Ataxia Telangiectasia Mutated Protein Research Articles

ATM: a protein involved in glucose and lipid metabolism in the heart

Abstract Backround Post-mitotic cells, such as neurons and cardiomyocytes, cannot repair DNA lesions with DNA replication and rely for their survival on efficient sensors and effectors that orchestrate the DNA damage response (DDR). The Ataxia Telangiectasia Mutated (ATM) protein kinase is the most important sensor of oxidative stress and the DNA damage response (DDR) and is implicated in cellular metabolism. It is believed that while transient DNA damage and DDR can temporarily improve cardiovascular function, persistent activation of DDR (and ATM) might promote the onset and development of heart failure (HF). Purpose We hypothesised that ATM might control and regulate energy metabolism in the heart under stress to promote DNA repair. Methods We analyzed the effects of ATM inactivation on cardiomyocyte hypertrophy, cardiac function, DDR and metabolism in hearts from wild-type (Atm+/+) or Atm-mutated (Atm-/-) mice under sham conditions or after pressure overload by transverse aortic constriction (TAC). Results ATM inactivation in Atm-/- mice induced cardiomyocyte hypertrophy, fetal gene expression re-activation and a specific metabolomic signature in the heart, characterized by significant accumulation of pyruvate, branched chain amino-acids, short-medium acyl-carnitines and metabolites of tricarboxylic acid cycle. The levels of the glycolytic enzymes, hexokinase-2 (HK2) and phosphofructokinase (PFK), were elevated. pyruvate was trapped in the cytosol because mitochondrial carriers were suppressed and the enzymes that process pyruvate were dysregulated. Because of pyruvate metabolic block, fatty acids oxidation was inefficient and resulted in the accumulation of acyl-carnitines and insulin resistance. These metabolic changes were amplified by TAC, which rapidly induced heart failure in Atm-/- mice. ATM inactivation also increased basal and TAC-induced genomic stress in cardiomyocytes, as shown by the levels of p-γ-H2AX, 8-oxodG glycosylase (OGG1/2) and apurinic site nuclease (APE1). These results prove that ATM rewires the metabolism of cardiac cells by inducing glycolysis and fatty acids oxidation. ATM stimulates glycolysis to repair DNA lesions and protect the heart against stress-induced dysfunction. The metabolic block due to ATM inactivation accelerates heart failure. Conclusions Our data suggest that ATM favors the metabolic flexibility of the heart by stimulating glucose entry and balancing glucose catabolism with fatty acid oxidation, thus preventing mechanical stress-induced cardiac systolic dysfunction.

Association of ATM and ARID1A in gastric carcinoma

BackgroundThe ataxia telangiectasia mutated (ATM) gene is involved in the repair of double-stranded DNA breaks and a component of the DNA damage repair pathway. Tumors with mutations or low expression of both ARID1A and ATM exhibit increased numbers of tumor-infiltrating lymphocytes and a favorable prognosis. However, the relationship between ATM and ARID1A in gastric carcinoma (GC) is unclear. MethodsWe used the mRNA expression data from the Asian Cancer Research Group to construct tissue microarrays (N = 249). Next-generation sequencing (NGS) databases of Samsung Medical Center (SMC) (N = 813) were used to compare genetic alterations. Tissue microarrays were used for ATM and ARID1A immunohistochemistry, and expressions were categorized as “low” and “high.” NGS data from TCGA-STAD (N = 431) were used as independent cohorts for genetic alterations validation. ResultsIn GCs, 32.1 % (80/249) of the cases showed low ATM protein expression (ATMlow) and 20.9 % (52/249) showed low ARID1A expression (ARID1Alow). ATMlow was significantly associated with older age (P <.01), gross type of tumor (P =.02), histology (P <. 01), lower incidence of perineural invasion (P =.04), lower disease stage (P <.01), microsatellite instability-high (P <.01), and ARID1Alow (P <.01). Furthermore, GCs in the SMC NGS database showed that ATM mutations were significantly correlated with ARID1A mutations (P <.01), and this finding remained significant in TCGA-STAD validation cohort (P <.01). ConclusionATMlow in GCs shows a characteristic clinicopathological feature that correlates strongly with ARID1Alow. ATM mutation was also associated with ARID1A mutations, highlighting the interactions between ATM and ARID1A in GC and suggesting a potential therapeutic target.

Regulation of transcription patterns, poly(ADP-ribose), and RNA-DNA hybrids by the ATM protein kinase

The ataxia telangiectasia mutated (ATM) protein kinase is a master regulator of the DNA damage response and also an important sensor of oxidative stress. Analysis of gene expression in ataxia-telangiectasia (A-T) patient brain tissue shows that large-scale transcriptional changes occur in patient cerebellum that correlate with the expression level and guanine-cytosine (GC) content of transcribed genes. In human neuron-like cells in culture, we map locations of poly(ADP-ribose) and RNA-DNA hybrid accumulation genome-wide with ATM inhibition and find that these marks also coincide with high transcription levels, active transcription histone marks, and high GC content. Antioxidant treatment reverses the accumulation of R-loops in transcribed regions, consistent with the central role of reactive oxygen species in promoting these lesions. Based on these results, we postulate that transcription-associated lesions accumulate in ATM-deficient cells and that the single-strand breaks and PARylation at these sites ultimately generate changes in transcription that compromise cerebellum function and lead to neurodegeneration over time in A-T patients.

Abstract A148: The radiosensitizing effects of the novel brain penetrant and potent ATM inhibitor WSD0628 in glioblastoma and melanoma patient derived xenografts

Abstract Radiation therapy (RT) is an important non-surgical clinical treatment for glioblastoma (GBM) and brain metastases, but inherent radio-resistance can limit the efficacy in many patients. The Ataxia Telangiectasia Mutated (ATM) protein critically regulates radiotherapy-mediated DNA damage repair pathways, and defects in this kinase can lead to profound radiosensitivity. In this study, the efficacy of the highly potent ATM inhibitor WSD0628 was evaluated in GBM and melanoma brain metastasis patient derived xenografts (PDXs). In short term explant cultures of three PDXs, WSD0628 robustly suppressed RT-induced autophosphorylation of ATM at serine 1981 and ATM-mediated phosphorylation of Chk2 at threonine-68 and KAP1 at serine-824, with maximal inhibition at 100 nM. Similarly, RT-induced gH2AX foci were significantly reduced when combined with WSD0628 in the GBM43 cells. Consistent with the importance of ATM in the DNA damage response after radiation, WSD0628 significantly increased the radiosensitivity of U251 cells in clonogenic survival assays (0nM vs. 30nM at 5Gy, p&amp;lt;0.01). To evaluate the optimal duration of drug exposure, WSD0628 was removed from U251 cells at various intervals following irradiation with 5 Gy. Using clonogenic survival as a readout, a 10-fold increase in cytotoxicity was observed with an 8 hour drug exposure, with progressively increasing cytotoxicity gains with exposures up to 24 hours. In vivo efficacy of WSD0628 was evaluated in multiple intracranial PDX models. In an initial dose-ranging study, robust radiosensitization was observed in GBM43 treated with 2 Gy x 5 fractions combined with 5 mg/kg WSD0628 (20 day survival extension) or 10mg/kg WSD0628 (39 day with 10 mg/kg; p&amp;lt;0.01 for both dose levels). Moreover, a single dose of 12.5 Gy combined with 10 mg/kg WSD0628 had profound impact on treatment efficacy in the melanoma brain metastasis M12 PDX: at 180 days post-treatment, all mice in the combination group were electively euthanized, while median survival for sham or RT alone was 17d and 49d, respectively. Histologic analysis identified large intracranial tumors in all sham and RT-only treated mice, but only a small accumulation of melanotic cells without obvious tumor in the combination-treated mice. Immunohistochemical staining of NeuN and GFAP in the combination-treated mice showed preserved neuronal density at the 180-post treatment timepoint and minimal reactive gliosis within the ‘tumor scar’. An ongoing study is comparing this single-fraction 12.5 Gy regimen to a 2.4 Gy x 10 fraction regimen alone or in combination with WSD0628. Collectively, this study demonstrates the potential for profound radiosensitizing effects of WSD0628 in combination without obvious neuronal toxicity and has provided the scientific rational for the first-in-man study of this combination in recurrent GBM (NCT05917145) at Mayo Clinic. Citation Format: Zhiyi Xue, Ann C. Mladek, Sneha Rathi, Danielle M. Burgenske, Shiv K. Gupta, Brett L. Carlson, Zeng Hu, Lauren L Ott, Katrina K. Bakken, Rachael A. Vaubel, William F Elmquist, Wei Zhong, Jann N Sarkaria. The radiosensitizing effects of the novel brain penetrant and potent ATM inhibitor WSD0628 in glioblastoma and melanoma patient derived xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A148.

Radiation Therapy for Ocular Melanoma – a Narrative Review with Insides from TRIUMF, Canada’s Only Proton Beam Therapy Center

"Ocular melanoma (OM) originates from melanocytes in the eye, predominantly in the uvea, particularly the choroid. The yearly incidence is around six cases per million. OM is not primarily driven by ultraviolet exposure like skin melanoma, but is usually caused by mutations in GNAQ or GNA11. Symptoms like blurry vision and visual field defects appear late. Diagnosis is often made via eye exams, specialized ultrasound, and rarely biopsy. This narrative review describes the radiation treatment modalities of OM and highlights the landscape of proton beam irradiation in Canada. Historically, enucleation was the standard of care for OM. However, current strategies consider tumor size, location, patient age, visual potential, and metastatic presence. Primary treatments include radiation therapy and surgery. Radiation therapy includes plaque brachytherapy (PB), proton beam irradiation (PBI), stereotactic radiosurgery (SRS), and stereotactic radiotherapy (SRT). Surgery includes endoresection, exoresection, and enucleation. Tebentafusp-tebn has been FDA-approved for metastatic cases. PB, the most common radiation therapy for OM, involves radioisotopes delivering radiation into the tumor. Comparable survival rates between PB and enucleation for medium choroidal melanoma have made PB the standard of care. PB has certain limitations, mainly surgical complications. PBI uses a particle accelerator for focused, high-energy proton radiation, yielding high tumor control and survival rates, though the availability of proton facilities is a significant limitation. Vancouver is the only center in Canada for PBI, administered not in a healthcare facility but at TRIUMF (Tri-University Meson Facility). TRIUMF, the world's largest cyclotron particle accelerator, in partnership with BC Cancer and UBC Department of Ophthalmology and Eye Care Center, has treated over 200 ocular melanoma patients between 1995 and 2017, achieving a 91% tumor control rate and 82% five-year survival rate. Emerging combination therapies like Ataxia Telangiectasia Mutated (ATM) protein kinase inhibition before PBI show potential, possibly reducing radiation dose and resistance."

Integrated Multi-Tissue Transcriptome Profiling Characterizes the Genetic Basis and Biomarkers Affecting Reproduction in Sheep (Ovis aries).

The heritability of litter size in sheep is low and controlled by multiple genes, but the research on its related genes is not sufficient. Here, to explore the expression pattern of multi-tissue genes in Chinese native sheep, we selected 10 tissues of the three adult ewes with the highest estimated breeding value in the early study of the prolific Xinggao sheep population. The global gene expression analysis showed that the ovary, uterus, and hypothalamus expressed the most genes. Using the Uniform Manifold Approximation and Projection (UMAP) cluster analysis, these samples were clustered into eight clusters. The functional enrichment analysis showed that the genes expressed in the spleen, uterus, and ovary were significantly enriched in the Ataxia Telangiectasia Mutated Protein (ATM) signaling pathway, and most genes in the liver, spleen, and ovary were enriched in the immune response pathway. Moreover, we focus on the expression genes of the hypothalamic-pituitary-ovarian axis (HPO) and found that 11,016 genes were co-expressed in the three tissues, and different tissues have different functions, but the oxytocin signaling pathway was widely enriched. To further explore the differences in the expression genes (DEGs) of HPO in different sheep breeds, we downloaded the transcriptome data in the public data, and the analysis of DEGs (Xinggao sheep vs. Sunite sheep in Hypothalamus, Xinggao sheep vs. Sunite sheep in Pituitary, and Xinggao sheep vs. Suffolk sheep in Ovary) revealed the neuroactive ligand-receptor interactions. In addition, the gene subsets of the transcription factors (TFs) of DEGs were identified. The results suggest that 51 TF genes and the homeobox TF may play an important role in transcriptional variation across the HPO. Altogether, our study provided the first fundamental resource to investigate the physiological functions and regulation mechanisms in sheep. This important data contributes to improving our understanding of the reproductive biology of sheep and isolating effecting molecular markers that can be used for genetic selection in sheep.

Toward an Early Diagnosis for Alzheimer's Disease Based on the Perinuclear Localization of the ATM Protein.

Alzheimer's disease (AD) is the most common neurodegenerative dementia, for which the molecular origins, genetic predisposition and therapeutic approach are still debated. In the 1980s, cells from AD patients were reported to be sensitive to ionizing radiation. In order to examine the molecular basis of this radiosensitivity, the ATM-dependent DNA double-strand breaks (DSB) signaling and repair were investigated by applying an approach based on the radiation-induced ataxia telangiectasia-mutated (ATM) protein nucleoshuttling (RIANS) model. Early after irradiation, all ten AD fibroblast cell lines tested showed impaired DSB recognition and delayed RIANS. AD fibroblasts specifically showed spontaneous perinuclear localization of phosphorylated ATM (pATM) forms. To our knowledge, such observation has never been reported before, and by considering the role of the ATM kinase in the stress response, it may introduce a novel interpretation of accelerated aging. Our data and a mathematical approach through a brand-new model suggest that, in response to a progressive and cumulative stress, cytoplasmic ATM monomers phosphorylate the APOE protein (pAPOE) close to the nuclear membrane and aggregate around the nucleus, preventing their entry in the nucleus and thus the recognition and repair of spontaneous DSB, which contributes to the aging process. Our findings suggest that pATM and/or pAPOE may serve as biomarkers for an early reliable diagnosis of AD on any fibroblast sample.

Exploration of clinical and genetic findings in Ataxia-Telangiectasia (AT) patients from the Indian subcontinent

BackgroundAtaxia-Telangiectasia (AT) is a rare autosomal recessive neurodegenerative disorder. It is caused by mutations in the Ataxia-Telangiectasia mutated (ATM) gene, which codes for protein ATM serine/threonine kinase. ObjectiveWe aim to describe the clinical and radiological findings in children and adolescents of 20 molecularly confirmed cases of AT. We aim to correlate these findings with the genotype identified among them. MethodsThis retrospective study included 20 patients diagnosed clinically and genetically with AT over 10 years. The clinical, radiological and laboratory data were extracted from the hospital's electronic medical records. Molecular testing was done using next generation sequencing and Sanger sequencing. In silico predictions were performed for the variants identified by applying Cryp-Skip, Splice site prediction by Neural Network, Mutation Taster and Hope prediction tool. ResultsConsanguinity was documented in nearly half of the patients. Telangiectasia was absent in 10%. Microcephaly was seen in 40% cases. The incidence of malignancy in our study population was low. Molecular testing done in the 18 families (20 patients) identified 23 variants of which ten were novel. Biallelic homozygous variants were noted in 13 families and compound heterozygous in 5 families. Out of the 13 families who were homozygous, 8 families (61.5%) (9 patients) have history of consanguinity. In silico prediction of novel missense variants, NM_000051.4 (ATM_v201): c.2702T > C showed disruption of the α-helix of ATM protein and NM_000051.4 (ATM_v201): c.6679C > G is expected to disturb the rigidity of protein structure in the FAT domain. The four novel splice site variants and two intronic variants result in exon skipping as predicted by Cryp-Skip. ConclusionsAT should be confirmed by molecular testing in young-onset cerebellar ataxia, even without telangiectasia. Awareness of this rare disease will facilitate study of larger cohorts from Indian population to characterize variants and determine its prevalence in this population.

Abstract 6192: MSI2 regulates DNA damage response signaling and tumor progression in murine NSCLC

Abstract Lung cancer is one of the most frequently diagnosed cancers and is the leading cause of cancer-related death worldwide, accounting for &amp;gt;2.2 million of new cancer diagnoses and 1.8 million cancer-related deaths in 2020. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, including adenocarcinomas and squamous cell carcinomas. Many lung adenocarcinomas have activating driver mutations in KRAS (~32% tumors), often co-occurring with mutations inactivating the TP53 tumor suppressor. Expression of the RNA-binding protein Musashi-2 (MSI2) MSI2 regulates target mRNA translation, and its expression is progressively elevated along with tumor stage in lung cancers. We previously showed that MSI2 actively promotes lung cancer growth, invasion, and metastasis, in part through promoting expression and activity of the TGFβR1/SMAD3 and EGFR signaling cascades. We now find that genetic MSI2 deficiency in a murine lung tumor model, Krasmut/Trp53KO/Msi2KO (KPM2) decreases both the total tumor number and tumor burden in comparison to control Krasmut/Trp53KO (KP) mice. Using a panel of KPM2 and KP cell lines we established from tumors from these mice, we found KPM2 cell lines demonstrate a significant decrease in proliferation compared to KP cell lines. Interestingly, reverse phase protein array (RPPA) comparison analysis of KPM2 and KP cell lines showed that MSI2 strongly and positively regulates expression of Ataxia-Telangiectasia Mutated (ATM) protein, with lower levels observed in KPM2 cells. The ATM serine/threonine protein kinase is a key transducer of DDR signaling in cases of double-strand breaks; ATM activation causes G1/S cell cycle arrest, contributes to efficiency of DNA repair, and triggers apoptosis in cases of repair failure. Notably, KPM2 cells showed significantly higher levels of γH2AX, suggesting inefficient DDR. Finally, untreated or cisplatin-treated KPM2 cells demonstrated G2/M arrest and had increased level of apoptosis as demonstrated by increase in caspase 3 cleavage, consistent with ATM loss and compensatory activation of Ataxia-telangiectasia and Rad3-related protein (ATR). Taken together, our data suggests that one of the consequences of MSI2 upregulation in NSCLC includes maintenance of an efficient DDR. Targeting MSI2 and its downstream signaling would be of therapeutic interest. Citation Format: Igor Bychkov, Alexander Deneka, Iuliia Topchu, Rajendra P. Pangeni, Christopher Lengner, Evgeny Izumchenko, Jyoti Patel, John Karanicolas, Petr Makhov, Yanis Boumber. MSI2 regulates DNA damage response signaling and tumor progression in murine NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6192.

Abstract 6180: Antitumor activity of the ATR inhibitor Elimusertib in patient-derived xenograft models with DNA damage response pathway alterations

Abstract Introduction: The ataxia telangiectasia and RAD3-related (ATR) kinase is a key component of the DNA damage response (DDR) and functions in conjunction with ataxia telangiectasia mutated (ATM). ATM loss or functional deficiency may enhance reliance on ATR signaling. Preclinical and clinical studies have pointed to a potential synthetic lethality of ATR inhibition and ATM loss. In this study, we sought to further elaborate potential candidates for ATR inhibition by testing the anti-tumor effect of elimusertib (BAY 1895344) in patient-derived tumor xenograft (PDX) models with DDR alterations including ATM loss. Methods: Patient derived xenograft (PDX) models were implanted into athymic nu/nu mice. Once tumors reached an appropriate size of approximately 200-400mm3 treatments were started. Mice were randomized (N= 3-8) to different treatment arms for assessment of anti-tumor activity of elimusertib as monotherapy and in combination therapy with copanlisib. Monotherapy treatment was performed with 20 mg/kg and 40 mg/kg doses, applied twice daily for 3 days with 4 days treatment break (BID 3 days on/4 days off). Efficacy read-out was either Event-Free Survival (EFS-2), defined as time for doubling of tumor volume relative to baseline, or response based on RECIST criteria:(partial response [PR]= &amp;gt;30% decrease, stable disease [SD]= between 20% increase and 30% decrease, and progressive disease [PD]= &amp;gt;20% increase). For assessment of pharmacodynamic effects, tumors were harvested 10 days after treatment start and analyzed by immunohistochemistry (IHC) and RPPA (Reverse Phase Proteomics Array). Results: Elimusertib was tested in 21 PDX models with various DDR alterations. Of those 11 showed a statistically significant prolongation of event free survival compared to control. In 4 PDX models elimusertib reached a partial response (PR), and another 4 models showed stable disease (SD). Amongst the 5 models with ATM protein Ioss (IHC), one was PR and another one SD. In 3 of 5 PARP inhibitor resistant there was statistically significant prolongation of EFS upon elimusertib treatment vs control. We found an increase in DNA damage in tumors, as indicated by increased level of ƳH2AX (on IHC and RPPA). We also detected an increase in PI3K/mTOR pathway signaling as determined by p-MTOR and pS6 (RPPA) in 2 of 4 models (PR and PD model) with different sensitivity to ATRi and PARPi alone. We tested the combination of elimusertib with the PI3K inhibitor copanlisib achieved a statistically significant increase of EFS compared to monotherapy in 3 of 11 models tested. Conclusion ATR inhibition shows potent monotherapy activity in selected PDX models that harbor DDR defects, including models with intrinsic and acquired PARPi resistance. Further work is needed to identify markers predicting sensitivity or resistance that providing rationale for synergistic combination therapies. Citation Format: Kaushik Varadarajan, Christian X. Pico, Kurt Evans, Maria G. Raso, Yasmeen Rizvi, Xiaofeng Zheng, Timothy P. Diperi, Bailiang Wang, Stephen Scott, Ming Zhao, Argun Akcakanat, Antje M. Wengner, Timothy A. Yap, Funda Meric-Bernstam. Antitumor activity of the ATR inhibitor Elimusertib in patient-derived xenograft models with DNA damage response pathway alterations. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6180.

Liposomal ATM siRNA delivery for enhancing triple-negaitive breast cancer immune checkpoint blockade therapy.

Triple-negative breast cancer (TNBC), which accounts for 10%-20% of breast cancer cases, is characterized by a higher metastasis rate, higher recurrence risk, and worse prognosis. Traditional treatments such as chemotherapy, surgery, and radiotherapy have limited therapeutic effects. Although immune checkpoint blockade (ICB) therapy represented by anti-programmed death 1 (aPD-1) antibody has made further progress in treating TNBC, its therapeutic effect is still not optimistic. Ataxia telangiectasia mutated (ATM) is a critical factor in the DNA damage response (DDR) pathway, which is associated with the development of tumors. Recent studies have found that it can regulate the tumor immune microenvironment, affecting ICB responsiveness. Inhibition of ATM could enhance ICB therapy by promoting mitochondrial DNA cytoplasmic leakage and activating the innate immune signaling pathway. To explore the effect of ATM siRNA(siATM) on the ICB responsiveness of TNBC, we designed and synthesized nanoparticles using 1,2-dioleoyl-glycero-3-phosphatidylcholine (DOPC) liposomes to deliver siATM. In vitro and in vivo experiments demonstrated that DOPC/siATM could enhance the ability of siRNA to enter tumor cells and effectively inhibit the expression of ATM protein. Our study found that nanoparticles carrying siATM could activate cytotoxic T lymphocytes and regulate the immunosuppressive tumor microenvironment (ITM) by activating the cGAS-STING pathway. Its combination with aPD-1 may be a potential way to improve the efficacy of TNBC.

HSA-MIR-183-3P TARGETING ATAXIA-TELANGIECTASIA MUTATED PROTEIN REGULATION OF NF-ΚB SIGNALING PATHWAY AFFECTS CELLULAR SENESCENCE CAUSED BY DNA DAMAGE IN LUMBAR DISC DEGENERATION.

To test the effect of Hsa-miR-183-3p on cell aging and disc degeneration in lumbar intervertebral disc. This study combined clinical research with basic cell experiment, analyzing clinical data from patients with lumbar disc degeneration and traumatic lumbar spine fracture, as well as the differences in baseline data. The degree of lumbar disc injury in patients of different ages was also compared. Differentially expressed miRNAs were predicted via GEO database, and qPCR confirmation was determined by collecting cartilage endplates from two groups. ACAN, Col2A1, p16, p21, and p53 were detected by immunofluorescence, Western blot and qPCR in human nucleus pulposus cells. Changes of cell senescence were detected. The binding of Hsa-miR-183-3p to ataxia-telangiectasia mutated protein was confirmed by dual luciferase reporter assay. Degenerative discs showed elevated expression of hsa-miR-183-3p, which may be upregulated by TNF-α via NF-κB signaling pathway and target ataxia-telangiectasia mutated protein regulation. Degeneration of the intervertebral disc can be accelerated by TNF-α. Additionally, Hsa-miR-183-3p passed NF-κB signaling pathway is blocked via up-regulation of TNF-α to reduce inflammation via targeting ataxia-telangiectasia mutated protein. As a result, this negative feedback mechanism may assist in maintaining a low degenerative load and preserving chronic disc degeneration.

RPRM negatively regulates ATM levels through its nuclear translocation on irradiation mediated by CDK4/6 and IPO11

SummaryHow the ataxia telangiectasia mutated (ATM) protein kinase, a core protein in DNA damage response, is regulated at post-transcription level remains unclear. Here it is identified that protein Reprimo (RPRM) downregulates ATM protein levels, resulting in impaired DNA repair and enhanced cellular radiosensitivity. Mechanistically, although primarily localized in the cytoplasm, RPRM translocates to the nucleus shortly after induced by X-irradiation, interacts with ATM and promotes its nuclear export and proteasomal degradation. The RPRM nuclear translocation involves its phosphorylation at serine 98 mediated by cyclin-dependent kinases 4/6 (CDK4/6), and requires Importin-11 (IPO11). Of importance, IPO11-regulated RPRM nuclear import upon irradiation is essential for its regulation on ATM. Thus, RPRM overexpression and its phosphorylation inhibition sensitize cells to genotoxic agents such as irradiation, whereas RPRM deficiency significantly increases resistance to radiation-induced damage both in vitro and in vivo. These findings establish a crucial regulatory mechanism in which ATM is negatively modulated by RPRM.

Minigene-based splicing analysis and ACMG/AMP-based tentative classification of 56 ATM variants.

The ataxia telangiectasia‐mutated (ATM) protein is a major coordinator of the DNA damage response pathway. ATM loss‐of‐function variants are associated with 2‐fold increased breast cancer risk. We aimed at identifying and classifying spliceogenic ATM variants detected in subjects of the large‐scale sequencing project BRIDGES. A total of 381 variants at the intron–exon boundaries were identified, 128 of which were predicted to be spliceogenic. After further filtering, we ended up selecting 56 variants for splicing analysis. Four functional minigenes (mgATM) spanning exons 4–9, 11–17, 25–29, and 49–52 were constructed in the splicing plasmid pSAD. Selected variants were genetically engineered into the four constructs and assayed in MCF‐7/HeLa cells. Forty‐eight variants (85.7%) impaired splicing, 32 of which did not show any trace of the full‐length (FL) transcript. A total of 43 transcripts were identified where the most prevalent event was exon/multi‐exon skipping. Twenty‐seven transcripts were predicted to truncate the ATM protein. A tentative ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)‐based classification scheme that integrates mgATM data allowed us to classify 29 ATM variants as pathogenic/likely pathogenic and seven variants as likely benign. Interestingly, the likely pathogenic variant c.1898+2T>G generated 13% of the minigene FL‐transcript due to the use of a noncanonical GG‐5’‐splice‐site (0.014% of human donor sites). Circumstantial evidence in three ATM variants (leakiness uncovered by our mgATM analysis together with clinical data) provides some support for a dosage‐sensitive expression model in which variants producing ≥30% of FL‐transcripts would be predicted benign, while variants producing ≤13% of FL‐transcripts might be pathogenic. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract CT006: Phase Ib expansion trial of the safety and efficacy of the oral ataxia telangiectasia and Rad3-related (ATR) inhibitor elimusertib in advanced solid tumors with DNA damage response (DDR) defects

Abstract Introduction: ATR kinase is a critical component of DDR machinery and is activated by DNA damage or replication stress. Elimusertib is a selective ATR inhibitor with promising antitumor activity in pts with advanced solid tumors with ataxia telangiectasia mutated (ATM) protein loss and/or ATM putative deleterious alterations (Yap et al. Cancer Discov 2021). We report here the safety and efficacy of elimusertib in expansion cohorts of pts with a range of cancer types and DDR deficiencies and/or ATM loss. We also explored an alternative dose schedule in pts with ATM aberrations. Methods: Pts with advanced solid tumors resistant or refractory to standard treatment were screened for pathogenic DDR defects by next-generation sequencing or ATM protein loss by immunohistochemistry (IHC) analysis of baseline tumor tissue. Pts were assigned to cohorts: colorectal cancer (CRC); castration-resistant prostate cancer (CRPC); HER2− breast cancer (BC); gynecologic (GYN, mainly ovarian and endometrial); and advanced cancers with ATM IHC loss. Pts were treated with elimusertib 40 mg twice daily (BID) 3 days on/4 days off (3 on/4 off). A schedule of 3 on/11 off was also explored by dose escalation in pts with ATM loss or inactivating mutation. Results: 143 pts received ≥1 dose of elimusertib 40 mg BID 3 on/4 off, including 24 CRC, 19 CRPC, 19 BC, 45 GYN, and 36 ATM loss. 56% of pts had ≥4 prior therapy lines. Drug-related grade 3 and 4 treatment-emergent adverse events (TEAEs) were observed in 69% and 15% of pts, respectively, mainly anemia, leukopenia/neutropenia, and thrombocytopenia leading to dose modification but not withdrawal. 32 pts were treated 3 on/11 off with doses from 60 to 120 mg BID. Compared with 3 on/4 off, drug-related grade ≥3 hematologic TEAEs and dose modifications improved at lower doses, with 80 mg 3 on/11 off determined as the recommended dose. In pts receiving elimusertib 3 on/4 off, 5 RECIST partial responses were observed: 1 in ovarian (BRCA1 heterozygous mutation), 1 in BC (BRCA2 mutation), and 3 in ATM IHC loss: 2 CRPC pts both with ATM mutations (1 heterozygous, 1 homozygous), including 1 with FANCA mutation; 1 esophageal with ATM mutation. In pts with ATM IHC loss, objective response rate (ORR) was 9% and disease control rate (DCR) was 65%. A similar ORR was seen on 3 on/11 off in pts with ATM aberrations. In GYN pts, ORR was 2.3% and DCR was 73%. Conclusions: Elimusertib monotherapy demonstrated clinical activity in pts with DDR defects. Overall safety was manageable and hematologic toxicity improved on the 3 on/11 off schedule. Further biomarker analysis is underway to identify potential gene signatures associated with response. Clinical development of elimusertib in combination with checkpoint inhibitors and chemotherapy is ongoing (NCT04095273, NCT04514497). Citation Format: Timothy A. Yap, David S. Tan, Anastasios Stathis, Geoffrey I. Shapiro, Satoru Iwasa, Markus Joerger, Jingsong Zhang, Ruth Plummer, Michael Sawyer, Aaron C. Tan, Vincent Castonguay, Nashat Gabrail, Nobuaki Matsubara, Gary Wilkinson, Matthias Ludwig, Yinghui Zhou, Claudia Merz, Joseph Hreiki, Neelesh Sharma, Johan deBono. Phase Ib expansion trial of the safety and efficacy of the oral ataxia telangiectasia and Rad3-related (ATR) inhibitor elimusertib in advanced solid tumors with DNA damage response (DDR) defects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT006.

Predicting acute severe toxicity for head and neck squamous cell carcinomas by combining dosimetry with a radiosensitivity biomarker: a pilot study.

Radiotherapy (RT) against head and neck squamous cell carcinomas (HNSCC) may lead to severe toxicity in 30-40% of patients. The normal tissue complication probability (NTCP) models, based on dosimetric data refined the normal tissue dose/volume tolerance guidelines. In parallel, the radiation-induced nucleoshuttling (RIANS) of the Ataxia-Telangiectasia Mutated protein (pATM) is a predictive approach of individual intrinsic radiosensitivity. Here, we combined NTCP with RADIODTECT©, a blood assay derived from the RIANS model, to predict RT toxicity in HNSCC patients. RADIODTECT© cutoff values (i.e. 57.8 ng/mL for grade⩾2 toxicity and 46 ng/mL for grade⩾3 toxicity) have been previously assessed. Validation was performed on a prospective cohort of 36 HNSCC patients treated with postoperative RT. Toxicity was graded with the Common Terminology Criteria for Adverse Events (CTCAE) scale and two criteria were considered: grade⩾2 oral mucositis (OM2), grade⩾3 mucositis (OM3) and grade⩾2 dysphagia (DY2), grade⩾3 dysphagia (DY3). pATM quantification was assessed in lymphocytes of HNSCC patients. The discrimination power of the pATM assay was evaluated through the Area Under the Receiver Operator Characteristics Curve (AUC-ROC). Two previously described NTCP models were considered, including the dose to the oral cavity and the mean dose to the parotid glands (OM2 and OM3) and the dose to the oral cavity, to the larynx and the volume of pharyngeal constrictor muscles (DY2 and DY3). Combining NTCP models with RADIODTECT© blood test improved the AUC-ROC. Considering the prediction of mucositis, AUC-ROCNTCP+RADIODTECT©=0.80 was for OM2, and AUC-ROCNTCP+RADIODTECT©=0.78 for OM3. Considering the prediction of acute dysphagia, AUC-ROCNTCP+RADIODTECT©=0.71 for DY2 and for DY3. Combining NTCP models with a radiosensitivity biomarker might significantly improve the prediction of toxicities for HNSCC patients.

The potential efficacy and mechanism of bendamustine in entra-nodal NK/T cell lymphoma.

Bendamustine has been shown to have anti-tumor activities in hematological malignancies, but the role of bendamustine in natural killer (NK)/T cell lymphoma (NKTCL) treatment is unclear. Our study has shown that bendamustine had potent growth-inhibitory and apoptosis-inducing effects on NKTCL cells. Interestingly, we noticed that the combination of either gemcitabine or etoposide results in additive or synergistic cytotoxicity. Bendamustine induced mitochondria-mediated apoptosis in concentration- and time-dependent manners in NKTCL cells, shown as down-regulation of Bcl-2 and activation of cleavage of caspases 3, 7, 9 and poly adenosinediphosphate-ribose polymerase (PARP). Bendamustine arrested NKTCL cells in G2/M phase, with downregulation of expression of cyclin B1 and upregulation of expression of p-cdc2, p-cdc25c and p-P53. Furthermore, we confirmed that bendamustine activated DNA damage response (DDR) directly or through Ataxia Telangiectasia Mutated Protein (ATM)/Chk2 and ATR/Chk1 pathway and increased the intracellular reactive oxygen species (ROS) level in NKTCL cells, which caused G2/M phase arrest and apoptosis. Bendamustine also inhibited phosphorylation of transcriptional factor STAT3, contributing to cell apoptosis and proliferation inhibition. Finally, we verified the effect of bendamustine on NKTCL cells in vivo. It showed that bendamustine dramatically inhibited the growth of the subcutaneous tumor, with no obvious impact on mice weight. These findings demonstrate that bendamustine activates DDR pathway, induces the accumulation of intracellularROS level as well as inhibition of STAT3, leading to cell apoptosis and G2/M cell cycle arrest in NKTCL cells, which indicates that bendamustine dramatically suppressed NKTCL both in vitro and in vivo and provides a potential therapeutic strategy in the treatment of NK/T lymphoma.

Oxidative Damage Induced Telomere Mediated Genomic Instability in Cells from Ataxia Telangiectasia Patients.

Our cellular genome is susceptible to cytotoxic lesions which include single strand breaks and double strand breaks among other lesions. Ataxia telangiectasia mutated (ATM) protein was one of the first DNA damage sensor proteins to be discovered as being involved in DNA repair and as well as in telomere maintenance. Telomeres help maintain the stability of our chromosomes by protecting the ends from degradation. Cells from ataxia telangiectasia (AT) patients lack ATM and accumulate chromosomal alterations. AT patients display heightened susceptibility to cancer. In this study, cells from AT patients (called as AT -/- and AT +/- cells) were characterized for genome stability status and it was observed that AT -/- cells show considerable telomere attrition. Furthermore, DNA damage and genomic instability were compared between normal (AT +/+ cells) and AT -/- cells exhibiting increased frequencies of spontaneous DNA damage and genomic instability markers. Both AT -/- and AT +/- cells were sensitive to sodium arsenite (1.5 and 3.0 μg/ml) and ionizing radiation-induced (2 Gy, gamma rays) oxidative stress. Interestingly, telomeric fragments were detected in the comet tails as revealed by comet-fluorescence in situ hybridization analysis, suggestive of telomeric instability in AT -/- cells upon exposure to sodium arsenite or radiation. Besides, there was an increase in the number of chromosome alterations in AT -/- cells following arsenite treatment or irradiation. In addition, complex chromosome aberrations were detected by multicolor fluorescence in situ hybridization in AT -/- cells in comparison to AT +/- and normal cells. Telomere attrition and chromosome alterations were detected even at lower doses of sodium arsenite. Peptide nucleic acid - FISH analysis revealed defective chromosome segregation in cells lacking ATM proteins. The data obtained in this study substantiates the role of ATM in telomere stability under oxidative stress.

Cell Death and Survival Pathways Involving ATM Protein Kinase.

Cell death is the ultimate form of cellular dysfunction, and is induced by a wide range of stresses including genotoxic stresses. During genotoxic stress, two opposite cellular reactions, cellular protection through DNA repair and elimination of damaged cells by the induction of cell death, can occur in both separate and simultaneous manners. ATM (ataxia telangiectasia mutated) kinase (hereafter referred to as ATM) is a protein kinase that plays central roles in the induction of cell death during genotoxic stresses. It has long been considered that ATM mediates DNA damage-induced cell death through inducing apoptosis. However, recent research progress in cell death modality is now revealing ATM-dependent cell death pathways that consist of not only apoptosis but also necroptosis, ferroptosis, and dysfunction of autophagy, a cellular survival mechanism. In this short review, we intend to provide a brief outline of cell death mechanisms in which ATM is involved, with emphasis on pathways other than apoptosis.

Chemical screen identifies shikonin as a broad DNA damage response inhibitor that enhances chemotherapy through inhibiting ATM and ATR

DNA damage response (DDR) is a highly conserved genome surveillance mechanism that preserves cell viability in the presence of chemotherapeutic drugs. Hence, small molecules that inhibit DDR are expected to enhance the anti-cancer effect of chemotherapy. Through a recent chemical library screen, we identified shikonin as an inhibitor that strongly suppressed DDR activated by various chemotherapeutic drugs in cancer cell lines derived from different origins. Mechanistically, shikonin inhibited the activation of ataxia telangiectasia mutated (ATM), and to a lesser degree ATM and RAD3-related (ATR), two master upstream regulators of the DDR signal, through inducing degradation of ATM and ATR-interacting protein (ATRIP), an obligate associating protein of ATR, respectively. As a result of DDR inhibition, shikonin enhanced the anti-cancer effect of chemotherapeutic drugs in both cell cultures and in mouse models. While degradation of ATRIP is proteasome dependent, that of ATM depends on caspase- and lysosome-, but not proteasome. Overexpression of ATM significantly mitigated DDR inhibition and cell death induced by shikonin and chemotherapeutic drugs. These novel findings reveal shikonin as a pan DDR inhibitor and identify ATM as a primary factor in determining the chemo sensitizing effect of shikonin. Our data may facilitate the development of shikonin and its derivatives as potential chemotherapy sensitizers through inducing ATM degradation.