Decreased DNA Repair Research Articles

Chemoproteogenomic stratification of the missense variant cysteinome

Cancer genomes are rife with genetic variants; one key outcome of this variation is widespread gain-of-cysteine mutations. These acquired cysteines can be both driver mutations and sites targeted by precision therapies. However, despite their ubiquity, nearly all acquired cysteines remain unidentified via chemoproteomics; identification is a critical step to enable functional analysis, including assessment of potential druggability and susceptibility to oxidation. Here, we pair cysteine chemoproteomics—a technique that enables proteome-wide pinpointing of functional, redox sensitive, and potentially druggable residues—with genomics to reveal the hidden landscape of cysteine genetic variation. Our chemoproteogenomics platform integrates chemoproteomic, whole exome, and RNA-seq data, with a customized two-stage false discovery rate (FDR) error controlled proteomic search, which is further enhanced with a user-friendly FragPipe interface. Chemoproteogenomics analysis reveals that cysteine acquisition is a ubiquitous feature of both healthy and cancer genomes that is further elevated in the context of decreased DNA repair. Reference cysteines proximal to missense variants are also found to be pervasive, supporting heretofore untapped opportunities for variant-specific chemical probe development campaigns. As chemoproteogenomics is further distinguished by sample-matched combinatorial variant databases and is compatible with redox proteomics and small molecule screening, we expect widespread utility in guiding proteoform-specific biology and therapeutic discovery.

DNA repair efficiency in young hypertensive: Is lifestyle a prooxidant factor?

Background: High prevalence of hypertension (HTN) is of major concern among the middle-aged population of Kerala. Understudied in the context of HTN is oxidative damage to DNA, which is caused by free radical assaults. The modern lifestyle associated with an unhealthy diet and lack of exercise plays a key role in oxidative stress induction. Aims and Objectives: The objective of this study was to assess the significance of oxidative stress and the effectiveness of DNA repair mechanisms in young hypertensives along with how it correlates with aspects of lifestyle. Materials and Methods: This prospective case–control study enrolled clinically proven hypertensive patients referred from the outpatient department of Hridayalaya, Institute of Preventive Cardiology, Thiruvananthapuram, to Genetika, a research center for Cytogenetic Studies in South Kerala. Hypertensive patients (n=180) within the age group of 18–39 were compared with matched healthy controls (n=140). Associated lifestyle factors were determined. Results: In comparison to controls, cases had noticeably higher mean levels of malondialdehyde (MDA) and mean break per cell (P<0.05). There was a difference that was statistically significant (P<0.05) between the patients and controls in terms of diet, exercise, physical activity, obesity, mental stress, alcohol, and tobacco use. On comparing MDA and mean break per cell values with lifestyle factors, a statistically significant difference was detected (P<0.05). Observed a significant positive correlation of break per cell value with MDA (P<0.05). Conclusion: The current study demonstrated increased DNA damage and decreased DNA repair efficiency in young hypertensive and was strongly associated with lifestyle factors. Appropriate measures such as regular BP checkups, regular exercises, and abstinence from alcohol and smoking are to be taken for the prevention and control of HTN.

NBS1 lactylation is required for efficient DNA repair and chemotherapy resistance

The Warburg effect is a hallmark of cancer that refers to the preference of cancer cells to metabolize glucose anaerobically rather than aerobically1,2. This results in substantial accumulation of lacate, the end product of anaerobic glycolysis, in cancer cells3. However, how cancer metabolism affects chemotherapy response and DNA repair in general remains incompletely understood. Here we report that lactate-driven lactylation of NBS1 promotes homologous recombination (HR)-mediated DNA repair. Lactylation of NBS1 at lysine 388 (K388) is essential for MRE11–RAD50–NBS1 (MRN) complex formation and the accumulation of HR repair proteins at the sites of DNA double-strand breaks. Furthermore, we identify TIP60 as the NBS1 lysine lactyltransferase and the ‘writer’ of NBS1 K388 lactylation, and HDAC3 as the NBS1 de-lactylase. High levels of NBS1 K388 lactylation predict poor patient outcome of neoadjuvant chemotherapy, and lactate reduction using either genetic depletion of lactate dehydrogenase A (LDHA) or stiripentol, a lactate dehydrogenase A inhibitor used clinically for anti-epileptic treatment, inhibited NBS1 K388 lactylation, decreased DNA repair efficacy and overcame resistance to chemotherapy. In summary, our work identifies NBS1 lactylation as a critical mechanism for genome stability that contributes to chemotherapy resistance and identifies inhibition of lactate production as a promising therapeutic cancer strategy.

Microwave hyperthermia enhances radiosensitization by decreasing DNA repair efficiency and inducing oxidative stress in PC3 prostatic adenocarcinoma cells

Purpose Radiotherapy (RT) is the primary treatment for prostate cancer (PCa); however, the emergence of castration-resistant prostate cancer (CRPC) often leads to treatment failure and cancer-related deaths. In this study, we aimed to explore the use of microwave hyperthermia (MW-HT) to sensitize PCa to RT and investigate the underlying molecular mechanisms. Methods We developed a dedicated MW-HT heating setup, created an in vitro and in vivo MW-HT + RT treatment model for CRPC. We evaluated PC3 cell proliferation using CCK-8, colony experiments, DAPI staining, comet assay and ROS detection method. We also monitored nude mouse models of PCa during treatment, measured tumor weight, and calculated the tumor inhibition rate. Western blotting was used to detect DNA damage repair protein expression in PC3 cells and transplanted tumors. Results Compared to control, PC3 cell survival and clone formation rates decreased in RT + MW-HT group, demonstrating significant increase in apoptosis, ROS levels, and DNA damage. Lower tumor volumes and weights were observed in treatment groups. Ki-67 expression level was reduced in all treatment groups, with significant decrease in RT + MW-HT groups. The most significant apoptosis induction was confirmed in RT + MW-HT group by TUNEL staining. Protein expression levels of DNA-PKcs, ATM, ATR, and P53/P21 signaling pathways significantly decreased in RT + MW-HT groups. Conclusion MW-HT + RT treatment significantly inhibited DNA damage repair by downregulating DNA-PKcs, ATM, ATR, and P53/P21 signaling pathways, leading to increased ROS levels, aggravate DNA damage, apoptosis, and necrosis in PC3 cells, a well-established model of CRPC.

Abstract 4686: Identification of ART3 as a novel synthetic lethal partner of the BRCA1 mutation in breast cancer

Abstract Breast cancer (BC), the most prevalent malignancy in women, ranks fourth in cancer mortality as of 2022. Mutations in the BRCA genes significantly elevate BC risk and are associated with markedly reduced survival compared to non-carriers. These genes are crucial in homologous recombination, a mechanism essential for error-free DNA repair. Despite advancements, treatment options for BRCA mutation-driven cancers remain limited. Synthetic lethality (SL) has emerged as a promising strategy, with poly(ADP-ribose) polymerase inhibitors (PARPi) being the first developed drugs based on this principle. However, PARPi's effectiveness is limited, as nearly 40% of BRCA-mutated BCs are unresponsive, and resistance often develops in initial responders. Therefore, novel mutation-specific targeted therapies are urgently needed. In this study, we employed the novel computational method 'Mining Synthetic Lethals (MiSL)' to analyze TCGA pan-cancer primary tumor data, identifying ART3 as a potential SL partner of the BRCA1 germline mutation. Our approach focused on the intersection of genetic interactions and cancer vulnerabilities, providing a unique insight into SL relationships. siRNA-mediated silencing of ART3 in BRCA1-mutated cells consistently led to decreased cell viability, apoptosis induction, and G2/M cell cycle arrest. Following stable transfection with an inducible ART3 shRNA construct, we confirmed significant reduction in cell viability in BRCA1-deficient cells through colony formation assays. RNA sequencing of ART3 knockdown cells revealed transcriptional changes in genes involved in cell cycle progression, apoptosis, and growth and proliferation pathways compared with control cells. Immunofluorescent staining revealed increased DNA damage foci with decreased DNA repair in ART3 knockdown BRCA1-deficient cells. In conclusion, our study identifies ART3 as a novel SL partner of the BRCA1 mutation in BC. This discovery paves the way for developing new therapeutic strategies for BRCA1-mutated breast cancers and novel chemoprevention approaches for individuals carrying germline BRCA1 mutations. Future research will focus on further elucidating the molecular mechanisms underlying this SL interaction and exploring its therapeutic potential in clinical settings. Citation Format: Hannah Neiger, Xinyu Pei, Anh Nguyen, Wenjia Wang, Emily L. Siegler, James Zhou, Kristie Lau, Alexandra Tan, Yihui Shi. Identification of ART3 as a novel synthetic lethal partner of the BRCA1 mutation in breast 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 4686.

Inhibition of demethylase by IOX1 modulates chromatin accessibility to enhance NSCLC radiation sensitivity through attenuated PIF1

Chromatin accessibility is a critical determinant of gene transcriptional expression and regulated by histones modification. However, the potential for manipulating chromatin accessibility to regulate radiation sensitivity remains unclear. Our findings demonstrated that the histone demethylase inhibitor, 5-carboxy-8-hydroxyquinoline (IOX1), could enhance the radiosensitivity of non-small cell lung cancer (NSCLC) in vitro and in vivo. Mechanistically, IOX1 treatment reduced chromatin accessibility in the promoter region of DNA damage repair genes, leading to decreased DNA repair efficiency and elevated DNA damage induced by γ irradiation. Notably, IOX1 treatment significantly reduced both chromatin accessibility and the transcription of phytochrome interacting factor 1 (PIF1), a key player in telomere maintenance. Inhibition of PIF1 delayed radiation-induced DNA and telomeric DNA damage repair, as well as increased radiosensitivity of NSCLC in vitro and in vivo. Further study indicated that the above process was regulated by a reduction of transcription factor myc-associated zinc finger protein (MAZ) binding to the distal intergenic region of the PIF1. Taken together, IOX1-mediated demethylase inactivation reduced chromatin accessibility, leading to elevated telomere damage which is partly due to PIF1 inhibition, thereby enhancing NSCLC radiosensitivity.

Decreased DNA repair capacity caused by exposure to metal mixtures is modulated by the PARP1 rs1136410 variant in newborns from a polluted metropolitan area

BackgroundDNA damage caused by exposure to metal mixtures and the potential modulating role of genes involved in DNA repair and the antioxidant response have not been evaluated in newborns. AimThe aim was to evaluate the association between prenatal exposure to metal mixtures and DNA repair capacity (DRC) in newborns from the Metropolitan Area of Mexico City (MAMC), a heavily polluted area, and the impact of variants in genes involved in DNA repair and the antioxidant response on this association. MethodsWe analyzed cord blood samples obtained at delivery from 125 healthy newborns from the MAMC. Twenty-four elements were determined by inductively coupled plasma mass spectrometry (ICP‒MS), but only 12 (Cu, I, Se, Zn, As, Ba, Cs, Mn, Sb, Sr, Pb, and Ti) were quantified in most samples. DRC was assessed by the challenge-comet assay, and OGG1, PARP1, and NFE2L2 genotyping was performed with TaqMan probes. Metal mixtures were identified and analyzed using principal component analysis (PCA) and weighted quantile sum (WQS) regression. Independent adjusted linear regression models were used to evaluate the associations. ResultsA null DRC was observed in 46% of newborns. The metals with the highest concentrations were Mn, Sr, Ti, and Pb. Essential elements showed normal levels. Only the mixture characterized by increased As, Cs, Cu, Se, and Zn levels was inversely associated with DRC. As was the principal contributor (37.8%) in the negative direction in the DRC followed by Ba and Sb, according to the WQS regression. Newborns carrying of the derived (G) allele of the PARP1 rs1136410 variant showed decreased DRC by exposure to some potentially toxic metals (PTMs) (As, Cs, and Ba). ConclusionPrenatal exposure to metal mixtures negatively affected DRC in newborns, and the PARP1 rs1136410 variant had a modulating role in this association.

Androgen signaling stabilizes genomes to counteract senescence by promoting XRCC4 transcription.

Aging is accompanied by a decreased DNA repair capacity, which might contribute to age-associated functional decline in multiple tissues. Disruption in hormone signaling, associated with reproductive organ dysfunction, is an early event of age-related tissue degeneration, but whether it impacts DNA repair in nonreproductive organs remains elusive. Using skin fibroblasts derived from healthy donors with a broad age range, we show here that the downregulation of expression of XRCC4, a factor involved in nonhomologous end-joining (NHEJ) repair, which is the dominant pathway to repair somatic double-strand breaks, is mediated through transcriptional mechanisms. We show that the androgen receptor (AR), whose expression is also reduced during aging, directly binds to and enhances the activity of the XRCC4 promoter, facilitating XRCC4 transcription and thus stabilizing the genome. We also demonstrate that dihydrotestosterone (DHT), a powerful AR agonist, restores XRCC4 expression and stabilizes the genome in different models of cellular aging. Moreover, DHT treatment reverses senescence-associated phenotypes, opening a potential avenue to aging interventions in the future.

Genomic Instability and Epigenetic Changes during Aging.

Aging is considered the deterioration of physiological functions along with an increased mortality rate. This scientific review focuses on the central importance of genomic instability during the aging process, encompassing a range of cellular and molecular changes that occur with advancing age. In particular, this revision addresses the genetic and epigenetic alterations that contribute to genomic instability, such as telomere shortening, DNA damage accumulation, and decreased DNA repair capacity. Furthermore, the review explores the epigenetic changes that occur with aging, including modifications to histones, DNA methylation patterns, and the role of non-coding RNAs. Finally, the review discusses the organization of chromatin and its contribution to genomic instability, including heterochromatin loss, chromatin remodeling, and changes in nucleosome and histone abundance. In conclusion, this review highlights the fundamental role that genomic instability plays in the aging process and underscores the need for continued research into these complex biological mechanisms.

P19-01: Prenatal exposure to mixtures of potentially toxic and essential metals decreased DNA repair capacity in newborns from a polluted area

P19-01: Prenatal exposure to mixtures of potentially toxic and essential metals decreased DNA repair capacity in newborns from a polluted area

Augmented oxidative stress, accumulation of DNA damage and impaired DNA repair mechanisms in thrombotic primary antiphospholipid syndrome

Antiphospholipid syndrome (APS) is a rare autoimmune disorder with complex pathogenesis. Studies have shown that oxidative stress may contribute to APS pathophysiology. In peripheral blood mononuclear cells (PBMCs) from thrombotic Primary APS (thrPAPS) patients and age/sex-matched healthy controls (HC), as well as a control group of asymptomatic antiphospholipid antibody (aPL) positive individuals without APS (aPL+/non-APS), we examined oxidative stress, abasic (apurinic/apyrimidinic) sites, and DNA damage response (DDR)-associated parameters, including endogenous DNA damage (single- and double-strand breaks) and DNA repair mechanisms, namely nucleotide excision repair (NER) and double-strand breaks repair (DSB/R). We found that thrPAPS patients exhibited significantly higher levels of endogenous DNA damage, increased oxidative stress and abasic sites, as well as lower NER and DSB/R capacities versus HC (all P < 0.001) and versus aPL+/non-APS subjects (all P < 0.05). Our findings demonstrate that oxidative stress and decreased DNA repair mechanisms contribute to the accumulation of endogenous DNA damage in PBMCs from thrPAPS patients and, if further validated, may be exploited as therapeutic targets and potential biomarkers.

Loss of the DNA repair gene RNase H2 and a unique subset of DDR-deficient leiomyosarcomas.

11549 Background: Targeting the DNA damage response (DDR)/Homologous Recombination (HR) pathway is an emerging therapeutic approach for leiomyosarcoma (LMS). Loss of RNase H2 decreases DNA repair via the Non-Homologous End-Joining (NHEJ) pathway, leading to increased double stranded breaks, replication stress, and increased cell death. Therefore, we developed an assay to screen for RNase H2 loss in STS patient samples to determine its prevalence and prognostic significance, particularly in LMS patients. Methods: RNASEH2B homozygous deletion (HomDel) calls from TCGA samples were based on the Allele-Specific Copy number Analysis of Tumors (ASCAT2) algorithm. Immunohistochemistry (IHC) of RNase H2 was performed on tissue microarrays (TMAs) of uterine (U-LMS) and soft tissue (ST-LMS) leiomyosarcoma samples from MD Anderson Cancer Center (MDACC) using a selective antibody developed by Repare Therapeutics. RNase H2 loss was defined as &lt; = 10% cells without nuclear IHC staining and scored by experienced pathologists in 2 separate cohorts. Genomic analysis was performed using SNiPDx™, a targeted-NGS assay designed to detect biallelic loss of function in select DDR related genes and whole genome sequencing (WGS). MDACC TMA samples were clinically annotated by retrospective review. Results: Using TCGA data, RNASEH2B HomDels were seen in 6% (5/80) of all LMS cases, with a higher proportion in U-LMS (15%; 4/27) as compared to ST-LMS (2%; 1/53). In a pan-tumor TMA, RNase H2 loss by IHC was found in 3.8% (32/843) of samples overall, and in 10% (9/88) of LMS samples. This proportion of RNase H2 loss was higher in a larger MDACC LMS cohort, where negative staining of RNase H2 was found in 30% (33/110) of U-LMS and 38% (39/102) of ST-LMS cases. 30 MDACC LMS cases (15 U-LMS and 15 ST-LMS) were analyzed for RNASEH2B HomDels by SNiPDx. In U-LMS, RNASEH2B HomDels were detected in 64% (7/11) of RNase H2 IHC loss cases vs. 0% (0/3) in RNase H2 IHC intact cases. No RNASEH2B HomDels were detected in ST-LMS cases (10 IHC loss, 5 intact). The median overall survival (mOS) of MDACC U-LMS patients (n = 109) was 4.3 years. No significant mOS difference was seen in RNase H2 IHC intact cases versus loss (mOS 4.4 v 3.3 years, p = 0.54). In a separate cohort, 45 U-LMS samples were screened by IHC; 22% (10/45) had RNase H2 loss. RNASEH2B HomDels were detected in 70% (7/10) of RNase H2 IHC loss cases using SNiPDx and confirmed using WGS. In contrast, RNASEH2B HomDels were detected in 3% (1/33) of RNase H2 IHC intact cases. In the combined U-LMS cohort with IHC and SNiPDx results (n = 71), the diagnostic accuracy, sensitivity and specificity of RNase H2 IHC for detecting RNASEH2B HomDels was 76%, 93% and 71% respectively. Conclusions: RNase H2 loss via RNASEH 2B HomDels is prevalent in U-LMS and is unique from other DDR pathway alterations. RNase H2 IHC is an effective screening tool and is being developed for future clinical trials targeting DDR in LMS.

MP20-02 27 HYDROXYCHOLESTEROL AND DNA DAMAGE REPAIR: IMPLICATIONS IN PROSTATE CANCER

You have accessJournal of UrologyCME1 Apr 2023MP20-02 27 HYDROXYCHOLESTEROL AND DNA DAMAGE REPAIR: IMPLICATIONS IN PROSTATE CANCER Cecilia G. Galvan, Nadine A. Friedrich, Sanjay Das, James P. Daniels, Michael R. Freeman, Ryusuke Suzuki, Sergio Sanders, Sara Pollan, Shweta Dambal, and Stephen J. Freedland Cecilia G. GalvanCecilia G. Galvan More articles by this author , Nadine A. FriedrichNadine A. Friedrich More articles by this author , Sanjay DasSanjay Das More articles by this author , James P. DanielsJames P. Daniels More articles by this author , Michael R. FreemanMichael R. Freeman More articles by this author , Ryusuke SuzukiRyusuke Suzuki More articles by this author , Sergio SandersSergio Sanders More articles by this author , Sara PollanSara Pollan More articles by this author , Shweta DambalShweta Dambal More articles by this author , and Stephen J. FreedlandStephen J. Freedland More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Recent studies from our group have reported that cholesterol homeostasis in prostate cancer (PC) is controlled by 27-hydroxycholesterol (27HC) and that CYP27A1, the enzyme that converts cholesterol to 27HC, acts as a cellular cholesterol sensor in prostate cells. Furthermore, the loss of the CYP27A1/27HC axis is correlated with PC pathogenesis. Herein, we sought to better understand the mechanism of 27HC-mediated anti-PC effects. METHODS: LNCaP (AR+) and DU145 (AR-) cells were treated with 27HC or control. Transcriptome profiling was done with Affymetrix GeneChip™ microarray assay and key changes validated by qPCR. Differential expression and gene set enrichment analysis was done using the GSEA software with hallmark gene sets from MSigDB. Human transcriptome signatures were analyzed from the Prostate Cancer Transcriptome Atlas (PCTA). DNA damage was assessed via comet assays and cell growth using proliferation assays. RESULTS: In a transcriptome analysis, 27HC treatment resulted in downregulation of Hallmark pathways related to DNA damage repair, decreased expression of key cell cycle genes, and induced BRCAness (Figure 1). FEN1 and RAD51 decreased expression were confirmed by qPCR. Using publicly available data, we found a correlation between higher CYP27A1 expression (i.e. higher intracellular 27HC) and decreased DNA repair gene expression among 1,321 human transcriptomes. While testing the clinical implications of this, we noted that 27HC treatment increased DNA damage in PC cells via neutral comet assay. Effects were reversible by adding back cholesterol, but not androgens. Finally, in combination with Olaparib, a PARP inhibitor, we showed additive anti-tumor activity. CONCLUSIONS: Our research suggests 27HC treatment, via cholesterol depletion, induces BRCAness and leads to increased DNA damage. This DNA damage when combined with PARP inhibitors leads to additive anti-PC effects and may be a potential effective therapy in PC. Additional studies are needed to directly test whether 27HC creates a synthetic lethality to PARP inhibitors and other DNA damaging agents. Source of Funding: Dr. Galvan is funded by the Department of Defense Health Disparities Research Fellowship. Dr. Das was supported by the Patient Centered Outcomes in Urological and Gynecological Cancers (PCORT UroGynCan). Dr. Daniels was funded by the NIH grants TL1 DK132768 and U2C DK129496. Dr. Friedrich was funded by NIH grant T32 CA240172-03. Other funds: Department of Defense Prostate Cancer Research Program © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e275 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Cecilia G. Galvan More articles by this author Nadine A. Friedrich More articles by this author Sanjay Das More articles by this author James P. Daniels More articles by this author Michael R. Freeman More articles by this author Ryusuke Suzuki More articles by this author Sergio Sanders More articles by this author Sara Pollan More articles by this author Shweta Dambal More articles by this author Stephen J. Freedland More articles by this author Expand All Advertisement PDF downloadLoading ...

Transcriptome analysis of high mortality phenomena in polyploid fish embryos: An allotriploid embryo case study in hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂)

Triploidization of diploid fish is an ideal means for achieving polyploid breeding. During the preparation of allotriploid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂), the allotriploid embryos (T) had a much higher morality from the high blastula to middle gastrula stage compared to the normal hybrid diploids (D). Therefore, the internal regulation of gene expression in allotriploid fertilized grouper eggs was explored by comparing the transcriptional profiles of T and D mapped at the high blastula (1), middle gastrula (2), and crystal (3) stages using the Illumina RNA-seq technology. There were 2357 differentially expressed genes (DEGs) found only in T1_vs_T2 and 1585 DEGs found only in T2_vs_T3. According to the GO functional enrichment analysis, when T developed from the high blastula to middle gastrula stage (i.e., pre-embryonic development; T1_vs_T2), the DEGs showed significant enrichment in terms associated with the cell cycle and cytoskeleton, indicating that cell proliferation was inhibited. KEGG pathway analysis of DEGs showed significant enrichment in mRNA processing and translation and in pathways associated with programmed cell death (PCD). Cell differentiation can trigger PCD and the combined effects of up-regulated ripk1, dnm1l, pgam5, Slc25a5, and vdac1 expression and down-regulated fadd expression during pre-embryonic development may have been the main cause of mass mortality in embryos up until the middle gastrula stage. A two-by-two comparison of D2_vs_T2 DEGs at the middle gastrula stage to the aforementioned DEGs expressed only in T revealed that the Parp4 and pole were down-regulated in the pre-development of T, which might lead to decreased DNA repair efficiency. These results could have reflected either (1) the altered physiological state of the cells due to cold-shock treatment or (2) the sensitivity of pre-embryonic development to an elevated number of chromosomes, which induced disrupted gene expression and abnormal cell division. In conclusion, the above study helps explain why allotriploid grouper exhibit high mortality at the middle gastrula stage, improves our understanding of the regulation of triploid embryo development, and provides basic information for reducing mortality at the middle gastrula stage.

Age, sex, and apolipoprotein E isoform alter contextual fear learning, neuronal activation, and baseline DNA damage in the hippocampus

Age, female sex, and apolipoprotein E4 (E4) are risk factors to develop Alzheimer’s disease (AD). There are three major human apoE isoforms: E2, E3, and E4. Compared to E3, E4 increases while E2 decreases AD risk. However, E2 is associated with increased risk and severity of post-traumatic stress disorder (PTSD). In cognitively healthy adults, E4 carriers have greater brain activation during learning and memory tasks in the absence of behavioral differences. Human apoE targeted replacement (TR) mice display differences in fear extinction that parallel human data: E2 mice show impaired extinction, mirroring heightened PTSD symptoms in E2 combat veterans. Recently, an adaptive role of DNA double strand breaks (DSBs) in immediate early gene expression (IEG) has been described. Age and disease synergistically increase DNA damage and decrease DNA repair. As the mechanisms underlying the relative risks of apoE, sex, and their interactions in aging are unclear, we used young (3 months) and middle-aged (12 months) male and female TR mice to investigate the influence of these factors on DSBs and IEGs at baseline and following contextual fear conditioning. We assessed brain-wide changes in neural activation following fear conditioning using whole-brain cFos imaging in young female TR mice. E4 mice froze more during fear conditioning and had lower cFos immunoreactivity across regions important for somatosensation and contextual encoding compared to E2 mice. E4 mice also showed altered co-activation compared to E3 mice, corresponding to human MRI and cognitive data, and indicating that there are differences in brain activity and connectivity at young ages independent of fear learning. There were increased DSB markers in middle-aged animals and alterations to cFos levels dependent on sex and isoform, as well. The increase in hippocampal DSB markers in middle-aged animals and female E4 mice may play a role in the risk for developing AD.

CTP synthase 2 predicts inferior survival and mediates DNA damage response via interacting with BRCA1 in chronic lymphocytic leukemia

BackgroundCytidine triphosphate synthase 2 (CTPS2) is an essential metabolic enzyme that catalyzes the biosynthesis of CTP. CTP synthases contribute to lymphocytes proliferation and tumorigenesis, but the role of CTPS2 in chronic lymphocytic leukemia (CLL) remains undefined.MethodsIn silico analysis was performed to quantified the expression and clinical analysis of CTPS2 and BRCA1. The expression was then validated on the internal sets. Loss-and gain-of-function assays were conducted to investigate the physiological phenotypes in CLL. RNA-seq was employed to probe the molecular mechanism of CTPS2.ResultsHerein, significant elevated expression of CTPS2 was observed in CLL patients compared to normal CD19 + B cells, which was verified in three independent cohorts. Furthermore, overexpression of CTPS2 was closely associated with undesired prognostic indicators, including unmutated IGHV status and chromosome 11q23 deletion. Additionally, elevated CTPS2 expression predicted adverse overall survival and treatment-free survival with independent prognostic significance. Downregulation of CTPS2 in CLL cells exhibited attenuated cell proliferation, arrested G2/M cell cycle and increased apoptosis. The addition of CTP or glutamine could reverse the above effects. Since RNA-seq showed the enrichment in DNA damage and response signaling, we subsequently found that silence of CTPS2 remarkably elevated DNA damage and decreased DNA repair. It was demonstrated that CTPS2 mediated DNA damage response via interacting with Breast Cancer 1 (BRCA1) protein in CLL through CoIP assays and rescued experiments.ConclusionsCollectively, our study generated the novel findings that CTPS2 promoted CLL progression via DNA damage response and repair pathway. Targeting nucleotide metabolism potentially became an attractive strategy for treatment against CLL.

Complementary anti-cancer pathways triggered by inhibition of sideroflexin 4 in ovarian cancer

DNA damaging agents are a mainstay of standard chemotherapy for ovarian cancer. Unfortunately, resistance to such DNA damaging agents frequently develops, often due to increased activity of DNA repair pathways. Sideroflexin 4 (SFXN4) is a little-studied inner mitochondrial membrane protein. Here we demonstrate that SFXN4 plays a role in synthesis of iron sulfur clusters (Fe-S) in ovarian cancer cells and ovarian cancer tumor-initiating cells, and that knockdown of SFXN4 inhibits Fe-S biogenesis in ovarian cancer cells. We demonstrate that this has two important consequences that may be useful in anti-cancer therapy. First, inhibition of Fe-S biogenesis triggers the accumulation of excess iron, leading to oxidative stress. Second, because enzymes critical to multiple DNA repair pathways require Fe-S clusters for their function, DNA repair enzymes and DNA repair itself are inhibited by reduction of SFXN4. Through this dual mechanism, SFXN4 inhibition heightens ovarian cancer cell sensitivity to DNA-damaging drugs and DNA repair inhibitors used in ovarian cancer therapy, such as cisplatin and PARP inhibitors. Sensitization is achieved even in drug resistant ovarian cancer cells. Further, knockout of SFXN4 decreases DNA repair and profoundly inhibits tumor growth in a mouse model of ovarian cancer metastasis. Collectively, these results suggest that SFXN4 may represent a new target in ovarian cancer therapy.

Impact of Nuclear De Novo NAD+ Synthesis via Histone Dynamics on DNA Repair during Cellular Senescence To Prevent Tumorigenesis.

NAD+ synthesis is a fundamental process in living cells. The effects of local metabolite production on chromatin influence the epigenetic status of chromatin in DNA metabolism. We have previously shown that K5 acetylation of H2AX by TIP60 is required for the ADP ribosylation activity of PARP-1, for histone H2AX exchange at DNA damage sites. However, the detailed molecular mechanism has remained unclear. Here, we identified de novo NAD synthetase 1 (NAD syn1) as a novel binding partner to H2AX. The enzymatic activity of NAD syn1 is crucial for the ADP ribosylation activity of PARP-1 for the H2AX dynamics at sites of DNA damage. Inhibition of the NAD synthetase activity in the cell nucleus decreased the overall cellular NAD+ concentration, leading to cellular senescence. Accordingly, the acetylation-dependent H2AX dynamics and homologous recombination repair were suppressed, leading to increased tumorigenesis. Our findings have revealed the importance of de novo NAD+ production in the cell nucleus for protection against the decreased DNA repair capacity caused by cellular senescence and thus against tumorigenesis.

RF07 | PSUN384 Hepatocyte Deletion of IGF2 Prevents DNA Damage and Tumor Formation in Hepatocellular Carcinoma

Abstract Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide with a 18% 5-year survival rate. Mice lacking the splicing factor SRSF3 in hepatocytes (SKO mice) overexpress IGF2 and are predisposed to developing HCC with age. Loss of Igf2 in the SKO background prevented hepatic fibrosis and inflammation, and completely prevented tumor formation. This was associated with decreased proliferation, apoptosis and DNA damage, and restored DNA repair enzyme expression. Mechanistically, IGF2 treatment of HepG2 cells in vitro caused DNA damage and decreased DNA repair enzyme expression, and tumors from the SKO mice show mutational signatures consistent with double strand break and defective mismatch repair. Looking at human data, HCC patients with high IGF2 mRNA expression had worse survival compared to patients with normal IGF2 expression. The patients also showed a switch in IGF2 promoter usage that correlated with increased expression. HCC patients having high SRSF3 mRNA expression also showed poor survival as did patients with alterations in known SRSF3-dependent splicing events. The level of SRSF3 protein was decreased 6-fold in human HCC tissues compared with normal liver tissues. The results indicate that IGF2 overexpression in conjunction with reduced SRSF3 splicing activity could be a major cause of DNA damage and driver of liver cancer. Presentation: Saturday, June 11, 2022 1:18 p.m. - 1:23 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

LMO2 expression is frequent in T-lymphoblastic leukemia and correlates with survival, regardless of T-cell stage

AbstractT- lymphoblastic leukemia/lymphoma (T-LL) is an aggressive malignancy of immature T-cells with poor overall survival (OS) and in need of new therapies. LIM-domain only 2 (LMO2) is a critical regulator of hematopoietic cell development that can be overexpressed in T-LL due to chromosomal abnormalities. Deregulated LMO2 expression contributes to T-LL development by inducing block of T-cell differentiation and continuous thymocyte self-renewal. However, LMO2 expression and its biologic significance in T-LL remain largely unknown. We analyzed LMO2 expression in 100 initial and follow-up biopsies of T-LL from 67 patients, including 31 (46%) early precursor T-cell (ETP)-ALL, 26 (39%) cortical and 10 (15%) medullary type. LMO2 expression was present in 50 (74.6%) initial biopsies with an average of 87% positive tumor cells (range 30–100%). LMO2 expression in ETP, medullary and cortical T-LLs was not statistically different. In patients with biopsies after initial therapy, LMO2 expression was stable. LMO2 expression was associated with longer OS (p = 0.048) regardless of T-lymphoblast stage or other clinicopathologic features. These findings indicate that LMO2 is a promising new prognostic marker that could predict patients' outcomes and potentially be targeted for novel chemotherapy, i.e. PARP1/2 inhibitors, which have been shown to enhance chemotherapy sensitivity in LMO2 expressing diffuse large B cell lymphoma (DLBCL) tumors by decreasing DNA repair efficiency.