Investigating the DNA Damage Response Elicited by Transposable Element Integration

Introduction Senescence is a tumor-suppressive mechanism characterized by a halt in cellular proliferation. It can be triggered by various genotoxic sources, such as exposure to ionizing radiation during anticancer therapies to prevent the proliferation of cells with DNA damage. These cells adopt a senescence-associated secretory phenotype (SASP), which consists of hundreds of molecules, including cytokines, chemokines, and other pro-inflammatory factors. These secretions have beneficial anti-proliferative effects. However, their long-term accumulation can lead to detrimental outcomes, such as aging and tumorigenesis, driven by chronic inflammation. The senescence mechanism develops progressively in multiple steps over several days following genotoxic stress. In our model of irradiation-induced senescence (IR-10Gy) in human fibroblasts (BJ, HCA2), IR activates DNA damage response (DDR) pathways. These cells enter senescence 7 to 10 days post-exposure and develop their SASP. It is well established that there is a link between DDR activation and SASP induction. The genetic depletion of key DDR players (ATM, CHK2, NBS1) results in SASP suppression. However, the timing of these processes does not coincide. While DDR pathways are activated within minutes of DNA damage, the SASP takes several days to develop. Thus, the mechanism by which DDR influences SASP activation remains poorly understood. We identified a late non-canonical branch of the DDR (ncDDR) that appears to activate several days after the initial stress and is associated with the late pro-inflammatory SASP. In contrast, the canonical DDR pathway seems responsible for early non-inflammatory secretions. These findings suggest that the composition of senescent cell secretions evolves over time. Objective: We hypothesize that during the multi-step process of senescence establishment, the composition of the SASP evolves based on the activation or deactivation of DDR pathways. Modulating DDR activation through irradiation or genetic/pharmacological approaches will allow us to understand its impact on SASP. Our objective is to determine how the activation of different DDR pathways drives SASP induction and how these pathways influence its composition over time. Results: We irradiated fibroblasts with increasing doses of irradiation (10, 20, 40, 80 Gy) to sustain a high level of canonical DDR activation. At day 10 post-IR, we observed: • a dose-dependent decrease in the number of cells with genomic instability, • an almost complete suppression of the pro-inflammatory factor IL-6, • a significant increase in the secretion of GDF15, another SASP factor. Interestingly, these results were reversed when analyzing the secretome 24 hours post-irradiation. In conclusion, increased DDR activation induced a significant increase in various components of the early SASP and demonstrated distinct regulation between the early and late SASP. Citation Format: Jordan Desmul, Nicolas Malaquin, Francis Rodier. Senescence and secreted factors: implication of DNA damage response pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 300.

The DNA damage response (DDR) is a signaling cascade that is vital to ensuring the fidelity of the host genome in the presence of genotoxic stress. Growing evidence has emphasized the importance of both activation and repression of the host DDR by diverse DNA and RNA viruses. Previous work has shown that HIV-1 is also capable of engaging the host DDR, primarily through the conserved accessory protein Vpr. However, the extent of this engagement has remained unclear. Here, we show that HIV-1 and HIV-2 Vpr directly induce DNA damage and stall DNA replication, leading to the activation of several markers of double- and single-strand DNA breaks. Despite causing damage and activating the DDR, we found that Vpr represses the repair of double-strand breaks (DSB) by inhibiting homologous recombination (HR) and nonhomologous end joining (NHEJ). Mutational analyses of Vpr revealed that DNA damage and DDR activation are independent from repression of HR and Vpr-mediated cell cycle arrest. Moreover, we show that repression of HR does not require cell cycle arrest but instead may precede this long-standing enigmatic Vpr phenotype. Together, our data uncover that Vpr globally modulates the host DDR at at least two independent steps, offering novel insight into the primary functions of lentiviral Vpr and the roles of the DNA damage response in lentiviral replication.IMPORTANCE The DNA damage response (DDR) is a signaling cascade that safeguards the genome from genotoxic agents, including human pathogens. However, the DDR has also been utilized by many pathogens, such as human immunodeficiency virus (HIV), to enhance infection. To properly treat HIV-positive individuals, we must understand how the virus usurps our own cellular processes. Here, we have found that an important yet poorly understood gene in HIV, Vpr, targets the DDR at two unique steps: it causes damage and activates DDR signaling, and it represses the ability of cells to repair this damage, which we hypothesize is central to the primary function of Vpr. In clarifying these important functions of Vpr, our work highlights the multiple ways human pathogens engage the DDR and further suggests that modulation of the DDR is a novel way to help in the fight against HIV.

Differential expression of TP53 associated genes in Fanconi anemia cells after mitomycin C and hydroxyurea treatment

DNA damage response (DDR) is a coordinated network of diverse cellular processes including the detection, signaling, and repair of DNA lesions, the adjustment of metabolic network and cell fate determination. To deal with the unavoidable DNA damage caused by either endogenous or exogenous stresses, the cells need to reshape the gene expression profile to allow efficient transcription and translation of DDR-responsive messenger RNAs (mRNAs) and to repress the nonessential mRNAs. A predominant method to adjust RNA fate is achieved by modulating the 3'-end oligo(A) or poly(A) length via the opposing actions of polyadenylation and deadenylation. Poly(A)-specific ribonuclease (PARN) and the carbon catabolite repressor 4 (CCR4)-Not complex, the major executors of deadenylation, are indispensable to DDR and genomic integrity in eukaryotic cells. PARN modulates cell cycle progression by regulating the stabilities of mRNAs and microRNA (miRNAs) involved in the p53 pathway and contributes to genomic stability by affecting the biogenesis of noncoding RNAs including miRNAs and telomeric RNA. The CCR4-Not complex is involved in diverse pathways of DDR including transcriptional regulation, signaling pathways, mRNA stabilities, translation regulation, and protein degradation. The RNA targets of deadenylases are tuned by the DDR signaling pathways, while in turn the deadenylases can regulate the levels of DNA damage-responsive proteins. The mutual feedback between deadenylases and the DDR signaling pathways allows the cells to precisely control DDR by dynamically adjusting the levels of sensors and effectors of the DDR signaling pathways. Here, the diverse functions of deadenylases in DDR are summarized and the underlying mechanisms are proposed according to recent findings. This article is categorized under: RNA Processing > 3' End Processing RNA in Disease and Development > RNA in Disease RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms.

DNA double strand breaks (DSBs) result in activation of several key DNA damage response (DDR) kinases including ATM, ATR, and DNA-PK. These protein kinases not only promote DNA damage-induced checkpoint control, but also facilitate DSB repair in humans. Thus, these DDR kinases have become promising drug targets for cancer therapy. However, the benefits of targeting DDR kinases remain to be realized, in part due to the lack of predictive biomarkers. By undertaking CRISPR screens with inhibitors targeting key DDR kinases, we obtained a global and unbiased view of genetic interactions with DDR inhibition. Additionally, we compared the synergistic effects of combining different DDR inhibitors and found that ATM and PARP inhibitors showed remarkable synergy, which depends on the dominant negative function of ATM inhibition. Moreover, to provide comprehensive and unbiased perspective of DDR signaling pathways, we performed 30 fluorescence-activated cell sorting–based genome-wide CRISPR screens with antibodies recognizing distinct endogenous DNA damage–signaling proteins to identify new regulators involved in DNA damage response (DDR). We discovered that proteasome-mediated processing is an early and prerequisite event for cells to trigger camptothecin- and etoposide-induced DDR signaling. Furthermore, we identified PRMT1 and PRMT5 as new modulators that regulate ATM protein level. Additionally, we discovered that GNB1L is a master regulator of DDR signaling via its role as a co-chaperone for PIKK proteins. Collectively, these screens offer a rich resource for further investigation of DDR, which may provide insight into strategies of targeting these DDR pathways to improve therapeutic outcomes. More recently, we have adopted dTAG technology for the investigation of many essential DDR and replication proteins, which provide mechanistic insights into the roles of these essential proteins in genome maintenance. Additionally, we have expanded FACS-based screens for the studies of other cancer-associated pathways and explored key biological processes critical for cancer progression in vivo. These studies will facilitate the development of better therapies for cancer patients. Citation Format: Junjie Chen. Targeting DNA damage responsive pathways in cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr A009.

DNA damage and the DNA damage response have been identified in human atherosclerosis, including in vascular smooth muscle cells (VSMCs). However, although double-stranded breaks (DSBs) are hypothesized to promote plaque progression and instability, in part, by promoting cell senescence, apoptosis, and inflammation, the direct effects of DSBs in VSMCs seen in atherogenesis are unknown. To determine the presence and effect of endogenous levels of DSBs in VSMCs on atherosclerosis. Human atherosclerotic plaque VSMCs showed increased expression of multiple DNA damage response proteins in vitro and in vivo, particularly the MRE11/RAD50/NBS1 complex that senses DSB repair. Oxidative stress-induced DSBs were increased in plaque VSMCs, but DSB repair was maintained. To determine the effect of DSBs on atherosclerosis, we generated 2 novel transgenic mice lines expressing NBS1 or C-terminal deleted NBS1 only in VSMCs, and crossed them with apolipoprotein E(-/-) mice. SM22α-NBS1/apolipoprotein E(-/-) VSMCs showed enhanced DSB repair and decreased growth arrest and apoptosis, whereas SM22α-(ΔC)NBS1/apolipoprotein E(-/-) VSMCs showed reduced DSB repair and increased growth arrest and apoptosis. Accelerating or retarding DSB repair did not affect atherosclerosis extent or composition. However, VSMC DNA damage reduced relative fibrous cap areas, whereas accelerating DSB repair increased cap area and VSMC content. Human atherosclerotic plaque VSMCs show increased DNA damage, including DSBs and DNA damage response activation. VSMC DNA damage has minimal effects on atherogenesis, but alters plaque phenotype inhibiting fibrous cap areas in advanced lesions. Inhibiting DNA damage in atherosclerosis may be a novel target to promote plaque stability.

BackgroundWe developed a novel system for quantifying DNA damage response (DDR) to help diagnose and predict the risk of Alzheimer’s disease (AD).MethodsWe thoroughly estimated the DDR patterns in AD patients Using 179 DDR regulators. Single-cell techniques were conducted to validate the DDR levels and intercellular communications in cognitively impaired patients. The consensus clustering algorithm was utilized to group 167 AD patients into diverse subgroups after a WGCNA approach was employed to discover DDR-related lncRNAs. The distinctions between the categories in terms of clinical characteristics, DDR levels, biological behaviors, and immunological characteristics were evaluated. For the purpose of choosing distinctive lncRNAs associated with DDR, four machine learning algorithms, including LASSO, SVM-RFE, RF, and XGBoost, were utilized. A risk model was established based on the characteristic lncRNAs.ResultsThe progression of AD was highly correlated with DDR levels. Single-cell studies confirmed that DDR activity was lower in cognitively impaired patients and was mainly enriched in T cells and B cells. DDR-related lncRNAs were discovered based on gene expression, and two different heterogeneous subtypes (C1 and C2) were identified. DDR C1 belonged to the non-immune phenotype, while DDR C2 was regarded as the immune phenotype. Based on various machine learning techniques, four distinctive lncRNAs associated with DDR, including FBXO30-DT, TBX2-AS1, ADAMTS9-AS2, and MEG3 were discovered. The 4-lncRNA based riskScore demonstrated acceptable efficacy in the diagnosis of AD and offered significant clinical advantages to AD patients. The riskScore ultimately divided AD patients into low- and high-risk categories. In comparison to the low-risk group, high-risk patients showed lower DDR activity, accompanied by higher levels of immune infiltration and immunological score. The prospective medications for the treatment of AD patients with low and high risk also included arachidonyltrifluoromethane and TTNPB, respectively,ConclusionsIn conclusion, immunological microenvironment and disease progression in AD patients were significantly predicted by DDR-associated genes and lncRNAs. A theoretical underpinning for the individualized treatment of AD patients was provided by the suggested genetic subtypes and risk model based on DDR.

Aging-Related Gene Expression Signatures Define Likelihood of Disease Progression in Patients with Lower-Risk Myelodysplastic Syndromes

The genome of an organism is under constant attack from endogenous and exogenous DNA damaging factors, such as reactive radicals, radiation, and genotoxins. Therefore, DNA damage response systems to sense DNA damage, arrest cell cycle, repair DNA lesions, and/or induce programmed cell death are crucial for maintenance of genomic integrity and survival of the organism. Genome sequences revealed that, although plants possess many of the DNA damage response factors that are present in the animal systems, they are missing some of the important regulators, such as the p53 tumor suppressor. These observations suggest differences in the DNA damage response mechanisms between plants and animals. In this review the DNA damage responses in plants and animals are compared and contrasted. In addition, the function of SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a plant-specific transcription factor that governs the robust response to DNA damage, is discussed.

Tumor promoting role of the DNA damage response

10574 Background: Approximately 10% of cancers stem from inherited germline pathogenic variants (gPV), predominantly in DNA repair genes including ATM and BRCA1 or BRCA2 . Identification of a gPV in high-risk families can guide management, however the clinical implications of variants of uncertain significance (VUS) identified by genetic testing remain unclear. Our aim is to develop a DNA damage response (DDR) activity profile of high-risk populations with/without cancer with ATM or BRCA2 gPV to assist in determining the relevance of ATM or BRCA2 gPV/VUS. Methods: We profiled several key proteins that play a role in DDR using a Luminex-based Multianalyte immunoassay (hereafter referred to as DDR xMAP) in peripheral blood monocytes (PBMCs) derived from whole blood. The standardized DDR xMAP assay was first applied to PBMC specimens from sporadic colorectal cancer (CRC, n = 95) patients and cancer-free age-matched controls (n = 47) at baseline. The DDR xMAP was used to profile seven DDR proteins, phosphorylated Chk1 S345 , Chk2 T68 , γH2AX S139 , p53 S15 and total ATR, MDM2, p21. Univariate classification and regression tree analysis was used to identify statistically significant cut points in DDR analyte levels. We then measured the DDR analyte levels in individuals with BRCA2 gPVs with (n = 11) and without a diagnosis of cancer (n = 11) as well as in cancer-free non-carrier controls (n = 15) at baseline. We compared these values using the two-sided Mann-Whitney test and the Benjamini-Hochberg false discovery rate method to account for multiple markers. Results: Using the initial set of CRC cases and healthy controls, we identified statistically significant cut points in multiple DDR analyte levels including total ATR ( > 81.8), Chk1 S345 ( > 28.0) and γH2AX S139 ( > 51.3) that can individually distinguish between CRC cases and cancer-free controls (P < 0.001). Next, in preliminary analysis of patients with BRCA2 gPV with and without cancer had increased levels of all DDR analytes (P < 0.05) compared to non-carrier cancer-free controls, except for γH2AX S139 . Levels of proteins involved in replication stress response, ATR and downstream Chk1 S345 , were elevated. Total MDM2 levels, negative regulator of p53, were highly elevated in BRCA2 gPV carriers. There was not a significant difference between DDR analytes for those with BRCA2 gPV with and without cancer. Conclusions: Profiling key DDR markers can significantly distinguish between CRC patients and cancer-free controls. DDR analytes are significantly increased in PBMC specimens from individuals with BRCA2 gPV (with or without a diagnosis of cancer) compared to non-carrier cancer-free controls. Ongoing research in high-risk groups will establish cut points for this assay, improving our understanding of DDR activity in individuals with ATM/BRCA2 gPV and clarifying the clinical significance of gPV/VUS.

Arrhythmogenic cardiomyopathy (ACM) encompasses a genetically heterogeneous group of myocardial diseases whose manifestations are sudden cardiac death, cardiac arrhythmias, heart failure, and in a subset fibro-adipogenic infiltration of the myocardium. Mutations in the TMEM43 gene, encoding transmembrane protein 43 (TMEM43) are known to cause ACM. The purpose of the study was to gain insights into the molecular pathogenesis of ACM caused by TMEM43 haploinsufficiency. The Tmem43 gene was specifically deleted in cardiac myocytes by crossing the Myh6-Cre and floxed Tmem43 mice. Myh6-Cre:Tmem43W/F mice showed an age-dependent phenotype characterized by an increased mortality, cardiac dilatation and dysfunction, myocardial fibrosis, adipogenesis, and apoptosis. Sequencing of cardiac myocyte transcripts prior to and after the onset of cardiac phenotype predicted early activation of the TP53 pathway. Increased TP53 activity was associated with increased levels of markers of DNA damage response (DDR), and a subset of senescence-associated secretary phenotype (SASP). Activation of DDR, TP53, SASP, and their selected downstream effectors, including phospho-SMAD2 and phospho-SMAD3 were validated by alternative methods, including immunoblotting. Expression of SASP was associated with epithelial-mesenchymal transition and age-dependent expression of myocardial fibrosis and apoptosis in the Myh6-Cre:Tmem43W/F mice. TMEM43 haploinsufficiency is associated with activation of the DDR and the TP53 pathways, which lead to increased expression of SASP and an age-dependent expression of a pro-fibrotic cardiomyopathy. Given that TMEM43 is a nuclear envelope protein and our previous data showing deficiency of another nuclear envelope protein, namely lamin A/C, activates the DDR/TP53 pathway, we surmise that DNA damage is a shared mechanism in the pathogenesis of cardiomyopathies caused by mutations involving nuclear envelope proteins.

The recent finding that MYC-driven cancers are sensitive to inhibition of the DNA damage response (DDR) pathway, prompted us to investigate the role of DDR pathway as therapeutic target in diffuse large B-cell lymphoma (DLBCL), which frequently overexpresses the MYC oncogene. In a preliminary immunohistochemical study conducted on 99 consecutive DLBCL patients, we found that about half of DLBCLs showed constitutive expression of the phosphorylated forms of checkpoint kinases (CHK) and CDC25c, markers of DDR activation, and of phosphorylated histone H2AX (γH2AX), marker of DNA damage and genomic instability. Constitutive γH2AX expression correlated with c-MYC levels and DDR activation, and defined a subset of tumors characterised by poor outcome. Next, we used the CHK inhibitor PF-0477736 as a tool to investigate whether the inhibition of the DDR pathway might represent a novel therapeutic approach in DLBCL. Submicromolar concentrations of PF-0477736 hindered proliferation in DLBCL cell lines with activated DDR pathway. These results were fully recapitulated with a different CHK inhibitor (AZD-7762). Inhibition of checkpoint kinases induced rapid DNA damage accumulation and apoptosis in DLBCL cell lines and primary cells. These data suggest that pharmacologic inhibition of DDR through targeting of CHK kinases may represent a novel therapeutic strategy in DLBCL.

The DNA damage response (DDR) has a critical role in maintaining genome integrity and serves as a barrier to tumorigenesis by promoting cell-cycle arrest, DNA repair, and apoptosis. The DDR is activated not only by genotoxic agents that induce DNA damage, but also during aberrant cell-division cycles caused by activated oncogenes and inactivated tumor suppressors. Here we use RNAi and cDNA overexpression screens in human cells to identify genes that, when deregulated, lead to activation of the DDR. The RNAi screen identified 73 genes that, when silenced in at least two cell types, cause DDR activation. Silencing several of these genes also caused an increased frequency of micronuclei, a marker of genetically unstable cells. The cDNA screen identified 97 genes that when overexpressed induce DDR activation in the absence of any exogenous genotoxic agent, with an overrepresentation of genes linked to cancer. Secondary RNAi screens identified CDK2-interacting protein (CINP) as a cell-cycle checkpoint protein. CINP interacts with ATR-interacting protein and regulates ATR-dependent signaling, resistance to replication stress, and G2 checkpoint integrity.

Introduction: Cigarette smoking is an important risk factor for the development pancreatic ductal adenocarcinoma (PDAC). Nicotine and nitrosamines cause pancreatic cell injury and contribute to a cascade of oncogenic events. Among multiple tobacco specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is considered to be the most carcinogenic and induces DNA double strand-breaks (DSBs). However, identification of potential prognostic biomarkers and experimental models of smoking-induced PDAC remain unknown. We have studied phosphorylated histone H2AX (γH2AX), a marker for double-stranded DNA breaks, as a new biomarker for detecting cigarette smoke-induced pathogenesis of PDAC. Experimental Procedure: We have characterized the expression of γH2AX protein in human pancreatic tissues, human pancreatic duct epithelial (HPDE)6, PDAC cell lines, and PanIn, primary PDAC (PDA) and liver metastasis (LMP) cell lines generated from a genetically engineered mice model of PDAC. Kaplan-Meier survival analysis was performed to determine the significance of γH2AX expression amongst smokers and non-smokers with PDAC. HPDE6 and PanIN cells were cultured for 4 days in the presence or absence of cigarette smoke (CS) (0-25μg/ml) or NNK (0-10μM). Cell cycle analysis and apoptosis were assessed through flow cytometry with the markers propidium iodide and annexin V respectively. HPDE6 and PanIN cells treated with CS or NNK for up to 50 days were analyzed for rapid activation of DNA damage response (DDR) pathway proteins γH2AX, RAD51, BRCA1, BRCA2, phospho-CHK2 and phospho-CHK1. Cells were treated with either CS or NNK along with a CHK2 inhibitor and analyzed for activation of γH2AX. To determine DNA integrity, accumulation of γH2AX was measured by ELISA in cells treated with CS or NNK. Immunofluorescence confocal microscopy was utilized to examine the role of γH2AX on nuclear foci formation with DNA repair complexes by quantifying the amount of co-localization. To replicate chronic smoke exposure, we treated HPDE6 and PanIn cells with 1 and 10 µg/ml CS or 10nM and 1µM NNK for 10 passages, and examined these cell lines for DSBs by comet assay, apoptosis markers by protein array and in vitro tumorigenicity by colony formation assay. Results: γH2AX expression was significantly higher (p<0.0001) in smokers when compared to non-smokers. Overall survival of patients with high γH2AX expression in their primary tumor was associated with significantly decreased median survival (11 months) when compared to low γH2AX expression (36 months, p=0.0007). HPDE6 cells exposed to CS or NNK, showed more than 80% survival. The surviving cells had altered morphology, acquired mesenchymal characteristics and displayed increased tumorigenicity suggesting that these cells acquire resistance to apoptosis after CS or NNK exposure. In addition, exposure of cells to CS or NNK resulted in generation of γH2AX and induction of phospho-CHK2 expression. RAD51 and phospho-CHK2 co-localize with γH2AX after DNA damage with CS or NNK exposure. Inhibition of CHK2 reduced expression of γH2AX. The product of the tumor suppressor gene BRCA2 co-localized with γH2AX indicating that γH2AX is necessary for the recruitment of DNA repair factors to the site of DNA damage. BRCA2 mutant cell line CAPAN1 exhibited a substantially reduced ability to form co-localization with RAD51 and γH2AX, consistent with a role of γH2AX in DNA damage repair. Conclusion: This work supports the role of γH2AX as a new molecular biomarker to assess the contribution of cigarette smoking-induced PDAC. γH2AX is necessary for DNA damage repair in PDAC and its activation is dependent on CHK2. Therapies that can block γH2AX expression by inhibiting upstream kinases may be a new potential therapeutic approach to treat and/or prevent PDAC. Citation Format: Kumaraswamy Honnenahally, Chanjuan Shi, Xi Chen, Jason Castellanos, Nipun Merchant, Timothy Blackwell, Nagaraj Nagathihalli. γH2AX: A molecular marker of DNA damage response in smoking-induced pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A33.