DNA Repair-related Genes Research Articles

Abstract P1-01-15: Clinical relevance of TRIM37 gene expression in breast cancer

Abstract Background: Tripartite motif-containing protein 37 (TRIM37) is an oncogenic histone H2A ubiquitin ligase that is overexpressed in a subset of breast cancers. TRIM37 was suggested to be associated with chemotherapy resistance and metastasis of triple-negative breast cancer (TNBC) in in vivo and in vitro studies. Breast cancer with TRIM37 amplification is sensitive to polo-like kinase 4 (PLK4) inhibition. On the other hand, clinical relevance of TRIM37 in breast cancer was never investigated. Material and Methods: Total of 6836 breast cancer patients from three large patient cohorts (Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), The Cancer Genome Atlas (TCGA), GSE96058, GSE25066 and GSE20194. Gene set enrichment analysis (GSEA) was performed. The high and low expression of TRIM37 gene were divided by median. Results: TRIM37 expression was significantly associated with worse survival (disease-free (DFS), disease-specific (DSS), and overall survival (OS)) in ER-positive/HER2-negative breast cancer (p=0.012, p=0.011, and p=0.003, respectively), but not in the other subtypes. TRIM37 expression was higher in HER2-positive among the subtypes. TRIM37 expression correlated with Nottingham histological grade (p<0.001), and with MKI-67 expression (p<0.001) consistently in METABRIC and GSE96058 cohorts. TRIM37 high expression enriched cell proliferation-related gene sets; E2F targets, G2M checkpoints, mitotic spindle, and MYC targets v1 and v2, as well as DNA repair gene sets regardless of subtypes. TRIM37 high expression was also associated with RAD51C and/or PLK4 expression, which are DNA repair related genes. In agreement, silent and non-silent mutation rate and altered fraction were all significantly elevated in TRIM37 high tumors regardless of subtypes. TRIM37 expression correlated with response to Cisplatin, Paclitaxel, and Tamoxifen in breast cancer cell line study (r=0.655, r=0.446, and r=0.9, respectively). TRIM37 high tumors demonstrated significantly worse pathological complete response after neoadjuvant chemotherapy in ER-positive/HER2-negative patients (p=0.002), but the opposite in TNBC (p=0.025) in GSE25066 cohort, but this result was not validated by GSE20194, a cohort with very small samples size. Interestingly, TRIM37 low expression enriched immune related gene sets; inflammatory response, IL2 signaling, IL6 signaling, TNF-a signaling, and allograft rejection in ER-positive/HER2-negative patients, but not in TNBC. Indeed, CD8 central memory T cells and CD4 effector memory T cells are highly infiltrated in TRIM37 low tumors consistently in both METABRIC and GSE96058 in ER-positive/HER2-negative patients, but not in TNBC. Taken together, TRIM37 high expression was associated with cell proliferation regardless of subtypes, but TRIM37 low expression was associated with high tumor infiltrating lymphocytes and immune response that may have contributed to the survival difference in ER-positive/HER2-negative patients, but not in TNBC. Conclusions: In conclusion, TRIM37 expression is associated with cell proliferation and DNA repair, less tumor infiltrating lymphocytes and immune response, and with worse survival in ER-positive/HER2-negative breast cancer. Citation Format: Junko Tsuchida, Rongrong Wu, Maiko Endo, Kazuki Moro, Chie Toshikawa, Yu Koyama, Hiroshi Ichikawa, Takaaki Hanyu, Yamato Takabe, Kazuyasu Takizawa, Yoshifumi Shimada, Takashi Kobayashi, Takashi Ishikawa, Jun Sakata, Toshifumi Wakai, Kazuaki Takabe. Clinical relevance of TRIM37 gene expression in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-01-15.

BRD4 inhibition induces synthetic lethality in ARID2-deficient hepatocellular carcinoma by increasing DNA damage.

Hepatocellular carcinoma (HCC) has emerged as the third cause of cancer-related death owing to lacking effective systemic therapies. Genomic DNA sequencing revealed the high frequency of loss-of-function mutations in ARID2, which encodes a subunit of SWI/SNF chromatin remodeling complex, however, the therapeutic strategy for the HCC patients with ARID2 mutations is still completely unclear. In this study, we first performed a high-throughput screening approach using a compound library consisting of 2 180 FDA-approved drugs and other compounds, to elicit the potential drugs for synthetic lethality to target ARID2-deficient HCC cells. Interestingly, JQ1, a selective inhibitor of bromodomain protein BRD4, uniquely suppressed the growth of ARID2- deficient HCC cells. Next JQ1 is further confirmed to predominantly induce cell lethality upon ARID2 depletion through exacerbating DNA damage, especially double strand breaks (DSBs). Functional assays demonstrated that both BRD4 inhibition and ARID2 deficiency synergistically impede two main DNA damage repair pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ), through attenuating the transcription of BRCA1, RAD51, and 53BP1, which encode the core molecules responsible for DSB repair. Mechanistically, both ARID2 and BRD4 exert a synergistic effect for maintaining transcriptional enhancer-promoter loops of these genes within chromatin conformation. However, as both ARID2 and BRD4 are disrupted, the expression of these DNA repair-related genes in response to DNA damage are hindered, resulting in DSB accumulation and cell apoptosis. Taken together, this study discloses that BRD4 inhibition may induce synthetic lethality in ARID2-deficient HCC cells, which might provide a potential therapeutic strategy for HCC patients with ARID2 mutations.

Identification of DNA Repair-Related Genes Predicting Clinical Outcome for Thyroid Cancer.

Recent studies have demonstrated the utility and superiority of DNA repair-related genes as novel biomarkers for cancer diagnosis, prognosis, and therapy. Here, we aimed to screen the potential survival-related DNA repair-related genes in thyroid cancer (TC). TCGA datasets were utilized to analyze the differentially expressed DNA repair-related genes between TC and nontumor tissues. The K–M approach and univariate analysis were employed to screen survival-related genes. RT-PCR was employed to examine the expression of DNA repair-related genes in TC samples and matched noncancer samples. CCK-8 analyses were used to determine cellular proliferation. Herein, our team discovered that the expression of four DNA repair-related genes was remarkably upregulated in TC samples in contrast to noncancer samples. Survival assays identified 14 DNA repair-related genes. In our cohort, we observed that the expression of TAF13 and DCTN4 was distinctly elevated in TC specimens in contrast to nontumor specimens. Moreover, knockdown of TAF13 and DCTN4 was observed to inhibit the TC cellular proliferation. Overall, the upregulation of TAF13 and DCTN4 is related to decreased overall survival in TC patients. Therefore, the assessment of TAF13 and DCTN4 expression may be useful for predicting prognosis in these patients.

DNA repair-related genes and adipogenesis: Lessons from congenital lipodystrophies.

Classical and progeroid congenital lipodystrophies are a collection of rare diseases displaying a large genetic heterogeneity. They occur due to pathogenic variants in genes associated with adipogenesis, DNA repair pathways, and genome stability. Subjects with lipodystrophy exhibit an impairment in the homeostasis of subcutaneous white adipose tissue (sWAT), resulting in low leptin and adiponectin levels, insulin resistance (IR), diabetes, dyslipidemia, ectopic fat deposition, inflammation, mitochondrial and endoplasmic reticulum commitments, among others. However, how pathogenic variants in adipogenesis-related genes modulate DNA repair in some classical congenital lipodystrophies has not been elucidated. In the same way, no data is clarifying how pathogenic variants in DNA repair genes result in sWAT loss in different types of progeroid lipodystrophies. This review will concentrate on the main molecular findings to understand the link between DNA damage/repair and adipogenesis in human and animal models of congenital lipodystrophies. We will focus on classical and progeroid congenital lipodystrophies directly or indirectly related to DNA repair pathways, highlighting the role of DNA repair-related proteins in maintaining sWAT homeostasis.

Strategies for accelerating osteogenesis through nanoparticle-based DNA/mitochondrial damage repair.

The efficiency of gene therapy is often dictated by the gene delivery system. Cationic polymers are essential elements of gene delivery systems. The relatively cheap cationic polymer, polyethyleneimine, has high gene delivery efficiency and is often used for gene delivery. However, the efficiency of gene therapy with polyethyleneimine-pDNA polyplex (PEI) is low. Human mesenchymal stem cells transfected with polyethyleneimine and a plasmid carrying the important osteogenic differentiation gene runt-related transcription factor 2 (RUNX2) accumulated DNA double-strand breaks and mitochondrial damage proportional to the amount of polyethyleneimine, reducing viability. Genomic/cellular stabilizer mediating RUNX2 delivery (GuaRD), a new reagent incorporating RS-1 NPs developed in this study, promoted DNA repair and prevented the accumulation of cell damage, allowing the delivery of pRUNX2 into hMSCs. while maintaining genome and mitochondrial stability. DNA damage was significantly lower and the expression of DNA repair-related genes significantly higher with GuaRD than with PEI. In addition, GuaRD improved mitochondrial stability, decreased the level of reactive oxygen species, and increased mitochondrial membrane potential. Osteogenic extracellular matrix (ECM) expression and calcification were higher with GuaRD than with PEI, suggesting improved osteogenic differentiation. These results indicate that lowering the cytotoxicity of PEI and improving cell stability are key to overcoming the limitations of conventional gene therapy, and that GuaRD can help resolve these limitations.

Incorporating genetic diversity to improve alignment of mouse models to human Alzheimer’s disease

Previous work by our labs, and others, reveal the benefits of incorporating diverse mouse strains to better model human Alzheimer's disease (AD). For instance, mouse models of AD on genetically distinct wild-derived mouse strains demonstrated robust differences in immune response to amyloid and neurodegeneration, better representing the phenotypic spectrum of AD than previous models. To further investigate the importance of genetic diversity, as part of MODEL-AD, we generated AD-relevant mouse models on Collaborative Cross (CC) lines background, a recombinant inbred mouse panel created from eight highly diverse founder strains.C57BL/6J (B6J) mice homozygous for humanized APOE4 allele and carrying mutant APP and PS1 alleles (APPswe , PS1de9 ) were crossed to five CC strains that were selected based on variation in AD-relevant genes. Cohorts of male and female B6JCCnF1.APOE4.APP/PS1 mice and controls were aged to 8 months (a mid-point for amyloid deposition in B6.APP/PS1 mice) and brain hemispheres processed for RNA-Seq and neuropathological assessment. Expectation Maximization for Allele Specific Expression (EMASE) tool was used to quantify transcriptome-wide expression at allelic level. Differential expression and KEGG pathway enrichment analysis were performed to determine altered molecular processes in CC strains. Strain specific transcriptional signatures were mapped to co-expression gene modules of human Late-Onset AD (LOAD) from the AMP-AD consortium. Neuropathological assessment included characterization of plaque deposition, Tau pathology, glial activation, neurodegeneration and cerebrovascular health.RNA-Seq analysis revealed up-regulation of neuroinflammation related pathways (lysosome, complement and coagulation cascades) in all CC strains as an effect of mutant APP and PS1 transgenes in presence of APOE4 allele. On the other hand, we observed significant CC strain and sex specific variation in immune (Fc gamma R-mediated phagocytosis) and metabolic (glycosphingolipid biosynthesis) response to amyloid pathogenesis. Alignment with human LOAD signatures revealed high strain and sex specific variation in correlation with synaptic signaling, cell cycle and DNA repair related gene modules. Overall, novel AD mouse models that incorporated CC lines demonstrated increased alignment to human AD-like transcriptomic signatures compared to B6J.This work builds on previous work highlighting the strengths of including multiple diverse mouse strains to better model complex neurodegenerative disorders such as AD and related dementias.

Excessive DNA damage mediates ECM degradation via the RBBP8/NOTCH1 pathway in sporadic aortic dissection

Stanford type A aortic dissection (TA-AD) is a life-threatening disease. Most cases of aortic dissection (AD) are sporadic rather than inherited. Unlike that of inherited AD, the pathogenesis of sporadic AD is still unclear. In the current study, we aimed to explore the pathogenesis of sporadic AD through transcriptome sequencing data analyses. We downloaded sporadic TA-AD transcriptome profiles from Gene Expression Omnibus (GEO) and found response to DNA damage stimulus was activated in AD. Furthermore, by conducting mouse AD tissue single cell RNA sequencing and immunostaining, we found that DNA damage mainly occurred in smooth muscle cells (SMCs) and fibroblasts. Next, we examined the repair patterns in response to DNA damage and found the linker molecules RBBP8/NOTCH1 between DNA damage/repair and extracellular matrix (ECM) organization through protein–protein interaction analysis. Thus, we proposed that DNA damage could contribute to AD by regulating ECM changes. To explore the underlying mechanism, we knocked down the DNA repair-related gene RBBP8 in aortic SMCs, which could exacerbate DNA damage, and observed decreased expression level of NOTCH1. Inhibition of NOTCH1 with crenigacestat in vivo accelerated β-aminopropionitrile-induced formation of AD and increased mortality. Meanwhile, phenotype switching of SMCs was induced by Notch1 knockdown or inhibition; this switching occurred via a pathway involving downregulation of contractile marker gene expression and upregulation of MMP2 expression, which might aggravate ECM degradation. In conclusion, excessive DNA damage is a characteristic pathological change of sporadic aortic dissection, which might contribute to ECM changes and AD development via action on the NOTCH1 pathway.

Integrative analysis of transcriptomics and metabolomics reveals the hepatotoxic mechanism of thiamethoxam on male Coturnix japonica

Thiamethoxam (TMX), a representative neonicotinoids, is widely used for seed coating. The consumption of TMX-coated seeds posed threat to birds during crop sowing. The hepatotoxicity of TMX has been reported in mammals, however, no clear evidence showed TMX-induced toxic effects on bird liver. In this study, male Japanese quails (Coturnix japonica) were exposed to 20 or 200 mg/kg TMX-treated bird feed for 28 days. Results showed that Clothianidin (CLO), a TMX metabolite preferred to accumulate in quail plasma and liver, and inflammatory cell infiltration was found in quail livers. Oxidative stress-related biological processes were significantly enriched in both TMX treatment groups through transcriptomics analysis. Moreover, integrative analysis of transcriptomics and metabolomics indicated ferroptosis and DNA damage was implicated in hepatotoxicity caused by high- and low-concentration of TMX exposure, respectively. High-dose TMX treatment decreased CAT activity and GSH concentration and increased expression of the ferroptosis-related gene. In addition, the up-regulation of 8-OHdG concentration and DNA repair-related genes expression demonstrated low-dose TMX triggered oxidative DNA damage. The present results highlight the toxicity of TMX to bird livers and contribute to a better understanding of the TMX toxic mechanism in birds.

Identification of New Tumor-Related Gene Mutations in Chinese Gastrointestinal Stromal Tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. As the main GIST drivers, gain-of-function mutations in KIT or PDGFRA are closely associated with not only tumor development and progression but also therapeutic response. In addition to the status of KIT and PDGFRA, little is known about other potential GIST-related genes. In this study, we identified the mutation profiles in 49 KIT-mutated GIST tumors using the whole exome sequencing (WES) method. Furthermore, some representative mutations were further validated in an independent GIST cohort using the SNaPshot SNP assay. We identified extensive and diverse mutations of KIT in GIST, including many undescribed variants. In addition, we revealed some new tumor-related gene mutations with unknown pathogenicity. By enrichment analyses of gene function and protein-protein interaction network construction, we showed that these genes were enriched in several important cancer- or metabolism-related signaling pathways, including PI3K-AKT,RTK-RAS, Notch, Wnt, Hippo, mTOR, AMPK, and insulin signaling. In particular, DNA repair-related genes, including MLH1, MSH6, BRCA1, BRCA2, and POLE, are frequently mutated in GISTs, suggesting that immune checkpoint blockade may have promising clinical applications for these GIST subpopulations. In conclusion, in addition to extensive and diverse mutations of KIT, some genes related to DNA-repair and cell metabolism may play important roles in the development, progression and therapeutic response of GIST.

Fanconi Anemia Complementation Group E, a DNA Repair-Related Gene, Is a Potential Marker of Poor Prognosis in Hepatocellular Carcinoma

Introduction: Fanconi anemia complementation group E (FANCE) is a Fanconi anemia (FA) pathway gene that regulates DNA repair. We evaluated the clinical relevance of FANCE expression in hepatocellular carcinoma (HCC). Methods: First, the associations between the expression of FA pathway genes including FANCE and clinical outcomes in HCC patients were analyzed in 2 independent cohorts: The Cancer Genome Atlas (TCGA, n = 373) and our patient cohort (n = 53). Localization of FANCE expression in HCC tissues was observed by immunohistochemical staining. Gene set enrichment analysis (GSEA) and gene network analysis (SiGN_BN) were conducted using the TCGA dataset. Next, an in vitro proliferation assay was performed using FANCE-knockdown HCC cell lines (HuH7 and HepG2). The association between mRNA expression of FANCE and that of DNA damage response genes in HCC was analyzed using TCGA and Cancer Cell Line Encyclopedia datasets. Finally, the association between FANCE mRNA expression and overall survival (OS) in various digestive carcinomas was analyzed using TCGA data. Results: FANCE was highly expressed in HCC cells. Multivariate analysis indicated that high FANCE mRNA expression was an independent factor predicting poor OS. GSEA revealed a positive relationship between enhanced FANCE expression and E2F and MYC target gene expression in HCC tissues. FANCE knockdown attenuated the proliferation of HCC cells, as well as reduced cdc25A expression and elevated histone H3 pSer10 expression. SiGN_BN revealed that FANCE mRNA expression was positively correlated with DNA damage response genes (H2A histone family member X and checkpoint kinase 1) in HCC tissues. Significant effects of high FANCE expression on OS were observed in hepatobiliary pancreatic carcinomas, including HCC. Conclusions: FANCE may provide a potential therapeutic target and biomarker of poor prognosis in HCC, possibly by facilitating tumor proliferation, which is mediated partly by cell cycle signaling activation.

The roles of epigenetic modifications in neurodegenerative diseases

In neuronal system, epigenetic modifications are essential for neuronal development, the fate determination of neural stem cells and neuronal function. The dysfunction of epigenetic regulation is closely related to occurrence and development of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease. Abnormally elevated DNA methylation inhibits the expression of some DNA repair-related genes and affects the progression of Huntington's disease. In the brain of Alzheimer's disease patients, the levels of H3K27ac and H3K9ac histone modifications increased. In addition, the alteration of RNA methylation in animal models of Alzheimer's disease and Parkinson's disease showed discrepancy trends. Therefore, epigenetic modifications may serve as potential therapeutic targets for neurodegenerative diseases. Here, we summarize the recent progress of the roles of epigenetic modifications in neurodegenerative diseases.

Exome sequencing of familial adenomatous polyposis-like individuals identifies both known and novel causative genes.

Inherited polyposis syndromes are predominantly caused by pathogenic variants in APC and are linked to familial adenomatous polyposis (FAP). However, after clinical screening, 20%-30% of individuals diagnosed with FAP do not carry a pathogenic variant in APC (often categorised as FAP-like). Other known inherited adenomatous polyposis syndromes such as MUTYH, POLD1/E, or NTHL1-associated polyposis only account for, 3 a fraction of the remaining cases. A cohort of 48 individuals clinically diagnosed with a FAP-like phenotype was selected based on a strong family history of colorectal cancer and no previous pathogenic variant found in APC and/or MUTYH, by genetic screening. Using whole exome sequencing, FAP-like patients were found to carry pathogenic variants in MUTYH, APC, POLE and TP53, as well as DNA-repair genes and inflammation related genes. Additionally, a comprehensive assessment of copy number variation revealed two loci of interest that appeared to be associated with polyposis risk. In total, 6 out of 48 polyposis were explained through re-sequencing. This study highlights the potential role of DNA-repair as well as inflammation-related variants towards polyp development.

Synephrine and caffeine combination promotes cytotoxicity, DNA damage and transcriptional modulation of apoptosis-related genes in human HepG2 cells

The abusive consumption of thermogenic supplements occurs worldwide and deserves special attention due to their use to stimulate weight loss and prevent obesity. Thermogenic formulations usually contain Synephrine (SN) and Caffeine (CAF), stimulating compounds extracted from natural sources, but no genetic toxicology studies have predicted this hazardous combination potential. This study examined the toxicogenomic responses induced by SN and CAF, either alone or in combination, in the human hepatic cell line HepG2 in vitro. SN (0.03–30 μM) and CAF (0.6–600 μM) alone did neither decrease cell viability nor induce DNA damage, as assessed using the MTT and comet assays, respectively. SN (3 μM) and CAF (30–600 μM) were combined at concentrations similar to those found in commercial dietary supplements. SN/CAF at 3:90 and 3:600 μM ratios significantly decreased cell viability and increased DNA damage levels in HepG2 cells. CAF (600 μM) and the SN/CAF association at 3:60, 3:90, and 3:600 μM ratios promoted cell death by apoptosis, as demonstrated by flow cytometry. Similar results were observed in gene expression (RT-qPCR): SN/CAF up-regulated the expression of apoptosis- (BCL-2 and CASP9) and DNA repair-related (XPC) genes. SN/CAF at 3:90 μM also downregulated the expression of cell cycle control (CDKN1A) genes. In conclusion, the SN/CAF combination reduces cell viability by inducing apoptosis, damages DNA, and modulates the transcriptional expression of apoptosis-, cell cycle-, and DNA repair-related genes in human hepatic (HepG2) cells in vitro. These effects can be worrisome to consumers of thermogenic supplements.

Abstract 2072: DNA repair pathway score is associated with cancer aggressiveness and worse survival in hepatocellular carcinoma (HCC)

Abstract Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies and a leading cause of cancer-related deaths worldwide. A prognostic biomarker based on cancer biology can tailor treatments according to patient prognosis. DNA repair mechanisms activated following DNA damage are essential to suppress carcinogenesis. Hence, deficiencies in DNA damage repair pathways promote carcinogenesis. The role of DNA repair genes in HCC is actively being investigated, but contradictory reports on some DNA repair-related genes have complicated our overall understanding of DNA repair in HCC progression. Though it is important to study the function of each individual DNA repair-related gene, it is more important to understand the role of the entire DNA repair pathway in HCC progression. We hypothesize that the enhancement of the DNA repair pathway is associated with worse survival in HCC patients. Method: A total of 749 HCC patients from 5 cohorts (TCGA, GSE6764, GSE89377, GSE56545, GSE76427) were studied to examine the relationships between enhancement of DNA repair and cancer aggressiveness, tumor immune microenvironment, and patient survival in HCC utilizing a DNA repair pathway score, which was calculated by Gene Set Variation Analysis (GSVA) with Hallmark DNA repair gene set. Results: We found that DNA repair pathway was enhanced by stepwise carcinogenic process of HCC and significantly enhanced in higher grade HCC compared with low grade tumor in two cohorts (both p < 0.001). DNA repair high HCC was associated with worse survival (disease-free survival: p < 0.001, disease-specific survival: p = 0.002, and overall survival: p < 0.001), elevated intratumor heterogeneity (p = 0.036) and mutation load (p < 0.001), but not with fraction of immune cell infiltration nor immune response in the TCGA cohort. HCC tumors with DNA repair high score enriched proliferation-related (E2F targets, G2M checkpoint, mitotic spindle, MYC target v1 and v2) and other cancer aggressiveness-related (unfolded protein response, PI3K/AKT/MTOR signaling, MTORC1, notch signaling, and WNT-β catenin signaling) gene sets. Interestingly, these features were more pronounced in early HCC compared to advanced HCC. DNA repair high HCC was associated with elevated mutation load, enriched proliferation- and other cancer aggressiveness-related gene sets and was associated with worse survival in early (grade 1 and grade 2) but not in advanced (grade 3) HCC. To our knowledge, this is the first study to use DNA repair pathway-related gene set expression data to examine and validate the clinical relevance of DNA repair pathway activity in HCC. Conclusion: The DNA repair score may be used to better understand and predict prognosis in HCC. Citation Format: Masanori Oshi, Tae Hee Kim, Yoshihisa Tokumaru, Li Yan, Ryusei Matsuyama, Itaru Endo, Leonid Cherkassky, Kazuaki Takabe. DNA repair pathway score is associated with cancer aggressiveness and worse survival in hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2072.

A Single Gene-Mediated Perturbation of the Human Genome Leads to Malignant Transformation in a Murine Transgenic Model

We hypothesized that induction or activation of a gene that directly affects DNA integrity can provide a tool for ongoing genomic changes in a cell and may eventually lead to oncogenesis. In the human genome, apurinic/apyrimidinic (AP) sites are created by number of external and internal factors including exposure to chemicals, UV radiation, DNA damaging agents, reactive oxygen species (ROS), DNA glycosylases and/or spontaneous hydrolysis by other mechanisms. AP sites are pre-mutagenic lesions that can also affect normal DNA replication. An evolutionarily conserved mechanism “base excision repair (BER)” has been established to identify and repair such sites. As part of BER, AP nucleases cleave the phosphodiester backbone at 5' to the AP site, prior to its repair. Although APEX 1 and 2 are the nucleases required for this repair, their hyperactivity has been connected with increased DNA breaks. We have previously observed that over expression of either APEX 1 or APEX 2 leads to malignant transformation with number of genomic changes including copy number alterations, single nucleotide variants (SNVs) and structural changes.In this study, we investigated if transgenic overexpression of APEX2 in B cells in mice will cause tumorogenesis. We specifically overexpressed APEX2 in B cells by generating Tg,CD19Cre+/-,APEX2+/- mouse lines and used Tg,CD19Cre+/- mouse (siblings of trail group) as control. The expression of APEX2 was detected by immunohistochemical (IHC) staining, which confirmed specific expression of APEX2 in lymph nodes. We next evaluated impact of APEX2 expression on B cell genome. We isolated B220+ B cells from spleen of 8 weeks old mice, and identified both increased single strand break (SSB) and double strand breaks (DSB). After 45 weeks of age and at variable times thereafter, 40% (23/58) of Tg,CD19Cre+/-,APEX2+/- mice showed enlargement of the lymph nodes and spleen. Histology showed effacement of normal tissue architecture by a nodular or diffuse population of atypical lymphoid cells in interfollicular areas. A distinct pattern of B220+BCL6+CD5- B-cell lymphomas were identified after IHC staining, which resemble human diffuse large B-cell lymphoma (DLBCL). To investigate the changes at genomic level, we performed whole genome sequencing of tumor tissues from 3 mice as well as heart tissues (with lowest APEX2 expression) from the same mice to be used as controls. From single nucleotide varients (SNVs) calling, we observed average of 24,388 SNVs in the tumor with acquisition of new copy number changes and significant number of structural variants including average of 43 translocations, 51 deletions, 21 duplications and 24 inversion. Indel frequency was variable with average of 348. We observed mutational hotspots on chr6, chr12 and chr16. Variant allele frequency (VAF) data shows clonality of chr11 related mutations. We observed most frequent T>G, and then T>C mutations in all 3 samples. Interestingly, the mutational signature analysis showed signature 17 accounting for an average of 60% of mutations followed by signature 9. According to the mutational signature profile of different diseases, both signature 9 and 17 are observed in human B cell lymphoma.To understand the genesis of the malignant transformation, we scarified Tg,CD19Cre+/-,APEX2+/- mice and control Tg,CD19Cre+/- mouse at 25 weeks after birth to see the pre-malignant changes in lymphoid organs. We observed enlarged and increased follicle numbers in lymph nodes and in spleen in Tg,CD19Cre+/-,APEX2+/- mice compared to control. A single cell sequencing is underway to detect clonal changes at an early stage.Our results demonstrate a single DNA-repair related gene responsible for inducing genomic instability with malignant transformation providing a model to study oncogenesis as well as possible preventative measures. DisclosuresAnderson:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; MedImmune: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Comprehensive Landscape of Ovarian Cancer Immune Microenvironment Based on Integrated Multi-Omics Analysis.

Epithelial ovarian cancer has a low response rate to immunotherapy and a complex immune microenvironment that regulates its treatment outcomes. Understanding the immune microenvironment and its molecular basis is of great clinical significance in the effort to improve immunotherapy response and outcomes. To determine the characteristics of the immune microenvironment in ovarian cancer, we stratified ovarian cancer patients into three immune subtypes (C1, C2, and C3) using immune-related genes based on gene expression data from The Cancer Genome Atlas and found that these three subtypes had significant differences in immune characteristics and prognosis. Methylation and copy number variant analysis showed that the immune checkpoint genes that influenced immune response were significantly hypermethylated and highly deleted in the immunosuppressive C3 subtype, suggesting that epigenetic therapy may be able to reverse the efficacy of immunotherapy. In addition, the mutation frequencies of BRCA2 and CDK12 were significantly higher in the C2 subtype than in the other two subtypes, suggesting that mutation of DNA repair-related genes significantly affects the prognosis of ovarian cancer patients. Our study further elucidated the molecular characteristics of the immune microenvironment of ovarian cancer, which providing an effective hierarchical method for the immunotherapy of ovarian cancer patients, and has clinical relevance to the design of new immunotherapies and a reasonable combination strategies.

BBIT20 inhibits homologous DNA repair with disruption of the BRCA1-BARD1 interaction in breast and ovarian cancer.

Advances in the treatment of triple-negative breast and ovarian cancer remain challenging. In particular, resistance to the available therapy, by restoring or overexpressing the DNA repair machinery, has often been reported. New strategies to improve the therapeutic outcomes of these cancers are needed. Herein, we disclose the dregamine 5-bromo-pyridin-2-ylhydrazone (BBIT20), a natural monoterpene indole alkaloid derivative, as an inhibitor of homologous DNA repair. To unveil BBIT20 antitumour activity and underlying molecular mechanism of action, two-dimensional (2D) and three-dimensional (3D) cell cultures, patient-derived cell lines and xenograft mouse models were used. BBIT20 disrupted the BRCA1-BARD1 interaction, triggering nuclear-to-cytoplasmic BRCA1 translocation, cell cycle arrest and downregulation of homologous DNA repair-related genes and proteins, with subsequent enhancement of DNA damage, reactive oxygen species generation and apoptosis, in triple-negative breast and ovarian cancer cells. BBIT20 also displayed pronounced antitumour activity in patient-derived cells and xenograft mouse models of ovarian cancer, with low toxicity in non-malignant cells and undetectable side effects in mice. Additionally, it did not induce resistance in triple-negative breast and ovarian cancer and displayed marked synergistic effects with cisplatin and olaparib (a poly [ADP-ribose] polymerase inhibitor), on 2D and 3D models of these cancer cells. These findings add an inhibitor of the BRCA1-BARD1 interaction to the list of DNA-damaging agents. Importantly, either as a single agent or in combination therapy, BBIT20 reveals great potential in the personalized treatment of aggressive and resistant cancers, particularly triple-negative breast and advanced ovarian cancer.

Downregulation of both mismatch repair and non-homologous end-joining pathways in hypoxic brain tumour cell lines.

Glioblastoma, a grade IV astrocytoma, has a poor survival rate in part due to ineffective treatment options available. These tumours are heterogeneous with areas of low oxygen levels, termed hypoxic regions. Many intra-cellular signalling pathways, including DNA repair, can be altered by hypoxia. Since DNA damage induction and subsequent activation of DNA repair mechanisms is the cornerstone of glioblastoma treatment, alterations to DNA repair mechanisms could have a direct influence on treatment success. Our aim was to elucidate the impact of chronic hypoxia on DNA repair gene expression in a range of glioblastoma cell lines. We adopted a NanoString transcriptomic approach to examine the expression of 180 DNA repair-related genes in four classical glioblastoma cell lines (U87-MG, U251-MG, D566-MG, T98G) exposed to 5 days of normoxia (21% O2), moderate (1% O2) or severe (0.1% O2) hypoxia. We observed altered gene expression in several DNA repair pathways including homologous recombination repair, non-homologous end-joining and mismatch repair, with hypoxia primarily resulting in downregulation of gene expression. The extent of gene expression changes was dependent on hypoxic severity. Some, but not all, of these downregulations were directly under the control of HIF activity. For example, the downregulation of LIG4, a key component of non-homologous end-joining, was reversed upon inhibition of the hypoxia-inducible factor (HIF). In contrast, the downregulation of the mismatch repair gene, PMS2, was not affected by HIF inhibition. This suggests that numerous molecular mechanisms lead to hypoxia-induced reprogramming of the transcriptional landscape of DNA repair. Whilst the global impact of hypoxia on DNA repair gene expression is likely to lead to genomic instability, tumorigenesis and reduced sensitivity to anti-cancer treatment, treatment re-sensitising might require additional approaches to a simple HIF inhibition.

A Novel Analysis Method for Evaluating the Interplay of Oxygen and Ionizing Radiation at the Gene Level.

Whilst the impact of hypoxia and ionizing radiations on gene expression is well-understood, the interplay of these two effects is not. To better investigate this aspect at the gene level human bladder, brain, lung and prostate cancer cell lines were irradiated with photons (6 Gy, 6 MV LINAC) in hypoxic and normoxic conditions and prepared for the whole genome analysis at 72 h post-irradiation. The analysis was performed on the obtained 20,000 genes per cell line using PCA and hierarchical cluster algorithms to extract the most dominant genes altered by radiation and hypoxia. With the help of the introduced novel radiation-in-hypoxia and oxygen-impact profiles, it was possible to overcome cell line specific gene regulation patterns. Based on that, 37 genes were found to be consistently regulated over all studied cell lines. All DNA-repair related genes were down-regulated after irradiation, independently of the oxygen state. Cell cycle-dependent genes showed up-regulation consistent with an observed change in cell population in the S and G2/M phases of the cell cycle after irradiation. Genes behaving oppositely in their regulation behavior when changing the oxygen concentration and being irradiated, were immunoresponse and inflammation related genes. The novel analysis method, and by consequence, the results presented here have shown how it is important to consider the two effects together (oxygen and radiation) when analyzing gene response upon cancer radiation treatment. This approach might help to unrevel new gene patterns responsible for cancer radioresistance in patients.

Identification of hub molecules of FUS-ALS by Bayesian gene regulatory network analysis of iPSC model: iBRN

Fused in sarcoma/translated in liposarcoma (FUS) is a causative gene of amyotrophic lateral sclerosis (ALS). Mutated FUS causes accumulation of DNA damage and cytosolic stress granule (SG) formation, thereby motor neuron (MN) death. However, key molecular aetiology remains unclear. Here, we applied a novel platform technology, iBRN, "Non- biased" Bayesian gene regulatory network analysis based on induced pluripotent stem cell (iPSC)-derived cell model, to elucidate the molecular aetiology using transcriptome of iPSC-derived MNs harboring FUSH517D. iBRN revealed "hub molecules", which strongly influenced transcriptome network, such as miR-125b-5p-TIMELESS axis and PRKDC for the molecular aetiology. Next, we confirmed miR-125b-5p-TIMELESS axis in FUSH517D MNs such that miR-125b-5p regulated several DNA repair-related genes including TIMELESS. In addition, we validated both introduction of miR-125b-5p and knocking down of TIMELESS caused DNA damage in the cell culture model. Furthermore, PRKDC was strongly associated with FUS mis-localization into SGs by DNA damage under impaired DNA-PK activity. Collectively, our iBRN strategy provides the first compelling evidence to elucidate molecular aetiology in neurodegenerative diseases.