NHEJ Pathway Research Articles

Glycyrrhizin ameliorates colorectal cancer progression by regulating NHEJ pathway through inhibiting HMGB1-induced DNA damage response

As one of the most common malignancies, colorectal cancer (CRC) usually starts with a benign lesion and accumulates DNA damage as it progresses to full-fledged cancer. Glycyrrhizin (GL) has been found to alleviate tumor growth and inflammation, while the role of GL influences DNA damage response (DDR) in colorectal cancer remains unclear. GL exposure significantly reduced cell colony formation and viability with a concomitant increase in DNA fragmentation in CRC, meanwhile GL induced apoptosis by activating caspase-3. Moreover, GL induced cell cycle arrest in CRC cells at S phase, which was associated with decreased cyclin D1 in vitro. GL treatment significantly ameliorated tumor growth and promoted DDR in vivo. Mechanism analysis revealed that GL significantly downregulated the NHEJ pathway via inhibiting HMGB1. Finally, the expression of HMGB1 was abnormal regulated in CRC tissue than in adjacent normal tissues and associated with TNM stage and overall survival. Our findings indicate that HMGB1 may be a novel therapeutic target in CRC, a result that GL may serve as a promising drug for CRC treatment.

Cytotoxicity, genotoxicity and mutagenicity of mixed ternary mononuclear Mg complex based on valproic acid with 1,10-phenanthroline in Saccharomyces cerevisiae and V79 cells.

Valproic acid (VA) is a widely used drug for the treatment of diseases affecting the central nervous system. Due to its epigenetic modulatory potential, it has been studied for possible therapeutic application in anticancer therapies. However, the VA exhibits different side effects in its application. Thus, synthetic coordination complexes with valproate can generate promising candidates for new active drugs with reduced toxicity. In this sense, we investigated the genotoxic and mutagenic potential of the sodium valproate and of the mixed ternary mononuclear Mg complex based on VA with 1,10-phenanthroline (Phen) ligand - [Mg (Valp)2Phen], in Saccharomyces cerevisiae and V79 cells. The MTT and clonal survival assays in V79 cells indicated that the Mg complex has higher cytotoxicity than sodium valproate. A similar cytotoxicity profile is observed in yeast. This fact is possibly due to the intercalation capacity of [Mg(Valp)2Phen], inducing DNA strand breaks, as observed in the comet assay and micronucleus test. In this sense, members of the NER, HR, NHEJ and TLS repair pathways are required for the repair of DNA lesions induced by [Mg(Valp)2Phen]. Interestingly, BER proteins apparently increase the cytotoxic potential of the drug. Furthermore, the [Mg(Valp)2Phen] showed higher cytotoxicity in V79 cells and yeast when compared to sodium valproate indicating applicability as a cytotoxic agent.

Prognostic factors of 87 ovarian yolk sac tumor (OYST) patients and molecular characteristics of persistent and recurrent OYST

ObjectiveWe aimed to explore the characteristics of OYST, particularly for persistent and recurrent OYST, in order to explore potential treatment options and thereby improve patient outcomes. MethodsWe retrospectively reviewed the clinical records of all patients with OYST at Fudan university Shanghai Cancer Center from December 3, 2005 to November 27, 2020. Furthermore, and performed whole-exome sequencing on 17 paired OYST (including 8 paired persistent and recurrent OYST) tumor and blood samples to elucidate the aberrant molecular features. ResultsTotally, 87 OYST patients were included between 2007/03/13 and 2020/11/17. With a median follow-up of 73 [3–189] months, 22 patients relapsed or disease persisted. Overall, 17 patients died with a median overall survival of 21 [3–54] months. Univariate and multivariate analysis revealed tumor histology and residual lesions were independently associated with event free survival and overall survival, cycles to AFP normalization were another independent risk factor for overall survival. For the 8 persistent and recurrent OYST: cancer driver genes including ANKRD36, ANKRD62, DNAH8, MUC5B, NUP205, RYR2, STARD9, MUC16, TTN, ARID1A and PIK3CA were frequently mutated; cell cycle, ABC transporters, HR, NHEJ and AMPK signal pathway demonstrated as the most significantly enriched pathways; TMB, DNA MMR gene mutation and MSI were significantly higher. Mutation signature 11, 19 and 30 were the dominant contributors in persistent and recurrent OYST mutation. ConclusionPersistent and recurrent OYST associated with poor prognosis, and probably susceptible to immune checkpoint blockade therapy. Molecular characteristics contributed to predict the persistence and recurrence of OYST.

PRKDC-Mediated NHEJ May Play a Crucial Role in Aneuploidy of Chromosome 8-Driven Progression of Ovarian Cancer.

High malignancy is a prominent characteristic of epithelial ovarian cancer (EOC), emphasizing the necessity for further elucidation of the potential mechanisms underlying cancer progression. Aneuploidy and copy number variation (CNV) partially contribute to the heightened malignancy observed in EOC; however, the precise features of aneuploidy and their underlying molecular patterns, as well as the relationship between CNV and aneuploidy in EOC, remain unclear. In this study, we employed single-cell sequencing data along with The Cancer Genome Atlas (TCGA) to investigate aneuploidy and CNV in EOC. The technique of fluorescence in situ hybridization (FISH) was employed using specific probes. The copy number variation within the genomic region of chromosome 8 (42754568-47889815) was assessed and utilized as a representative measure for the ploidy status of individual cells in chromosome 8. Differential expression analysis was performed between different subgroups based on chromosome 8 ploidy. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI), and hub-gene analyses were subsequently utilized to identify crucial genes involved. By classifying enriched tumor cells into distinct subtypes based on chromosome 8 ploidy combined with TCGA data integration, we identified key genes driving chromosome 8 aneuploidy in EOC, revealing that PRKDC gene involvement through the mediated non-homologous end-joining pathway may play a pivotal role in disease progression. Further validation through analysis of the GEO and TCGA database and survival assessment, considering both mRNA expression levels and CNV status of PRKDC, has confirmed its involvement in the progression of EOC. Further functional analysis revealed an upregulation of PRKDC in both ovarian EOC cells and tissues, with its expression showing a significant correlation with the extent of copy number variation (CNV) on chromosome 8. Taken together, CNV amplification and aneuploidy of chromosome 8 are important characteristics of EOC. PRKDC and the mediated NHEJ pathway may play a crucial role in driving aneuploidy on chromosome 8 during the progression of EOC.

Abstract LB015: Differential expression of deubiquitinating enzymes and its related DNA damage response genes in neuroblastoma heterogeneity

Abstract SK-N-AS and SK-N-DZ are two cell lines of pediatric solid tumors neuroblastomas well characterized to evaluate heterogeneity between different neuroblastomas and are presumed as models for differences between different neuroblastoma. Using this model system, we recently evaluated if radiation could improve efficacy of radiation therapy and to follow these studies later with animal models to determine decrease of tumor burden. Differences between the N-type and S-type neuroblastoma cell lines SK-N-DZ and SK-N-AS, respectively were observed to be high and their responses to radiation stress were equally heterogeneous. Ubiquitin signaling plays a key role during the DNA damage response in protein recruitment and protein interactions. Deubiquitinating enzymes (DUBs) deconjugate ubiquitin from ubiquitylated substrate proteins to control their activity and stability and are shown to regulate various biological processes. DUBs may play a role in regulating the radiosensitivity of cancer cells by modulating DNA damage response and repair pathways. Determination of specific DUBs that are involved in regulating radiosensitivity in cancer cells will provide opportunity for drug targeting and visa-vis enhance the efficacy of radiation therapy in neuroblastoma cancer treatment. Here, using bioinformatic tools, the differentially regulated DUBs were identified. The relationship between DEGs-DUBs with the genes involved in various DNA repair pathways (NHEJ, HR and Fanconi anemia pathway; KEGG pathway database) are described. We identified 32 DEGs (14 upregulated and 18 downregulated) with these neuroblastoma cell lines. USP22 and USP41 DUBs and its differential expression in these cell lines denotes the importance of such analysis. Especially considering USP22 a potentially biomarker for predicting the outcome of patients with neuroblastoma. These DEG-DUBs were likewise correlated with 91 DNA damage response (DDR) genes belonging to three different pathways (NHEJ, HR and Fanconi anemia pathway). Out of which 44 of the DDR genes showed significant correlation with DEG-DUBs. Further studies are needed to determine the mechanisms underlying the complex relationship of these DUBs and to determine the critical DUBs for drug targeting and to enhance the efficacy of radiation therapy in cancer treatment. Citation Format: Vaibhav Vaibhav, Fahika Nazeerulla, Tatjana Paunesku, Ekta Tripathi, Gayle Woloschak, Prashanthi Karyala, Rao V. Papineni. Differential expression of deubiquitinating enzymes and its related DNA damage response genes in neuroblastoma heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB015.

GCN5 mediates DNA-PKcs crotonylation for DNA double-strand break repair and determining cancer radiosensitivity.

DNA double-strand break (DSB) induction and repair are important events for determining cell survival and the outcome of cancer radiotherapy. The DNA-dependent protein kinase (DNA-PK) complex functions at the apex of DSBs repair, and its assembly and activity are strictly regulated by post-translation modifications (PTMs)-associated interactions. However, the PTMs of the catalytic subunit DNA-PKcs and how they affect DNA-PKcs's functions are not fully understood. Mass spectrometry analyses were performed to identify the crotonylation sites of DNA-PKcs in response to γ-ray irradiation. Co-immunoprecipitation (Co-IP), western blotting, in vitro crotonylation assays, laser microirradiation assays, in vitro DNA binding assays, in vitro DNA-PK assembly assays and IF assays were employed to confirm the crotonylation, identify the crotonylase and decrotonylase, and elucidate how crotonylation regulates the activity and function of DNA-PKcs. Subcutaneous xenografts of human HeLa GCN5 WT or HeLa GCN5 siRNA cells in BALB/c nude mice were generated and utilized to assess tumor proliferation in vivo after radiotherapy. Here, we reveal that K525 is an important site of DNA-PKcs for crotonylation, and whose level is sharply increased by irradiation. The histone acetyltransferase GCN5 functions as the crotonylase for K525-Kcr, while HDAC3 serves as its dedicated decrotonylase. K525 crotonylation enhances DNA binding activity of DNA-PKcs, and facilitates assembly of the DNA-PK complex. Furthermore, GCN5-mediated K525 crotonylation is indispensable for DNA-PKcs autophosphorylation and the repair of double-strand breaks in the NHEJ pathway. GCN5 suppression significantly sensitizes xenograft tumors of mice to radiotherapy. Our study defines K525 crotonylation of DNA-PKcs is important for the DNA-PK complex assembly and DSBs repair activity via NHEJ pathway. Targeting GCN5-mediated K525 Kcr of DNA-PKcs may be a promising therapeutic strategy for improving the outcome of cancer radiotherapy.

Abstract 6013: VIO-01, a pan-DDR DNA decoy mediating DNA repair abrogation and unleashing the anti-tumor immune response

Abstract Background: Targeting specific DNA Damage Response (DDR) proteins has been worldwide studied and developed, with the example of PARP inhibitors being the only approved treatments in this field. Here, we describe the antineoplastic and immunomodulatory effects of VIO-01, a first-in-class DNA decoy-cholesterol conjugate, that operates as a pan repair proteins decoy, resulting in constitutive exhaustion of the DNA damage response. Methods: VIO-01-induced DDR proteins trapping and cell cytotoxicity were examined in both homologous recombination proficient (HRP) and deficient (HRD) cancer cells. DNA repair efficacy was monitored by analyzing repair protein recruitment to damage sites. RNAseq analysis in HRP/HRD ovarian cancer cells was employed to uncover the molecular mechanisms underlying VIO-01 effects. Effects on the innate and adaptive immune responses were assessed by following T-cell mediated anti-tumor cytotoxicity. VIO-01 antitumor efficacy and biodistribution were also assessed. Additionally, the interest of cholesterol as a vector for VIO-01 was analyzed compared to other ligands like the folate receptor ligand. Results: VIO-01 bounds to several DDR proteins, such as PARP1, KU70/80, MRN complex and MSH2/MSH3 with high affinity, resulting in an abrogation of single- and double-strand break repair. In line with this, and through global transcriptome analysis, VIO-01 treatment resulted in notable downregulation of Base Excision Repair, NHEJ and Nucleotide Excision Repair pathways in both HRP/HRD cell lines. Additionally, VIO-01 elicited the activation of the immune system and inflammatory responses in ovarian cancer cells. Conjointly, VIO-01 treatment induced a significant differentiation of monocytes into non-classical monocytes, leading to a shaping of the tumor microenvironment via CD8+ T cell recruitment in early stage of activation. The activity of VIO-01 was specific to tumor cells, while sparing healthy and immune cells, at odds with PARP inhibitors. In line with in vitro results, VIO-01 mediated antitumor efficacy in vivo coupled to tumor-targeting T-cell responses. These interesting effects were driven by a favorable ADME/PK profile, showing a long-lasting VIO-01 residence time VIO-01 into tumors (at least 3 days post treatment), coupled to a clear hijacking from the liver and a rapid blood clearance, ensuring a minimal toxicity. Moreover, regulatory toxicology studies demonstrated a favorable safety profile of VIO-01 in non-human primate with the major findings being a transient increase in complement factors induced by the binding of VIO-01 to factor H, which was not observed in human serum, predicting favorable safety profile in humans. Conclusions: Our results provide a preclinical rationale for using VIO-01 to trigger DNA damage exhaustion and an antitumor immune response, paving the way for rapid clinical application in patients bearing HRD or HRP tumors. Citation Format: Vlada Zakharova, Chloe Doizelet, Nicolas Babault, Marie-Christine Lienafa, Fetta Mazed, Megane Debiais, Pierre Vilela, Emilie Perroud, Wael Jdey. VIO-01, a pan-DDR DNA decoy mediating DNA repair abrogation and unleashing the anti-tumor immune response [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 6013.

Abstract A007: Ser/Thr kinase BUB1 stabilizes DNAPKcs in response to radiation induced DNA double strand break repair

Abstract Ser/Thr kinase BUB1 is known to regulate chromosomal segregation during mitosis. However, its exact role in DNA damage repair in response to radiotherapy remained elusive. Here, we demonstrate that inhibition of BUB1 kinase activity (BAY1816032) or genomic depletion (CRISPR) lead to radiation sensitization in triple negative breast cancer and lung cancer cell lines and in TNBC tumor xenograft models. Mechanistic studies demonstrated reduced DNA-DSB repair (γ-H2AX foci assay), and effect on the NHEJ pathway by the bioluminescent DNA repair reporter (BLRR). Gene expression analysis show that BUB1 expression correlates tightly with key members of the NHEJ machinery including H2AX, ATM, 53BP1, DNAPKcs, Ku70, Ku80, Lig4 among others. Cycloheximide-chase experiments demonstrate that BUB1 ablation (pharmacological or genomic) stabilizes DNAPKcs after irradiation. The half-lives (t1/2) of total-DNAPK, phospho-DNAPK could not be estimated in BUB1 ablated cells post radiotherapy. Conversely, we observed BUB1 stabilization following radiotherapy alone while pre-treatment with BUB1i or DNAPKi caused robust degradation of BUB1 (significantly shorter t1/2). Nuclear and chromatin fractionation assays revealed an increase in the phospho- and total- DNAPK, ATM, and KAP1 in nuclear fractions and in chromatin enriched fractions in BUB1 inhibited or depleted cells. There was no significant change in the levels of Ku70 and Ku80 in the nuclear fractions or in the chromatin enriched fractions. This data indicates that BUB1 may be involved in the activation and recruitment of key NHEJ proteins (ATM, DNAPKcs) to DSBs. Further experiments are underway to determine how BUB1 stabilizes DNAPKcs and regulate chromatin loading of key NHEJ factors following radiation. Additionally, we demonstrate that BUB1 mediate apoptotic cell death of cancer cells post-radiation. Our findings suggest that BUB1 may serve as a novel molecular target for radiosensitization in various solid cancers including TNBC. Citation Format: Sushmitha Sriramulu, Shivani Thoidingjam, Stephen Brown, Farzan Siddiqui, Benjamin Movsas, Michael Green, Corey Speers, Shyam Nyati. Ser/Thr kinase BUB1 stabilizes DNAPKcs in response to radiation induced DNA double strand break repair [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A007.

SMYD3 promotes endometrial cancer through epigenetic regulation of LIG4/XRCC4/XLF complex in non-homologous end joining repair

Endometrial cancer (EC) stands as one of the most prevalent malignancies affecting the female genital tract, witnessing a rapid surge in incidence globally. Despite the well-established association of histone methyltransferase SMYD3 with the development and progression of various cancers, its specific oncogenic role in endometrial cancer remains unexplored. In the present study, we report that the expression level of SMYD3 is significantly upregulated in EC samples and associated with EC progression. Through meticulous in vivo and in vitro experiments, we reveal that depletion of SMYD3 curtails cell proliferation, migration, and invasion capabilities, leading to compromised non-homologous end joining repair (NHEJ) and heightened sensitivity of EC cells to radiation. Furthermore, our pathway enrichment analysis underscores the pivotal involvement of the DNA damage repair pathway in regulating EC progression. Mechanistically, in response to DNA damage, SMYD3 is recruited to these sites in a PARP1-dependent manner, specifically methylating LIG4. This methylation sets off a sequential assembly of the LIG4/XRCC4/XLF complex, actively participating in the NHEJ pathway and thereby fostering EC progression. Notably, our findings highlight the promise of SMYD3 as a crucial player in NHEJ repair and its direct correlation with EC progression. Intriguingly, pharmacological intervention targeting SMYD3 with its specific inhibitor, BCI-121, emerges as a potent strategy, markedly suppressing the tumorigenicity of EC cells and significantly enhancing the efficacy of radiotherapy. Collectively, our comprehensive data position SMYD3 as a central factor in NHEJ repair and underscore its potential as a promising pharmacological target for endometrial cancer therapy, validated through both in vitro and in vivo systems.

ROCK inhibition reduces the sensitivity of mutant p53 glioblastoma to genotoxic stress through a Rac1-driven ROS production.

Resistance to radio and chemotherapy in Glioblastoma (GBM) is correlated with its malignancy, invasiveness, and aggressiveness. The Rho GTPase pathway plays important roles in these processes, but its involvement in the GBM response to genotoxic treatments remains unsolved. Inhibition of this signaling pathway has emerged as a promising approach for the treatment of CNS injuries and diseases, proving to be a strong candidate for therapeutic approaches. To this end, Rho-associated kinases (ROCK), classic downstream effectors of small Rho GTPases, were targeted for pharmacological inhibition using Y-27632 in GBM cells, expressing the wild-type or mutated p53 gene, and exposed to genotoxic stress by gamma ionizing radiation (IR) or cisplatin (PT). The use of the ROCK inhibitor (ROCKi) had opposite effects in these cells: in cells expressing wild-type p53, ROCKi reduced survival and DNA repair capacity (reduction of γH2AX foci and accumulation of strand breaks) after stress promoted by IR or PT; in cells expressing the mutant p53 protein, both treatments promoted longer survival and more efficient DNA repair, responses further enhanced by ROCKi. The target DNA repair mechanisms of ROCK inhibition were, respectively, an attenuation of NHEJ and NER pathways in wild-type p53 cells, and a stimulation of HR and NER pathways in mutant p53 cells. These effects were accompanied by the formation of reactive oxygen species (ROS) induced by genotoxic stress only in mutant p53 cells but potentiated by ROCKi and reversed by p53 knockdown. The p53-dependent actions of ROCKi were completely abolished by N-acetyl-L-cysteine (NAC) treatment or Rac1 knockdown, as Rac-GTP levels were specifically elevated by ROCK inhibition only in mutant p53 cells. Combining IR or PT and ROCKi treatments broadens our understanding of the sensitivity and resistance of, respectively, GBM expressing wild-type or mutant p53 to genotoxic agents. Our proposal may be a determining factor to improve the efficiency and assertiveness of CNS antitumor therapies based on ROCK inhibitors. SIGNIFICANCE: The use of ROCK inhibitors in association with radio or chemotherapy modulates GBM resistance and sensitivity depending on the p53 activity, suggesting the potential value of this protein as therapeutic target for tumor pre-sensitization strategies.

Transcriptomic Analysis to Uncover the Mechanism of Radiosensitization of AR-Positive Triple Negative Breast Cancers with AR Inhibition

The androgen receptor (AR) has been shown to drive tumor growth in triple negative breast cancers (TNBC), and previous work demonstrated AR inhibition as a strategy for radiosensitization in AR-positive (AR+) TNBC. Despite its role in radioresistance, the mechanistic role of AR in response to radiation therapy (RT) remains unknown, as does the benefit of 2nd generation anti-androgens in this context. We hypothesized that all 2nd generation anti-AR therapy would radiosensitize similarly and that canonical AR transcriptional function was responsible for radioresistance in these models. Radiosensitization was assessed using 2nd generation AR antagonists (apalutamide, enzalutamide, and darolutamide) using clonogenic survival assays in MDA-MB-453, SUM185, MFM-223, and MDA-MB-231 cells at 2-6Gy. Cellular fractionation experiments were performed and quantitated to determine the location of the AR protein in cells treated with AR agonists +/- RT. RNA Seq was performed and transcriptomic approaches were used (Advaita iPathway analysis) to investigate AR-mediated effects in response to RT. Inhibition with the 2nd generation anti-androgens enzalutamide and apalutamide is sufficient to radiosensitize AR+ TNBC models (rER: 1.34-1.41); while darolutamide had no effect on radiosensitivity (rER: 0.96-1.11). Additionally, TNBC cells with low AR expression were not radiosensitized by AR inhibition with any drug (rER: 0.96-1.03). While stimulation with the synthetic androgen methyltrienolone R1881 is sufficient to induce nuclear translocation of AR in AR+ TNBC cells, AR inhibition with enzalutamide, apalutamide, or darolutamide blocked AR nuclear translocation under growth conditions with charcoal stripped serum or fetal bovine serum. When cells are treated with R1881+RT, nuclear translocation of AR was induced at similar or greater levels compared to R1881 alone in AR+ TNBC cells. Combination treatment of RT with enzalutamide in the presence of hormones reduced nuclear localization of AR (32-39% reduction) compared to RT alone. RNA-sequencing after RT identified transcriptional changes potentially regulated by AR+RT, including changes in the NHEJ pathway genes. Additionally, pathway analyses in these models demonstrated changes in the MAPK/ERK signaling pathway, among others, that may regulate RT resistance in AR+ TNBC models. Most 2nd generation anti-androgens confer radiosensitization in AR+ TNBC models with cellular localization changes of AR noted after RT. The known structural differences amongst 2nd generation anti-androgens may account for differences in radiosensitization noted. Furthermore, AR-mediated radioresistance may be due, at least in part, to downstream MAPK/ERK signaling. This work builds on the mechanistic understanding of AR-mediated radioresistance in AR+ TNBC and may expose vulnerabilities to overcome resistance to combination treatment with AR inhibition and RT.

Combination of BET Inhibition with Radiation Induces Anti-Tumor Immune Response in Pre-Clinical Models of ER-Positive Breast Cancer

Despite advances in multidisciplinary care, the risk of late recurrence in Estrogen Receptor-positive (ER+) breast cancer (BC) patients following standard-of-care treatment ranges from 20-52% at 20 years. While immune checkpoint inhibitors (CPI), have shown enormous potential in inducing long term remission in some cases, patients with ER+ BC show poor response to these agents. The objective of this study is to develop effective radiation therapy (RT)-based combination regimens that induce anti-tumor immunity against immunologically cold ER+ breast tumors. We have previously shown that pharmacological inhibition of bromodomain and extraterminal domain (BET) family of epigenetic regulator proteins overcomes both endocrine therapy resistance and radiation resistance in pre-clinical models of ER+ BC by reversing pathological reprogramming of transcriptional and DNA repair pathways, respectively. In this study, we explore the potential of RT+BET inhibition combination to elicit anti-tumor immunity against ER+ BC. A unique ER+ BC model, MXT+, was used to generate tumors in female, BDF1 mice. PD-L1 expression following RT or RT+OTX015 (a BET inhibitor) was assessed by Western blot analysis. Comet assay was used to assess the degree of unrepaired DNA damage following treatment with RT alone, OTX015 alone and RT+OTX015. Multiplex IHC was used to assess changes in tumor microenvironment after various treatments. Tumor growth characteristics were established following treatment with vehicle, RT alone (8 Gy x 3), OTX015 alone (100 mg/kg x 3 days) and RT+OTX015. Using murine ER+ BC model, MXT+, we show that RT is a potent inducer of PD-L1 expression. Combination of RT+OTX015 completely ablated RT-mediated PD-L1 induction at the transcription level. OTX015 also accentuated RT-induced DNA damage through inhibition of NHEJ pathway, resulting in increased micronuclei formation (which activate cGAS/STING pathway, a critical bridge between innate and adaptive immunity). Combination of OTX015 with RT also caused a statistically significant (on unpaired t test) increase tumor infiltrating lymphocytes. In in vivo therapeutic studies, combination of OTX015 with RT (8 Gy x 3) resulted in extraordinary synergy and caused complete inhibition of long-term tumor growth. Treatment with RT alone (8 Gy x 3) or OTX015 alone (on 3 consecutive days) only caused modest inhibition of tumor growth. ANOVA with Dunnett's test was used to adjust for multiple comparisons and showed statistically significant difference in tumor growth between OTX015+RT arm and OTX alone as well as OTX015+RT arm and RT alone. Patients with ER+ BC face high risk of late recurrence after definitive treatment and show poor response to immune CPIs. We show that combination of BET inhibition with RT induces anti-tumor immunity and completely prevents tumor growth. Clinical validation of this regimen is warranted to prevent late recurrences in ER+ BC patients.

Tanshinone I suppresses hepatocellular carcinoma cells growth through targeting DNA double-strand break repair

ABSTRACT Hepatocellular carcinoma (HCC) is one of the most common types of malignant tumors with increasing incidence rates and high mortality rates. The currently available methods for treating HCC include surgery, radiotherapy or chemotherapy, but all of them have limitations. Therefore, developing novel therapeutic methods for HCC is in great need. Here, in this study, we found that tanshinone I, a small molecule compound, inhibited the proliferation of HCC cells in a dose-dependent manner. We also observed that Tanshinone I destabilized genomes by inhibiting both NHEJ and HR repair pathways, which are responsible for repairing DNA double strand breaks (DSBs). Mechanistically, this compound suppressed the expression of 53BP1, and the recruitment of RPA2 to DNA damage sites. Importantly, we demonstrated that combining Tanshinone I with radiotherapy exhibited better therapeutic potential for treating HCC.

SMYD2 inhibition-mediated hypomethylation of Ku70 contributes to impaired nonhomologous end joining repair and antitumor immunity.

DNA damage repair (DDR) is a double-edged sword with different roles in cancer susceptibility and drug resistance. Recent studies suggest that DDR inhibitors affect immune surveillance. However, this phenomenon is poorly understood. We report that methyltransferase SMYD2 plays an essential role in nonhomologous end joining repair (NHEJ), driving tumor cells adaptive to radiotherapy. Mechanically, in response to DNA damage, SMYD2 is mobilized onto chromatin and methylates Ku70 at lysine-74, lysine-516, and lysine-539, leading to increased recruitment of Ku70/Ku80/DNA-PKcs complex. Knockdown of SMYD2 or its inhibitor AZ505 results in persistent DNA damage and improper repair, which sequentially leads to accumulation of cytosolic DNA, and activation of cGAS-STING pathway and triggers antitumor immunity via infiltration and activation of cytotoxic CD8+ T cells. Our study reveals an unidentified role of SMYD2 in regulating NHEJ pathway and innate immune responses, suggesting that SMYD2 is a promising therapeutic target for cancer treatment.

Abstract B054: Inherited mutations of DNA damage repair genes in a racially diverse cohort of men with prostate cancer

Abstract Introduction: DNA damage repair genes (DDRGs) play an important role in genomic stability and mutations in DDRGs contribute to clinically significant prostate cancer (PCa). However, the association of germline mutations in DDRGs with PCa progression and therapeutic stratification remain to be defined in African American (AA) men. Our objective was to genomically profile all known DDRGs and provide ancestry specific findings, focusing on PCa patients with African ancestry. Methods: Germline mutations in all DDRGs (N=276) was evaluated by whole genome sequence (WGS) analysis of archived blood DNA samples from 600 PCa patients (300 AA and 300 CA) who underwent primary treatment at Walter Reed National Military Medical Center. The WGS mean coverage exceeded 37x. Principal Component Analysis using the Peddy program was conducted, and ancestry prediction was based on a machine learning approach using 1000G as reference. Variant frequencies in CPDR PCa patients were compared to variant frequencies available from the Exome Aggregation Consortium (ExAC) controls with no PCa. Data analysis approaches included single variant association, gene-based total frequency test and clinico-pathological associations for germline mutations. All statistical tests were two-sided, and FDR adjusted. FDR < 0.05 was set as significance threshold. Results: Comprehensive analysis of 276 DDRGs revealed that germline mutation (Pathogenic/Likely Pathogenic; P/LP) carrier rate was 23.5%, which is three times higher than reported before in both AA and CA men, due to our unbiased WGS approach. We found that only 13 of the 46 DDRGs identified with P/LP mutations were common to AA and CA men, demonstrating clear disparity along ancestry lines. Importantly, RAD family genes (RAD54L, RAD54B, RAD51), which are potentially targetable, as well as PMS2 and BRCA1, were among the most frequently mutated DDRGs in AA, but not in CA men. Germline mutations in RAD51 and PMS2 were enriched in AA cohort as compared to CA men (P 0.01 and 0.03 respectively), while germline mutations in FANCA were significantly enriched in CA cohort (P < 0.01). Pathway analysis showed that the HR pathway (with 15 mutations) was associated with faster disease progression to biochemical recurrence (BCR) (P <0.05), while the NHEJ pathway (with three mutations) was found to be marginally associated with metastasis (p <0.05). Additionally, we found that germline mutations in DDRGs was associated with shorter time to BCR (P <0.05) in AA patients, but not in CA men. Conclusion: Our findings highlight distinct DDRG germline mutation profile across AA and CA prostate cancer men. This may help refine patient stratification for specific targeted therapeutic options especially for under-studied AA men. Future replication studies in an independent patient cohort are warranted to validate our findings. Citation Format: Indu Kohaar, Xijun Zhang, Gregory Chesnut, Clifton Dalgard, Matthew D. Wilkerson, Gyorgy Petrovics. Inherited mutations of DNA damage repair genes in a racially diverse cohort of men with prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B054.

A novel NHEJ gene signature based model for risk stratification and prognosis prediction in hepatocellular carcinoma

BackgroundNon-homologous DNA end joining (NHEJ) is the predominant DNA double-strand break (DSB) repair pathway in human. However, the relationship between NHEJ pathway and hepatocellular carcinoma (HCC) is unclear. We aimed to explore the potential prognostic role of NHEJ genes and to develop an NHEJ-based prognosis signature for HCC.MethodsTwo cohorts from public database were incorporated into this study. The Kaplan–Meier curve, the Least absolute shrinkage and selection operator (LASSO) regression analysis, and Cox analyses were implemented to determine the prognostic genes. A NHEJ-related risk model was created and verified by independent cohorts. We derived enriched pathways between the high- and low-risk groups using Gene Set Enrichment Analysis (GSEA). CIBERSORT and microenvironment cell populations-counter algorithm were used to perform immune infiltration analysis. XRCC6 is a core NHEJ gene and immunohistochemistry (IHC) was further performed to elucidate the prognostic impact. In vitro proliferation assays were conducted to investigate the specific effect of XRCC6.ResultsA novel NHEJ-related risk model was developed based on 6 NHEJ genes and patients were divided into distinct risk groups according to the risk score. The high-risk group had a poorer survival than those in the low-risk group (P < 0.001). Meanwhile, an obvious discrepancy in the landscape of the immune microenvironment also indicated that distinct immune status might be a potential determinant affecting prognosis as well as immunotherapy reactiveness. High XRCC6 expression level associates with poor outcome in HCC. Moreover, XRCC6 could promote HCC cell proliferation in vitro.ConclusionsIn brief, this work reveals a novel NHEJ-related risk signature for prognostic evaluation of HCC patients, which may be a potential biomarker of HCC immunotherapy.

RAD51 and RAD51B Play Diverse Roles in the Repair of DNA Double Strand Breaks in Physcomitrium patens.

RAD51 is involved in finding and invading homologous DNA sequences for accurate homologous recombination (HR). Its paralogs have evolved to regulate and promote RAD51 functions. The efficient gene targeting and high HR rates are unique in plants only in the moss Physcomitrium patens (P. patens). In addition to two functionally equivalent RAD51 genes (RAD1-1 and RAD51-2), other RAD51 paralogues were also identified in P. patens. For elucidation of RAD51's involvement during DSB repair, two knockout lines were constructed, one mutated in both RAD51 genes (Pprad51-1-2) and the second with mutated RAD51B gene (Pprad51B). Both lines are equally hypersensitive to bleomycin, in contrast to their very different DSB repair efficiency. Whereas DSB repair in Pprad51-1-2 is even faster than in WT, in Pprad51B, it is slow, particularly during the second phase of repair kinetic. We interpret these results as PpRAD51-1 and -2 being true functional homologs of ancestral RAD51 involved in the homology search during HR. Absence of RAD51 redirects DSB repair to the fast NHEJ pathway and leads to a reduced 5S and 18S rDNA copy number. The exact role of the RAD51B paralog remains unclear, though it is important in damage recognition and orchestrating HR response.

TGFβ pathway is required for viable gestation of Fanconi anemia embryos.

Overexpression of the TGFβ pathway impairs the proliferation of the hematopoietic stem and progenitor cells (HSPCs) pool in Fanconi anemia (FA). TGFβ promotes the expression of NHEJ genes, known to function in a low-fidelity DNA repair pathway, and pharmacological inhibition of TGFβ signaling rescues FA HSPCs. Here, we demonstrate that genetic disruption of Smad3, a transducer of the canonical TGFβ pathway, modifies the phenotype of FA mouse models deficient for Fancd2. We observed that the TGFβ and NHEJ pathway genes are overexpressed during the embryogenesis of Fancd2-/- mice and that the Fancd2-/-Smad3-/- double knockout (DKO) mice undergo high levels of embryonic lethality due to loss of the TGFβ-NHEJ axis. Fancd2-deficient embryos acquire extensive genomic instability during gestation which is not reversed by Smad3 inactivation. Strikingly, the few DKO survivors have activated the non-canonical TGFβ-ERK pathway, ensuring expression of NHEJ genes during embryogenesis and improved survival. Activation of the TGFβ-NHEJ axis was critical for the survival of the few Fancd2-/-Smad3-/- DKO newborn mice but had detrimental consequences for these surviving mice, such as enhanced genomic instability and ineffective hematopoiesis.

Structural basis for the inactivation of cytosolic DNA sensing by the vaccinia virus

Detection of cytosolic DNA is a central element of the innate immunity system against viral infection. The Ku heterodimer, a component of the NHEJ pathway of DNA repair in the nucleus, functions as DNA sensor that detects dsDNA of viruses that replicate in the cytoplasm. Vaccinia virus expresses two proteins, C4 and C16, that inactivate DNA sensing and enhance virulence. The structural basis for this is unknown. Here we determine the structure of the C16 – Ku complex using cryoEM. Ku binds dsDNA by a preformed ring but C16 sterically blocks this access route, abrogating binding to a dsDNA end and its insertion into DNA-PK, thereby averting signalling into the downstream innate immunity system. C4 replicates these activities using a domain with 54% identity to C16. Our results reveal how vaccinia virus subverts the capacity of Ku to recognize viral DNA.

Significance of the Wnt canonical pathway in radiotoxicity via oxidative stress of electron beam radiation and its molecular control in mice

Purpose Radiation triggers cell death events through signaling proteins, but the combined mechanism of these events is unexplored The Wnt canonical pathway, on the other hand, is essential for cell regeneration and cell fate determination. Aim The relationship between the Wnt pathway's response to radiation and its role in radiotoxicity is overlooked, even though it is a critical molecular control of the cell. The Wnt pathway has been predicted to have radioprotective properties in some reports, but the overall mechanism is unknown. We intend to investigate how this combined cascade works throughout the radiation process and its significance over radiotoxicity. Materials and methods Thirty adult mice were irradiated with electron beam radiation, and 5 served as controls. Mice were sacrificed after 24 h and 30 days of irradiation. We assessed DNA damage studies, oxidative stress parameters, mRNA profiles, protein level (liver, kidney, spleen, and germ cells), sperm viability, and motility. Observation The mRNA profile helps to understand how the combined cascade of the Wnt pathway and NHEJ work together during radiation to combat oxidative response and cell survival. The quantitative examination of mRNA uncovers unique critical changes in all mRNA levels in all cases, particularly in germ cells. Recuperation was likewise seen in post-30 day’s radiation in the liver, spleen, and kidney followed by oxidative stress parameters, however not in germ cells. It proposes that reproductive physiology is exceptionally sensitive to radiation, even at the molecular level. It also suggests the suppression of Lef1/Axin2 could be the main reason for the permanent failure of the sperm function process. Post-irradiation likewise influences the morphology of sperm. The decrease in mRNA levels of Lef1, Axin2, Survivin, Ku70, and XRCC6 levels suggests radiation inhibits the Wnt canonical pathway and failure in DNA repair mechanisms in a coupled manner. An increase in Bax, Bcl2, and caspase3 suggests apoptosis activation followed by the decreased expression of enzymatic antioxidants. Conclusion Controlled several interlinked such as the Wnt canonical pathway, NHEJ pathway, and intrinsic apoptotic pathway execute when the whole body is exposed to radiation. These pathways decide the cell fate whether it will survive or will go to apoptosis which may further be used in a study to counterpart and better comprehend medication focus on radiation treatment