Involving DNA Repair Research Articles

Identifying therapeutic targets for primary ovarian insufficiency through integrated genomic analyses

BackgroundPrimary ovarian insufficiency (POI) is a disorder characterized by the premature decline in ovarian function, leading to significant fertility and health impacts on women under 40. The unclear etiology of POI hinders the development of effective treatments, highlighting the need for novel therapeutic targets.MethodsThis study employed genome-wide association analysis (GWAS) integrated with expression quantitative trait loci (eQTL) data from the GTEx and eQTLGen databases. Mendelian randomization (MR) and colocalization analyses were conducted to investigate causal relationships between genetic variants and POI and to identify potential therapeutic targets.ResultsWe identified 431 genes with available index cis-eQTL signals, of which four (HM13, FANCE, RAB2A, and MLLT10) were significantly associated with POI. Colocalization analysis revealed strong evidence for FANCE and RAB2A, indicating their potential as therapeutic targets. Subsequent druggability assessments identified FANCE and RAB2A as promising candidates for POI treatment, supported by their involvement in DNA repair and autophagy regulation, respectively.ConclusionsOur study establishes a causal link between specific genes and POI, highlighting FANCE and RAB2A as potential drug targets. These findings provide a foundation for future research and therapeutic development, aiming to improve outcomes for women with POI. Validation in further trials is necessary to confirm these potential targets.

Roles of Growth Hormone-Dependent JAK-STAT5 and Lyn Kinase Signaling in Determining Lifespan and Cancer Incidence.

In rodents, loss of growth hormone (GH) or its receptor is associated with extended lifespan. We aimed to determine the signaling process resulting in this longevity using GH receptor (GHR)-mutant mice with key signaling pathways deleted and correlate this with cancer incidence and expression of genes associated with longevity. GHR uses both canonical janus kinase (JAK)2-signal transducer and activator of transcription (STAT) signaling as well as signaling via the LYN-ERK1/2 pathway. We used C57BL/6 mice with loss of key receptor tyrosines and truncation resulting in 1) loss of most STAT5 response to GH; 2) total inability to generate STAT5 to GH; 3) loss of Box1 to prevent activation of JAK2 but not LYN kinase; or 4) total knockout of the receptor. For each mutant we analyzed lifespan, histopathology to determine likely cause of death, and hepatic gene and protein expression. The extended lifespan is evident in the Box1-mutant males (retains Lyn activation), which have a median lifespan of 1016 days compared to 890 days for the Ghr-/- males. In the females, GhrBox1-/- mice have a median lifespan of 970 days compared to 911 days for the knockout females. Sexually dimorphic GHR-STAT5 is repressive for longevity, since its removal results in a median lifespan of 1003 days in females compared to 734 days for wild-type females. Numerous transcripts related to insulin sensitivity, oxidative stress response, and mitochondrial function are regulated by GHR-STAT5; however, LYN-responsive genes involve DNA repair, cell cycle control, and anti-inflammatory response. There appears to be a yin-yang relationship between JAK2 and LYN that determines lifespan.

Deciphering the phospho-signature induced by hepatitis B virus in primary human hepatocytes.

Phosphorylation is a major post-translation modification (PTM) of proteins which is finely tuned by the activity of several hundred kinases and phosphatases. It controls most if not all cellular pathways including anti-viral responses. Accordingly, viruses often induce important changes in the phosphorylation of host factors that can either promote or counteract viral replication. Among more than 500 kinases constituting the human kinome only few have been described as important for the hepatitis B virus (HBV) infectious cycle, and most of them intervene during early or late infectious steps by phosphorylating the viral Core (HBc) protein. In addition, little is known on the consequences of HBV infection on the activity of cellular kinases. The objective of this study was to investigate the global impact of HBV infection on the cellular phosphorylation landscape early after infection. For this, primary human hepatocytes (PHHs) were challenged or not with HBV, and a mass spectrometry (MS)-based quantitative phosphoproteomic analysis was conducted 2- and 7-days post-infection. The results indicated that while, as expected, HBV infection only minimally modified the cell proteome, significant changes were observed in the phosphorylation state of several host proteins at both time points. Gene enrichment and ontology analyses of up- and down-phosphorylated proteins revealed common and distinct signatures induced by infection. In particular, HBV infection resulted in up-phosphorylation of proteins involved in DNA damage signaling and repair, RNA metabolism, in particular splicing, and cytoplasmic cell-signaling. Down-phosphorylated proteins were mostly involved in cell signaling and communication. Validation studies carried out on selected up-phosphorylated proteins, revealed that HBV infection induced a DNA damage response characterized by the appearance of 53BP1 foci, the inactivation of which by siRNA increased cccDNA levels. In addition, among up-phosphorylated RNA binding proteins (RBPs), SRRM2, a major scaffold of nuclear speckles behaved as an antiviral factor. In accordance with these findings, kinase prediction analysis indicated that HBV infection upregulates the activity of major kinases involved in DNA repair. These results strongly suggest that HBV infection triggers an intrinsic anti-viral response involving DNA repair factors and RBPs that contribute to reduce HBV replication in cell culture models.

High expression of PPP1CC promotes NHEJ-mediated DNA repair leading to radioresistance and poor prognosis in nasopharyngeal carcinoma.

Protein phosphatase 1 catalytic subunit gamma (PPP1CC) promotes DNA repair and tumor development and progression, however, its underlying mechanisms remain unclear. This study investigated the molecular mechanism of PPP1CC's involvement in DNA repair and the potential clinical implications. High expression of PPP1CC was significantly correlated with radioresistance and poor prognosis in human nasopharyngeal carcinoma (NPC) patients. The mechanistic study revealed that PPP1CC bound to Ku70/Ku80 heterodimers and activated DNA-PKcs by promoting DNA-PK holoenzyme formation, which enhanced nonhomologous end junction (NHEJ) -mediated DNA repair and led to radioresistance. Importantly, BRCA1-BRCA2-containing complex subunit 3 (BRCC3) interacted with PPP1CC to enhance its stability by removing the K48-linked polyubiquitin chain at Lys234 to prevent PPP1CC degradation. Therefore, BRCC3 helped the overexpressed PPP1CC to maintain its high protein level, thereby sustaining the elevation of DNA repair capacity and radioresistance. Our study identified the molecular mechanism by which PPP1CC promotes NHEJ-mediated DNA repair and radioresistance, suggesting that the BRCC3-PPP1CC-Ku70 axis is a potential therapeutic target to improve the efficacy of radiotherapy.

Comprehensive pan-cancer analysis: essential role of ABCB family genes in cancer.

The adenosine triphosphate-binding-cassette (ABC) transporter orchestrates the transmembrane transport of diverse substrates with the aid of ATP as an energy source. ABC transporter constitutes a widespread superfamily of transporters prominently present on the cellular membrane of organisms. Advancements in understanding have unveiled additional roles beyond mere intracellular or extracellular transport functions for the ABC protein family, encompassing involvement in DNA repair, protein translation, and gene expression regulation. Yet its role in tumors is still unknown. This study drew support from multiple databases, including Gene Expression Omnibus (GEO), European Genome-phenome Archive (EGA), The Cancer Genome Atlas (TCGA), and employed multidimensional bioinformatics analyses, incorporating online databases and the R-project. Through a comprehensive analysis, we seek to discern transcriptional-level disparities among genes and their consequential impacts on prognosis, tumor microenvironment (TME), stemness score, immune subtypes, clinical characteristics, and drug sensitivity across human cancers. ABC transporter subfamily B (ABCB) family genes exhibited heightened expression across diverse tumors, demonstrating a significant correlation with overall prognosis in pan-cancer contexts. Notably, gene expression levels manifested substantial associations with TME, stemness score, immune subtypes, clinical characteristics, and drug sensitivity in specific cancers, including kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), and pancreatic adenocarcinoma (PAAD). Within this subset, transporter associated with antigen processing 1 (TAP1), TAP2, and ABCB6 emerged as noteworthy oncogenes. The outcomes of this study contribute to a comprehensive understanding of the implications of ABCB family genes in tumor progression, offering insights into potential therapeutic targets for cancer. Notably, the identification of ABCB6 as a significant oncogene suggests promising avenues for targeted therapies in KIRP, LIHC, and PAAD.

Abstract 5611: Unraveling the role of F-actin in resistant GBM: Insights into DNA repair, GSC phenotype, and therapeutic implications

Abstract Glioblastoma (GBM), a highly aggressive brain cancer, poses a formidable challenge marked by its resistance to conventional therapies. F-actin, a crucial cytoskeletal protein, influences DNA repair and impacts genomic stability. F-actin dynamics also shape the glioma stem cell (GSC) phenotype, affecting self-renewal and migration. Altered F-actin organization contributes to GBM resistance to conventional therapies. Understanding F-actin's multifaceted role is vital for developing targeted strategies to overcome treatment resistance and enhance outcomes for GBM patients. This study goes deeper into the intricate interplay between the F-actin cytoskeleton and the acquisition of resistance in GBM cells. Our primary goal was to explore the effects of pharmacological or genetic F-actin depolymerization on the reversal of acquired resistance to ionizing radiation (IR) and temozolomide (TMZ) in GBM cells, particularly focusing on the underlying mechanisms involving DNA repair, the glioma stem cell (GSC) phenotype, and therapeutic implications. To establish resistant sublines, GBM U87-MG cells underwent cycles of IR and TMZ exposure, revealing a significant increase in IC50 values for TMZ and ID50 values for IR through viability assays. Subsequent characterization of these sublines growth was in both adherent and spheroid cultures uncovering a distinct sphere-forming capacity. Notably, alterations in DNA repair and the Rho GTPase pathway were observed, with evident disorganized F-actin polymerization in resistant cells, especially at the periphery of spheroids. Elevated expression of stemness markers (Nestin, CD133, and CD44) highlighted a GSC-like profile in the resistant cells, a characteristic further accentuated in spheroid cultures. To assess the relevance of the actin cytoskeleton in maintaining the resistant phenotype, cells were treated with Rho-GTPase inhibitor (C3) or actin polymerization inhibitor (Cytochalasin D). Both treatments resulted in reduced TMZ IC50 and IR ID50 values, partially attributed to the negative modulation of DNA repair capacity - observed by alkaline comet and host-cell reactivation assays. Additionally, F-actin disassembly was found to impact the expression of stemness markers, further reinforcing the connection between cytoskeletal dynamics and the GSC phenotype. Overall, our data strongly suggest that F-actin cytoskeleton dynamics plays a pivotal role in the development and maintenance of resistance in GBM. Targeting these cytoskeletal components sees a promising pharmacological strategy for resensitizing recurrent and resistant GBM tumors, not only by diminishing DNA repair capabilities but also by influencing the GSC-like phenotype, thus opening new avenues for therapeutic interventions. Citation Format: Yuli Thamires Magalhães, Fábio Luís Forti. Unraveling the role of F-actin in resistant GBM: Insights into DNA repair, GSC phenotype, and therapeutic implications [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 5611.

XRCC1 and hOGG1 polymorphisms and endometrial carcinoma: A meta-analysis.

Endometrial carcinoma's (EC) etiology is complex and involves DNA repair gene polymorphisms like XRCC1-Arg399Gln and hOGG1-Ser326Cys, but their association with the disease is unclear. Following PRISMA, we conducted a systematic review and meta-analysis, collecting data from four databases. The studies needed to be population-based case-control studies examining the association between the named polymorphisms and EC. Quality was assessed with the Newcastle-Ottawa Scale. Pooled odds ratios (OR) and 95% confidence intervals (CI) were calculated, and subgroup analyses were conducted based on ethnicity. Seven studies were included. Both polymorphisms were found to significantly increase EC risk, particularly in Caucasians. XRCC1-Arg399Gln showed a dominant model OR of 1.14 (95% CI: 1.01-1.29) and a homozygous model OR of 1.59 (95% CI: 1.12-2.25). The heterozygote model OR for hOGG1-Ser326Cys was 1.29 (95% CI: 1.02-1.63), and the allele OR was 1.31 (95% CI: 1.07-1.60). XRCC1-Arg399Gln and hOGG1-Ser326Cys may increase EC risk, primarily in Caucasian women, emphasizing the role of DNA repair in disease susceptibility. More extensive studies are needed to validate these findings in diverse ethnicities and investigate other DNA repair gene polymorphisms.

HJURP is recruited to double-strand break sites and facilitates DNA repair by promoting chromatin reorganization.

HJURP is overexpressed in several cancer types and strongly correlates with patient survival. However, the mechanistic basis underlying the association of HJURP with cancer aggressiveness is not well understood. HJURP promotes the loading of the histone H3 variant, CENP-A, at the centromeric chromatin, epigenetically defining the centromeres and supporting proper chromosome segregation. In addition, HJURP is associated with DNA repair but its function in this process is still scarcely explored. Here, we demonstrate that HJURP is recruited to DSBs through a mechanism requiring chromatin PARylation and promotes epigenetic alterations that favor the execution of DNA repair. Incorporation of HJURP at DSBs promotes turnover of H3K9me3 and HP1, facilitating DNA damage signaling and DSB repair. Moreover, HJURP overexpression in glioma cell lines also affected global structure of heterochromatin independently of DNA damage induction, promoting genome-wide reorganization and assisting DNA damage response. HJURP overexpression therefore extensively alters DNA damage signaling and DSB repair, and also increases radioresistance of glioma cells. Importantly, HJURP expression levels in tumors are also associated with poor response of patients to radiation. Thus, our results enlarge the understanding of HJURP involvement in DNA repair and highlight it as a promising target for the development of adjuvant therapies that sensitize tumor cells to irradiation.

Interferon-Gamma-Inducible Protein 16 Inhibits Hepatocellular Carcinoma via Interferon Regulatory Factor 3 on Chemosensitivity.

Previous reports have suggested IFI16 as a tumor suppressor in hepatocellular carcinoma (HC). Nonetheless, the biological significance of IFI16 and its mechanism concerning resistance to cisplatin (DDP) in HC requires further exploration. Samples of tumor and corresponding para-carcinoma tissues were acquired from patients with HC. Furthermore, DDP-resistant cell lines of HC, specifically HCC, Huh7 and Hepatoblastoma, HepG3, were generated by gradually increasing the concentration of DDP. Cell apoptosis and DNA damage were evaluated by utilizing flow cytometry assay and TUNEL staining. The interaction between IFI16 and interferon regulatory factor 3 (IRF3) proteins were analyzed using Co-Immunoprecipitation (Co-IP) assay. In vivo assays were conducted by establishing HC subcutaneous xenograft tumor models. The study found a reduction in IFI16 expression in both HC tissues and DDP-resistant HC cell lines. The binding of IFI16 to IRF3 regulated DNA damage-associated markers in vitro. Overexpression of IFI16 heightened the susceptibility of DDP-induced apoptosis and DNA damage, which was counteracted by IRF3 knockdown, while strengthened by IRF3 overexpression. Moreover, overexpression of IFI16 diminished in vivo DDP-resistant HC tumorigenicity. In summary, our findings suggest that IFI16 serves as a tumor suppressor in HC by promoting DNA damage via its interaction with IRF3, thereby reversing DDP resistance.

Prime editing: Mechanism insight and recent applications in plants.

Prime editing (PE) technology utilizes an extended prime editing guide RNA (pegRNA) to direct a fusion peptide consisting of nCas9 (H840) and reverse transcriptase (RT) to a specific location in the genome. This enables the installation of base changes at the targeted site using the extended portion of the pegRNA through RT activity. The resulting product of the RT reaction forms a 3' flap, which can be incorporated into the genomic site through a series of biochemical steps involving DNA repair and synthesis pathways. PE has demonstrated its effectiveness in achieving almost all forms of precise gene editing, such as base conversions (all types), DNA sequence insertions and deletions, chromosomal translocation and inversion and long DNA sequence insertion at safe harbour sites within the genome. In plant science, PE could serve as a groundbreaking tool for precise gene editing, allowing the creation of desired alleles to improve crop varieties. Nevertheless, its application has encountered limitations due to efficiency constraints, particularly in dicotyledonous plants. In this review, we discuss the step-by-step mechanism of PE, shedding light on the critical aspects of each step while suggesting possible solutions to enhance its efficiency. Additionally, we present an overview of recent advancements and future perspectives in PE research specifically focused on plants, examining the key technical considerations of its applications.

Polymerase θ inhibition steps on the cGAS pedal.

Deficiencies in homologous recombination (HR) repair lead to an accumulation of DNA damage and can predispose individuals to cancer. Polymerase theta (Pol θ, encoded by POLQ) is overexpressed by HR-deficient cancers and promotes cancer cell survival by mediating error-prone double-stranded break (DSB) repair and facilitating resistance against poly-ADP ribose polymerase inhibitor treatment. In this issue of the JCI, Oh, Wang, et al. report on the impact of Pol θ inhibition on activation of antitumor immunity. The authors used pancreatic ductal adenocarcinoma (PDAC) cell and mouse models characterized by HR-associated gene alterations and POLQ overexpression. POLQ knockdown showed synthetic lethality in combination with gene mutations involving DNA repair, including BRCA1, BRCA2, and ATM. Notably, Pol θ deficiency or inhibition suppressed tumor growth, increased the accumulation of unrepaired DNA damage, and enhanced T cell infiltration via the cGAS/STING pathway. These findings suggest a broader scope for Pol θ inhibition in HR-deficient cancers.

Genetic analyses of DNA repair pathway associated genes implicate new candidate cancer predisposing genes in ancestrally defined ovarian cancer cases.

Not all familial ovarian cancer (OC) cases are explained by pathogenic germline variants in known risk genes. A candidate gene approach involving DNA repair pathway genes was applied to identify rare recurring pathogenic variants in familial OC cases not associated with known OC risk genes from a population exhibiting genetic drift. Whole exome sequencing (WES) data of 15 OC cases from 13 families tested negative for pathogenic variants in known OC risk genes were investigated for candidate variants in 468 DNA repair pathway genes. Filtering and prioritization criteria were applied to WES data to select top candidates for further analyses. Candidates were genotyped in ancestry defined study groups of 214 familial and 998 sporadic OC or breast cancer (BC) cases and 1025 population-matched controls and screened for additional carriers in 605 population-matched OC cases. The candidate genes were also analyzed in WES data from 937 familial or sporadic OC cases of diverse ancestries. Top candidate variants in ERCC5, EXO1, FANCC, NEIL1 and NTHL1 were identified in 5/13 (39%) OC families. Collectively, candidate variants were identified in 7/435 (1.6%) sporadic OC cases and 1/566 (0.2%) sporadic BC cases versus 1/1025 (0.1%) controls. Additional carriers were identified in 6/605 (0.9%) OC cases. Tumour DNA from ERCC5, NEIL1 and NTHL1 variant carriers exhibited loss of the wild-type allele. Carriers of various candidate variants in these genes were identified in 31/937 (3.3%) OC cases of diverse ancestries versus 0-0.004% in cancer-free controls. The strategy of applying a candidate gene approach in a population exhibiting genetic drift identified new candidate OC predisposition variants in DNA repair pathway genes.

WHOLE EXOME SEQUENCING OF A MULTIPLEX CROHN’S DISEASE FAMILY IDENTIFIES RARE ALTERATIONS IN PYGB ANDXRN2 AS POTENTIALLY HIGH IMPACT RISK VARIANTS OF DISEASE

Abstract BACKGROUND The study of multiplex Crohn’s disease (CD) families has played a critical role in our understanding of the disease, including the identification of the first recognized CD risk variants in NOD2, which remain among the highest impact genetic predispositions known. Since then, genome wide association studies have revealed over 200 additional inflammatory bowel disease (IBD) risk alleles. However, such technologies may miss low prevalence, high impact variants. We therefore have been studying multiplex CD families to identify additional novel variants of disease. METHODS A 3 generation family, including 5 CD subjects, 12 relatives, and 5 controls (spouses) underwent whole exome sequencing on the Illumina platform. Single nucleotide polymorphisms (SNPs) were identified, whose minor allele frequencies were <0.1% in all gnomAD subpopulations and were also predicted to be damaging, deleterious, or disease-causing by at least one of the pathogenicity prediction tools (PhyloP, SIFT, PolyPhen2, MutationTaster, LRT, and CADD). We selected the 5 CD diagnosed individuals, as well as a first degree relative of all 5 individuals (but who did not have CD) as the putative variant-carrying group. SNPs that were carried in this group, but were not found in the controls, were identified. Subjects for whom we did not have sufficient DNA to complete whole exome sequencing were excluded from analysis (Figure 1). RESULTS We found 32 rare, pathogenic SNPs shared among the variant-carrying group. After removing those that were also found in any of the 5 controls, we were left with 4 variants as described in Table 1. Of these, rs145974526 was also found in 3 non-CD relatives (1 adult and 2 children). rs369261067 was also identified in 3 non-CD relatives (2 adults and 1 child). The remaining two variants (rs150582502 and rs149199982) were present in only 1 additional relative, the adult offspring of a CD-affected subject. Both SNPs are found on chromosome 20, and these risk alleles are located in the PYGB and XRN2 genes, respectively. Of note, NOD2 variants were not identified in this family. DISCUSSION Based on our analysis, we identify SNPs rs150582502 and rs149199982 as possible private, rare, high impact variants for Crohn’s disease. PYGB (glycogen phosphorylase B) has been shown to promote cellular proliferation in multiple tumor types by regulation of the Wnt/β-Catenin pathway. XRN2 encodes 5'-3' exoribonuclease 2, which is involved in transcription termination. Studies of XRN2 have demonstrated its involvement in DNA repair, and alterations in the gene have been identified in various cancers. Additional functional work will be performed to further validate the role of these mutations. We are also in the process of analyzing additional multiplex IBD families and datasets for these SNPs, as well as for other potential novel variants of disease.

Genetic alterations of GI-NECs involving three main signaling pathways.

Gastrointestinal (GI)-neuroendocrine neoplasms (NENs) are subclassified in neuroendocrine tumors (NETs), neuroendocrine carcinomas (NECs), and mixed neuroendocrine-non-neuroendocrine neoplasms (MiNENs). The genetic characteristics of GI-NEN has been a hot issue in recent years, but more studies are needed to provide further details. This study aims to provide additional data about genomic characteristics of GI-NENs and the genetic differences between NETs and NECs. Thirteen samples were selected for next-generation sequencing (NGS) analysis with a 425-gene panel. Microsatellite instability (MSI) and tumor mutational burden (TMB) were calculated as well as immunohistochemistry (IHC) was used to test for protein expression. Genetic alterations were very common in NECs, but rare in NETs. The average TMB of NETs and NECs was 2.3 and 6.9, respectively. The TMB of NECs was significantly higher compared to NETs. The TP53 mutation rate was significantly higher in NECs than in NETs (100% vs. 20%), other mutations involved MTOR (n= 2, 15.4%), DDR2 (n= 3, 23.1%), ERBB4 (n= 1, 7.7%), BRCA1 (n= 1, 7.7%), BRCA2 (n= 1, 7.7%), ATM (n= 1, 7.7%), and SMAD4 (n= 1, 7.7%). Deep loss of SMAD4 (1/3, 33.3%), SDHB (1/3, 33.3%), RB1 (1/3, 33.3%), and BRCA2 (1/3, 33.3%), high-level amplification of CRKL (1/3, 33.3%), CCNE1(1/3, 33.3%), and MCL1(1/3, 33.3%) were found in NECs. The integrated analysis found these genetic alterations frequently involve DNA repair and cell cycle, PI3K/AKT/mTOR and TGF-β/SMAD4 signaling pathways. Genetic alterations were very common in NECs and rare in NETs, and frequently involved three main signaling pathways. NEC patients harboring these genetic alterations may benefit from targeted therapy and PD-1/PD-L1 immunotherapy.

Stabilization of DNA fork junctions by Smc5/6 complexes revealed by single-molecule imaging.

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.

PLK1-mediated phosphorylation of PPIL2 regulates HR via CtIP.

Homologous recombination (HR) is an error-free DNA double-strand break (DSB) repair pathway, which safeguards genome integrity and cell viability. Human C-terminal binding protein (CtBP)—interacting protein (CtIP) is a central regulator of the pathway which initiates the DNA end resection in HR. Ubiquitination modification of CtIP is known in some cases to control DNA resection and promote HR. However, it remains unclear how cells restrain CtIP activity in unstressed cells. We show that the ubiquitin E3 ligase PPIL2 is recruited to DNA damage sites through interactions with an HR-related protein ZNF830, implying PPIL2’s involvement in DNA repair. We found that PPIL2 interacts with and ubiquitinates CtIP at the K426 site, representing a hereunto unknown ubiquitination site. Ubiquitination of CtIP by PPIL2 suppresses HR and DNA resection. This inhibition of PPIL2 is also modulated by phosphorylation at multiple sites by PLK1, which reduces PPIL2 ubiquitination of CtIP. Our findings reveal new regulatory complexity in CtIP ubiquitination in DSB repair. We propose that the PPIL2-dependent CtIP ubiquitination prevents CtIP from interacting with DNA, thereby inhibiting HR.

Abstract 3493: Comprehensive analysis of the DNA repair enzyme signature in tumor and blood cells from head and neck cancer patients and correlation with clinical data from a 18-months follow-up study

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer worldwide. It is often associated with a history of smoking/alcohol consumption or exposure to the human papilloma virus (HPV). Beyond surgery, treatment usually includes DNA damaging treatments i.e. cisplatin/5FU combined or not with radiation therapy. Treatment failure rates are still high due to intrinsic and acquired mechanisms of resistance, largely involving DNA repair mechanisms. Here we precisely examined the main DNA repair mechanisms susceptible to drive tumor progression and resistance to treatment, using a miniaturized comprehensive functional approach on biochip. Enzymatic DNA repair profiles were compared for lymphocytes and tumor biopsies taken before treatment from 38 patients in a prospective clinical study. We simultaneously investigated double strand break repair pathways (HR, NHEJ, SSA, alt-EJ), excision/synthesis repair mechanisms (BER, NER, ICLR) and several glycosylases/AP endonuclease activities, using lysates prepared from the samples. Results were correlated with physiologic and lifestyle/risk factors, TNM tumor classification, treatment-induced adverse effects, and disease progression or death at 18 months. Analysis of blood cells and biopsies provided different and complementary information. Indeed, the prediction of treatment-induced severe toxicity was effective on blood cells. Several risk factors significantly affected specific repair activities of tumor cells. HPV positive and negative tumors displayed distinct DNA repair profiles and, cancer progression and tumor staging correlated with deregulated repair activities in tumors. Interestingly, the most affected DNA repair activities concerned double strand break repair, repair of cisplatin adducts, and repair of oxidative damage. This accurate DNA repair profiling represents an innovative strategy to reveal tumor diversity and better understand the impact of risk factors. It improved our understanding of the role of DNA repair in the development and progression of cancer. In addition, the use of a panel of DNA repair-based enzymatic biomarkers is more accurate than the single parameter approach in stratifying patients into different groups, thus allowing for more effective therapeutic strategies. Citation Format: Sylvie Sauvaigo, Giovanna Muggiolu, Sarah Libert, Bertrand Treillard, Gersende Alphonse, Christian A. Righini, Philippe Ceruse, Pierre Philouze, Claire Rodriguez-Lafrasse. Comprehensive analysis of the DNA repair enzyme signature in tumor and blood cells from head and neck cancer patients and correlation with clinical data from a 18-months follow-up study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3493.

Allele-informed copy number evaluation of plasma DNA samples from metastatic prostate cancer patients: the PCF_SELECT consortium assay.

Sequencing of cell-free DNA (cfDNA) in cancer patients’ plasma offers a minimally-invasive solution to detect tumor cell genomic alterations to aid real-time clinical decision-making. The reliability of copy number detection decreases at lower cfDNA tumor fractions, limiting utility at earlier stages of the disease. To test a novel strategy for detection of allelic imbalance, we developed a prostate cancer bespoke assay, PCF_SELECT, that includes an innovative sequencing panel covering ∼25 000 high minor allele frequency SNPs and tailored analytical solutions to enable allele-informed evaluation. First, we assessed it on plasma samples from 50 advanced prostate cancer patients. We then confirmed improved detection of genomic alterations in samples with <10% tumor fractions when compared against an independent assay. Finally, we applied PCF_SELECT to serial plasma samples intensively collected from three patients previously characterized as harboring alterations involving DNA repair genes and consequently offered PARP inhibition. We identified more extensive pan-genome allelic imbalance than previously recognized in prostate cancer. We confirmed high sensitivity detection of BRCA2 allelic imbalance with decreasing tumor fractions resultant from treatment and identified complex ATM genomic states that may be incongruent with protein losses. Overall, we present a framework for sensitive detection of allele-specific copy number changes in cfDNA.

Therapeutic Opportunities of Disrupting Genome Integrity in Adult Diffuse Glioma.

Adult diffuse glioma, particularly glioblastoma (GBM), is a devastating tumor of the central nervous system. The existential threat of this disease requires on-going treatment to counteract tumor progression. The present outcome is discouraging as most patients will succumb to this disease. The low cure rate is consistent with the failure of first-line therapy, radiation and temozolomide (TMZ). Even with their therapeutic mechanism of action to incur lethal DNA lesions, tumor growth remains undeterred. Delivering additional treatments only delays the inescapable development of therapeutic tolerance and disease recurrence. The urgency of establishing lifelong tumor control needs to be re-examined with a greater focus on eliminating resistance. Early genomic and transcriptome studies suggest each tumor subtype possesses a unique molecular network to safeguard genome integrity. Subsequent seminal work on post-therapy tumor progression sheds light on the involvement of DNA repair as the causative contributor for hypermutation and therapeutic failure. In this review, we will provide an overview of known molecular factors that influence the engagement of different DNA repair pathways, including targetable vulnerabilities, which can be exploited for clinical benefit with the use of specific inhibitors.

Large dataset of octocoral mitochondrial genomes provides new insights into mt-mutS evolution and function

All studied octocoral mitochondrial genomes (mt-genomes) contain a homologue of the Escherichia coli mutS gene, a member of a gene family encoding proteins involved in DNA mismatch repair, other types of DNA repair, meiotic recombination, and other functions. Although mutS homologues are found in all domains of life, as well as viruses, octocoral mt-mutS is the only such gene found in an organellar genome. While the function of mtMutS is not known, its domain architecture, conserved sequence, and presence of several characteristic residues suggest its involvement in mitochondrial DNA repair. This inference is supported by exceptionally low rates of mt-sequence evolution observed in octocorals. Previous studies of mt-mutS have been limited by the small number of octocoral mt-genomes available. We utilized sequence-capture data from the recent Quattrini et al. 2020 study [Nature Ecology & Evolution 4:1531–1538] to assemble complete mt-genomes for 94 species of octocorals. Combined with sequences publicly available in GenBank, this resulted in a dataset of 184 complete mt-genomes, which we used to re-analyze the conservation and evolution of mt-mutS. In our analysis, we discovered the first case of mt-mutS loss among octocorals in one of the two Pseudoanthomastus spp. assembled from Quattrini et al. data. This species displayed accelerated rate and changed patterns of nucleotide substitutions in mt-genome, which we argue provide additional evidence for the role of mtMutS in DNA repair. In addition, we found accelerated mt-sequence evolution in the presence of mt-mutS in several octocoral lineages. This accelerated evolution did not appear to be the result of relaxed selection pressure and did not entail changes in patterns of nucleotide substitutions. Overall, our results support previously reported patterns of conservation in mt-mutS and suggest that mtMutS is involved in DNA repair in octocoral mitochondria. They also indicate that the presence of mt-mutS contributes to, but does not fully explain, the low rates of sequence evolution in octocorals