Transcription Of DNA Repair Genes Research Articles

Abstract 5646: Altered intronic polyadenylation by mutant p53 impairs transcription of DNA repair genes in lung cancer

Abstract Introduction: Changes in alternative pre-mRNA processing have been found in many cancers. Aberrant intronic polyadenylation (IPA) causes a stop of normal transcription resulting in the production of truncated mRNA isoform or lncRNA. We sought to examine the role of mutant p53 in pre-mRNA processing, especially in IPA regulation in lung cancer. Methods: We established stable cell lines transfected with mutant p53 (R273H, R175H, H179Q, C238Y, and C242F) or vector control using the NSCLC cell line H1299 with a homozygous deletion of the endogenous p53. Total RNA was used to build libraries enriched in sequences near polyadenylation sites (PASs) using the 3’mRNA-Seq REV library kit (Lexogen; Greenland, NH). Sequencing reads were mapped to the human genome (GRCh37), and PASs with ≥2 supporting reads and ≥0.05 usage in any experiment were identified. Counts for each Intronic Polyadenylation (IPA) and total reads in the terminal exon were compiled into matrices for each condition. DEXseq was applied to detect changes in the relative abundance of each exon part normalized to all exons within the gene. Gene Ontology (GO) analysis was implemented using the enrichR package. Results: We identified 32,752 IPA sites in 5,718 genes, and 26,416 distal PASs (dPASs) in the 3’UTRs of 10,298 genes. Across all five cell lines, when compared individually to the vector control, a comprehensive shift in IPA events was observed, with a concurrent decrease in dPASs. Many of IPA isoforms exhibited consistent patterns of increased or decreased abundance changes. Upon closer examination of each individual cell line, such as the p53-R273H cell line (vs. vector), significant usage changes were identified for 2,389 IPA sites, of which 1,210 were increased and 1,128 terminal exons were altered, with 650 being suppressed. At the gene level, a total of 1,183 genes had at least one significantly increased IPA isoform, a significantly decreased distal isoform, or both. GO analysis showed that these genes were highly enriched in DNA repair-related functional terms such as DNA repair, double-strand break repair, and nucleotide excision repair. The observed IPA changes in DNA-repair genes were consistently observed across the other 4 mutant lines (vs. vector). This observation was further supported by analyzing data from patients with lung adenocarcinoma (LUAD) in the TCGA cohort, where we selected samples carrying nonsense/frameshift mutations, and/or a deep deletion as the p53-null control group, and those carrying missense mutations as the p53-mutant group. Conclusions: Our study illustrated a novel role of p53 mutants in the regulation of intronic polyadenylation processing that contributes to deficient DNA repair. These observations offer a fresh perspective on the multifaceted roles of p53 in cellular homeostasis. Acknowledgment: This work was supported by NCI Cancer Center Support Grant P30CA012197 and NIH/NCI grant R03CA256100. Citation Format: Liang Liu, Elizabeth Forbes, Wei Zhang. Altered intronic polyadenylation by mutant p53 impairs transcription of DNA repair genes in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5646.

Dynamic reprogramming of H3K9me3 at hominoid-specific retrotransposons during human preimplantation development.

Reprogramming of H3K9me3-dependent heterochromatin is required for early development. How H3K9me3 is involved in early human development remains, however, largely unclear. Here, we resolve the temporal landscape of H3K9me3 during human preimplantation development and its regulation for diverse hominoid-specific retrotransposons. At the 8-cell stage, H3K9me3 reprogramming at hominoid-specific retrotransposons termed SINE-VNTR-Alu (SVA) facilitates interaction between certain promoters and SVA-derived enhancers, promoting the zygotic genome activation. In trophectoderm, de novo H3K9me3 domainsprevent pluripotent transcription factors from binding to hominoid-specific retrotransposons-derived regulatory elements for inner cell mass (ICM)-specific genes. H3K9me3 re-establishment at SVA elements in the ICM is associated with higher transcription of DNA repair genes, when compared with naive human pluripotent stem cells. Our data demonstrate that species-specific reorganization of H3K9me3-dependent heterochromatin at hominoid-specific retrotransposons plays important roles during early human development, shedding light on how the epigenetic regulation for early development has evolved in mammals.

ZL-n-91, a specific Phosphodiesterase-4 inhibitor, suppresses the growth of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that frequently develops resistance to chemotherapy. A new approach to treating TNBC is required to improve patient survival. Phosphodiesterase-4 (PDE4) is an enzyme that is predominantly involved in the modulation of intracellular signaling mediated by cAMP. Although the efficacy of PDE4 inhibitors in several human inflammatory diseases is well documented, their clinical utility has been limited by side effects, including nausea and emesis. Recently, PDE4 has been used as a potential therapeutic target for different cancer types. In the present study, we investigated the anticancer effects of a novel PDE4 inhibitor ZL-n-91 on TNBC and the underlying mechanism. We showed that ZL-n-91 inhibited the proliferation of TNBC cells, induced cell apoptosis, and caused cell cycle arrest. Western blot analysis showed that ZL-n-91 increased Bax level and reduced Bcl-2 expression. Furthermore, downregulation of the cell cycle-related proteins, such as CDK2, CDK4, cyclin D1, PCNA, p-RB, and ZL-n-91, significantly inhibited the transcription of DNA repair genes and triggered an intracellular DNA damage response. Moreover, ZL-n-91 prevented the growth of the transplanted MDA-MB-231 tumor xenograft in nude mice and increased the γ-H2AX expression. These data demonstrate the anticancer effects of ZL-n-91 on TNBC cells and suggest its potential use in anticancer therapy.

Evolutionary Divergence in DNA Damage Responses among Fungi.

Cell cycle checkpoints and DNA repair pathways contribute to maintaining genome integrity and are thought to be evolutionarily ancient and broadly conserved. For example, in the yeast Saccharomyces cerevisiae and humans, DNA damage induces activation of a checkpoint effector kinase, Rad53p (human homolog Chk2), to promote cell cycle arrest and transcription of DNA repair genes. However, recent studies have revealed variation in the DNA damage response networks of some fungi. For example, Shor et al. (mBio 11:e03044-20, 2020, https://doi.org/10.1128/mBio.03044-20) demonstrate that in comparison to S. cerevisiae, the fungal pathogen Candida glabrata has reduced activation of Rad53p in response to DNA damage. Consequently, some downstream targets that contribute to S. cerevisiae genome maintenance, such as DNA polymerases, are transcriptionally downregulated in C. glabrata Downregulation of genome maintenance genes likely contributes to higher rates of mitotic failure and cell death in C. glabrata This and other recent findings highlight evolutionary diversity in eukaryotic DNA damage responses.

Dun1, a Chk2-related kinase, is the central regulator of securin-separase dynamics during DNA damage signaling.

The DNA damage checkpoint halts cell cycle progression in G2 in response to genotoxic insults. Central to the execution of cell cycle arrest is the checkpoint-induced stabilization of securin-separase complex (yeast Pds1-Esp1). The checkpoint kinases Chk1 and Chk2 (yeast Chk1 and Rad53) are thought to critically contribute to the stability of securin-separase complex by phosphorylation of securin, rendering it resistant to proteolytic destruction by the anaphase promoting complex (APC). Dun1, a Rad53 paralog related to Chk2, is also essential for checkpoint-imposed arrest. Dun1 is required for the DNA damage-induced transcription of DNA repair genes; however, its role in the execution of cell cycle arrest remains unknown. Here, we show that Dun1′s role in checkpoint arrest is independent of its involvement in the transcription of repair genes. Instead, Dun1 is necessary to prevent Pds1 destruction during DNA damage in that the Dun1-deficient cells degrade Pds1, escape G2 arrest and undergo mitosis despite the presence of checkpoint-active Chk1 and Rad53. Interestingly, proteolytic degradation of Pds1 in the absence of Dun1 is mediated not by APC but by the HECT domain-containing E3 ligase Rsp5. Our results suggest a regulatory scheme in which Dun1 prevents chromosome segregation during DNA damage by inhibiting Rsp5-mediated proteolytic degradation of securin Pds1.

Genomic analysis of hepatocellular carcinoma (HCC) with active hepatitis B virus (HBV) replication.

e15593 Background: HBV replication contributes to HCC initiation and is associated with worse patient outcomes. Prior tumor genomic studies of HBV-positive and -negative (HBV+/-) HCC have used detection of HBV surface antigen (HBsAg) in serum to annotate HBV status. However, a substantial proportion of HBsAg+ patients lack HBV replication in tumor, suggesting a potentially distinct patient subset. In this study, we determined HBV status by measuring tumor HBV RNA, a proxy for active replication. We then investigated HBV RNA+/- association with somatic mutations, gene sets, homologous recombination deficiency (HRD) and tumor mutation burden (TMB). Methods: RNA-Seq data for 371 HCC tumors were obtained from TCGA. Tumors were classified as HBV RNA+ if they harbored more than 1 HBV RNA read per million human reads, as measured using GATK PathSeq software. Associations between HBV RNA status and somatic mutations, gene sets, HRD and TMB were investigated. HRD score was calculated as the sum of 3 independent HRD measures (large scale state transitions, loss of heterozygosity and telomeric allelic imbalance). Results: HBV RNA+ status was associated with a higher rate of nonsynonymous somatic mutations in multiple genes, including the tumor suppressors TP53, CDKN2A, CHD5 and TET1, as well as AXIN2 and the proto-oncogene BCL11A ( p < 0.05 for all), while HBV RNA- status was associated with a higher rate of nonsynonymous mutations in the chromatin modifier BAP1 ( p = 0.03). In gene set enrichment analysis of normalized RNA-Seq expression data, HBV RNA+ status was associated with increased transcription of DNA repair genes, as well as genes upregulated by mTORC1 and MYC (FDR < 0.03 for all). HBV RNA status was also associated with HRD score (22.19 for HBV RNA+ vs. 15.97 for HBV RNA-, p = 1e-6), but not with TMB. A substantial subset of HBV RNA+ patients (33/100) were not annotated as HBV+ in the TCGA clinical database. Conclusions: HBV status based on tumor HBV RNA detection identifies a genetically distinct subset within all HBV-infected HCC patients that is associated with nonsynonymous somatic mutations in several genes and differential transcription of gene sets, some of which have not been previously reported, as well as with HRD score. These findings suggest potential for differential responsiveness to targeted therapies.

Abstract 324: SIK2 inhibitors regulate DNA repair pathway and sensitize ovarian cancer to PARP1 inhibitors

Abstract Genomic instability is a recognized hallmark of cancer. Germline mutations in critical DNA-repair and DNA-damage response genes, including BRCA1and BRCA2, predispose to cancer development, but also create vulnerabilities that can be exploited for cancer therapy. The successful development of PARP inhibitors for cancers with BRCA1/2 mutations and a deficiency in homologous recombination DNA repair has provided proof of concept. Inhibition of kinases that promote transcription of DNA repair genes might provide targets that enhance the activity of PARP-inhibitors. One novel candidate, salt-inducible kinase 2 (SIK2), is overexpressed in approximately 30% of high grade serous ovarian cancers. Inhibition of SIK2 induces tetraploidy, triggers apoptotic cell death, prevents metastasis, reduces AKT/survivin signaling and decreases phosphorylation of class-IIa histone deacetylases (HDACs) enhancing inhibition of gene expression. In contrast to other HDACs, class-IIa HDACs function as signal-responsive transcriptional corepressors for the myocyte enhancer factor-2 (MEF2) family of transcription factors. MEF2s play important roles in regulating growth factor responses, neuronal survival, T-cell apoptosis and, importantly, B-cell DNA double strand break (DSB) repair. Here we ask whether inhibition of SIK2 can enhance HDAC class-IIa inhibition of MEF activity, decrease DNA repair and enhance sensitivity to the PARP inhibitor Olaparib in ovarian cancer cells. Treatment with SIK2 inhibitors (ARN3236 or ARN3261) enhanced sensitivity to Olaparib in each of 8 ovarian cancer cell lines without BRCA1/2 mutations (OC316, OVCAR8, IGROV1, A2780, HCC5030, HCC5032, SKOv3 and OVCAR5) and significantly reduced the IC50 of Olaparib in two PARP inhibitor-resistant ovarian cancer cell lines (A2780CP-R and UWB1-289-10R). Synergistic cytotoxicity, judged with a Chou-Talaylay combination index (CI), was observed in all 8 cell lines. Treatment with a SIK2 inhibitor decreased the phosphorylation of class-IIa HDAC4/5/7, abolished class-IIa HDAC4/5/7-associated transcriptional activity of MEF2 and decreased MEF2 binding to regulatory regions with high-chromatin accessibility in DNA repair genes, resulting in repression of critical gene expression in DNA repair pathways. DNA damage, judged by rH2AX expression was enhanced by SIK2 inhibition. These observations not only provide new insights into the transcriptional regulation of DNA repair gene expression, but also have important implications for enhancing sensitivity of high grade serous ovarian cancers to PARP inhibition, with or without BRCA1 or BRCA2 germline mutations. Citation Format: Zhen Lu, Wequn Mao, Lan Pang, Janice M. Santiago-O'Farrill, Haling Yang, Ahmed Ahmed, Hariprasad Vankayalapati, Robert C. Bast. SIK2 inhibitors regulate DNA repair pathway and sensitize ovarian cancer to PARP1 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 324.

TP53 Regulates Transcription of DNA Repair Genes

TP53 Regulates Transcription of DNA Repair Genes

Hormones in Repair and Resistance

Radiotherapy resistance in prostate cancer is promoted by androgen receptor-stimulated transcription of DNA repair genes.

DNA repair in Mycoplasma gallisepticum

BackgroundDNA repair is essential for the maintenance of genome stability in all living beings. Genome size as well as the repertoire and abundance of DNA repair components may vary among prokaryotic species. The bacteria of the Mollicutes class feature a small genome size, absence of a cell wall, and a parasitic lifestyle. A small number of genes make Mollicutes a good model for a “minimal cell” concept.ResultsIn this work we studied the DNA repair system of Mycoplasma gallisepticum on genomic, transcriptional, and proteomic levels. We detected 18 out of 22 members of the DNA repair system on a protein level. We found that abundance of the respective mRNAs is less than one per cell. We studied transcriptional response of DNA repair genes of M. gallisepticum at stress conditions including heat, osmotic, peroxide stresses, tetracycline and ciprofloxacin treatment, stationary phase and heat stress in stationary phase.ConclusionsBased on comparative genomic study, we determined that the DNA repair system M. gallisepticum includes a sufficient set of proteins to provide a cell with functional nucleotide and base excision repair and mismatch repair. We identified SOS-response in M. gallisepticum on ciprofloxacin, which is a known SOS-inducer, tetracycline and heat stress in the absence of established regulators. Heat stress was found to be the strongest SOS-inducer. We found that upon transition to stationary phase of culture growth transcription of DNA repair genes decreases dramatically. Heat stress does not induce SOS-response in a stationary phase.

ATR cooperates with CTC1 and STN1 to maintain telomeres and genome integrity inArabidopsis

The CTC1/STN1/TEN1 (CST) complex is an essential constituent of plant and vertebrate telomeres. Here we show that CST and ATR (ataxia telangiectasia mutated [ATM] and Rad3-related) act synergistically to maintain telomere length and genome stability in Arabidopsis. Inactivation of ATR, but not ATM, temporarily rescued severe morphological phenotypes associated with ctc1 or stn1. Unexpectedly, telomere shortening accelerated in plants lacking CST and ATR. In first-generation (G1) ctc1 atr mutants, enhanced telomere attrition was modest, but in G2 ctc1 atr, telomeres shortened precipitously, and this loss coincided with a dramatic decrease in telomerase activity in G2 atr mutants. Zeocin treatment also triggered a reduction in telomerase activity, suggesting that the prolonged absence of ATR leads to a hitherto-unrecognized DNA damage response (DDR). Finally, our data indicate that ATR modulates DDR in CST mutants by limiting chromosome fusions and transcription of DNA repair genes and also by promoting programmed cell death in stem cells. We conclude that the absence of CST in Arabidopsis triggers a multifaceted ATR-dependent response to facilitate maintenance of critically shortened telomeres and eliminate cells with severe telomere dysfunction.

Oxidative stress and 8-oxoguanine repair are enhanced in colon adenoma and carcinoma patients

Oxidative stress is involved in the pathogenesis of colon cancer. We wanted to elucidate at which stage of the disease this phenomenon occurs. In the examined groups of patients with colorectal cancer (CRC, n = 89), benign adenoma (AD, n = 77) and healthy volunteers (controls, n = 99), we measured: vitamins A, C and E in blood plasma, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanine (8-oxoGua) in leukocytes and urine, leukocyte 8-oxoGua excision activity, mRNA levels of APE1, OGG1, 8-oxo-7,8-dihydrodeoxyguanosine 5'-triphosphate pyrophosphohydrolase (MTH1) and OGG1 polymorphism. The vitamin levels decreased gradually in AD and CRC patients. 8-OxodG increased in leukocytes and urine of CRC and AD patients. 8-OxoGua was higher only in the urine of CRC patients. 8-OxoGua excision was higher in CRC patients than in controls, in spite of higher frequency of the OGG1 Cys326Cys genotype, encoding a glycosylase with decreased activity. mRNA levels of OGG1 and APE1 increased in CRC and AD patients, which could explain increased 8-oxoGua excision rate in CRC patients. MTH1 mRNA was also higher in CRC patients. The results suggest that oxidative stress occurs in CRC and AD individuals. This is accompanied by increased transcription of DNA repair genes, and increased 8-oxoGua excision rate in CRC patients, which is, however, insufficient to counteract the increased DNA damage.