BRCA1 safeguards genome integrity by activating chromosome asynapsis checkpoint to eliminate recombination-defective oocytes.

In the meiotic prophase, programmed DNA double-strand breaks are repaired by meiotic recombination. Recombination-defective meiocytes are eliminated to preserve genome integrity in gametes. BRCA1 is a critical protein in somatic homologous recombination, but studies have suggested that BRCA1 is dispensable for meiotic recombination. Here we show that BRCA1 is essential for meiotic recombination. Interestingly, BRCA1 also has a function in eliminating recombination-defective oocytes. Brca1 knockout (KO) rescues the survival of Dmc1 KO oocytes far more efficiently than removing CHK2, a vital component of the DNA damage checkpoint in oocytes. Mechanistically, BRCA1 activates chromosome asynapsis checkpoint by promoting ATR activity at unsynapsed chromosome axes in Dmc1 KO oocytes. Moreover, Brca1 KO also rescues the survival of asynaptic Spo11 KO oocytes. Collectively, our study not only unveils an unappreciated role of chromosome asynapsis in eliminating recombination-defective oocytes but also reveals the dual functions of BRCA1 in safeguarding oocyte genome integrity.

Combination of EZH2 and ATM inhibition in BAP1-deficient mesothelioma.

BACKGROUND: More than half of mesothelioma tumours show alterations in the tumour suppressor gene BAP1. BAP1-deficient mesothelioma is shown to be sensitive to EZH2 inhibition in preclinical settings but only showed modest efficacy in clinical trial. Adding a second inhibitor could potentially elevate EZH2i treatment efficacy while preventing acquired resistance at the same time. METHODS: A focused drug synergy screen consisting of 20 drugs was performed by combining EZH2 inhibition with a panel of anti-cancer compounds in mesothelioma cell lines. The compounds used are under preclinical investigation or already used in the clinic. The synergistic potential of the combinations was assessed by using the Bliss model. To validate our findings, in vivo xenograft experiments were performed. RESULTS: Combining EZH2i with ATMi was found to have synergistic potential against BAP1-deficient mesothelioma in our drug screen, which was validated in clonogenicity assays. Tumour growth inhibition potential was significantly increased in BAP1-deficient xenografts. In addition, we observe lower ATM levels upon depletion of BAP1 and hypothesise that this might be mediated by E2F1. CONCLUSIONS: We demonstrated the efficacy of the combination of ATM and EZH2 inhibition against BAP1-deficient mesothelioma in preclinical models, indicating the potential of this combination as a novel treatment modality using BAP1 as a biomarker.

Characterization of the signal transduction cascade for inflammatory gene expression in fibroblasts with ATM-ATR deficiencies after Ionizing radiation.

BACKGROUND AND PURPOSE: Ionizing radiation (IR) induces DNA double-strand breaks (DSBs), leading to micronuclei formation, which has emerged as a key mediator of inflammatory responses after IR. This study aimed to investigate the signaling cascade in inflammatory gene expression using fibroblasts harboring DNA damage response deficiency after exposure to IR. MATERIALS AND METHODS: Micronuclei formation was examined in human dermal fibroblasts derived from patients with deficiencies in ATM, ATR, MRE11, XLF, Artemis, or BRCA2 after IR. RNA-sequencing analysis was performed to assess gene expression, pathway mapping, and the balance of transcriptional activity using the transcription factor-based downstream gene expression mapping (TDEM) method developed in this study. RESULTS: Deficiencies in ATM, ATR, or MRE11 led to increased micronuclei formation after IR compared to normal cells. RNA-seq analysis revealed significant upregulation of inflammatory expression in cells deficient in ATM, ATR, or MRE11 following IR. Pathway mapping analysis identified the upregulation of RIG-I, MDA-5, IRF7, IL6, and interferon stimulated gene expression after IR. These changes were pronounced in cells deficient in ATM, ATR, or MRE11. TDEM analysis suggested the differential activation of STAT1/3-pathway between ATM and ATR deficiency. CONCLUSION: Enhanced micronuclei formation upon ATM, ATR, or MRE11 deficiency activated the cGAS/STING, RIG-I-MDA-5-IRF7-IL6 pathway, resulting in its downstream interferon stimulated gene expression following exposure to IR. Our study provides comprehensive information regarding the status of inflammation-related gene expression under DSB repair deficiency after IR. The generated dataset may be useful in developing functional biomarkers to accurately identify patients sensitive to radiotherapy.

Causative mechanisms and clinical impact of immunoglobulin deficiencies in ataxia telangiectasia.

BACKGROUND: Ataxia telangiectasia (AT) is characterized by cerebellar ataxia, telangiectasia, immunodeficiency, and increased cancer susceptibility and is caused by mutations in the ataxia telangiectasia mutated (ATM) gene. The immunodeficiency comprises predominantly immunoglobulin deficiency, mainly IgA and IgG2, with a variable severity. So far, the exact mechanisms underlying the immunoglobulin deficiency, especially the variable severity, remain unelucidated. OBJECTIVE: We characterized the clinical impact of immunoglobulin deficiencies in AT and elucidated their mechanisms in AT. METHODS: We analyzed long-term immunoglobulin levels, immunophenotyping, and survival time in our cohort (n = 87, median age 16 years; maximum 64 years). Somatic hypermutation and class-switch junctions in B cells were analyzed by next-generation sequencing. Furthermore, an in vitro class-switching induction assay was performed, followed by RNA sequencing, to assess the effect of ATM inhibition. RESULTS: Only the hyper-IgM AT phenotype significantly worsened survival time, while IgA or IgG2 deficiencies did not. The immunoglobulin levels showed predominantly decreased IgG2 and IgA. Moreover, flow cytometric analysis demonstrated reduced naive B and T lymphocytes and a deficiency of class-switched IgG2 and IgA memory B cells. Somatic hypermutation frequencies were lowered in IgA- and IgG2-deficient patients, indicating hampered germinal center reaction. In addition, the microhomology of switch junctions was elongated, suggesting alternative end joining during class-switch DNA repair. The in vitro class switching and proliferation were negatively affected by ATM inhibition. RNA sequencing analysis showed that ATM inhibitor influenced expression of germinal center reaction genes. CONCLUSION: Immunoglobulin deficiency in AT is caused by disturbed development of class-switched memory B cells. ATM deficiency affects both germinal center reaction and choice of DNA-repair pathway in class switching.

ATM immunohistochemistry as a potential marker for the differential diagnosis of no specific molecular profile subtype and POLE-mutation subtype endometrioid carcinoma.

Ancillary immunohistochemical tools can facilitate an integrated diagnosis of endometrial pathology. According to The Cancer Genome Atlas classification, endometrial cancers are of four molecular subtypes: mismatch repair (MMR)-deficient (MMR-d), p53 mutation (p53mut), DNA polymerase epsilon (POLE) mutation (POLEmut), and no specific molecular profile (NSMP). As the specific histological and immunohistochemical features of POLEmut and NSMP subtypes are unknown, these cancers are categorized based on molecular analysis. In this study, we analyzed POLEmut-subtype endometrioid carcinoma (EC) using a custom-made cancer gene panel and the Catalog of Somatic Mutations in Cancer (COSMIC) database, extracted a characteristic genome profile, and identified an immunohistochemical marker that could be used as a diagnostic tool. The results indicated that the POLEmut-subtype EC exhibited nonsense mutations in the ataxia telangiectasia mutated (ATM) gene and a subsequent loss of ATM expression, which was monitored through immunohistochemical analysis. Moreover, analyses using the COSMIC database indicated that POLEmut-subtype EC cases often harbored similar ATM nonsense mutations. These results suggest that ATM expression is a potential immunohistochemical marker for the differential diagnosis of POLEmut- and NSMP-subtype EC. DATA AVAILABILITY: The data supporting the findings of this study are available upon request from the corresponding author. The data are not publicly available because of privacy or ethical restrictions.

De novo deletions and duplications at recombination hotspots in mouse germlines.

Numerous DNA double-strand breaks (DSBs) arise during meiosis to initiate homologous recombination. These DSBs are usually repaired faithfully, but here, we uncover a distinct type of mutational event in which deletions form via joining of ends from two closely spaced DSBs (double cuts) within a single hotspot or at adjacent hotspots on the same or different chromatids. Deletions occur in normal meiosis but are much more frequent when DSB formation is dysregulated in the absence of the ATM kinase. Events between chromosome homologs point to multi-chromatid damage and aborted gap repair. Some deletions contain DNA from other hotspots, indicating that double cutting at distant sites creates substrates for insertional mutagenesis. End joining at double cuts can also yield tandem duplications or extrachromosomal circles. Our findings highlight the importance of DSB regulation and reveal a previously hidden potential for meiotic mutagenesis that is likely to affect human health and genome evolution.

Cellular functions of the protein kinase ATM and their relevance to human disease.

The protein kinase ataxia telangiectasia mutated (ATM) is a master regulator of double-strand DNA break (DSB) signalling and stress responses. For three decades, ATM has been investigated extensively to elucidate its roles in the DNA damage response (DDR) and in the pathogenesis of ataxia telangiectasia (A-T), a human neurodegenerative disease caused by loss of ATM. Although hundreds of proteins have been identified as ATM phosphorylation targets and many important roles for this kinase have been identified, it is still unclear how ATM deficiency leads to the early-onset cerebellar degeneration that is common in all individuals with A-T. Recent studies suggest the existence of links between ATM deficiency and other cerebellum-specific neurological disorders, as well as the existence of broader similarities with more common neurodegenerative disorders. In this Review, we discuss recent structural insights into ATM regulation, and possible aetiologies of A-T phenotypes, including reactive oxygen species, mitochondrial dysfunction, alterations in transcription, R-loop metabolism and alternative splicing, defects in cellular proteostasis and metabolism, and potential pathogenic roles for hyper-poly(ADP-ribosyl)ation.

Survival and Functional Immune Reconstitution After Haploidentical Stem Cell Transplantation in Atm-Deficient Mice.

Hematopoietic stem cell transplantation (HSCT) has been proposed as a promising therapeutic opportunity to improve immunity and prevent hematologic malignancies in Ataxia-telangiectasia (A-T). However, experience in the transplantation strategy for A-T patients is still scarce. The aim of this study was to investigate whether different approaches of HSCT are feasible in regard to graft versus host response and sufficient concerning functional immune reconstitution. Atm-deficient mice were treated with a clinically relevant non-myeloablative host-conditioning regimen and transplanted with CD90.2-depleted, green fluorescent protein (GFP)-expressing, and ataxia telangiectasia mutated (ATM)-competent bone marrow donor cells in a syngeneic, haploidentical or allogeneic setting. Like syngeneic HSCT, haploidentical HSCT, but not allogeneic HSCT extended the lifespan of Atm-deficient mice through the reduction of thymic tumors and normalized T-cell numbers. Donor-derived splenocytes isolated from transplanted Atm-deficient mice filled the gap of cell loss in the naïve T-cell population and raised CD4 cell functionality up to wild-type level. Interestingly, HSCT using heterozygous donor cells let to a significantly improved survival of Atm-deficient mice and increased CD4 cell numbers as well as CD4 cell functionality equivalent to HSCT using with wild-type donor cells. Our data provided evidence that haploidentical HSCT could be a feasible strategy for A-T, possibly even if the donor is heterozygous for ATM. However, this basic research cannot substitute any research in humans.

The Fanconi anemia ubiquitin E3 ligase complex as an anti-cancer target.

Agents that induce DNA damage can cure some cancers. However, the side effects of chemotherapy are severe because of the indiscriminate action of DNA-damaging agents on both healthy and cancerous cells. DNA repair pathway inhibition provides a less toxic and targeted alternative to chemotherapy. A compelling DNA repair target is the Fanconi anemia (FA) E3 ligase core complex due to its critical-and likely singular-role in the efficient removal of specific DNA lesions. FA pathway inactivation has been demonstrated to specifically kill some types of cancer cells without the addition of exogenous DNA damage, including cells that lack BRCA1, BRCA2, ATM, or functionally related genes. In this perspective, we discuss the genetic and biochemical evidence in support of the FA core complex as a compelling drug target for cancer therapy. In particular, we discuss the genetic, biochemical, and structural data that could rapidly advance our capacity to identify and implement the use of FA core complex inhibitors in the clinic.

FLYWCH1, a Multi-Functional Zinc Finger Protein Contributes to the DNA Repair Pathway.

Over recent years, several Cys2-His2 (C2H2) domain-containing proteins have emerged as critical players in repairing DNA-double strand breaks. Human FLYWCH1 is a newly characterised nuclear transcription factor with (C2H2)-type zinc-finger DNA-binding domains. Yet, our knowledge about FLYWCH1 is still in its infancy. This study explores the expression, role and regulation of FLYWCH1 in the context of DNA damage and repair. We provide evidence suggesting a potential contribution of FLYWCH1 in facilitating the recruitment of DNA-damage response proteins (DDRPs). We found that FLYWCH1 colocalises with γH2AX in normal fibroblasts and colorectal cancer (CRC) cell lines. Importantly, our results showed that enforced expression of FLYWCH1 induces the expression of γH2AX, ATM and P53 proteins. Using an ATM-knockout (ATMKO) model, we indicated that FLYWCH1 mediates the phosphorylation of H2AX (Ser139) independently to ATM expression. On the other hand, the induction of DNA damage using UV-light induces the endogenous expression of FLYWCH1. Conversely, cisplatin treatment reduces the endogenous level of FLYWCH1 in CRC cell lines. Together, our findings uncover a novel FLYWCH1/H2AX phosphorylation axis in steady-state conditions and during the induction of the DNA-damage response (DDR). Although the role of FLYWCH1 within the DDR machinery remains largely uncharacterised and poorly understood, we here report for the first-time findings that implicate FLYWCH1 as a potential participant in the DNA damage response signaling pathways.

Influence of the microenvironment on modulation of the host response by typhoid toxin.

Bacterial genotoxins cause DNA damage in eukaryotic cells, resulting in activation of the DNA damage response (DDR) in vitro. These toxins are produced by Gram-negative bacteria, enriched in the microbiota of inflammatory bowel disease (IBD) and colorectal cancer (CRC) patients. However, their role in infection remains poorly characterized. We address the role of typhoid toxin in modulation of the host-microbial interaction in health and disease. Infection with a genotoxigenic Salmonella protects mice from intestinal inflammation. We show that the presence of an active genotoxin promotes DNA fragmentation and senescence in vivo, which is uncoupled from an inflammatory response and unexpectedly associated with induction of an anti-inflammatory environment. The anti-inflammatory response is lost when infection occurs in mice with acute colitis. These data highlight a complex context-dependent crosstalk between bacterial-genotoxin-induced DDR and the host immune response, underlining an unexpected role for bacterial genotoxins.

Elevated inflammatory responses and targeted therapeutic intervention in a preclinical mouse model of ataxia-telangiectasia lung disease.

Ataxia-telangiectasia (A-T) is an autosomal recessive, multisystem disorder characterized by cerebellar degeneration, cancer predisposition, and immune system defects. A major cause of mortality in A-T patients is severe pulmonary disease; however, the underlying causes of the lung complications are poorly understood, and there are currently no curative therapeutic interventions. In this study, we examined the lung phenotypes caused by ATM-deficient immune cells using a mouse model of A-T pulmonary disease. In response to acute lung injury, ATM-deficiency causes decreased survival, reduced blood oxygen saturation, elevated neutrophil recruitment, exaggerated and prolonged inflammatory responses and excessive lung injury compared to controls. We found that ATM null bone marrow adoptively transferred to WT recipients induces similar phenotypes that culminate in impaired lung function. Moreover, we demonstrated that activated ATM-deficient macrophages exhibit significantly elevated production of harmful reactive oxygen and nitrogen species and pro-inflammatory cytokines. These findings indicate that ATM-deficient immune cells play major roles in causing the lung pathologies in A-T. Based on these results, we examined the impact of inhibiting the aberrant inflammatory responses caused by ATM-deficiency with reparixin, a CXCR1/CXCR2 chemokine receptor antagonist. We demonstrated that reparixin treatment reduces neutrophil recruitment, edema and tissue damage in ATM mutant lungs. Thus, our findings indicate that targeted inhibition of CXCR1/CXCR2 attenuates pulmonary phenotypes caused by ATM-deficiency and suggest that this treatment approach represents a viable therapeutic strategy for A-T lung disease.

Poly-ADP-ribosylation drives loss of protein homeostasis in ATM and Mre11 deficiency.

Loss of the ataxia-telangiectasia mutated (ATM) kinase causes cerebellum-specific neurodegeneration in humans. We previously demonstrated that deficiency in ATM activation via oxidative stress generates insoluble protein aggregates in human cells, reminiscent of protein dysfunction in common neurodegenerative disorders. Here, we show that this process is driven by poly-ADP-ribose polymerases (PARPs) and that the insoluble protein species arise from intrinsically disordered proteins associating with PAR-associated genomic sites in ATM-deficient cells. The lesions implicated in this process are single-strand DNA breaks dependent on reactive oxygen species, transcription, and R-loops. Human cells expressing Mre11 A-T-like disorder mutants also show PARP-dependent aggregation identical to ATM deficiency. Lastly, analysis of A-T patient cerebellum samples shows widespread protein aggregation as well as loss of proteins known to be critical in human spinocerebellar ataxias that is not observed in neocortex tissues. These results provide a hypothesis accounting for loss of protein integrity and cerebellum function in A-T.

Tumor genotype dictates radiosensitization after Atm deletion in primary brainstem glioma models.

Diffuse intrinsic pontine glioma (DIPG) kills more children than any other type of brain tumor. Despite clinical trials testing many chemotherapeutic agents, palliative radiotherapy remains the standard treatment. Here, we utilized Cre/loxP technology to show that deleting Ataxia telangiectasia mutated (Atm) in primary mouse models of DIPG can enhance tumor radiosensitivity. Genetic deletion of Atm improved survival of mice with p53-deficient but not p53 wild-type gliomas after radiotherapy. Similar to patients with DIPG, mice with p53 wild-type tumors had improved survival after radiotherapy independent of Atm deletion. Primary p53 wild-type tumor cell lines induced proapoptotic genes after radiation and repressed the NRF2 target, NAD(P)H quinone dehydrogenase 1 (Nqo1). Tumors lacking p53 and Ink4a/Arf expressed the highest level of Nqo1 and were most resistant to radiation, but deletion of Atm enhanced the radiation response. These results suggest that tumor genotype may determine whether inhibition of ATM during radiotherapy will be an effective clinical approach to treat DIPGs.

SIRT3, a metabolic target linked to ataxia-telangiectasia mutated (ATM) gene deficiency in diffuse large B-cell lymphoma.

Inactivation of Ataxia-telangiectasia mutated (ATM) gene results in an increased risk to develop cancer. We show that ATM deficiency in diffuse large B-cell lymphoma (DLBCL) significantly induce mitochondrial deacetylase sirtuin-3 (SIRT3) activity, disrupted mitochondrial structure, decreased mitochondrial respiration, and compromised TCA flux compared with DLBCL cells expressing wild type (WT)-ATM. This corresponded to enrichment of glutamate receptor and glutamine pathways in ATM deficient background compared to WT-ATM DLBCL cells. ATM-/- DLBCL cells have decreased apoptosis in contrast to radiosensitive non-cancerous A-T cells. In vivo studies using gain and loss of SIRT3 expression showed that SIRT3 promotes growth of ATM CRISPR knockout DLBCL xenografts compared to wild-type ATM control xenografts. Importantly, screening of DLBCL patient samples identified SIRT3 as a putative therapeutic target, and validated an inverse relationship between ATM and SIRT3 expression. Our data predicts SIRT3 as an important therapeutic target for DLBCL patients with ATM null phenotype.

Radiation-induced DNA damage and repair effects on 3D genome organization.

The three-dimensional structure of chromosomes plays an important role in gene expression regulation and also influences the repair of radiation-induced DNA damage. Genomic aberrations that disrupt chromosome spatial domains can lead to diseases including cancer, but how the 3D genome structure responds to DNA damage is poorly understood. Here, we investigate the impact of DNA damage response and repair on 3D genome folding using Hi-C experiments on wild type cells and ataxia telangiectasia mutated (ATM) patient cells. We irradiate fibroblasts, lymphoblasts, and ATM-deficient fibroblasts with 5 Gy X-rays and perform Hi-C at 30 minutes, 24 hours, or 5 days after irradiation. We observe that 3D genome changes after irradiation are cell type-specific, with lymphoblastoid cells generally showing more contact changes than irradiated fibroblasts. However, all tested repair-proficient cell types exhibit an increased segregation of topologically associating domains (TADs). This TAD boundary strengthening after irradiation is not observed in ATM deficient fibroblasts and may indicate the presence of a mechanism to protect 3D genome structure integrity during DNA damage repair.

Maintenance Therapy for ATM-Deficient Pancreatic Cancer by Multiple DNA Damage Response Interferences after Platinum-Based Chemotherapy.

Personalized medicine in treating pancreatic ductal adenocarcinoma (PDAC) is still in its infancy, albeit PDAC-related deaths are projected to rise over the next decade. Only recently, maintenance therapy with the PARP inhibitor olaparib showed improved progression-free survival in germline BRCA1/2-mutated PDAC patients after platinum-based induction for the first time. Transferability of such a concept to other DNA damage response (DDR) genes remains unclear. Here, we conducted a placebo-controlled, three-armed preclinical trial to evaluate the efficacy of multi-DDR interference (mDDRi) as maintenance therapy vs. continuous FOLFIRINOX treatment, implemented with orthotopically transplanted ATM-deficient PDAC cell lines. Kaplan-Meier analysis, cross-sectional imaging, histology, and in vitro analysis served as analytical readouts. Median overall survival was significantly longer in the mDDRi maintenance arm compared to the maintained FOLFIRINOX treatment. This survival benefit was mirrored in the highest DNA-damage load, accompanied by superior disease control and reduced metastatic burden. In vitro analysis suggests FOLFIRINOX-driven selection of invasive subclones, erased by subsequent mDDRi treatment. Collectively, this preclinical trial substantiates mDDRi in a maintenance setting as a novel therapeutic option and extends the concept to non-germline BRCA1/2-mutant PDAC.

Kinetochore protein MAD1 participates in the DNA damage response through ataxia-telangiectasia mutated kinase-mediated phosphorylation and enhanced interaction with KU80.

Objective: Mitotic arrest-deficient protein 1 (MAD1) is a kinetochore protein essential for the mitotic spindle checkpoint. Proteomic studies have indicated that MAD1 is a component of the DNA damage response (DDR) pathway. However, whether and how MAD1 might be directly involved in the DDR is largely unknown. Methods: We ectopically expressed the wild type, or a phosphorylation-site--mutated form of MAD1 in MAD1 knockdown cells to look for complementation effects. We used the comet assay, colony formation assay, immunofluorescence staining, and flow cytometry to assess the DDR, radiosensitivity, and the G2/M checkpoint. We employed co-immunoprecipitation followed by mass spectrometry to identify MAD1 interacting proteins. Data were analyzed using the unpaired Student's t-test. Results: We showed that MAD1 was required for an optimal DDR, as knocking down MAD1 resulted in impaired DNA repair and hypersensitivity to ionizing radiation (IR). We found that IR-induced serine 214 phosphorylation was ataxia-telangiectasia mutated (ATM) kinase-dependent. Mutation of serine 214 to alanine failed to rescue the phenotypes of MAD1 knockdown cells in response to IR. Using mass spectrometry, we identified a protein complex mediated by MAD1 serine 214 phosphorylation in response to IR. Among them, we showed that KU80 was a key protein that displayed enhanced interaction with MAD1 after DNA damage. Finally, we showed that MAD1 interaction with KU80 required serine 214 phosphorylation, and it was essential for activation of DNA protein kinases catalytic subunit (DNA-PKcs). Conclusions: MAD1 serine 214 phosphorylation mediated by ATM kinase in response to IR was required for the interaction with KU80 and activation of DNA-PKCs.

Heart failure and diabetes: role of ATM.

Heart failure is a leading cause of death in the United States. Diabetes, also known as diabetes mellitus (DM), exponentially increases the risk of heart failure. The increase in oxidative stress and metabolic dysfunction caused by DM can lead to DNA damage and the development of diabetic cardiomyopathy. Ataxia telangiectasia mutated kinase (ATM) is a DNA damage response protein with a primary nuclear function to regulate cell cycle progression in response to double-strand DNA breaks, acts as a redox sensor, and facilitates DNA repair. ATM deficiency associates with the development of insulin resistance and DM. Consequently, patients with Ataxia telangiectasia, a rare autosomal recessive disorder, have an increased risk of developing heart failure. The main objective of this review is to summarize the shared metabolic and cardiac abnormalities associated with DM and ATM deficiency, with a focus on the development of heart failure.

Genomic and Clinicopathologic Characterization of ATM-deficient Prostate Cancer.

PURPOSE: The ATM (ataxia telangiectasia mutated) gene is mutated in a subset of prostate cancers, and ATM mutation may confer specific therapeutic vulnerabilities, although ATM-deficient prostate cancers have not been well-characterized. EXPERIMENTAL DESIGN: We genetically validated a clinical grade IHC assay to detect ATM protein loss and examined the frequency of ATM loss among tumors with pathogenic germline ATM mutations and genetically unselected primary prostate carcinomas using tissue microarrays (TMAs). Immunostaining results were correlated with targeted somatic genomic sequencing and clinical outcomes. RESULTS: ATM protein loss was found in 13% (7/52) of primary Gleason pattern 5 cancers with available sequencing data and was 100% sensitive for biallelic ATM inactivation. In a separate cohort with pathogenic germline ATM mutations, 74% (14/19) had ATM protein loss of which 70% (7/10) of evaluable cases had genomic evidence of biallelic inactivation, compared with zero of four of cases with intact ATM expression. By TMA screening, ATM loss was identified in 3% (25/831) of evaluable primary tumors, more commonly in grade group 5 (17/181; 9%) compared with all other grades (8/650; 1%; P < 0.0001). Of those with available sequencing, 80% (4/5) with homogeneous ATM protein loss and 50% (6/12) with heterogeneous ATM protein loss had detectable pathogenic ATM alterations. In surgically treated patients, ATM loss was not significantly associated with clinical outcomes in random-effects Cox models after adjusting for clinicopathologic variables. CONCLUSIONS: ATM loss is enriched among high-grade prostate cancers. Optimal evaluation of ATM status requires both genomic and IHC studies and will guide development of molecularly targeted therapies.