysregulation mainly involving pathways related to transcription regulation, cell proliferation, and chromatin remodeling. For the first time, our work provides the underlying molecular basis for synthetic lethality due to cohesin mutations and suggests that targeting the WNT may be a promising therapeutic approach for tumors carrying mutated cohesin.

Cornelia De Lange Syndrome with left heart hypoplasia: a case study

Cornelia de Lange syndrome (CdLS) is characterized by slow growth that can lead to short stature. Despite mutations in the NIPBL, RAD21 or SMC3 genes, CdLS is thought to be inherited in an autosomal dominant manner. Findings include intellectual disability and bone abnormalities in the upper extremities and distinct facial features. It has similar characteristics to autism spectrum disorder, which is a developmental condition that affects communication and social interaction. The physical and clinical findings of a one-and-a-half-year-old girl patient diagnosed with CdLS were reported. In addition to NIPBL gene mutation, atrioventricular septal defect (AVSD), an overriding aorta, and a hypoplastic left heart (HLH) were observed in our case. This study revealed the patient's physical and clinical findings consistent with the diagnosis of CdLS. Additionally emphasized HLH. The importance of performing corrective surgery immediately following the evaluation of congenital heart diseases in CdLS patients can be emphasized.

Genetically driven predisposition leads to an unusually genomic unstable renal cell carcinoma

Renal cell carcinoma originates from the lining of the proximal convoluted renal tubule and represents the most common type of kidney cancer. Risk factors and comorbidities might be associated to renal cell carcinoma, while a small fraction of 2–3% emerges from patients with predisposing cancer syndromes, typically associated to hereditary mutations in VHL, folliculin, fumarate hydratase or MET genes. Here, we report a case of renal cell carcinoma in patient with concurrent germline mutations in BRCA1 and RAD51 genes. This case displays an unusual high mutational burden and chromosomal aberrations compared to the typical profile of renal cell carcinoma. Mutational analysis on whole genome sequencing revealed an enrichment of the MMR2 mutational signature, which is indicative of impaired DNA repair capacity. Overall, the tumor displayed a profile of unusual high genomic instability which suggests a possible origin from germline predisposing mutations in the DNA repair genes BRCA1 and RAD51. While BRCA1 and RAD51 germline mutations are well-characterised in breast and ovarian cancer, their role in renal cell carcinoma is still largely unexplored. The genomic instability detected in this case of renal cell carcinoma, along with the presence of unusual mutations, might offer support to clinicians for the development of patient-tailored therapies.

The Association between Inefficient Repair of DNA Double-Strand Breaks and Common Polymorphisms of the HRR and NHEJ Repair Genes in Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation affecting up to 2.0% of adults around the world. The molecular background of RA has not yet been fully elucidated, but RA is classified as a disease in which the genetic background is one of the most significant risk factors. One hallmark of RA is impaired DNA repair observed in patient-derived peripheral blood mononuclear cells (PBMCs). The aim of this study was to correlate the phenotype defined as the efficiency of DNA double-strand break (DSB) repair with the genotype limited to a single-nucleotide polymorphism (SNP) of DSB repair genes. We also analyzed the expression level of key DSB repair genes. The study population contained 45 RA patients and 45 healthy controls. We used a comet assay to study DSB repair after in vitro exposure to bleomycin in PBMCs from patients with rheumatoid arthritis. TaqMan SNP Genotyping Assays were used to determine the distribution of SNPs and the Taq Man gene expression assay was used to assess the RNA expression of DSB repair-related genes. PBMCs from patients with RA had significantly lower bleomycin-induced DNA lesion repair efficiency and we identified more subjects with inefficient DNA repair in RA compared with the control (84.5% vs. 24.4%; OR 41.4, 95% CI, 4.8-355.01). Furthermore, SNPs located within the RAD50 gene (rs1801321 and rs1801320) increased the OR to 53.5 (95% CI, 4.7-613.21) while rs963917 and rs3784099 (RAD51B) to 73.4 (95% CI, 5.3-1011.05). These results were confirmed by decision tree (DT) analysis (accuracy 0.84; precision 0.87, and specificity 0.86). We also found elevated expression of RAD51B, BRCA1, and BRCA2 in PBMCs isolated from RA patients. The findings indicated that impaired DSB repair in RA may be related to genetic variations in DSB repair genes as well as their expression levels. However, the mechanism of this relation, and whether it is direct or indirect, needs to be elucidated.

Targeting FBXO22 enhances radiosensitivity in non-small cell lung cancer by inhibiting the FOXM1/Rad51 axis

Radioresistance is a major constraint on the efficacy of lung cancer radiotherapy, but its mechanism has not been fully elucidated. Here, we found that FBXO22 was aberrantly highly expressed in lung cancer and that FBXO22 knockdown increased the radiosensitivity of lung cancer cells. Mechanistically, FBXO22 promoted Rad51 gene transcription by increasing the level of FOXM1 at the Rad51 promoter, thereby inducing the formation of lung cancer radioresistance. Furthermore, we found that deguelin, a potential inhibitor of FBXO22, enhanced radiosensitivity in an FBXO22/Rad51-dependent manner and was safely tolerated in vivo. Collectively, our results illustrate that FBXO22 induces lung cancer radioresistance by activating the FOXM1/Rad51 axis and provide preclinical evidence for the clinical translation of this critical target.

Uncovering the clinical relevance of unclassified variants in DNA repair genes: a focus on BRCA negative Tunisian cancer families.

Introduction: Recent advances in sequencing technologies have significantly increased our capability to acquire large amounts of genetic data. However, the clinical relevance of the generated data continues to be challenging particularly with the identification of Variants of Uncertain Significance (VUSs) whose pathogenicity remains unclear. In the current report, we aim to evaluate the clinical relevance and the pathogenicity of VUSs in DNA repair genes among Tunisian breast cancer families. Methods: A total of 67 unsolved breast cancer cases have been investigated. The pathogenicity of VUSs identified within 26 DNA repair genes was assessed using different in silico prediction tools including SIFT, PolyPhen2, Align-GVGD and VarSEAK. Effects on the 3D structure were evaluated using the stability predictor DynaMut and molecular dynamics simulation with NAMD. Family segregation analysis was also performed. Results: Among a total of 37 VUSs identified, 11 variants are likely deleterious affecting ATM, BLM, CHEK2, ERCC3, FANCC, FANCG, MSH2, PMS2 and RAD50 genes. The BLM variant, c.3254dupT, is novel and seems to be associated with increased risk of breast, endometrial and colon cancer. Moreover, c.6115G>A in ATM and c.592+3A>T in CHEK2 were of keen interest identified in families with multiple breast cancer cases and their familial cosegregation with disease has been also confirmed. In addition, functional in silico analyses revealed that the ATM variant may lead to protein immobilization and rigidification thus decreasing its activity. We have also shown that FANCC and FANCG variants may lead to protein destabilization and alteration of the structure compactness which may affect FANCC and FANCG protein activity. Conclusion: Our findings revealed that VUSs in DNA repair genes might be associated with increased cancer risk and highlight the need for variant reclassification for better disease management. This will help to improve the genetic diagnosis and therapeutic strategies of cancer patients not only in Tunisia but also in neighboring countries.

SOG1 and BRCA1 Interdependently Regulate RAD54 Expression for Repairing Salinity-Induced DNA Double-Strand Breaks in Arabidopsis.

As sessile organisms, land plants experience various forms of environmental stresses throughout their life span. Therefore, plants have developed extensive and complicated defense mechanisms, including a robust DNA damage response (DDR) and DNA repair systems for maintaining genome integrity. In Arabidopsis, the NAC [NO APICAL MERISTEM (NAM), ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR (ATAF), CUP-SHAPED COTYLEDON (CUC)] domain family transcription factor SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) plays an important role in regulating DDR. Here, we show that SOG1 plays a key role in regulating the repair of salinity-induced DNA double-strand breaks (DSBs) via the homologous recombination (HR) pathway in Arabidopsis. The sog1-1 mutant seedlings display a considerably slower rate of repair of salinity-induced DSBs. Accumulation of SOG1 protein increases in wild-type Arabidopsis under salinity stress, and it enhances the expression of HR pathway-related genes, including RAD51, RAD54 and BReast CAncer gene 1 (BRCA1), respectively, as found in SOG1 overexpression lines. SOG1 binds specifically to the AtRAD54 promoter at the 5'-(N)4GTCAA(N)3C-3' consensus sequence and positively regulates its expression under salinity stress. The phenotypic responses of sog1-1/atrad54 double mutants suggest that SOG1 functions upstream of RAD54, and both these genes are essential in regulating DDR under salinity stress. Furthermore, SOG1 interacts directly with BRCA1, an important component of the HR-mediated DSB repair pathway in plants, where BRCA1 appears to facilitate the binding of SOG1 to the RAD54 promoter. At the genetic level, SOG1 and BRCA1 function interdependently in modulating RAD54 expression under salinity-induced DNA damage. Together, our results suggest that SOG1 regulates the repair of salinity-induced DSBs via the HR-mediated pathway through genetic interactions with RAD54 and BRCA1 in Arabidopsis.

RAD50 Deficient in a Breast Cancer Model Predicts Sensitivity to PARP Inhibitors.

Breast and ovarian tumors with pathogenic variants in BRCA1 or BRCA2 genes are more sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi) treatment than wild-type tumors. Pathogenic variants in non-BRCA1/2 homologous recombination repair genes (HRR) also concede sensitivity to PARPi treatment. RAD50 participates in the Mre11-Rad50-Nbn (MRN) complex of the HRR pathway and plays an important role in DNA repair. The objective of this study is to evaluate whether RAD50 protein deficiency modulates the PARPi response in breast cancer cell lines. T47D breast cancer cell line was modified using small interfering RNA and CRISPR/Cas9 technology, to knockout the RAD50 gene. PARPi response (niraparib, olaparib and rucaparib alone or in combination with carboplatin), in T47D and T47D-edited clones, was evaluated by cell viability, cell cycle, apoptosis and protein expression analyses. Treatment with niraparib and carboplatin exerted a synergistic effect on T47D-RAD50 deficient cells and an antagonistic effect on T47D cells parental. Cell cycle analysis demonstrated an increase in the G2/M population in cells treated with niraparib or rucaparib alone or in combination with carboplatin. T47D-RAD50 deficient cells treated with rucaparib and carboplatin exhibited twofold levels in late apoptosis, also showing differences in PARP activation. All T47D RAD50 deficient clones treated with niraparib or rucaparib combined with carboplatin, or rucaparib alone showed increased levels of H2AX phosphorylation. T47D RAD50 deficient cells treated with PARP inhibitors alone or in combination with carboplatin showed cell cycle arrest in the G2/M phase, leading to death by apoptosis. Thus, RAD50 deficiency may be a good biomarker for predicting PARPi response.

Abstract C038: BRCA2 haploinsufficiency driven neoplastic events in the fallopian tube epithelia

Abstract One in 70 women will develop ovarian cancer over their lifetime in the US and epithelial ovarian cancer (EOC) remains the deadliest gynecological cancer. EOC is a heterogenous disease and high-grade serous carcinoma (HGSOC) is the most common histotype. It is genetically unstable due to pathogenic variants in BRCA1, BRCA2, PALB2, ATM, and RAD51 genes involved in DNA damage response and repair which are part of the hereditary breast and ovarian cancer syndrome (HBOC). In women with a BRCA1 or BRCA2 mutation undergoing risk-reducing surgery, 3-17% have pre-neoplastic and neoplastic lesions in their fallopian tubes. These lesions, known as serous tubal intra-epithelial carcinoma (STIC), display differential loss of heterozygosity of BRCA1/2. New insights into BRCA2 function could provide novel opportunities for preventative interventions in individuals with this mutation, and perhaps new treatment opportunities for HGSC associated with BRCA2 heterozygosity. We have identified distinct metabolic and mitochondrial deficits in BRCA2 mutation carriers compared to BRCA1 carriers and non-carriers. Fallopian tube epithelial cell lines derived from BRCA2 mutation carriers showed increased levels of mitochondrial respiration and activity followed by BRCA1 mutation carriers and non-carriers. Transmission electron microscopy of BRCA2 mutation carriers showed increased disrupted mitochondrial networks when compared to wild type, suggesting that mutation status may contribute to increased mitochondrial fission. These observations reveal an entirely new aspect of BRCA2 biology and further studies will disaggregate the effects of BRCA2 on mitochondrial function, versus DNA damage repair. Ultimately, this work will provide new insight into the adaptive changes within FT cells which promote neoplastic transformation and inform how high-grade serous carcinoma (HGSC) cells distinctively respond to microenvironmental stimuli. Citation Format: Jovanka Ravix, Iru Paudel, Ramlogan Sowamber, Gauthami Pulivendala, David Lombard, Sophia George. BRCA2 haploinsufficiency driven neoplastic events in the fallopian tube epithelia [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C038.

Tissue-specific toxic effects of nano-copper on zebrafish

Understanding the behavior and potential toxicity of copper nanoparticles (nano-Cu) in the aquatic environment is a primary way to assess their environmental risks. In this study, RNA-seq was performed on three different tissues (gills, intestines, and muscles) of zebrafish exposed to nano-Cu, to explore the potential toxic mechanism of nano-Cu on zebrafish. The results indicated that the toxic mechanism of nano-Cu on zebrafish was tissue-specific. Nano-Cu enables the CB1 receptor of the presynaptic membrane of gill cells to affect short-term synaptic plasticity or long-term synaptic changes (ECB-LTD) through DSI and DSE, causing dysfunction of intercellular signal transmission. Imbalance of de novo synthesis of UMP in intestinal cells and its transformation to UDP, UTP, uridine, and uracil, resulted in many functions involved in the pyrimidine metabolic pathway being blocked. Meanwhile, the toxicity of nano-Cu caused abnormal expression of RAD51 gene in muscle cells, which affects the repair of damaged DNA through Fanconi anemia and homologous recombination pathway, thus causing cell cycle disorder. These results provide insights for us to better understand the differences in toxicity of nano-Cu on zebrafish tissues and are helpful for a comprehensive assessment of nano-Cu's effects on aquatic organisms.

Exploring homologous recombination repair and base excision repair pathway genes for possible diagnostic markers in hematologic malignancies.

Hematologic malignancies (HMs) are a collection of malignant transformations, originating from the cells in the bone marrow and lymphoid organs. HMs comprise three main types; leukemia, lymphoma, and multiple myeloma. Globally, HMS accounts for approximately 10% of newly diagnosed cancer. DNA repair pathways defend the cells from recurrent DNA damage. Defective DNA repair mechanisms such as homologous recombination repair (HRR), nucleotide excision repair (NER), and base excision repair (BER) pathways may lead to genomic instability, which initiates HM progression and carcinogenesis. Expression deregulation of HRR, NER, and BER has been investigated in various malignancies. However, no studies have been reported to assess the differential expression of selected DNA repair genes combinedly in HMs. The present study was designed to assess the differential expression of HRR and BER pathway genes including RAD51, XRCC2, XRCC3, APEX1, FEN1, PARP1, and XRCC1 in blood cancer patients to highlight their significance as diagnostic/ prognostic marker in hematological malignancies. The study cohort comprised of 210 blood cancer patients along with an equal number of controls. For expression analysis, q-RT PCR was performed. DNA damage was measured in blood cancer patients and controls using the comet assay and LORD Q-assay. Data analysis showed significant downregulation of selected genes in blood cancer patients compared to healthy controls. To check the diagnostic value of selected genes, the Area under curve (AUC) was calculated and 0.879 AUC was observed for RAD51 (p < 0.0001) and 0.830 (p < 0.0001) for APEX1. Kaplan-Meier analysis showed that downregulation of RAD51 (p < 0.0001), XRCC3 (p < 0.02), and APEX1 (p < 0.0001) was found to be associated with a significant decrease in survival of blood cancer patients. Cox regression analysis showed that deregulation of RAD51 (p < 0.0001), XRCC2 (p < 0.02), XRCC3 (p < 0.003), and APEX1 (p < 0.00001) was found to be associated with the poor prognosis of blood cancer patients. Comet assay showed an increased number of comets in blood cancer patients compared to controls. These results are confirmed by performing the LORD q-assay and an increased frequency of lesions/Kb was observed in selected genes in cancer patients compared to controls. Our results showed significant downregulation of RAD51, XRCC2, XRCC3, APEX1, FEN1, PARP1, and XRCC1 genes with increased DNA damage in blood cancer patients. The findings of the current research suggested that deregulated expression of HRR and BER pathway genes can act as a diagnostic/prognostic marker in hematologic malignancies.