Gemcitabine-based chemotherapy remains the standard first-line treatment for cholangiocarcinoma (CCA), but acquired resistance presents a significant clinical challenge. Synthetic lethality approaches targeting double-strand break repair (DSBR) pathways offer promising therapeutic opportunities. Ataxia-telangiectasia mutated (ATM) kinase, a central regulator of homologous recombination (HR) and non-homologous end joining (NHEJ), is critical for maintaining genomic integrity following DNA damage. Here, we demonstrate that combining the ATM inhibitor AZD0156 with DNA-damaging agents (cisplatin or photon irradiation) significantly enhances cytotoxicity in gemcitabine-resistant intrahepatic CCA sublines (GR-iCCAs) while sparing gemcitabine-sensitive parental cells. This selective sensitization manifests in impaired colony formation, increased apoptosis, and persistent γ-H2AX nuclear accumulation. The magnitude of AZD0156 sensitization in GR cells substantially exceeds additive expectations, strongly suggesting synergistic interaction. Genetic ATM depletion in GR-iCCAs under genotoxic stress recapitulated these effects, confirming on-target specificity. Mechanistically, GR-iCCAs exhibit significantly reduced DNA ligase I (LIG1) expression, a critical component of the alternative NHEJ (alt-NHEJ) repair pathway, particularly under DNA damage conditions. Genetic restoration of LIG1 expression reversed AZD0156 sensitivity, establishing LIG1 deficiency as a key determinant modulating DNA repair pathway dependency. In xenograft models, AZD0156 combined with cisplatin substantially suppressed tumor growth compared to monotherapy, with acceptable tolerability profiles. These findings identify ATM inhibition as a promising strategy to overcome gemcitabine resistance in CCA, particularly in tumors with compromised alt-NHEJ repair capacity, providing a mechanistic rationale for clinical development of this combination therapy.

The Role of Genetic Testing in Localized Prostate Cancer.

Ribosomal DNA (rDNA) encodes the 18S, 5.8S, and 28S rRNA, accounting for ∼70% of cellular transcription. Despite its essential role and links to cancer and aging, quantifying rDNA instability in mammals remains challenging due to its repetitive organization and inherent heterogeneity. Here, we developed a murine rDNA FISH probe and genomic tools tailored for laboratory mouse strains. The results confirmed rDNA cluster locations, revealed substantial inter- and intra-strain as well as intercellular heterogeneity in rDNA organization within inbred mice and unstressed cells, and identified sources of spontaneous and replication-associated DNA double-strand breaks in the rDNA transcription termination region. Using mouse embryonic stem cells, we showed that BRCA1-mediated homologous recombination promotes rDNA instability, the non-homologous end joining factor XRCC1, but not Ku, suppresses intra-cluster deletions, and ATM kinase preserves rDNA cluster stability. Together, these findings establish a platform and tools for studying rDNA instability in animal models relevant to aging and cancer research.

Polygenic risk scores (PGS) have the potential to support enhanced, risk-based screening for breast cancer. Previous studies for many diseases found that genome-wide PGS (GW-PGS) outperform PGS derived by applying hard GWAS significance thresholds. To support future breast cancer risk predictions, we compared the predictive performance of two existing PGS (including PGS313, a leading hard-thresholding PGS) and five newly developed GW-PGS (applying different methods to recent GWAS). We evaluated the performance of PGS Z-scores and of predicted 5-year absolute breast cancer risks based on age alone or age and PGS, across three large cohorts from the UK (UK Biobank) and Australia (QSkin, Melbourne Collaborative Cohort Study). Performance was assessed using discrimination (AUC) and calibration metrics, with dedicated evaluations for European, South Asian and African genetic ancestry groups, different age groups and for UKB, by pre-baseline mammogram screening history. Z-scores from three GW-PGS (LDpred2, PRS-CS, PRS-CS2017) yielded improved discrimination over PGS313, especially in European and South Asian ancestry groups (AUC improvements 2-18%, p < 0.029). Incorporating PGS substantially improved absolute risk predictions compared to age-only models, with the strongest evidence in European-ancestry groups (AUC improvements 15-39%, p < 10⁻⁴) and similar trends in non-European groups. No PGS outperformed all others across all ancestry groups. Estimated relative risk for highest GW-PGS risk groups (e.g. top 5% LDpred2) was ~2.5-fold population-average risk, similar to previous estimates for individuals with pathogenic variants in ATM and CHEK2 genes. These findings support the potential of PGS for risk-based breast cancer screening, noting that current GW-PGS may not substantially improve breast cancer risk predictions compared to PGS313.

Pathogenic germline variants in the ATM gene are associated with a 20–30% lifetime risk of breast cancer. Crucially, a relevant fraction of loss-of-function variants in breast cancer susceptibility genes disrupts pre-mRNA splicing. We aimed to perform splicing analysis of ATM splice-site variants identified in the large-scale sequencing project BRIDGES (Breast Cancer After Diagnostic Gene Sequencing). To this end, we bioinformatically selected 47 splice-site variants across 17 exons that were genetically engineered into three minigenes and assayed in MCF-7 cells. Aberrant splicing was observed in 38 variants. Of these, 30 variants, including 7 missense, yielded no or negligible expression of the minigene full-length (mgFL) transcript. A total of 69 different transcripts were characterized, 48 of which harboured a premature termination codon. Some variants, such as c.2922-1G>A, generated complex patterns with up to 10 different transcripts. Alternative 3′ or 5′ splice-site usage was the predominant event. Integration of ATM minigene read-outs into the ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based specifications for the ATM gene enabled the classification of 30 ATM variants as pathogenic or likely pathogenic and 9 as likely benign. Overall, splicing assays provide key information for variant interpretation and the clinical management of patients.

Tumor resistance to radiotherapy continues to be a significant problem in improving cancer patient outcomes. To overcome radioresistance, drugs that sensitize cancer cells to ionizing radiation have been tested. In theory, radiosensitizers should increase irradiated tumor kill and improve patient outcomes. In practice, the clinical utility of such drugs is curtailed by radiosensitization of peri-tumoral normal tissues causing toxicities. To address these issues, we developed an activatable cell penetrating peptide-drug conjugate to deliver a small molecule radiosensitizer with spatial precision to tumors. The activatable cell penetrating peptide (ACPP) scaffold cloaks a cell penetrating peptide-drug conjugate until it is unmasked within tumors through matrix metalloproteinase cleavage. Using antibody-drug conjugate linker chemistry, we attached the potent ataxia-telangiectasia mutated (ATM) kinase inhibitor AZD0156 to ACPP and created ACPP-AZD0156. In immune-competent murine cancer models, tumor-targeted ACPP-AZD0156 in combination with ionizing radiation stimulated tumor immune infiltration by CD8+ T cells and increased tumor control when compared to non-targeted ATM inhibitor. Mechanistically, ACPP-AZD0156 radiosensitized tumor control was dependent on the adaptive arm of the immune system. Finally, the combination of radiotherapy and ACPP-AZD0156 potentiated immune checkpoint inhibitors that resulted in durable tumor control. The therapeutic synergies of ACPP targeted ATM inhibitor to radiosensitize and stimulate anti-tumor immune responses provides a rationale for developing tumor-targeted radiosensitizer drug conjugates that restrict radiosensitization to cancer cells that then engages anti-tumor immune responses to improve cancer patient outcomes.

The FANCD2-FANCI heterodimer coordinates chromatin openness and cell cycle progression throughout DNA double-strand break repair.

Newcastle disease (ND), caused by virulent strains of the Newcastle disease virus (NDV), is a highly contagious disease that poses significant economic burdens on the global poultry industry. The DNA damage response (DDR) is a critical cellular mechanism that detects and repairs genomic damage to maintain cellular integrity. While viral infections are known to modulate DDR pathways to either inhibit or enhance viral replication, the interaction between NDV and host DDR remains largely underexplored. Here, we demonstrate that NDV infection induces significant DNA damage in DF-1 cells and activates DDR signaling, primarily via the ataxia-telangiectasia mutated (ATM) kinase pathway, in a manner dependent on active viral replication. Pharmacological inhibition of ATM kinase, but not ataxia telangiectasia and Rad3-related (ATR) kinase, significantly suppresses NDV replication, alleviates virus-induced G1-phase cell cycle arrest, and modulates the host immune response. Moreover, short interfering RNA (siRNA)-mediated knockdown of Chk2 markedly reduced viral M gene expression and progeny production, indicating that Chk2 is required for efficient NDV replication. These findings suggest that NDV exploits the ATM–Chk2 DDR pathway to establish a replication-favorable environment. Our study provides new insights into NDV pathogenesis and highlights potential targets for antiviral interventions.

Ultra-low background radiation inhibits head and neck tumor via ATM downregulation mediated mitochondrial dysfunction.

Mitochondrial dysfunction is a critical driver of cardiac remodeling under conditions of chronic stress, such as hypertension and heart failure. Andrographolide, a bioactive diterpenoid from Andrographis paniculata, has demonstrated antioxidative and anti-inflammatory effects; however, its role in mitochondrial quality control within the heart remains unclear. In this study, a network pharmacology approach was applied to explore the molecular targets of andrographolide related to mitochondrial dysfunction in cardiac remodeling. A total of 1763 mitochondrial-associated cardiac remodeling genes were retrieved from CardGenes and intersected with 539 andrographolide-related targets identified using PharmMapper, SwissTargetPrediction, and the comparative toxicogenomics database. Fifty-four overlapping genes were subjected to protein–protein interaction analysis using STRING and Cytoscape. The top-ranked hub gene was identified as TP53. Functional enrichment indicated key involvement in the PI3K-Akt, mitogen-activated protein kinases (MAPK), Forkhead box protein (FOXO), and AGE-RAGE pathways, linking andrographolide to the modulation of oxidative stress, metabolism, and cell survival. Gene ontology (GO) terms supported roles in kinase activity and membrane-associated signaling. Molecular docking showed strong binding affinities between andrographolide and TP53 regulators, particularly ataxia telangiectasia mutated (ATM) (−8.63 kcal/mol, Kd 6.34 × 10−7 M) and checkpoint 2 (CHK2) (−8.47 kcal/mol, Kd 4.68 × 10−7 M). Machine learning predictions indicated favorable LELP and moderate bioactivity for ataxia telangiectasia Rad3-related (ATR), CHK2, and Sirtuin 1 (SIRT1). These findings suggest that andrographolide exerts cardioprotective effects by modulating mitochondrial stress signaling and p53 regulatory networks. Further experimental validation is warranted to confirm its therapeutic potential in cardiac remodeling-related diseases.

Objective. To evaluate the clinical efficacy of follow-up of healthy carriers of pathogenic germline variants associated with hereditary tumor syndromes and to determine the impact of personalized screening programs on early diagnosis of breast cancer. Material and methods. A prospective study included 986 participants who underwent genetic testing in the Loginov Moscow Clinical Scientific Center in 2018—2024. Among the participants, 551 people are healthy carriers of mutations (group A), 435 — patients with previously diagnosed malignant neoplasms (group B). Persons with germline mutations in BRCA1, BRCA2, CHEK2, PALB2, ATM, STK11 and TP53 genes were included. Monitoring was performed every 6 months, it included specialist consultations, mammography or magnetic resonance imaging, ultrasound examination of the mammary glands and pelvic organs. The primary outcome was the stage of detected breast cancer in mutation carriers compared to patients in whom disease has been diagnosed outside the screening program. Results. In healthy carriers of the mutations (group A, 551 people in total), 57 (10.3%) cases of cancer (predominantly breast cancer — 56 cases) have been detected during follow-up. Tumors were diagnosed at the stage I—II in 94.7% of the subjects, at the stage III — in 3.5%, cases of stage IV were not registered. Similar stages were revealed in 69.4% of the group B patients, the proportion of advanced stages (III—IV) amounted to 22%. BRCA1 c.5366dupC (p.Gln1777ProfsTer74) variant was the most frequent mutation. The proportion of triple-negative subtypes reached about 40% in both groups. Conclusion. Personalized follow-up and screening programs for the carriers of pathogenic germline variants ensure predominant early breast cancer detection and form an effective prevention model for high genetic risk groups.

Since 2014, a model of the individual response to ionizing radiation (IR), based on the radiation-induced nucleoshuttling of the ATM protein kinase (RIANS), has been developed by our lab: after irradiation, ATM dimers monomerize in cytoplasm and diffuse into the nucleus to trigger both recognition and repair of DNA double-strand breaks (DSB), the key-damage of IR response. Moderate radiosensitivity is generally caused by heterozygous mutations of ATM substrates (called X-proteins) that are over-expressed in cytoplasm and form complexes with ATM monomers, which reduces and/or delays the RIANS and DSB recognition. Here, we asked whether molecules, rather than X-proteins, can also influence RIANS. Caffeine was chosen as a potential “X-molecule” candidate. After incubation of cells with caffeine, cutaneous fibroblasts from an apparently healthy radioresistant donor, a patient suffering from Alzheimer’s disease (AD) and another suffering from neurofibromatosis type 1 (NF1) were exposed to X-rays. The functionality of ATM-dependent DSB repair and signaling was evaluated. We report here that caffeine molecule interaction with ATM leads to the inhibition of DSB recognition. This effect is significant in radioresistant cells. Conversely, in the AD and NF1 cells, the DSB recognition is already so low that caffeine does not provide any additional molecular effect.

PurposeThis study explores the therapeutic potential of sodium dodecyl sulphate (SDS)-based nanocarriers (NCs) for the targeted delivery of paclitaxel (PTX) to breast cancer (BC) cells, with a particular focus on the mechanisms governing their intracellular transport and biological activity.MethodsTwo types of SDS-based NCs differing in polyelectrolyte composition: poly-L-lysine (SDS/PLL) and poly-L-lysine with poly-L-glutamic acid (SDS/PLL/PGA), were prepared following the Layer-by-Layer (LbL) technique. Cellular uptake and distribution of Rhodamine B (RhoB)-labelled NCs were assessed via fluorescence microscopy and quantified by flow cytometry across three human cell lines: dermal microvascular endothelial cell line (HMEC-1), epithelial breast adenocarcinoma cell line (MCF-7), and triple-negative, mesenchymal-like BC cell line (MDA-MB-231). The cytotoxic and genotoxic effects of PTX-loaded NCs were evaluated using spectrophotometric and spectrofluorimetric assays. In parallel, DNA damage-responsive gene expression was examined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR).ResultsBoth NC formulations demonstrated comparable uptake efficiency, despite differences in fluorescence intensity. Inhibitor-based studies revealed distinct internalization pathways: SDS/PLL NCs entered via dynamin-dependent endocytosis and macropinocytosis, whereas SDS/PLL/PGA NCs relied predominantly on macropinocytosis. Genotoxicity of PTX-loaded NCs was confirmed by comet assay and H2A histone family member X (γH2AX) phosphorylation, particularly in MCF-7 and MDA-MB-231 cells. Cell cycle perturbations and transcriptional changes in ataxia-telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and cyclin-dependent kinase 1 (CDK1) genes accompanied these effects. Enzyme-linked immunosorbent assay (ELISA)-based analyses further demonstrated apoptosis-mediated cytotoxicity induced by both investigated formulations.ConclusionThese findings delineate the cellular uptake mechanisms and in vitro biological effects of the examined polyelectrolyte NCs for PTX delivery, with a particular focus on their genotoxicity. Collectively, these in vitro data provide a mechanistic basis to inform the rational design and preclinical optimization of SDS-based NCs, supporting subsequent in vivo evaluation.

Meiotic recombination is initiated by DNA double-strand breaks (DSBs); factors that control DSB frequencies are important to produce viable progeny. In many organisms, the ATM (Tel1) protein kinase prevents excessive meiotic DSBs, especially nearby DSBs on the same chromatid. Normally, two close DSBs are less frequent than expected from independence, a feature called DSB interference, which is lost in tel1Δ mutants. In the fission yeast Schizosaccharomyces pombe, high-level DSB formation depends on linear elements, Hop1, and meiotic cohesin complexes; we show here that these complexes impart competition between nearby DSB sites. When these complexes are impaired, Tel1 substantially represses DSB formation, and in its absence, two close DSBs on the same chromatid occur frequently and manifest high negative interference. After mitotic DNA damage, the conserved Mre11–Rad50–Nbs1 (MRN) complex is required for DNA resection, and the Tel1 kinase activity is needed to complete DSB repair. We found that during meiosis mre11Δ and rad50Δ mutants, like tel1Δ mutants, lack DSB interference and display highly negative DSB interference in meiotic complex mutants. Thus, MRN at a DSB site appears critical for Tel1 function in meiosis and reveals a complex interplay of positive and negative factors controlling meiotic DSB formation.

Ataxia-telangiectasia (A-T), caused by biallelic mutations in the ATM gene, leads to multiple disease phenotypes, including cerebellar neurodegeneration, radiosensitivity, cancer predisposition, immunodeficiency, insulin resistance, and pulmonary inflammation. ATM plays a central role in regulating cellular responses to DNA breakage [M. B. Kastan, J. Bartek, Nature 432, 316-323 (2004)], but several cellular and physiologic abnormalities associated with ATM dysfunction suggest the possibility of noncanonical roles for ATM as well. Herein, we identified the HSP90 paralogue, GRP94, as an ATM interactor/substrate and found that ATM influences N-glycosylation of GRP94 and its subsequent activation/translocation to the plasma membrane, where it serves as a scaffold protein and stabilizer for several membrane proteins, including receptor tyrosine kinases (RTKs), such as EGFR and IGF1-R. In selected cell types, ATM loss/inhibition resulted in increased cell surface expression of RTKs and overactivation of RTK pathways, alterations that were rescued by specific inhibition of cell surface GRP94. This ATM/GRP94 pathway also regulated the activation of microglial cells, manifest as increased cytokine production and phagocytosis activity associated with ATM loss/inhibition and reversal of that activation with GRP94 inhibition. These results identified GRP94 as an ATM interactor and apparent substrate and demonstrated specific critical regulatory roles for ATM outside of DNA damage signaling. These insights provide potential explanations for several of the phenotypes associated with ATM dysfunction and potential opportunities for novel approaches to blunt clinical symptoms in A-T, and also suggest that other neurodegenerative and inflammatory disorders might benefit from selective inhibition of cell surface GRP94.

DNA double-strand breaks (DSB) represent one of the most severe forms of genomic damage. Thus, cells have evolved a complex network of DSB repair pathways, including homologous recombination, classical and alternative end joining, and single-strand annealing, which are tightly regulated by genetic and epigenetic factors. The selection and efficiency of these pathways influence genome integrity, oncogenesis, and therapeutic response. This comprehensive review synthesizes recent findings on the genetic regulation of DSB repair, with emphasis on pathway-specific regulators, chromatin context, and post-translational modifications. Moreover, this review integrates primary research from mammalian systems, including CRISPR-based studies, proteomics, and imaging, with a focus on publications from 2020 to 2025. We discuss the role of key players, such as MRE11-RAD50-NBS1 (MRN), ataxia telangiectasia mutated (ATM), mediator tumor suppressor p53-binding protein 1 (53BP1), breast cancer type 1 susceptibility protein (BRCA1), anti-silencing function 1 (ASF1), ring finger protein (RNF)8/168, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and RAD51 recombinase (RAD51), in orchestrating the associated pathway choice. Epigenetic modifications, RNA-mediated mechanisms, and chromatin remodeling dynamically influence the efficiency and fidelity of repair. Particular attention is provided to emerging regulators, including thyroid hormone receptor interactor 13 (TRIP13), ubiquitin-like with plant homeodomain (PHD) and RING finger domains 1 (UHRF1), Shieldin, and polymerase theta. This review highlights novel insights into transcription-associated DSB repair, the interplay of replication stress with repair pathway engagement, and context-dependent synthetic lethality. We also examine implications for cancer biology, including therapy resistance and biomarker development. Ultimately, understanding the genetic regulation of DSB repair pathways can provide critical insights into genome stability maintenance and reveal new therapeutic opportunities in cancer. Future work should focus on pathway crosstalk, phase-specific regulation, and integrating repair modulation into personalized medicine.

Aberrant innate immune responses contribute significantly to cellular senescence, yet the precise interplay between innate immunity and senescence remains poorly characterized. Here, we elucidate the pivotal role of nuclear respiratory factor 1 (NRF1) in orchestrating innate immune responses that drive senescence and the senescence-associated secretory phenotype (SASP). NRF1 deficiency delayed cellular senescence and ameliorated age-related deterioration in multiple organs. Mechanistically, NRF1 enhanced SASP by transcriptionally regulating TBK1 and IRF3, critical nodes in innate immunity essential for senescence induction. Conversely, NRF1 deficiency suppressed innate immune activation, thereby attenuating inflammation associated with senescence and aging. Additionally, DNA damage activated ATM kinase, which phosphorylated NRF1 at Ser393, augmenting the NRF1-TBK1/IRF3-type I interferon axis and exacerbating cellular senescence. Furthermore, NRF1 knockdown treatment effectively mitigated aging phenotypes and extended lifespan in aged mice. Collectively, our findings underscore the essential role of the ATM-NRF1-TBK1/IRF3-type I interferon axis in DNA damage-induced senescence, suggesting that targeted NRF1 modulation holds therapeutic promise for improving inflammaging.

There is a lack of knowledge in full-body skin examinations (FBSEs) in the context of pathogenic or likely pathogenic variants (PV/LPV) in skin cancer predisposition genes (CPGs). This study assessed the association between carrier status of PV/LPV in skin CPGs and FBSEs and described the patterns of family letter receipt, familial disclosure, and cascade testing among high-risk individuals. Between July and September 2023, we surveyed participants enrolled in the Chicago Cancer Prone Study. Carrier status of PV/LPV in skin CPGs was defined as having been told by a genetic counselor that they have a PV/LPV in CDKN2A, TP53, PTEN, BRIP1, PALB2, ATM, and/or CHEK2 genes. FBSEs were assessed by asking individuals whether they ever had an FBSE and modeled using logistic regression. Of 579 individuals (mean age, 59.5 years), 11.6% carry PV/LPV in skin CPGs and 63.8% ever had a FBSE. Carriers had greater odds of FBSEs than non-carriers (adjusted odds ratio, 2.15; 95% CI, 1.03-4.52). White race, older age, and female sex were associated with increased odds of FBSEs. Among carriers, 65.3% received a family letter from their genetic counselor after testing; 91.0% disclosed their PV/LPV-carrying status to any family members, and 62.7% of the individuals' family members underwent genetic testing. We found greater odds of FBSEs among PV/LPV carriers, notable disparities in FBSEs, and prevalent family letter receipt, familial disclosure, and cascade testing. This study highlights the role of PV/LPV-carrying status on FBSEs and the potential need for comprehensive guidelines beyond known risk factors to optimize skin cancer screening in high-risk populations.

Metastasis in breast cancer frequently spreads to the bones, significantly impacting patient outcomes and escalating mortality rates. The ataxia-telangiectasia mutated (ATM) kinase plays a pivotal role in regulating the DNA damage response (DDR) and has been linked to the invasion and spread of breast cancer. In this study we investigated the regulatory mechanisms of ATM in bone metastasis of breast cancer. The bone metastases models were constructed in female nude mice: The MDA-MB-231 tumor model was generated by implanting luciferase-tagged MDA-MB-231 cells into the left hind tibia and intra-caudal artery. For the SK-BR-3 tumor model, luciferase-tagged SK-BR-3 cells were injected through the intra-caudal artery. By conducting bioinformatics analyses and in vitro and in vivo experiments, we found that ATM expression was markedly elevated in bone metastasis samples compared to liver, lung or skin metastases. We demonstrated that ATM boosted the migrative and invasive abilities and pre-osteoclast differentiation of MDA-MB-231 and SK-BR-3 cell lines via expression of CCL2, an osteoclast-related cytokine. The regulation of ATM on CCL2 was found to be NFκB dependent. In vivo experiments confirmed that ATM knockout (ATM KO) or treatment with small-molecule ATM inhibitor KU55933 markedly inhibited osteoclastogenesis of SK-BR-3 cells and the progression of breast cancer bone metastasis. Our results underscore the pivotal role of ATM in regulating NFκB-CCL2 expression and promoting the progression of breast cancer bone metastasis.

ATM inhibitors in cancer radiotherapy: Mechanisms, clinical development, and future directions.