Role In DNA Repair Pathways Research Articles

The distinct roles of NEIL1 and XPA in limiting aflatoxin B₁-induced mutagenesis in mice.

Dietary exposure to aflatoxin B₁ (AFB₁) is a risk factor for the development of hepatocellular carcinomas (HCCs). Following metabolic activation, AFB₁ reacts with guanines to form covalent DNA adducts, which induce high-frequency G > T transversions. The molecular signature associated with these mutational events aligns with the single base substitution signature 24 (SBS24) in the Catalog of Somatic Mutations in Cancer (COSMIC) database. Deficiencies in either base excision repair (BER) due to the absence of Nei-like DNA glycosylase 1 (NEIL1) or nucleotide excision repair (NER) due to the absence of xeroderma complementation group A protein (XPA) contribute to HCCs in murine models. In the current study, ultra-low error duplex sequencing was used to characterize mutational profiles in liver DNAs of NEIL1-deficient, XPA-deficient, and DNA repair-proficient mice following neonatal injection of 1 mg/kg AFB₁. Analyses of AFB₁-induced mutations showed high cosine similarity to SBS24, regardless of repair proficiency status. The absence of NEIL1 resulted in an approximately 30% increase in the frequency of mutations, with distribution suggesting preferential NEIL1-dependent repair of AFB₁ lesions in open chromatin regions. A trend of increased mutagenesis was also observed in the absence of XPA. Consistent with the role of XPA in transcription-coupled repair, mutational profiles in XPA-deficient mice showed disruption of the transcriptional bias in mutations associated with SBS24. Implications: Our findings define the roles of DNA repair pathways in AFB₁-induced mutagenesis and carcinogenesis in murine models, with these findings having implications in human health for those with BER and NER deficiencies.

Site-specific acetylation of polynucleotide kinase 3'-phosphatase regulates its distinct role in DNA repair pathways.

Mammalian polynucleotide kinase 3'-phosphatase (PNKP), a DNA end-processing enzyme with 3'-phosphatase and 5'-kinase activities, is involved in multiple DNA repair pathways, including base excision (BER), single-strand break (SSBR), and double-strand break repair (DSBR). However, little is known as to how PNKP functions in such diverse repair processes. Here we report that PNKP is acetylated at K142 (AcK142) by p300 constitutively but at K226 (AcK226) by CBP, only after DSB induction. Co-immunoprecipitation analysis using AcK142 or AcK226 PNKP-specific antibodies showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP with DSBR proteins. Despite the modest effect of acetylation on PNKP's enzymatic activity in vitro, cells expressing non-acetylable PNKP (K142R or K226R) accumulated DNA damage in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This is consistent with the reported degradation of CBP, but not p300, in HD cells. Moreover, transcribed genomes of HD cells progressively accumulated DSBs. Chromatin-immunoprecipitation analysis demonstrated the association of Ac-PNKP with the transcribed genes, consistent with PNKP's role in transcription-coupled repair. Thus, our findings demonstrate that acetylation at two lysine residues, located in different domains of PNKP, regulates its distinct role in BER/SSBR versus DSBR.

Unraveling DNA Repair Processes In Vivo: Insights from Zebrafish Studies.

The critical role of the DNA repair system in preserving the health and survival of living organisms is widely recognized as dysfunction within this system can result in a broad range of severe conditions, including neurodegenerative diseases, blood disorders, infertility, and cancer. Despite comprehensive research on the molecular and cellular mechanisms of DNA repair pathways, there remains a significant knowledge gap concerning these processes at an organismal level. The teleost zebrafish has emerged as a powerful model organism for investigating these intricate DNA repair mechanisms. Their utility arises from a combination of their well-characterized genomic information, the ability to visualize specific phenotype outcomes in distinct cells and tissues, and the availability of diverse genetic experimental approaches. In this review, we provide an in-depth overview of recent advancements in our understanding of the in vivo roles of DNA repair pathways. We cover a variety of critical biological processes including neurogenesis, hematopoiesis, germ cell development, tumorigenesis, and aging, with a specific emphasis on findings obtained from the use of zebrafish as a model system. Our comprehensive review highlights the importance of zebrafish in enhancing our understanding of the functions of DNA repair systems at the organismal level and paves the way for future investigations in this field.

Abstract 3856: Evaluation of replication protein A inhibitor, NERx329 in combination with EGFR mutant targeted therapy

Abstract Lung cancer is the leading cause of cancer related deaths worldwide with the majority (~80%) of patients diagnosed with non-small lung cancer (NSCLC). Targeted therapies directed against tumor initiating growth factor receptors are effective treatment options for driver mutation positive NSCLC. Osimertinib is a third-generation EGFR-Tyrosine Kinase inhibitor (TKI) that has activity against EGFR exon 19, exon 21, and T790M mutations. Unfortunately, Osimertinib treated patients acquire resistance and median progression free survival is 18 months. Moreover, Osimertinib does not cure stage IV EGFR mutant NSCLC. The acquisition of secondary mutations leading to activation of bypass pathways, MET amplification, EMT, compensatory pathway activation and histological transformation to small cell phenotype are major causes of acquiring resistance to EGFR TKIs. Proliferating cancer cells regularly experience a low level of replication stress. Additionally, receptor tyrosine kinases such as EGFR are known to interact with DNA repair proteins and impact DNA damage repair following chemotherapy, radiation therapy, and EGFR TKI treatment. However, the role of DNA repair pathways in EGFR-TKI resistance is unknown and the interaction between EGFR pathway, DNA damage, and repair pathways has not been fully elucidated. At low-level replicative stress promotes genomic instability but at a high level through mitotic catastrophe it causes cell death. Replication protein A is a critical sensor of the DNA damage response detecting replication stress. The small molecule inhibitor of RPA, NERx 329, sequesters active RPA and induces replication catastrophe and cell death. Here we demonstrate that NERx329 in combination with Osimertinib enhanced Osimertinib mediated cell death. CCk-8 assays performed after 48h of NERx329 and Osimertinib combination treatment result in a robust decrease in IC50 compared to single agent therapy. Dissection of signaling pathways leading to this death-promoting effect of NERx329 showed a combination of bypass pathway marker (AXL) and compensatory pathway marker (STAT) inhibition in AXL-high expressing EGFR mutant lung cancer cells. Whereas inhibition of STAT pathway was responsible for the death-promoting effect of NERx329 in AXL-low expressing EGFR mutant lung cancer cells. AXL and STAT inhibition are associated with induced DNA damage and impaired DNA repair. From these data, we infer that DNA damage repair pathways could be involved in TKI resistance and NERx329 could be a promising drug candidate for combination targeted therapy. Citation Format: Reshma Bhowmick, John J. Turchi, Shadia I. Jalal. Evaluation of replication protein A inhibitor, NERx329 in combination with EGFR mutant targeted therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3856.

Abstract NG03: Multiomics in disparities research: Identifying African ancestry-specific mechanisms in the triple negative breast cancer tumor microenvironment

Abstract NG03: Multiomics in disparities research: Identifying African ancestry-specific mechanisms in the triple negative breast cancer tumor microenvironment

The Complex Roles of DNA Repair Pathways, Inhibitors, Hyperthermia, and Contact Inhibition in Cell Cycle Halts.

The cell cycle has the capacity to safeguard the cell's DNA from damage. Thus, cell cycle arrest can allow tumor cells to investigate their own DNA repair processes. Cancer cells become extremely reliant on G1-phase cyclin-dependent kinases due to mutated oncogenes and deactivated tumor suppressors, producing replication stress and DNA damage during the S phase and destroying checkpoints that facilitate progression through the S/G2/M phase. DNA damage checkpoints activate DNA repair pathways to prevent cell proliferation, which occurs when the genome is damaged. However, research on how cells recommence division after a DNA lesion-induced arrest is insufficient which is merely the result of cancer cells' susceptibility to cell cycle arrest. For example, defects in the G1 arrest checkpoint may cause a cancer cell to proliferate more aggressively, and attempts to fix these complications may cause the cell to grow more slowly and eventually die. Defects in the G2-M arrest checkpoint may enable a damaged cell to enter mitosis and suffer apoptosis, and attempts to boost the effectiveness of chemotherapy may increase its cytotoxicity. Alternatively, attempts to promote G2-M arrest have also been linked to increased apoptosis in the laboratory. Furthermore, variables, such as hyperthermia, contact inhibition, nucleotide shortage, mitotic spindle damage, and resting phase effects, and DNA replication inhibitors add together to halt the cell cycle. In this review, we look at how nucleotide excision repair, MMR, and other variables, such as DNA replication inhibitors, hyperthermia, and contact inhibition, contribute to the outlined processes and functional capacities that cause cell cycle arrest.

Role of DNA repair pathways in the recovery of a dried, radioresistant cyanobacterium exposed to high-LET radiation: implications for the habitability of Mars

AbstractIf life ever appeared on Mars and if it did refuge into sub-superficial environments when surface conditions turned too hostile, then it should have been periodically revived from the frozen, dormant state in order to repair the accumulated damage and reset the survival clock to zero for the next dormant phase. Thus, unravelling how long Earth dormant microorganisms can cope with high-LET radiation mimicking long-term irradiation is fundamental to get insights into the long-term resilience of a dormant microbial life in the Martian subsurface over geological timescales that might have taken advantage of periodically clement conditions that allowed the repair of the accumulated DNA damage. The exposure of dried cells of the radioresistant cyanobacterium Chroococcidiopsis sp. CCMEE 029 to 2 kGy of heavy-ion radiation (Fe ions) did not significantly reduce its survival, although DNA damage was accumulated. Upon rehydration, DNA lesions were repaired as suggested by the over-expression of genes involved in the repair of double strand breaks (DSBs), oxidized bases and apurinic-apyrimidinic sites. Indeed, the monitoring of repair genes upon rehydration suggested a key role of the RecF homologous recombination in repairing DSBs. While the fact that out of the eight genes of the BER system, only one was up-regulated, suggested the absence of DNA lesions generally induced by UV radiation. In conclusion, the non-significantly reduced survival of dried Chroococcidiopsis exposed to 2 kGy of Fe-ion radiation further expanded our appreciation of the resilience of a putative dormant life in the Martian subsurface. Moreover, it is also relevant when searching life on Europa and Enceladus where the radiation environment might critically affect the long-term survival of dormant, frozen life forms.

Enigmatic role of WRN-RECQL helicase in DNA repair and its implications in cancer

Werner (WRN) helicase belongs to the RECQL class of DNA helicases. Mutation in Werner (WRN) RECQL helicase leads to premature aging syndrome, Werner syndrome (WS), and predisposition to multiple cancers. WS patients exhibit heightened incidence of neoplasia, e.g., soft tissue sarcoma, osteosarcoma, malignant melanoma, meningioma, thyroid cancer, breast cancer, and leukemias. Extensive research on WRN helicase has revealed its important and diverse roles in DNA repair pathways, especially in double-strand break repair. Consequently, WRN deficiency is causally associated with genomic instability and cancer predispositions. In this review, we summarize recent studies unraveling the fundamental roles WRN helicase plays in DNA repair and genome stability and its implications in cancer therapy and resistance.

Identification of SNPs in hMSH3/MSH6 interaction domain affecting the structure and function of MSH2 protein.

MutS homolog 2 (MSH2) is a mismatch repair gene that plays a critical role in DNA repair pathways, and its mutations are associated with different cancers. The present study aimed to find out the single nucleotide polymorphisms (SNPs) of MSH2 protein associated with causing structural and functional changes leading to the development of cancer with the help of computational tools. Four different tools for predicting deleterious SNPs (SIFT, PROVEAN, PANTHER, and PolyPhen), two tools each for identifying disease association (PhD-SNP and SNP&GO) and estimating stability (I-mutant and MUPro) were employed. Homology modeling, energy minimization, and root mean square deviation calculation were used to estimate structural variations. Twenty-seven SNPs and five SNPs (double amino acid change) were identified based on a consensus approach that might be associated with the structural and functional change in MSH2 protein. Molecular docking reveals that six SNPs affect the interaction of MSH2 and MSH6. Twelve identified SNPs were reported to be linked with hereditary nonpolyposis, colorectal cancer, and Lynch syndrome. Further, selected SNPs need to be validated in an in vitro system for their precise association with cancer predisposition.

RAD51-UTRhaplotypegenetic polymorphisms and susceptibilityto breastcancer in women from Jordanian population.

Genetic predisposition to breast cancer (BC) has been extensively explored to achieve an enhanced understanding of the biology of BC. Targeting candidate genes to screen different genetic variants such as RAD51gene that plays a critical role in DNA repair pathways including the double-strand break repair system is an important task. To study several single nucleotide polymorphisms (SNPs) within RAD51-UTR gene and to find their relationship with BC risk and prognosis among Jordanian females. In this case-control study, DNA sequencing technique was used to screen SNPs within the untranslated region (UTR) of RAD51in 206cases and 185controls and the resulting data were statistically analyzed using different types of genetic analyses. Patients' clinical and pathological features were obtained from their medical records to perform genotype-phenotype association analysis. Our findings show a significant association between both SNPs rs528590644, rs1801320and BC risk (p=0.016). We estimated the correlation between many of BC prognostic factors and BC risk, and we found an association between rs1801321and age at first menstruation (p=0.032) in addition to a strong correlation between age at BC diagnosis and rs1801320 (p=0.008). RAD51-UTR polymorphisms may be involved in BC development and progression.

Studies of multifunctional DNA polymerase I from the extremely radiation resistant Deinococcus radiodurans: Recombinant expression, purification and characterization of the full-length protein and its large fragment

Deinococcus radiodurans is a bacterium with extreme resistance to desiccation and radiation. Although the origins of this extreme resistance have not been fully elucidated, an efficient DNA repair machinery that includes the enzyme DNA polymerase I, is potentially crucial as part of a protection mechanism.Here we have cloned and performed small, medium, and large-scale expression of full-length D. radiodurans DNA polymerase I (DrPolI) as well as the large/Klenow fragment (DrKlenow). We then carried out functional characterization of 5′ exonuclease, DNA strand displacement and polymerase activities of these proteins using gel-based and molecular beacon-based biochemical assays. With the same expression and purification strategy, we got higher yield in the production of DrKlenow than of the full-length protein, approximately 2.5 mg per liter of culture. Moreover, we detected a prominent 5′ exonuclease activity of DrPolI in vitro. This activity and, DrKlenow strand displacement and DNA polymerase activities are preferentially stimulated at pH 8.0–8.5 and are reduced by addition of NaCl. Interestingly, both protein variants are more thermostable at pH 6.0–6.5.The characterization of DrPolI's multiple functions provides new insights into the enzyme's role in DNA repair pathways, and how the modulation of these functions is potentially used by D. radiodurans as a survival strategy.

Transcriptome of DNA Repair Genes in CMML: Rationale for Targeting ERCC1 and CDKN1A As a Synthetic Lethality Approach

Background: Chronic myelomonocytic leukemia (CMML) is characterized by features of both myeloproliferative neoplasm and myelodysplastic syndrome (MDS). Despite the main role of DNA repair pathways in cancer, in myelodysplastic disorders, somatic mutations are not found in these pathways. The novel discovery of non-classical leukaemogenesis mediated by splicing defects, profound influence of epigenetic anomalies, deeper knowledge of the DNA repair network, and development of unbiased high-throughput sequencing approaches, prompted us to revisit whether these processes might be disrupted by non-mutational mechanisms in CMML and related myeloid disorders. Study aims: i) to screen the transcriptome of DNA repair genes in a CMML discovery cohort; ii) to validate their misregulation in an independent large CMML cohort; and iii) determining how those candidates behave through the spectrum of myeloid malignancies. Methods: RNA-seq was performed in the discovery cohort: 27 CMML bone marrow samples (BM) at diagnosis and 9 healthy BM samples, and in 7 patients treated with Azacitidine in BM collected before and after 4 cycles. RT-qPCR was used to validate 9 DEGs through the myeloid spectrum and in a CMML validation cohort: 74 additional CMML, 70 MDS, 66 AML , and 25 CML patients. Candidates were chosen based on clinical considerations: i) druggable oncogenes highly overexpressed ii) oncogenes infra-expressed with inhibitory molecules already being tested in myeloid neoplasms. Global pattern of DNA repair gene expression was compared with MDS and AML extracted from MILE study data. The DEGs methylation status was studied using available public data and we confirmed by pyrosequencing the methylation status of our main candidates. The CMML validation cohort was mutationally characterized by targeted sequencing of 18 CMML recurrently mutated genes. Alternative splice sites, skipped exons, mutually exclusive exons and retained introns were quantified using rMATS.Results: Of 27 CMML patients and 9 healthy donors, the expression of 30 genes was significantly different between the two groups (8 genes up-regulated and 22 down-regulated). Defects on genes predominantly unique to a single strand breaks repair pathway included: NEIL1 and OGG1 in base excision repair, XPA and MSS19 in nucleotide excision repair and RPA4 in mismatch repair. XRCC4 and MSH4 overexpression and PRKDC down-regulation were found as significant changes among the genes associated exclusively to double strand breaks repair. We next extracted the DNA repair transcriptional components from MILE study: 206 MDS, 47 ckAML and 73 healthy donors. MDS misregulation was characterized by upregulated genes while ckAML showed a global disruption, mostly downregulation of Homologous Recombination genes. Some genes showed opposite sense of misregulation depending on the myeloid entity: TDP1 was upregulated in CMML cases and downregulated in ckAML, and viceversa for BAP1 . CDK1 and EXO1 were upregulated in MDS cases but downregulated in ckAML cases. Statistically significant and direction-concordant dysregulation of CDKN1A, ERCC1, XRCC4, NEIL1, MSH4, PARP1 and BAP1 were confirmed in our independent CMML validation cohort. The methylation study showed a global demethylated status of our misregulated DNA repair genes in CMML. Either responder s (n=3) or non-responders (n=2) to azacitidine failed to achieve a substantial expression change in their DNA Repair DEGs. PARP1 infraexpresion was associated with TET2 mut cases. Lower CDKN1A and BAP1 expression was related to chromatin regulator mutations. SRSF2 mutations induced widespread aberrant splicing events in DNA repair genes in CMMLConclusions: We have identified by means of an unbiased high-throughput approach, and validated in an independent cohort, a subset of DNA repair genes consistently misregulated in CMML. The genes identified warrant further study as potential novel therapeutic targets, both directly and through modulation of associated compensatory pathways. CDKN1A and ERCC1 in particular emerge as realistic candidates for a synthetic lethality approach. Our findings suggest that the promise of PARP1 inhibition appears less pertinent to CMML than for other myeloid diseases. These differences might also partially explain the different genomic and phenotypic manifestations of different myeloid neoplasms, and shed insights into their distinctive biology. DisclosuresMaciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fees; Apellis Pharmaceuticals: Consultancy; Ra Pharma: Consultancy.

Mitochondrial genome stability in human: understanding the role of DNA repair pathways.

Mitochondria are semiautonomous organelles in eukaryotic cells and possess their own genome that replicates independently. Mitochondria play a major role in oxidative phosphorylation due to which its genome is frequently exposed to oxidative stress. Factors including ionizing radiation, radiomimetic drugs and replication fork stalling can also result in different types of mutations in mitochondrial DNA (mtDNA) leading to genome fragility. Mitochondria from myopathies, dystonia, cancer patient samples show frequent mtDNA mutations such as point mutations, insertions and large-scale deletions that could account for mitochondria-associated disease pathogenesis. The mechanism by which such mutations arise following exposure to various DNA-damaging agents is not well understood. One of the well-studied repair pathways in mitochondria is base excision repair. Other repair pathways such as mismatch repair, homologous recombination and microhomology-mediated end joining have also been reported. Interestingly, nucleotide excision repair and classical nonhomologous DNA end joining are not detected in mitochondria. In this review, we summarize the potential causes of mitochondrial genome fragility, their implications as well as various DNA repair pathways that operate in mitochondria.

Tumor protein expression of BRCA1 and development of lethal prostate cancer.

65 Background: DNA repair genes including BRCA1 are commonly altered in metastatic prostate tumors. However, mutations and copy number aberrations in these genes are rare in primary tumors. Instead, preliminary studies suggest that higher tumor expression of the BRCA1 protein may be associated with worse prognosis. Methods: We undertook a prospective study of tumor BRCA1 protein expression and lethal prostate cancer among men with clinically localized prostate cancer in the Health Professionals Follow-up Study. We performed immunohistochemical staining for BRCA1 on tumor tissue microarrays using a validated antibody and scored expression as positive or negative. We also assessed tumor proliferation by immunostaining for Ki67, angiogenesis by immunostaining for CD34, and apoptosis using a TUNEL assay. Proportional hazards regression was used to evaluate the association between BRCA1 protein expression and development of lethal prostate cancer (metastasis or cancer-specific death). Results: Ten percent of tumors (60 of 589) stained positive for the BRCA1 protein. BRCA1-positive tumors were characterized by higher Gleason scores, a higher proliferative index, and a higher apoptotic index. During a median follow-up of 14.3 years, 18 men (34%) in the BRCA1-positive group and 74 men (14%) in the BRCA1-negative group developed lethal prostate cancer. There was a strong positive association between BRCA1 protein expression and lethal prostate cancer in both unadjusted analyses (HR 2.71, 95% CI 1.73–4.26) and after adjusting for clinical factors (HR 2.00, 95% CI 1.26–3.18). The positive association with BRCA1 protein expression was also independent of proliferation index. Conclusions: Primary prostate tumors expressing the BRCA1 protein have a highly proliferative phenotype and are more likely to progress to lethal disease, independent of its higher proliferative index. Assessing tumor protein expression of BRCA1 may help elucidate the Janus-faced role of DNA repair pathways in prostate cancer progression.[Table: see text]

Abstract P5-03-08: Breast cancer genomic landscape according to germline BRCA1/2 pathogenicity

Abstract Background: BRCA1 and BRCA2 are tumor suppressor genes and their protein products play a key role in DNA repair pathway. Individuals harboring germline mutations in BRCA1/2 genes(gBRCA1/2) confer high risk of developing breast cancer. Although clinical features of BRCA-related breast cancer have been well established, the characteristic of tumor biology and specific somatic mutational profile has not been fully understood. Here we investigated tumor mutational landscape of germline BRCA-mutant or wild type breast cancer. Methods: We collected breast cancer patients who had been confirmed as pathogenic gBRCA1/2 mutation by blood test. Matched cohorts who had wild type or variants of unknown significance(VUS) in BRCA1/2 gene were also selected based on age, TNM stage, and molecular subtype. Formalin-fixed paraffin-embedded (FFPE) tumor samples from all participants were analyzed by Next Generation Sequencing(NGS)-based cancer panel. We used CancerSCAN panel from Samsung Genome Institute, which provides variant genomic data and its own class stratification. All the single nucleotide variants(SNVs) were reported based on classification of clinical importance. If there is no evidence of preclinical evidence, that variant is labeled as ‘novel’. When a variant has some relevance on tumor biology, it is categorized as class 1 to 5 based on evidence level. Results: Each of seven patients were found to be suitable for analysis in groups of ‘pathogenic gBRCA1/2’, ‘wild gBRCA1/2’, and ‘VUS gBRCA’, respectively. (N=21) Detected number of SNV in patients with pathogenic gBRCA1/2 were higher than wild gBRCA1/2. (19.3 vs 15.6 SNVs per tumor sample) The number of SNVs suspected to germline mutation due to high variant allele frequency(VAF) was higher in pathogenic BRCA1/2 group, but the difference was minimal. (11.1 vs 10.0 SNVs with VAF>30% per tumor sample) The mean VAF of all variants also had little difference. (34.2% vs 35.6%) The patients with VUS gBRCA1/2 showed highest number of SNVs and SNVs with elevated VAF. (21.0 SNVs 14.1 SNVs with VAF>30% per tumor sample, mean VAF 36.8%) The rate of SNVs denoted as class 1 to 5 were similar in pathogenic and wild type gBRCA1/2 patients. (20.1% vs 21.2%) In pathogenic group, nonsynonymous SNV of BRCA2 was the most common mutation, followed by BRCA1 and TP53. In wild type group, mutation in BRCA2 and TP53 were the two most common variants, followed by PIK3CA. The genomic copy number alteration was more prominent in wild type group. Discussion: In our study, the genomic variation according to germline BRCA1/2 pathogenicity was imperceptible, except for total number of SNVs per tumor sample. The patients with gBRCA1/2 VUS tended to similar to pathogenic group rather than wild type group. We will present more data from 50 patients on conference day. (This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &Welfare, Republic of Korea (Grant number: HI17C2206).) Citation Format: Ju Won Kim, Jung Yoon Choi, Soo Hyeon Lee, Won Jin Chang, Soo Youn Bae, Yoon Ji Choi, Seung Pil Jung, Kyu Ran Cho, Sang Won Shin, Yeul Hong Kim, Kyong Hwa Park. Breast cancer genomic landscape according to germline BRCA1/2 pathogenicity [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-03-08.

Identification of potential radiation-responsive biomarkers based on human orthologous genes with possible roles in DNA repair pathways by comparison between Arabidopsis thaliana and homo sapiens

Rapid and reliable ionization radiation (IR) exposure estimation has become increasingly important in environment due to the urgent requirement of medical evaluation and treatment in the event of nuclear accident emergency. Human DNA repair genes can be identified as important candidate biomarkers to assess IR exposure, while how to find the enough sensitive and specific biomarkers in the DNA repair networks is still challenged and not fully determined. The conserved features of DNA repair pathways may facilitate interdisciplinary studies that cross the traditional boundaries between animal and plant biology, with the aim of identifying undiscovered human DNA repair genes for potential radiation-responsive biomarkers. In this work, an in silico method of homologous comparison was performed to identify the human orthologues of A. thaliana DNA repair genes, and thereby to explore the sensitive and specific human radiation-responsive genes to evaluate the IR exposure levels. The results showed that a total of 16 putative candidate genes were involved in the human DNA repair pathways of homologous recombination (HR) and non-homologous end joining (NHEJ), and most of them were confirmed by previous experiments. Additionally, we analyzed the gene expression patterns of these 16 candidate genes in several human transcript microarray datasets with different IR treatments. The results indicated that most of the gene expression levels for these candidate genes were significantly changed under different radiation treatments. Based on these results, we integrated these putative human DNA repair genes into the DNA repair pathways to propose new insights of the HR and NHEJ pathways, which can also provide the potential targets for the development of radiation biomarkers. Notably, two putative DNA repair genes, named ERCC1 and ESCO2, were identified and were considered to be the sensitive and specific biomarkers in response to γ-ray exposures.

Can synthetic lethality approach be used with DNA repair genes for primary and secondary MDS?

Cancer-specific defects in DNA repair pathways create the opportunity to employ synthetic lethality approach. Recently, GEMA (gene expression and mutation analysis) approach detected insufficient expression of BRCA or NHEJ (non-homologous end joining) to predict PARP inhibitors response. We evaluated a possible role of DNA repair pathways using gene expression of single-strand break (XPA, XPC, XPG/ERCC5, CSA/ERCC8, and CSB/ERCC6) and double-strand break (ATM, BRCA1, BRCA2, RAD51, XRCC5, XRCC6, LIG4) in 92 patients with myelodysplastic syndrome (73 de novo, 9 therapy-related (t-MDS). Therapy-related MDS (t-MDS) demonstrated a significant downregulation of axis BRCA1-BRCA2-RAD51 comparing to normal controls (p = 0.048, p = 0.001, p = 0.001). XRCC6 showed significantly low expression in de novo MDS comparing to controls (p = 0.039) and for patients who presented chromosomal abnormalities (p = 0.047). Downregulation of LIG4 was consistently associated with poor prognostic markers in de novo MDS (hemoglobin < 8g/dL (p = 0.040), neutrophils < 800/mm3 (p < 0.001), patients with excess of blasts (p = 0.001), very high (p = 0.002)/high IPSS-R (p = 0.043) and AML transformation (p < 0.001). We also performed an evaluation of GEPIA Database in 30 cancer types and detected a typical pattern of downregulation as here presented in primary or secondary MDS. All these results suggest synthetic lethality approach can be tested with DNA repair genes (beyond that of BRCA1/2 status) for de novo and therapy-related myelodysplastic syndrome and may encourage clinical trials evaluating the use of PARP1 inhibitors in MDS.

Tiny Yet Indispensable Plant MicroRNAs Are Worth to Explore as Key Components for Combating Genotoxic Stresses.

Plants being sessile are always exposed to various stresses including biotic and abiotic stresses. Some of these stresses are genotoxic to cells causing DNA damage by forming lesions which include altered bases, cross-links, and breaking of DNA strands, which in turn hamper the genomic integrity. In order to survive through all these adverse conditions, plants have evolved different DNA repair mechanisms. As seen from the mammalian system and different human diseases, various microRNAs (miRNAs) can target the 3′-untranslated region of mRNAs that code for the proteins involved in DNA repair pathways. Since miRNAs play an important role in plant cells by regulating various metabolic pathways, it can also be possible that miRNAs play an important role in DNA repair pathways too. However, till date, only a handful of plant miRNAs have been identified to play important role in combating genotoxic stresses in plants. Limitation of information regarding involvement of miRNAs in DNA repair as well as in ROS scavenging prompted us to gather information about plant miRNAs specific for these tasks. This mini-review aims to present pertinent literature dealing with different genotoxic stresses that cause genome instability as well as plant specific responses to survive the damage. This is intertwined with the involvement of miRNAs in genotoxic stress in plants, challenges of applying miRNAs as a tool to combat DNA damage along with ways to overcome these challenges, and finally, the future prospective of these understudied aspects.

1439PD - BRCA1 expression level as prognostic factor for recurrence in resected NSCLC with adjuvant chemotherapy: SCAT Trial

BackgroundPost-operative platinum-based chemotherapy is the standard of care for resected NSCLC with nodal involvement. Expression of genes involved in DNA repair may have a prognostic role for the outcome. BRCA1 plays an important role in DNA repair pathways and could have a prognostic impact in this setting. The SCAT trial results found that for low BRCA1 levels subgroup Cis-Gem was superior to Cis-Doc and in high BRCA1 subgroup docetaxel single agent without platinum achieved similar survival to Cis-Doc. Risk of recurrence analysis according BRCA-1 levels has been performed. MethodsFrom Jun/2007 to May/2013 591 patients were screened and 500 were included, 108 in the control arm treated with Cis-Docetaxel and 392 in the experimental arms treated with Cis-Gem, Cis-Doc or docetaxel alone according to terciles of BRCA1 expression level. With a cut-off September 30th 2018 and a median follow-up of 60 months, recurrence patterns were analysed for the whole group according BRCA1 levels in tumor tissue and comparison were made for risk of recurrence, single/multiple recurrence, thoracic/extrathoracic and metastatic sites (liver, bone, brain). ResultsCumulative recurrence was evaluable in 232/456 patients (50.8%), 182/354 patients treated in the experimental arm and in 50/102 patients treated in the control arm (RR 1.04; 0.83-1.30) (p=0.672). The majority of recurrences 159/232 (68.5%) were single site intrathoracic recurrences in 121/232 (52%) while 111/232 were extrathoracic (47.8%). Overall recurrence was 56.5% (113/200 p) for low tercile BRCA1 vs 48.8% (63/129) for intermediate tercile vs 44% (56/127) for high tercile (p=0.025). No differences were seen between tercile groups for single site (p=0.35), multiple site (p=0.26), intrathoracic (p=0.36), or extrathoracic (p=0.38). More frequent distant metastatic sites were: bone (42 patients), brain (38 patients) and liver (11 patients). RIsk reduction was seen for brain metastases in patients with higher tercile BRCA1 (p=0.003). ConclusionsFor NSCLC resected patients with lymph node involvement risk of recurrence remains high with a cumulative rate > 50%. Relative risk of recurrence was lower for tumors with higher BRCA1 levels. Distant metastases were seen in 47.8% of patients. Brain metastases risk was significant lower for patients with low BRCA1 expression. BRCA1 levels acts as a prognostic factor in early stages NSCLC. Clinical trial identificationEudraCT: 2007-000067-15; NCT 00478699. Legal entity responsible for the studySpanish Lung Cancer Group - Grupo Español Cáncer de Pulmón. FundingSanofi Aventis. DisclosureAll authors have declared no conflicts of interest.

Abstract 2508: Genomic alterations of BRCA1/2 genes in Chinese solid tumor patients

Abstract Background: BRCA1 and BRCA2 are tumor suppressor genes that play an important role in DNA repair pathways. Germline mutations in BRCA1 and BRCA2 contribute to a significant number of familial and hereditary breast and/or ovarian cancers. Additionally, BRCA 1/2 alterations induce sensitivity to poly ADP ribose polymerase inhibitors (PARPi). Therefore, the detection of BRCA 1/2 mutations in solid tumor patients is essential to predict the sensitivity to PARPi therapy. Methods: FFPE tumor and matched blood samples of 5269 Chinese patients with solid tumor were collected for next generation sequencing (NGS) based assay. Genomic alterations including single nucleotide variations (SNV), short and long insertions/deletions (Indel), copy number variations (CNV) and gene rearrangements and fusions in selected genes were assessed. Results: Three hundred patients with BRCA1/2 mutations were identified in 5269 Chinese patients with solid tumor. The patients included 177 males and 123 females with a median age of 58. Highest yields of BRCA1/2 mutations were found in patients with ovarian (24.5%) and breast (12.3%) cancers. BRCA1/2 mutations were also identified in gastric cancer (8.9%), cholangiocarcinoma (8.8%), colorectal cancer (7.1%), esophageal cancer (6.1%), gallbladder cancer (5.1%), lung cancer (4.7%), soft tissue tumor (4.1%) and pancreatic cancer (4.0%). SNVs and short Indels (84.9%) were the most common variant types of BRCA1/2, while the percentages of gene rearrangements and fusions, CNV and long Indels were 12.0%, 1.6% and 1.5%, respectively. Among patients with BRCA1/2 mutations, 224 (74.7%) patients harbored somatic mutations, 73 (24.3%) patients harbored germline mutations, 3 (1.0%) patients harbored both somatic and germline mutations. Interestingly, all of the variant types of germline mutations were SNVs and short Indels. The mutation sites were distributed in the full length of BRCA1/2 genes. No hot spot mutation was observed. Conclusions: Our data revealed that BRCA1/2 mutations occurred in 5.7% of Chinese patients with solid tumor. NGS targeted sequencing provides comprehensive and accurate information of BRCA1/2 mutations. Beyond ovarian and breast cancers, BRCA1/2 mutations could be detected in other solid tumors, which suggests potential clinical benefits from PARPi therapy. Citation Format: Junjian Wang, Lingxiang Liu, Bin Ni, Xiaoqian Chen, Ling Li, Junping Shi, Jierong Chen, Ming Yao. Genomic alterations of BRCA1/2 genes in Chinese solid tumor patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2508.