DNA Repair Capacity Research Articles

SMAD4 Limits PARP1 dependent DNA Repair to Render Pancreatic Cancer Cells Sensitive to Radiotherapy

AbstractDysregulation of SMAD4 (i.e. somatic mutation) is strongly associated with poor pancreatic ductal adenocarcinoma (PDAC) prognosis, yet the molecular mechanisms remain underlying this relationship obscure. Previously, we discovered that SMAD4 mutation renders pancreatic cancer resistant to radiotherapy via promotion of autophagy. In the current work, we observed a downregulation of the protein level of SMAD4 in PDAC as compared with adjacent normal tissue, and that such SMAD4low PDAC failed to benefit from chemotherapy. Furthermore, we observed that SMAD4 depletion dramatically enhanced DNA repair capacity in response to irradiation (IR) or a radiomimetic chemical. Interestingly, we found the radiomimetic chemical having induced a robust translocation of SMAD4 into the nucleus, where a direct interaction was shown to occur between the MH1 domain of SMAD4 and the DBD domain of PARP1. Functionally, the SMAD4-PARP1 interaction was found to perturb the recruitment of PARP1 to DNA damage sites. Accordingly, the combination of olaparib and radiotherapy was indicated in vivo and in vitro to specifically reduce the growth of SMAD4-deficient PDAC by attenuating PARP1 activity. Collectively, our results revealed a novel molecular mechanism for the involvement of the SMAD4-PARP1 interaction in DNA repair with a vital role in radiotherapy response in PDAC. Based on our set of findings, our findings offer a new combined therapeutic strategy for SMAD4 deficient PDAC that can significantly reduce pancreatic cancer radiotherapy resistance.

Potential Strategies for Overcoming Drug Resistance Pathways Using Propolis and Its Polyphenolic/Flavonoid Compounds in Combination with Chemotherapy and Radiotherapy

Conventional cancer treatments include surgical resection, chemotherapy, hyperthermia, immunotherapy, hormone therapy, and locally targeted therapies such as radiation therapy. Standard cancer therapies often require the use of multiple agents, which can activate nuclear factor kappa B (NF-κB) in tumor cells, leading to reduced cell death and increased drug resistance. Moreover, the use of multiple agents also contributes to added toxicity, resulting in poor treatment outcomes. Cancer cells gradually develop resistance to almost all chemotherapeutics through various mechanisms, such as drug efflux, alterations in drug metabolism and transport, changes in signal transduction pathways, enhanced DNA repair capacity, evasion of apoptosis, increased mutations, reactivation of drug targets, interaction with the cancer microenvironment, cancer cell-stroma interactions, epithelial–mesenchymal transition (EMT)-mediated chemoresistance, epigenetic modifications, metabolic alterations, and the effect of cancer stem cells (CSCs). Developing new strategies to improve chemotherapy sensitivity while minimizing side effects is essential for achieving better therapeutic outcomes and enhancing patients’ quality of life. One promising approach involves combining conventional cancer treatments with propolis and its flavonoids. These natural compounds may enhance tumor response to treatment while reducing toxicity. Propolis and its components can sensitize cancer cells to chemotherapeutic agents, likely by inhibiting NF-κB activation, reprogramming tumor-associated macrophages (TAMs; an M2-like phenotype), and thereby reducing the release of matrix metalloproteinase (MMP)-9, cytokines, chemokines, and the vascular endothelial growth factor (VEGF). By reducing TAMs, propolis and its components may also overcome EMT-mediated chemoresistance, disrupt the crosstalk between macrophages and CSCs, inhibit the maintenance of stemness, and reverse acquired immunosuppression, thus promoting an antitumor response mediated by cytotoxic T-cells. This review highlights the potential of flavonoids to modulate the responsiveness of cancer to conventional treatment modalities. The evidence suggests that novel therapeutic strategies incorporating flavonoids could be developed to improve treatment outcomes. The positive effects of combining propolis with chemotherapeutics include reduced cytotoxicity to peripheral blood leukocytes, liver, and kidney cells. Therefore, polyphenolic/flavonoid components may hold potential for use in combination with chemotherapeutic agents in the clinical treatment of various types of cancers.

Roles of chromatin and genome instability in cellular senescence and their relevance to ageing and related diseases.

Ageing is a complex biological process in which a gradual decline in physiological fitness increases susceptibility to diseases such as neurodegenerative disorders and cancer. Cellular senescence, a state of irreversible cell-growth arrest accompanied by functional deterioration, has emerged as a pivotal driver of ageing. In this Review, we discuss how heterochromatin loss, telomere attrition and DNA damage contribute to cellular senescence, ageing and age-related diseases by eliciting genome instability, innate immunity and inflammation. We also discuss how emerging therapeutic strategies could restore heterochromatin stability, maintain telomere integrity and boost the DNA repair capacity, and thus counteract cellular senescence and ageing-associated pathologies. Finally, we outline current research challenges and future directions aimed at better comprehending and delaying ageing.

Changes in DNA repair compartments and cohesin loss promote DNA damage accumulation in aged oocytes.

Oocyte loss, a natural process that accelerates as women approach their mid-30s, poses a significant challenge to female reproduction. Recent studies have identified DNA damage as a primary contributor to oocyte loss, but the mechanisms underlying DNA damage accumulation remain unclear. Here, we show that aged oocytes have a lower DNA repair capacity and reduced mobility of DNA damage sites compared to young oocytes. Incomplete DNA repair in aged oocytes results in defective chromosome integrity and partitioning, thereby compromising oocyte quality. We found that DNA repair proteins are arranged in spatially distinct DNA repair compartments that form during the late stages of oocyte growth, accompanied by changes in the activity of DNA repair pathways. We demonstrate alterations in these compartments with age, including substantial changes in the levels of key DNA repair proteins and a shift toward error-prone DNA repair pathways. In addition, we show that reduced cohesin levels make aged oocytes more vulnerable to persistent DNA damage and cause changes in DNA repair compartments. Our study links DNA damage accumulation in aged oocytes, a leading cause of oocyte loss, to cohesin deterioration and changes in the organization, abundance, and response of DNA repair machinery.

Abstract A034: DNA repair capacity in metastatic castration-resistant prostate cancer patients using lymphocytes as surrogate markers

Abstract Prostate cancer (PCa) is one of the most commonly diagnosed cancers in men from the United States (US) and Puerto Rico (PR). In 2024, around 35,250 PCa-related deaths will occur in the US, making PCa the second leading cause of cancer-related mortality in men in the US. In Puerto Rican men, PCa represents the first cause of cancer incidence (38%) and mortality (19%). Metastatic castrate-resistant prostate cancer (mCRPC) remains among the most frequent causes of cancer-related mortality in males. Patients with mCRPC become progressively resistant to androgen deprivation therapy, and the median survival for these patients is around two years. Ortiz-Sanchez et al. (2022) previously reported that the nucleotide excision repair (NER) pathway is dysregulated in the lymphocytes from patients with indolent and aggressive PCa; nevertheless, mCRPC cases were not included in the study design. Therefore, this pilot study aimed to evaluate the DNA repair capacity (DRC) in Puerto Rican men with and without mCRPC through the CometChip assay using lymphocytes as surrogate markers. Variations in mean DRC values were expected among the study groups. DRC measurements through the NER pathway were performed in a cohort of 96 participants, including PCa cases (n=71) and controls (n=25). The mean DRC value for the control group was 17.63%, and for the cases, it was 7.90%. When comparing mCRPC (n=16), non-mCRPC (n=55), and controls, significant differences were observed between the controls and each cancer group. However, no significant differences were found between PCa cases stratified by disease aggressiveness. Currently, we are evaluating the functionality of the homologous recombination repair pathway to gain additional insights into the role of DRC in PCa. Our results represent the first effort to apply a phenotypic assay to evaluate the overall DRC in mCRPC. This may improve patient diagnosis and patient stratification in terms of disease progression. Citation Format: Jaime Matta, Jarline Encarnación-Medina, Gilberto Ruiz-Deya, Valerie Rodriguez-Baez, Carmen Ortiz-Sanchez. DNA repair capacity in metastatic castration-resistant prostate cancer patients using lymphocytes as surrogate markers [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr A034.

Abstract C028: Challenging PDAC adaptability: Uncovering therapeutic vulnerabilities in APE1 DNA repair mechanisms

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a therapeutically challenging malignancy notorious for its resilience to therapeutic strategies and ability to thrive under extreme conditions. The cancer's inherent ability to adaptively resist treatments underscores the need for novel and combinatorial therapeutic approaches. Our research focuses on elucidating the role of Apurinic-apyrimidinic endonuclease/Redox effector factor 1 (APE1/Ref-1), a critical multifunctional protein in PDAC pathobiology. APE1 serves two essential functions: as the principal endonuclease in the base excision repair (BER) pathway for DNA repair and as a redox regulator of transcription factors essential for cancer cell proliferation. While the therapeutic targeting of APE1’s redox function has been effective and continues to yield significant advances, the development of treatments aimed at its DNA repair function has proven more challenging with comparatively limited progress. Since standard PDAC treatment regimens involve DNA damaging agents potentially repaired via BER and APE1, we engineered three mutant cell lines with knock-in mutations resulting in a reduction of APE1’s BER activity while maintaining full redox activity. Biochemical evaluations confirmed the effects of the mutation, revealing a 30-fold reduction in DNA repair capacity. Surprisingly, with respect to growth and proliferation, the mutant and wild-type cell lines behaved similarly. Analyses of cytotoxic vulnerabilities revealed similar findings with the mutant lines exhibiting minimal phenotypic differences in sensitivity when challenged with cytotoxic and DNA damaging agents in proliferation-based assays. These findings led us to posit that the mutant cell lines were relying on compensatory DNA repair pathways which were providing sufficient compensation for the decrease in APE1 activity. We further hypothesized that the cell lines may still have hidden, long-term deficiencies in DNA damage processing which are not apparent during the initial stress response. Subsequent colony formation assays revealed that the mutant cell lines do exhibit poor long-term survival even after only short-term exposure to DNA damage. In vivo orthotopic mouse model experiments further corroborated these findings, showing a significant reduction in tumor size and decreased metastasis to the lungs and liver in mutant cell lines compared to wild-type controls. While APE1 is the primary endonuclease in the BER pathway, studies have demonstrated APE1-independent BER in rare, specific contexts. We are probing the hypothesis that these APE1-independent BER pathways function as the compensatory mechanism safeguarding these cells in short-term stress situations. Identifying and characterizing these backup pathways will not only offer a better understanding of APE1’s DNA repair capabilities within PDAC but will also provide a better understanding of how DNA repair mechanisms can be therapeutically targeted for novel therapeutic options or combinations. This opens the path to enhance standard of care treatments such as FOLFIRINOX. Citation Format: Eyram K Kpenu, Randall Wireman, Mahmut Mijiti, Silpa Gampala, Melissa L Fishel, Mark R Kelley. Challenging PDAC adaptability: Uncovering therapeutic vulnerabilities in APE1 DNA repair mechanisms [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C028.

NLRP4 renders pancreatic cancer resistant to olaparib through promotion of the DNA damage response and ROS-induced autophagy

Olaparib has been approved as a therapeutic option for metastatic pancreatic ductal adenocarcinoma patients with BRCA1/2 mutations. However, a significant majority of pancreatic cancer patients have inherent resistance or develop tolerance to olaparib. It is crucial to comprehend the molecular mechanism underlying olaparib resistance to facilitate the development of targeted therapies for pancreatic cancer. In this study, we conducted an analysis of the DepMap database to investigate gene expression variations associated with olaparib sensitivity. Our findings revealed that NLRP4 upregulation contributes to increased resistance to olaparib in pancreatic cancer cells, both in vitro and in vivo. RNA sequencing and Co-IP MS analysis revealed that NLRP4 is involved in the DNA damage response and autophagy pathway. Our findings confirmed that NLRP4 enhances the capacity for DNA repair and induces the production of significant levels of reactive oxygen species (ROS) and autophagy in response to treatment with olaparib. Specifically, NLRP4-generated mitochondrial ROS promote autophagy in pancreatic cancer cells upon exposure to olaparib. However, NLRP4-induced ROS do not affect DNA damage. The inhibition of mitochondrial ROS using MitoQ and autophagy using chloroquine (CQ) may render cells more susceptible to the effects of olaparib. Taken together, our findings highlight the significant roles played by NLRP4 in the processes of autophagy and DNA repair when pancreatic cancer cells are treated with olaparib, thereby suggesting the potential therapeutic utility of olaparib in pancreatic cancer patients with low NLRP4 expression.

Efficacy of FERscore in predicting sensitivity to ferroptosis inducers in breast cancer

Recent studies have highlighted the potential of ferroptosis in treating breast cancer. However, the efficacy of ferroptosis induction in the most common subtype, estrogen receptor-positive (ER + ) breast cancer, remains inadequately explored. This study unveils that both short-term and long-term treatment with ER-targeted endocrine agents sensitizes ER+ breast cancer cells to ferroptosis inducers, particularly the GPX4 inhibitor, revealing a non-mutational sensitization mechanism. Based on this finding, we introduce a 55-gene signature score (FERscore) tailored to assess ferroptosis susceptibility in breast cancer. Data from cell lines and primary tumors demonstrate significant lower FERscores in ER+ breast cancer compared to other subtypes; however, FERscores dramatically increase in endocrine-resistant ER+ tumor cells and residual tumors post-endocrine therapy. Furthermore, FERscore correlates positively with mesenchymal traits, stemness, immune cell infiltration, and cancer-associated fibroblasts enrichment, while inversely correlating with estrogen responsiveness and DNA repair capacity. Additionally, the FERscore proves effective in predicting therapeutic responses to anti-ER, anti-HER2, poly (ADP-ribose) polymerase inhibitor, and anti-angiogenesis therapies in breast cancer. In summary, ferroptosis induction emerges as a promising avenue in breast cancer therapy. The FERscore offers an innovative tool for identifying patients who may benefit from ferroptosis-inducing therapies, especially those responsive to GPX4 inhibitors.

Analysis of heterochromatin remodeling as a mechanism of radiotherapy (RT) resistance through molecular profiling of radioresistant (RR) head and neck (HNC) and prostate cancers (PCa).

137 Background: Resistance to RT remains a clinical challenge, given our limited understanding of the molecular pathways underpinning radioresistance. Here, we investigated for mechanisms linked to RR using paired experimental and clinico-molecular HNC and PCa datasets for discovery and validation of novel pathways that may be enriched in RR cancers. Methods: For experimental data, we generated RR HNC (FaDu, HK1) and PCa (22Rv1, DU145) cell lines following high-dose X-irradiation (90 Gy in 45 fr). Genotypic characterization of RR and wildtype (WT) cells was performed by genomic and transcriptomic sequencing using WES (100X) and RNAseq (Illumina, CA), respectively, followed by functional characterization by western blot (WB) and immunofluorescence (IF). For clinico-molecular data, we utilized two prospectively recruited RT cohorts, consisting of 311 patients with HNC (n=158) and PCa (n=153). Tumor transcriptomes were profiled using RNAseq and Affymetrix ST array (ThermoFisher, CA), respectively. Comparative analyses of molecular profiles were first performed between RR and WT in vitro models to identify dysregulated pathways that were linked to radioresistance, and subsequently tested for association with disease-free survival (DFS) in the clinical cohorts. Results: Comparative genomic analyses between RR and WT HNC and PCa models revealed an abundance of acquired mutations in the RR compared with WT models, with a higher mutation count observed in PCa than HNC cells (SNV counts: 2,158 [DU145-RR] and 1,387 [22Rv1-RR] vs 396 [FaDu-RR] and 25 [HK1-RR], P<0.0001). Mutational signature analyses indicated a common enrichment of DNA mismatch repair-related mutational signatures (SBS3, SBS14, and SBS21) across the RR HNC and PCa models. Transcriptomic profiling revealed an upregulation of the BAHD1 gene, which is involved in heterochromatin formation, across the 4 RR cell lines, corroborated by an enrichment of heterochromatin-related genesets. These results were consistent with functional characterization of RR versus WT cells by WB and IF indicating increased DNA repair capacity and heterochromatin response post-4 Gy irradiation. We further confirmed the dependency of the RR phenotype on the heterochromatin response with BAHD1 knockdown, resulting in reversal of these cellular responses. Finally, our results were supported by survival analyses indicating an inferior DFS post-RT in patients with HNC and PCa harboring a higher expression of heterochromatin-related geneset (HNC: HRhigh vs low 1.51 ; PCa: HRhigh vs low 1.37 ). Conclusions: Herein, by leveraging on paired experimental and clinico-molecular datasets, we have uncovered a novel BAHD1-dependent heterochromatin response that underpins resistance to RT in HNC and PCa, which may be amendable to systemic agents targeting chromatin remodeling.

Redox homeostasis and pH monitoring: a step forward to personalized medicine and dermatology

Skin is continually subjected to various environmental stressors, such as pollution, UV rays, and other oxidative damage factors. It is obligatory to ascertain the exact importance of redox status and ROS indicators, which may be assessed in diverse physiological fluids and tissues. Reactive oxygen species (ROS) can cause lipid peroxidation, which can compromise the integrity and functionality of skin cells by degrading membrane lipids. This may compromise the skin barrier, making the skin more prone to infections and environmental irritants, increasing water loss and dryness. Collagen in the dermis can covalently link to malondialdehyde (MDA), a byproduct of lipid peroxidation generated by oxidatively damaged skin lipids, to form MDA-collagen adducts. Several types of skin cancer are associated with a heightened vulnerability of DNA purine and pyrimidine bases to oxidative damage and altered DNA repair capacity (DRC). Damage from ROS may cause hyaluronate to become less viscous, which increases its susceptibility to degradation. Elastin hydrodynamic radius decreased the rate of self-assembly in response to ROS. Alterations in redox homeostasis can lead to various diseases and conditions, including vitiligo, actinic keratosis, psoriasis, contact dermatitis, systemic sclerosis, the onset of wrinkles, and skin aging. The most recent formulations' capacity to improve pH and Redox simultaneously can be attained by carefully selecting proposed antioxidant formulations. The combination of modern preparations is currently available and should be used in accordance with the suggested guidelines.

BRCA1 orchestrates the response to BI-2536 and its combination with alisertib in MYC-driven small cell lung cancer

PLK1 is currently at the forefront of mitotic research and has emerged as a potential target for small cell lung cancer (SCLC) therapy. However, the factors influencing the efficacy of PLK1 inhibitors remain unclear. Herein, BRCA1 was identified as a key factor affecting the response of SCLC cells to BI-2536. Targeting AURKA with alisertib, at a non-toxic concentration, reduced the BI-2536-induced accumulation of BRCA1 and RAD51, leading to DNA repair defects and mitotic cell death in SCLC cells. In vivo experiments confirmed that combining BI-2536 with alisertib impaired DNA repair capacity and significantly delayed tumor growth. Additionally, GSEA analysis and loss- and gain-of-function assays demonstrated that MYC/MYCN signaling is crucial for determining the sensitivity of SCLC cells to BI-2536 and its combination with alisertib. The study further revealed a positive correlation between RAD51 expression and PLK1/AURKA expression, and a negative correlation with the IC50 values of BI-2536. Manipulating RAD51 expression significantly influenced the efficacy of BI-2536 and restored the MYC/MYCN-induced enhancement of BI-2536 sensitivity in SCLC cells. Our findings indicate that the BRCA1 and MYC/MYCN-RAD51 axes govern the response of small cell lung cancer to BI-2536 and its combination with alisertib. This study propose the combined use of BI-2536 and alisertib as a novel therapeutic strategy for the treatment of SCLC patients with MYC/MYCN activation.

Markers of Mitochondrial Function and DNA Repair Associated with Physical Function in Centenarians.

Mitochondrial dysfunction and genomic instability are key hallmarks of aging. The aim of this study was to evaluate whether maintenance of physical capacities at very old age is associated with key hallmarks of aging. To investigate this, we measured mitochondrial bioenergetics, mitochondrial DNA (mtDNA) copy number and DNA repair capacity in peripheral blood mononuclear cells from centenarians. In addition, circulating levels of NAD+/NADH, brain-derived neurotrophic factor (BDNF) and carbonylated proteins were measured in plasma and these parameters were correlated to physical capacities. Centenarians without physical disabilities had lower mitochondrial respiration values including ATP production, reserve capacity, maximal respiration and non-mitochondrial oxygen-consumption rate and had higher mtDNA copy number than centenarians with moderate and severe disabilities (p < 0.05). In centenarian females, grip strength had a positive association with mtDNA copy number (p < 0.05), and a borderline positive trend for activity of the central DNA repair enzyme, APE 1 (p = 0.075), while a negative trend was found with circulating protein carbonylation (p = 0.07) in the entire cohort. Lastly, a trend was observed for a negative association between BDNF and activity of daily living disability score (p = 0.06). Our results suggest that mechanisms involved in maintaining mitochondrial function and genomic stability may be associated with maintenance of physical function in centenarians.

Prochlorococcus marinus responses to light and oxygen.

Prochlorococcus marinus, the smallest picocyanobacterium, comprises multiple clades occupying distinct niches, currently across tropical and sub-tropical oligotrophic ocean regions, including Oxygen Minimum Zones. Ocean warming may open growth-permissive temperatures in new, poleward photic regimes, along with expanded Oxygen Minimum Zones. We used ocean metaproteomic data on current Prochlorococcus marinus niches, to guide testing of Prochlorococcus marinus growth across a matrix of peak irradiances, photoperiods, spectral bands and dissolved oxygen. MED4 from Clade HLI requires greater than 4 h photoperiod, grows at 25 μmol O2 L-1 and above, and exploits high cumulative diel photon doses. MED4, however, relies upon an alternative oxidase to balance electron transport, which may exclude it from growth under our lowest, 2.5 μmol O2 L-1, condition. SS120 from clade LLII/III is restricted to low light under full 250 μmol O2 L-1, shows expanded light exploitation under 25 μmol O2 L-1, but is excluded from growth under 2.5 μmol O2 L-1. Intermediate oxygen suppresses the cost of PSII photoinactivation, and possibly the enzymatic production of H2O2 in SS120, which has limitations on genomic capacity for PSII and DNA repair. MIT9313 from Clade LLIV is restricted to low blue irradiance under 250 μmol O2 L-1, but exploits much higher irradiance under red light, or under lower O2 concentrations, conditions which slow photoinactivation of PSII and production of reactive oxygen species. In warming oceans, range expansions and competition among clades will be governed not only by light levels. Short photoperiods governed by latitude, temperate winters, and depth attenuation of light, will exclude clade HLI (including MED4) from some habitats. In contrast, clade LLII/III (including SS120), and particularly clade LLIV (including MIT9313), may exploit higher light niches nearer the surface, under expanding OMZ conditions, where low O2 relieves the stresses of oxidation stress and PSII photoinhibition.

O-038 Impact of maternal aging on the DNA repair landscape in mouse oocytes

Abstract Study question How does maternal aging impact DNA damage accumulation and DNA repair capacity in mammalian oocytes? Summary answer Age-related changes in DNA repair compartments and chromatin organisation drive DNA damage accumulation in aged oocytes, ultimately contributing to the decline in DNA repair capacity. What is known already Oocyte loss occurs progressively from birth and accelerates as women approach their mid-30s, posing a significant challenge to female reproductive health. Recent studies have highlighted the role of the DNA repair response in driving ovarian ageing and oocyte loss. Aged oocytes accumulate elevated levels of DNA damage. Unless repaired, persistent DNA damage activates DNA repair checkpoints, causing the elimination of defective oocytes. Although we know DNA double-strand breaks (DSBs) in oocytes increase with maternal age, surprisingly, little is known about why this damage is not repaired efficiently. Study design, size, duration Oocytes were isolated from ovaries of 8 weeks or 68-86-week-old CD1 mice, which correspond to reproductively young and old females respectively. Over 600 and 1000 oocytes from aged any young females were used to analyse DNA damage levels, DNA repair effciency, DNA repair compartments and for other live assays. Participants/materials, setting, methods DNA damage was induced in CD1 oocytes from young and aged mice using neocarzinostatin. Levels of DNA damage and DNA repair proteins were quantified using immunofluoresnce confocal/STED microscopy. High-resolution light sheet microscopy was used to perform live imaging of dynamics of DNA damage foci and chromosome segregation during meiosis. Main results and the role of chance Our work demonstrates that aged oocytes have a lower DNA repair capacity and reduced mobility of DNA damage sites compared to young oocytes. We further show that incomplete DNA repair results in defective chromosome integrity and partitioning, thereby compromising oocyte quality. To investigate the causes of the age-related decline in DNA repair, we systematically studied the localization and abundance of several DNA repair proteins in oocytes from young and aged mice. We found that DNA repair proteins localized in spatially distinct DNA repair compartments in oocyte nuclei, that form during the late stages of oocyte growth, accompanied by changes in the activity of DNA repair pathways. Several DNA repair proteins, their corresponding compartments, and their response to DNA damage were altered in aged oocytes. Lastly, we demonstrate that age-related cohesin loss increases DNA damage levels and reduces DNA repair capacity. We hypothesize that cohesin dissociation with maternal ageing may make chromosomes prone to accumulate DNA damage without proper DNA repair. Thus, age-related changes in DNA repair compartments and chromatin organisation drive DNA damage accumulation in aged oocytes, ultimately contributing to the decline in reproductive potential. Limitations, reasons for caution Neocarzinostatin, a radiomimetic drug used to induce DNA DSBs in mouse oocytes in vitro, may not fully represent sources of DNA damage during aging. The CD1 mouse model and ages were selected to recapitulate equivalent advanced maternal age in women. Further investigation is needed to confirm these findings in humans. Wider implications of the findings Overall, we propose that altered DNA repair machinery and cohesin loss contribute to persistent DNA damage and reduced DNA repair capacity in aged oocytes, which can cause chromosome defects and fragmentation, and eventually trigger oocyte death, thereby accelerating the loss of female fertility. Trial registration number Not Applicable

P-036 Female age affects the combined impact of DNA fragmentation and high DNA stainability on embryo development in intracytoplasmic sperm injection cycles

Abstract Study question To investigate the role of female age in regulating the co-effect of DNA fragmentation index (DFI) and high DNA stainability (HDS) on ICSI embryological outcomes. Summary answer When female age exceeded 35, co-exposure high DFI and middle HDS might delay embryonic development after ICSI treatment. What is known already Sperm DNA damage had a negative influence on embryonic development. Oocytes and embryos have adopted specific response and repair mechanisms to repair sperm DNA damage. However, the DNA repair capacity in the oocyte is limited and likely decline with increasing age. Study design, size, duration This historical cohort study was included 1334 couples underwent ICSI from January 2017 to December 2022. An unsupervised K-means clustering method was used to divide participants into three clusters base on DFI value and HDS value, as follow: cluster 1 with low DFI values and low HDS values (lDFI-lHDS group), cluster 2 with middle DFI values and high HDS values (mDFI-hHDS group), and cluster 3 with high DFI values and middle HDS values (hDFI-mHDS group). Participants/materials, setting, methods All participants were further stratified into female age&amp;lt;35 subgroup and female age≥35 subgroup. The univariate and multivariate linear regression model was used for statistical analysis. Multivariate model was adjusted for female factor (including age, AMH, basal FSH and the numbers of retrieved oocyte) and male age which were significantly correlated with embryo development in univariate models. The main outcome measure was D5 blastocyst development rate and D6 blastocyst development rate. Main results and the role of chance In the female age&amp;lt;35 subgroup, D5 blastocyst development rate in the mDFI-hHDS group (48.53% [25.00%, 66.67%] and the hDFI-mHDS group (50.00% [28.57%, 71.07%]) was significantly higher than that in the lDFI-lHDS group (40.00% [0.00%, 63.96%]). No significant differences were observed between the three groups regarding D6 blastocyst development rate. And univariate model and multivariate model showed that the mDFI-hHDS group (adjusted B value=0.36, 95% CI: 0.08∼0.64, P=0.010) and hDFI-mHDS group (adjusted B value=0.41, 95% CI: 0.12∼0.70, P=0.006) had higher D5 blastocyst development rate compared with lDFI-lHDS group. In the female age≥35 subgroup, D5 blastocyst development rate in the hDFI-mHDS (20.00% [0.00%, 50.00%]) group was significantly lower than that in the lDFI-lHDS group (36.93% [0.00%, 66.67%]) and the mDFI-hHDS group (41.43% [0.00%, 63.54%]). Accordingly, D6 blastocyst development rate in the hDFI-mHDS group (66.67% [33.33%, 100.00%]) was significantly higher than that in the lDFI-lHDS group (50.00% [16.67%, 80.00%]) and the mDFI-hHDS group (50.00% [33.33%, 66.67%]). And both model showed that hDFI-mHDS group (adjusted B value=0.73, 95% CI: 0.36∼1.11, P&amp;lt;0.001) had higher D6 blastocyst development rate compared with the lDFI-lHDS group. Although not statistically significant, hDFI-mHDS had negative correlation with D5 blastocyst development rate. Limitations, reasons for caution The main weakness of this study was the participants were not randomized. Wider implications of the findings hDFI-mHDS group were negatively associated with D5 blastocyst development rate and positively related with D6 blastocyst development rate in female age≥35 subgroup. These results suggested that co-exposure high DFI and middle HDS might be recommended as a predictor for the detection of delayed embryo development when female age exceeded 35. Trial registration number not applicable

Inhibition of AR and DNA-PK for radio-sensitization of AR-driven cancers.

e12593 Background: Breast cancer (BC) is the most diagnosed cancer globally with over 2.2 million new cases and 680,000 deaths annually. Radiotherapy (RT) is essential for curative BC treatment; however, 20% of BC cases are radio-resistant, with the underlying mechanisms remaining poorly understood. Previous studies in prostate cancer (PCa) have shown that androgen receptor (AR) promotes radio-resistance (RT-resistance) through transcriptional regulation and increased DNA repair capacity, including through upregulation of DNA repair proteins DNA-PK and PARP1. Although AR is expressed in 60% of BCs, the implications of AR in BC RT-resistance are not yet fully characterized. We hypothesized that AR may drive RT-resistance in BC through a similar AR-DNA repair loop. Methods: To characterize the repertoire of cofactors that cooperate with AR on the TF complex, we exposed a BC cell line to RT and performed rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) proteomics. We identified several candidate proteins that interact with AR to further investigate as targets for RT-sensitization. Survival analyses in BC and PCa cell lines were performed to identify RT-sensitizing agents. Molecular mechanisms of RT-sensitization were assessed using ATAC-seq and RNA-seq analyses. Results: Using RIME, we identified several DNA repair proteins including DNA-PK, PARP-1, and XRCC5 as differentially recruited to chromatin-bound AR. This suggests a cooperative role and provides potential druggable targets to improve RT-sensitivity. Based on these results, we also performed a survival analysis in AR-driven BC and PCa cell lines and showed that inhibition of either AR or DNA-PK led to RT-sensitization, and dual inhibition provided an additive effect. We sought to characterize the molecular mechanisms driving the RT-sensitizing effects of AR and DNA-PK inhibition and performed ATAC-seq and RNA-seq on treated and untreated cells. Combining enzalutamide with a DNA-PK inhibitor altered expression of several pathways implicated in RT-sensitization, although these pathways were largely distinct in BC and PCa. Further, we identified distinct genes that may be responsible for this phenotype in AR-driven BC. Additionally, we found a larger effect in chromatin rearrangement after treatment in PCa than BC. Conclusions: Identifying mechanisms of RT-resistance in hormone-driven cancers is essential to develop therapeutic strategies and improve patient outcomes. This study provides key evidence for AR-driven RT-resistance in BC and PCa, and identifies DNA-PK inhibition as a potential drug target. Distinct mechanisms appear to be driving this phenotype across BC and PCa, in our models. Understanding the cooperation of DNA repair and hormone signaling pathways will help support the use of DNA-damaging agents for hormone-driven cancers, and potentially inform rational design of novel drug-RT combinations.

Mechanism of Action of KL-50, a Candidate Imidazotetrazine for the Treatment of Drug-Resistant Brain Cancers.

Aberrant DNA repair is a hallmark of cancer, and many tumors display reduced DNA repair capacities that sensitize them to genotoxins. Here, we demonstrate that the differential DNA repair capacities of healthy and transformed tissue may be exploited to obtain highly selective chemotherapies. We show that the novel N3-(2-fluoroethyl)imidazotetrazine "KL-50" is a selective toxin toward tumors that lack the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT), which reverses the formation of O6-alkylguanine lesions. We establish that KL-50 generates DNA interstrand cross-links (ICLs) by a multistep process comprising DNA alkylation to generate an O6-(2-fluoroethyl)guanine (O6FEtG) lesion, slow unimolecular displacement of fluoride to form an N1,O6-ethanoguanine (N1,O6EtG) intermediate, and ring-opening by the adjacent cytidine. The slow rate of N1,O6EtG formation allows healthy cells expressing MGMT to reverse the initial O6FEtG lesion before it evolves to N1,O6EtG, thereby suppressing the formation of toxic DNA-MGMT cross-links and reducing the amount of DNA ICLs generated in healthy cells. In contrast, O6-(2-chloroethyl)guanine lesions produced by agents such as lomustine and the N3-(2-chloroethyl)imidazotetrazine mitozolomide rapidly evolve to N1,O6EtG, resulting in the formation of DNA-MGMT cross-links and DNA ICLs in healthy tissue. These studies suggest that careful consideration of the rates of chemical DNA modification and biochemical DNA repair may lead to the identification of other tumor-specific genotoxic agents.

The Mms22-Rtt107 axis dampens the DNA damage checkpoint by reducing the stability of the Rad9 checkpoint mediator.

The DNA damage checkpoint is a highly conserved signaling pathway induced by genotoxin exposure or endogenous genome stress. It alters many cellular processes such as arresting the cell cycle progression and increasing DNA repair capacities. However, cells can downregulate the checkpoint after prolonged stress exposure to allow continued growth and alternative repair. Strategies that can dampen the DNA damage checkpoint are not well understood. Here, we report that budding yeast employs a pathway composed of the scaffold protein Rtt107, its binding partner Mms22, and an Mms22-associated ubiquitin ligase complex to downregulate the DNA damage checkpoint. Mechanistically, this pathway promotes the proteasomal degradation of a key checkpoint factor, Rad9. Furthermore, Rtt107 binding to Mms22 helps to enrich the ubiquitin ligase complex on chromatin and target the chromatin-bound form of Rad9. Finally, we provide evidence that the Rtt107-Mms22 axis operates in parallel with the Rtt107-Slx4 axis, which displaces Rad9 from chromatin. We thus propose that Rtt107 enables a bifurcated "anti-Rad9" strategy to optimally downregulate the DNA damage checkpoint.

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.

Uncovering a novel DNA repair-related radiosensitivity model for evaluation of radiotherapy susceptibility in uterine corpus endometrial cancer

BackgroundUterine corpus endometrial cancer (UCEC) exhibit heterogeneity in their DNA repair capacity, which can impact their response to radiotherapy. Our study aimed to identify potential DNA repair-related biomarkers for predicting radiation response in UCEC. MethodsWe conducted a thorough analysis of 497 UCEC samples obtained from TCGA database. Using LASSO-COX regression analysis, we constructed a radiosensitivity signature and subsequently divided patients into the radiosensitive (RS) and the radioresistant (RR) groups based on their radiosensitivity index. The GSVA and GSEA were performed to explore functional annotations. The CIBERSORT and ESTIMATE algorithms were utilized to investigate the immune infiltration status of the two groups. Additionally, we utilized the Tumor Immune Dysfunction and Exclusion (TIDE), Immunophenotype Score (IPS), and pRRophetic algorithms to predict the effectiveness of different treatment modalities. ResultsWe constructed a radiosensitivity index consists of four DNA repair-related genes. Patients in the RS group demonstrated significantly improved prognosis compared to patients in the RR group when treated with radiotherapy. We observed that the RS group exhibited a higher proportion of the POLE ultra-mutated subtype, while the RR group had a higher proportion of the copy number high subtype. GSVA enrichment analysis revealed that the RS group exhibited enrichment in DNA damage repair pathways. Notably, the RS group demonstrated a higher proportion of naïve B cells and follicular helper T cells, while regulatory T cells (Tregs) and memory B cells were more abundant in the RR group. Furthermore, patients in the RS-PD-L1-high subgroup exhibited enrichment in immune-related pathways and increased sensitivity to immunotherapy, which is likely to contribute to their improved prognosis. Additionally, we conducted in vitro experiments to validate the expression of radiosensitivity genes in non-radioresistant (AN3CA) and radioresistant (AN3CA/IR) endometrial cancer cells. ConclusionsIn conclusion, our research successfully constructed a radiosensitivity signature with robust predictive capacity. These findings shed light on the association between immune activation, PD-L1 expression, and the response to immunotherapy in the context of radiotherapy.