Ataxia Telangiectasia Mutated Expression Research Articles

Age-related decline in the expression of BRG1, ATM and ATR are partially reversed by dietary restriction in the livers of female mice.

BRG1 (Brahma-related gene 1) is a member of the SWI/SNF (switch/sucrose nonfermentable) chromatin remodeling complex which utilizes the energy from ATP hydrolysis for its activity. In addition to its role of regulating the expression of a vast array of genes, BRG1 mediates DNA repair upon genotoxic stress and regulates senescence. During organismal ageing, there is accumulation of unrepaired/unrepairable DNA damage due to progressive breakdown of the DNA repair machinery. The present study investigates the expression level of BRG1 as a function of age in the liver of 5- and 21-month-old female mice. It also explores the impact of dietary restriction on BRG1 expression in the old (21-month) mice. Salient findings of the study are: Real-time PCR and Western blot analyses reveal that BRG1 levels are higher in 5-month-old mice but decrease significantly with age. Dietary restriction increases BRG1 expression in the 21-month-old mice, nearly restoring it to the level observed in the younger group. Similar expression patterns are observed for DNA damage response genes ATM (Ataxia Telangiectasia Mutated) and ATR (Ataxia Telangiectasia and Rad3-related) with the advancement in age and which appears to be modulated by dietary restriction. BRG1 transcriptionally regulates ATM as a function of age and dietary restriction. These results suggest that BRG1, ATM and ATR are downregulated as mice age, and dietary restriction can restore their expression. This implies that dietary restriction may play a crucial role in regulating BRG1 and related gene expression, potentially maintaining liver repair and metabolic processes as mice age.

Harmine alleviated STZ-induced rat diabetic nephropathy: A potential role via regulating AMPK/Nrf2 pathway and deactivating ataxia-telangiectasia mutated (ATM) signaling

Diabetic nephropathy (DN) is a serious kidney disorder driven by diabetes and affects people all over the world. One of the mechanisms promoting NF-κB-induced renal inflammation and injury has been theorized to be ATM signaling. On the other hand, AMPK, which can be activated by the naturally occurring alkaloid harmine (HAR), has been proposed to stop that action. As a result, the goal of this study was to evaluate the therapeutic effectiveness of HAR against streptozotocin (STZ)-induced DN in rats through AMPK-mediated inactivation of ATM pathways. Twenty male Wistar rats were grouped into 4 groups, as follow: CONT, DN, HAR (10 mg/kg), DN + HAR, where HAR was daily administered I.P. once for 2 weeks. The renal AMPK and PGC-1α expressions, as well as Sirt1 levels, were assessed. To ascertain the oxidative reactions, renal Nrf2 expression, HO-1, MDA, and TAC concentrations were measured. As parts of ATM pathways, ATM and p53 expressions, in addition to GSK-3β levels were determined. Renal expression of NEMO, TNF-α, and IL-6 levels were also estimated. Moreover, histopathological and immunohistochemical detection of Bcl-2, Bax, and caspase 3 were reported. Results indicated that HAR intake notably alleviated STZ-induced kidney damage by triggering AMPK and Sirt1, which in turn boosted PGC-1α, improved NRf2/HO-1 axis, and lowered ROS production. As a consequence, HAR blocked the ATM-triggered renal inflammation and minimized caspase-3 expression by repressing the Bax/Bcl2 ratio. Because of its ability to activate AMPK/Nrf2 axis, HAR may represent an emerging avenue for future DN therapy by blocking ATM pathways.

ATM deficiency confers specific therapeutic vulnerabilities in bladder cancer.

Ataxia-telangiectasia mutated (ATM) plays a central role in the cellular response to DNA damage and ATM alterations are common in several tumor types including bladder cancer. However, the specific impact of ATM alterations on therapy response in bladder cancer is uncertain. Here, we combine preclinical modeling and clinical analyses to comprehensively define the impact of ATM alterations on bladder cancer. We show that ATM loss is sufficient to increase sensitivity to DNA-damaging agents including cisplatin and radiation. Furthermore, ATM loss drives sensitivity to DNA repair-targeted agents including poly(ADP-ribose) polymerase (PARP) and Ataxia telangiectasia and Rad3 related (ATR) inhibitors. ATM loss alters the immune microenvironment and improves anti-PD1 response in preclinical bladder models but is not associated with improved anti-PD1/PD-L1 response in clinical cohorts. Last, we show that ATM expression by immunohistochemistry is strongly correlated with response to chemoradiotherapy. Together, these data define a potential role for ATM as a predictive biomarker in bladder cancer.

Invasion-Block and S-MARVEL: A high-content screening and image analysis platform identifies ATM kinase as a modulator of melanoma invasion and metastasis.

Robust high-throughput assays are crucial for the effective functioning of a drug discovery pipeline. Herein, we report the development of Invasion-Block, an automated high-content screening platform for measuring invadopodia-mediated matrix degradation as a readout for the invasive capacity of cancer cells. Combined with Smoothen-Mask and Reveal, a custom-designed, automated image analysis pipeline, this platform allowed us to evaluate melanoma cell invasion capacity posttreatment with two libraries of compounds comprising 3840 U.S. Food and Drug Administration (FDA)-approved drugs with well-characterized safety and bioavailability profiles in humans as well as a kinase inhibitor library comprising 210 biologically active compounds. We found that Abl/Src, PKC, PI3K, and Ataxia-telangiectasia mutated (ATM) kinase inhibitors significantly reduced melanoma cell invadopodia formation and cell invasion. Abrogation of ATM expression in melanoma cells via CRISPR-mediated gene knockout reduced 3D invasion in vitro as well as spontaneous lymph node metastasis in vivo. Together, this study established a rapid screening assay coupled with a customized image-analysis pipeline for the identification of antimetastatic drugs. Our study implicates that ATM may serve as a potent therapeutic target for the treatment of melanoma cell spread in patients.

ATM-AMPKα mediated LAG-3 expression suppresses T cell function in prostate cancer

Immunotherapy for prostate cancer (PCa) faces serious challenges. Therefore, the co-inhibitory receptors that regulate T cell function of PCa must be elucidated. Here we identified that the inhibitory receptor LAG3 was significantly induced in T cells from PCa patients. Gene array analysis revealed that insufficient ataxia telangiectasia mutated (ATM) gene expression in PCa T cells was responsible for the elevated LAG3 expression. Mechanistically, insufficient ATM expression impaired its ability to activate AMPKα signaling and CD4+ T cell functions, which further enhances the binding of the transcription factors XBP1 and EGR2 to LAG3 promoter. Reconstitution of ATM and inhibition of XBP1 or EGR2 in PCa T cells suppressed LAG3 expression and restored the effector function of CD4+ T cells from PCa. Our study revealed the mechanism of LAG3 upregulation in CD4+ T lymphocytes of PCa patients and may provide insights for the development of immunotherapeutic strategies for PCa treatment.

Reduced expression of phosphorylated ataxia-telangiectasia mutated gene is related to poor prognosis and gemcitabine chemoresistance in pancreatic cancer

BackgroundLoss of expression of the gene ataxia-telangiectasia mutated (ATM), occurring in patients with multiple primary malignancies, including pancreatic cancer, is associated with poor prognosis. In this study, we investigated the detailed molecular mechanism through which ATM expression affects the prognosis of patients with pancreatic cancer.MethodsThe levels of expression of ATM and phosphorylated ATM in patients with pancreatic cancer who had undergone surgical resection were analyzed using immunohistochemistry staining. RNA sequencing was performed on ATM-knockdown pancreatic-cancer cells to elucidate the mechanism underlying the invlovement of ATM in pancreatic cancer.ResultsImmunohistochemical analysis showed that 15.3% and 27.8% of clinical samples had low levels of ATM and phosphorylated ATM, respectively. Low expression of phosphorylated ATM substantially reduced overall and disease-free survival in patients with pancreatic cancer. In the pancreatic cancer cell lines with ATM low expression, resistance to gemcitabine was demonstrated. The RNA sequence demonstrated that ATM knockdown induced the expression of MET and NTN1. In ATM knockdown cells, it was also revealed that the protein expression levels of HIF-1α and antiapoptotic BCL-2/BAD were upregulated.ConclusionsThese findings demonstrate that loss of ATM expression increases tumor development, suppresses apoptosis, and reduces gemcitabine sensitivity. Additionally, loss of phosphorylated ATM is associated with a poor prognosis in patients with pancreatic cancer. Thus, phosphorylated ATM could be a possible target for pancreatic cancer treatment as well as a molecular marker to track patient prognosis.

Abstract 6208: Targeting DNA polymerase theta and ATM leads to synergistic killing of mantle cell lymphoma cells

Abstract Introduction: Rapid cell proliferation requires intact and faithful DNA damage repair mechanisms. DNA polymerase theta (POLQ) plays a key role in repairing DNA double-strand breaks through the microhomology-mediated end-joining (MMEJ), which is one of the three main pathways involved in repairing replication-induced double-strand breaks. Limited data have suggested that concurrent depletion of POLQ and ataxia-telangiectasia mutated (ATM) could be embryonic lethal. Hence, this phenomenon has the potential to be exploited for therapeutic benefit in cancers where ATM mutations are commonly seen. Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma marked by (11;14) translocation with ATM alterations seen in 40-50% of patients. There is an unmet need to find novel therapeutic strategies, especially in relapsed and/or refractory (R/R) MCL. Here, we investigated whether targeting POLQ and ATM could be a potential therapeutic strategy in MCL. Methods: In vitro studies were conducted by using MCL cell lines. CRISPR-Cas9 system was used to genetically deplete POLQ and ATM genes and sgRNAs co-expressing fluorescence markers were used to track the cell population with respective genotypes over time. Cell viability was assessed by CellTiter-Glo assay and flow cytometry. All cell lines were profiled for ATM expression and activity. A p-value of < 0.05 was considered statistically significant. The combination index of <1 was defined as synergistic. Results: CRISPR-Cas9-mediated depletion of POLQ significantly decreased cell proliferation in multiple MCL cell lines. In particular, Granta-519, which possesses a single copy of kinase-dead ATM that is reduced in expression, was most sensitive to POLQ depletion. Concurrent genetic depletion of ATM and POLQ resulted in a synergistic antiproliferative effect in ATM-proficient MCL cell lines. Subsequently, this cellular phenotype caused by the genetic intervention was recapitulated by using two POLQ inhibitors (novobiocin and ART558) and an ATM inhibitor (AZD0156). In vitro, single-agent treatment with novobiocin or ART558 caused a significant cytotoxic effect at physiologically relevant concentrations in ATM-deficient cells and co-treatment of novobiocin or ART558 with AZD0156 was synergistic in killing ATM-proficient MCL cells. Importantly, POLQ inhibitors significantly decreased the cell viability of MCIR1, which is an ibrutinib-resistant MCL cell line. Mechanistically, novobiocin or ART558 treatment induced gH2AX and cleaved PARP upregulation, which was further enhanced by ATM depletion, suggesting that co-inhibition of POLQ and ATM caused apoptosis due to the accumulation of unrepaired DNA damage. Conclusion: POLQ is a promising target in MCL, especially in ATM-deficient setting. In ATM-proficient MCL, targeting ATM and POLQ is synergistic. Our data has the potential to uncover novel biomarker-driven drug therapy of POLQ inhibitors in R/R MCL. Citation Format: Jithma Prasad Abeykoon, Shuhei Asada, Kalindi Parmar, Xiaosheng Wu, Thomas Witzig, Geoffrey Shapiro, Alan D. D'Andrea. Targeting DNA polymerase theta and ATM leads to synergistic killing of mantle cell lymphoma cells. [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 6208.

Ataxia-Telangiectasia Mutated Is Involved in Autolysosome Formation.

Ataxia-telangiectasia mutated (ATM), a master kinase of the DNA damage response (DDR), phosphorylates a multitude of substrates to activate signaling pathways after DNA double-strand breaks (DSBs). ATM inhibitors have been evaluated as anticancer drugs to potentiate the cytotoxicity of DNA damage-based cancer therapy. ATM is also involved in autophagy, a conserved cellular process that maintains homeostasis by degrading unnecessary proteins and dysfunctional organelles. In this study, we report that ATM inhibitors (KU-55933 and KU-60019) provoked accumulation of autophagosomes and p62 and restrained autolysosome formation. Under autophagy-inducing conditions, the ATM inhibitors caused excessive autophagosome accumulation and cell death. This new function of ATM in autophagy was also observed in numerous cell lines. Repression of ATM expression using an siRNA inhibited autophagic flux at the autolysosome formation step and induced cell death under autophagy-inducing conditions. Taken together, our results suggest that ATM is involved in autolysosome formation and that the use of ATM inhibitors in cancer therapy may be expanded.

Macamide B suppresses lung cancer progression potentially via the ATM signaling pathway.

Macamides are a class of bioactive natural products obtained from Lepidium meyenii (maca), which have been reported to exert inhibitory activity in cancer. However, their role in lung cancer is currently unknown. In the present study, macamide B was shown to inhibit the proliferation and invasion of lung cancer cells, as determined by Cell Counting Kit-8 and Transwell assays, respectively. By contrast, macamide B induced cell apoptosis, as determined by Annexin V-FITC assay. Moreover, combined treatment with macamide B and olaparib, an inhibitor of poly (ADP-ribose) polymerase, further suppressed the proliferation of lung cancer cells. At the molecular level, the expression of ataxia-telangiectasia mutated (ATM), RAD51, p53 and cleaved caspase-3 were significantly increased by macamide B, as determined by western blotting, whereas the expression levels of Bcl-2 were decreased. By contrast, when ATM expression was knocked down by small interfering RNA technology in A549 cells treated with macamide B, the expression levels of ATM, RAD51, p53 and cleaved caspase-3 were reduced, whereas those of Bcl-2 were increased. Consistently, cell proliferation and invasive ability were partially rescued by ATM knockdown. In conclusion, macamide B inhibits lung cancer progression by inhibiting cell proliferation and invasion, and inducing apoptosis. Furthermore, macamide B may participate in regulating the ATM signaling pathway. The present study provides a potential new natural drug for treating patients with lung cancer.

Transcriptomic Analysis of ATM Expression in Localized Prostate Cancer – Implications for Radiotherapy Treatment

<h3>Purpose/Objective(s)</h3> The prognostic impact and therapeutic implication of ATM (ataxia telangiectasia mutated) somatic and germline alterations have been previously characterized in prostate cancer. However, ATM expression, particularly in the context of radiotherapy (RT) for localized disease, has not been well documented. Herein, genomic expression analyses were performed to evaluate associations between ATM expression, biological pathways, clinical outcomes and RT treatment response. <h3>Materials/Methods</h3> Whole-transcriptome assays were used to interrogate 5,780 radical prostatectomy (RP) samples for ATM expression (5,000 from a prospective cohort was used to evaluate biological signatures and 780 samples from a retrospective cohort with long term follow-up data to evaluate ATM prognostic impact). Additionally, we assessed 245 patients (subset of the 780) who were treated with adjuvant or salvage RT after RP. A 1:1 matched cohort [n=98 treated with RT, n=98 with no RT] was generated to assess ATM expression in relation to RT response. We next investigated the relationship using Pearson's correlation tests between ATM expression and more than 200 genomic signatures, biological pathways, and a Genomic Classifier (GC) (molecular subtypes). ATM high, medium, and low were defined as the top 25%, middle 50%, and bottom 25% of ATM expression, respectively. The prognostic and predictive ability of ATM expression was examined for the metastasis-free survival (MFS) endpoint with hazard ratios (HRs) and 95% confidence intervals (CIs) from Cox Proportional Hazard model. <h3>Results</h3> In the prospective cohort, ATM was significantly overexpressed in high GC patients (molecular surrogate for poor outcome) (p<0.0001). ATM high (top 25%) was associated with worse MFS (HR = 1.16, 95% CI: 1.51-1.96, p=0.002). ATM expression was highly correlated to signatures related to DNA repair, angiogenesis, immune cell infiltration and genomic instability and highly correlated to CDK2 and PARP1 genes (R>0.5, p<0.0001 for all). In patients treated with RT, ATM high was associated with worse MFS compared to lower ATM (10-year MFS 30% vs 61%; p=0.009), whereas in the patients treated without RT, ATM high was not significantly associated with worse MFS (10-year MFS 52% vs 66%; p=0.26) <h3>Conclusion</h3> Prostate cancer patients harboring high ATM expression by transcriptomic analysis had poorer outcomes with a significantly higher risk of metastasis. Given the worse outcomes, particularly in patients treated with RT, a high ATM expression may be a marker of resistance to DNA damaging agents. Additional studies are needed to elucidate further the predictive ability of ATM overexpression for RT personalization.

A Novel ATM Antisense Transcript ATM-AS Positively Regulates ATM Expression in Normal and Breast Cancer Cells

ObjectiveThe ataxia telangiectasia mutated (ATM) gene is a master regulator in cellular DNA damage response. The dysregulation of ATM expression is frequent in breast cancer, and is known to be involved in the carcinogenesis and prognosis of cancer. However, the underlying mechanism remains unclear. The bioinformatic analysis predicted a potential antisense transcript ATM-antisense (AS) from the opposite strand of the ATM gene. The purpose of this study was to identify ATM-AS and investigate the possible effect of ATM-AS on the ATM gene regulation.MethodsSingle strand-specific RT-PCR was performed to verify the predicted antisense transcript ATM-AS within the ATM gene locus. qRT-PCR and Western blotting were used to detect the expression levels of ATM-AS and ATM in normal and breast cancer cell lines as well as in tissue samples. Luciferase reporter gene assays, biological mass spectrometry, ChIP-qPCR and RIP were used to explore the function of ATM-AS in regulating the ATM expression. Immunofluorescence and host-cell reactivation (HCR) assay were performed to evaluate the biological significance of ATM-AS in ATM-mediated DNA damage repair. Breast cancer tissue samples were used for evaluating the correlation of the ATM-AS level with the ATM expression as well as prognosis of the patients.ResultsThe ATM-AS significantly upregulated the ATM gene activity by recruiting KAT5 histone acetyltransferase to the gene promoter. The reduced ATM-AS level led to the abnormal downregulation of ATM expression, and impaired the ATM-mediated DNA damage repair in normal breast cells in vitro. The ATM-AS level was positively correlated with the ATM expression in the examined breast cancer tissue samples, and the patient prognosis.ConclusionThe present study demonstrated that ATM-AS, an antisense transcript located within the ATM gene body, is an essential positive regulator of ATM expression, and functions by mediating the binding of KAT5 to the ATM promoter. These findings uncover the novel mechanism underlying the dysregulation of the ATM gene in breast cancer, and enrich our understanding of how an antisense transcript regulates its host gene.

A phase I trial of elimusertib in combination with cisplatin or with cisplatin plus gemcitabine in advanced solid tumors with an emphasis on urothelial carcinoma.

TPS3174 Background: Cisplatin, a well-established backbone of combination therapy of various advanced solid tumors, inhibits DNA synthesis by forming DNA cross-links and adducts. Despite the activity of cisplatin, tumor cells can either be refractory or develop resistance to treatment. Cisplatin has been demonstrated to cause cell cycle G2/M arrest, which may allow for DNA damage response (DDR) and repair. Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad 3-related (ATR) protein kinases are key regulators of DDR, and contribute to maintaining genomic integrity in response to various exogenous and endogenous genotoxic insults like cytotoxic chemotherapy. In fact, cisplatin has been shown to transiently increase ATR expression. Inhibitors of ATR have been studied in combination with cisplatin both in vitro and in vivo, demonstrating enhanced activity. The oral small molecule ATR inhibitor elimusertib, which has been studied as a single agent in a Phase I study, has also demonstrated enhanced activity with cisplatin in vitro in lung cancer and bladder cancer cell lines. We sought to conduct a Phase I study evaluating the combination of elimusertib with cisplatin or with cisplatin and gemcitabine. Methods: In the first cohort, patients with histologically confirmed advanced solid tumors for which cisplatin-based therapy would be considered appropriate, who exhibit adequate organ function, and have received &lt; 300 mg/m2 of cisplatin previously are treated with cisplatin on Day (D) 1 and with elimusertib on D2 &amp; 9 of each 21-day cycle. The study follows a phase I queue (IQ) 3+3 dose escalation design, following standard practices for dose-limiting toxicity (DLT) impact on escalation, and when the maximum tolerated dose (MTD) is established with cisplatin alone, this will inform the first dose level of the next cohort, in which patients will be treated with cisplatin on D1, gemcitabine on D1 &amp; 8, and elimusertib on D2 &amp; 9 of each 21-day cycle. An expansion cohort will enroll urothelial carcinoma patients when the second MTD is established. The primary objective of the study is to evaluate the safety and MTD of elimusertib in combination with cisplatin, as well as in combination with cisplatin and gemcitabine. Secondary study objectives include evaluation of pharmacokinetics of elimusertib in these combinations, preliminary efficacy, and evaluating the association between ATM expression and responses to therapy. Currently, 6 patients have been enrolled to the first cohort of the study. Clinical trial information: NCT04491942.

The ataxia-telangiectasia mutated gene product regulates the cellular acid-labile sulfide fraction

The ataxia-telangiectasia mutated (ATM) protein regulates cell cycle checkpoints, the cellular redox state, and double-stranded DNA break repair. ATM loss causes the disorder ataxia-telangiectasia (A-T), distinguished by ataxia, telangiectasias, dysregulated cellular redox and iron responses, and an increased cancer risk. We examined the sulfur pool in A-T cells, with and without an ATM expression vector. While free and bound sulfide levels were not changed with ATM expression, the acid-labile sulfide faction was significantly increased. ATM expression also increased cysteine desulfurase (NFS1), NFU1 iron-sulfur cluster scaffold homolog protein, and several mitochondrial complex I proteins’ expression. Additionally, ATM expression suppressed cystathionine β-synthase and cystathionine γ-synthase protein expression, cystathionine γ-synthase enzymatic activity, and increased the reduced to oxidized glutathione ratio. This last observation is interesting, as dysregulated glutathione is implicated in A-T pathology. As ATM expression increases the expression of proteins central in initiating 2Fe-2S and 4Fe-4S cluster formation (NFS1 and NFU1, respectively), and the acid-labile sulfide faction is composed of sulfur incorporated into Fe-S clusters, our data indicates that ATM regulates aspects of Fe-S cluster biosynthesis, the transsulfuration pathway, and glutathione redox cycling. Thus, our data may explain some of the redox- and iron-related pathologies seen in A-T.

Ultraviolet-B Radiation Represses Primary Root Elongation by Inhibiting Cell Proliferation in the Meristematic Zone of Arabidopsis Seedlings.

In Arabidopsis thaliana plants, exposure to UV-B induces an inhibition of primary root elongation. Different mutants have been isolated that are deficient in this response; however, little is known about the cellular and molecular mechanisms that regulate inhibition of root elongation in seedlings exposed to UV-B. In this work, we investigated the effect UV-B irradiation of different organs on primary root elongation. Our results demonstrate that irradiation of the leaves and shoots only induce a partial inhibition of primary root elongation, while when only roots are exposed to this radiation, primary root inhibition is similar as that measured when the complete seedling is irradiated. The consequences of exposure at different root developmental stages and times after the end of the treatment was also studied. We here show that inhibition of primary root elongation is a consequence of a decrease in cell proliferation in the meristematic zone of the primary roots, while the elongation zone size is not affected by the treatment. The decrease in cell number after UV-B exposure is partially compensated by an increase in cell length in the root meristem; however, this compensation is not enough to maintain the meristem size. We also here demonstrate that, similarly as what occurs in developing leaves, GROWTH REGULATING FACTOR 3 (GRF3) transcription factor regulates cell proliferation in UV-B irradiated roots; however, and in contrast to what occurs in the leaves, this response does not depend on the presence of MITOGEN ACTIVATED PROTEIN KINASE 3 (MPK3). Inhibition of primary root elongation by UV-B under our experimental conditions is also independent of the UV-B photoreceptor UV RESISTANT LOCUS 8 (UVR8) or ATAXIA TELANGIECTASIA MUTATED (ATM); but a deficiency in ATM AND RAD3-RELATED (ATR) expression increases UV-B sensitivity in the roots. Finally, our data demonstrate that UV-B affects primary root growth in various Arabidopsis accessions, showing different sensitivities to this radiation.

Nuclear Ceramide Is Associated with Ataxia Telangiectasia Mutated Activation in the Neocarzinostatin-Induced Apoptosis of Lymphoblastoid Cells.

Ceramide is a bioactive sphingolipid that mediates ionizing radiation- and chemotherapy-induced apoptosis. Neocarzinostatin (NCS) is a genotoxic anti-cancer drug that induces apoptosis in response to DNA double-strand breaks (DSBs) through ataxia telangiectasia mutated (ATM) activation. However, the involvement of ceramide in NCS-evoked nuclear events such as DSB-activated ATM has not been clarified. Here, we found that nuclear ceramide increased by NCS-mediated apoptosis through the enhanced assembly of ATM and the meiotic recombination 11/double-strand break repair/Nijmengen breakage syndrome 1 (MRN) complex proteins in human lymphoblastoid L-39 cells. NCS induced an increase of ceramide production through activation of neutral sphingomyelinase (nSMase) and suppression of sphingomyelin synthase (SMS) upstream of DSB-mediated ATM activation. In ATM-deficient lymphoblastoid AT-59 cells compared with L-39 cells, NCS treatment showed a decrease of apoptosis even though ceramide increase and DSBs were observed. Expression of wild-type ATM, but not the kinase-dead mutant ATM, in AT-59 cells increased NCS-induced apoptosis despite similar ceramide accumulation. Interestingly, NCS increased ceramide content in the nucleus through nSMase activation and SMS suppression and promoted colocalization of ceramide with phosphorylated ATM and foci of MRN complex. Inhibition of ceramide generation by the overexpression of SMS suppressed NCS-induced apoptosis through the inhibition of ATM activation and assembly of the MRN complex. In addition, inhibition of ceramide increased by the nSMase inhibitor GW4869 prevented NCS-mediated activation of the ATM. Therefore, our findings suggest the involvement of the nuclear ceramide with ATM activation in NCS-mediated apoptosis. SIGNIFICANCE STATEMENT: This study demonstrates that regulation of ceramide with neutral sphingomyelinase and sphingomyelin synthase in the nucleus in double-strand break-mimetic agent neocarzinostatin (NCS)-induced apoptosis. This study also showed that ceramide increase in the nucleus plays a role in NCS-induced apoptosis through activation of the ataxia telangiectasia mutated/meiotic recombination 11/double-strand break repair/Nijmengen breakage syndrome 1 complex in human lymphoblastoid cells.

Expression and functional SNP loci screen of ATM from coal worker's pneumoconiosis

Objective: To detect of gene expression and genotype of the ataxia telangiectasia mutated (ATM) from coal workers' pneumoconiosis (CWP) , It is explored whether CWP is related to ATM gene. Methods: In October 2020, the relevant information of 264 subjects who received physical examination or medical treatment in the Department of occupational diseases of Guiyang public health treatment center from January 2019 to September 2020 was collected. Through the occupational health examination, 67 healthy people with no history of exposure to occupational hazards were selected as the healthy control group; The coal miners with more than 10 years of coal dust exposure history and small shadow in the lung but not up to the diagnostic criteria were the dust exposure control group, a total of 66 people; The patients with the same history of coal dust exposure and confirmed stage I were coal worker's pneumoconiosis stage I group, a total of 131 people. The expression of ATM was detected by QRT PCR. ATM rs189037 and rs1801516 were genotyped by massarray. Results: There was significant difference in the expression of ATM among the groups (P<0.05) ; Compared with the healthy control group, the expression of ATM in the dust exposed control group was significantly increased (P<0.05) . With the occurrence and development of CWP, the GG of rs189037 wild type decreased, the GA of mutant heterozygote and AA of homozygote increased, but the difference was not statistically significant (P>0.05) ; Rs1801516 wild type GG and mutant heterozygote GA had no significant changes (P>0.05) . There were significant differences in age, neutrophils and basophils among rs189037 groups (all P<0.05) . There were no significant differences in blood pressure, eosinophils, lymphocytes, monocytes, smoking and drinking history among rs189037 groups (all P>0.05) . Compared with wild-type GG, the or of mutant heterozygotes and homozygotes increased, but the differences were not statistically significant (P>0.05) . Conclusion: ATM gene may be one of the early activation genes of CWP and rs189037 may be the functional loci which affects gene expression. ATM gene is related to inflammatory response, Neutrophils and basophils have an impact on the development of CWP.

Acquired temozolomide resistance in MGMTlow gliomas is associated with regulation of homologous recombination repair by ROCK2

It was reported that MGMTlow gliomas may still be resistant to TMZ, while the mechanisms remain poorly understood. In this study, we demonstrated that rho-associated kinase 2 (ROCK2), a cytoskeleton regulator, was highly expressed in MGMTlow recurrent gliomas, and its expression strongly correlated with poor overall survival (OS) time in a subset of MGMTlow recurrent gliomas patients with TMZ therapy. And we also found that overactive ROCK2 enhanced homologous recombination repair (HR) in TMZ-resistant (TMZ-R) glioma cell lines with low MGMT expression. Silencing ROCK2 impaired HR repair, and induced double-strand break (DSB) and eradicated TMZ-R glioma cells in culture. Notably, in MGMTlow TMZ-R models, as a key factor of HR, ataxia telangiectasia-mutated (ATM) expression was upregulated directly by hyper-activation of ROCK2 to improve HR efficiency. ROCK2 enhanced the binding of transcription factor zinc finger E-box binding homeobox 1 (ZEB1) to ATM promoter for increasing ATM expression. Moreover, ROCK2 transformed ZEB1 into a gene activator via Yes-associated protein 1 (YAP1). These results provide evidence for the use of ROCK inhibitors in the clinical therapy for MGMTlow TMZ-resistant glioma. Our study also offered novel insights for improving therapeutic management of MGMTlow gliomas.

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 AvailabilityThe 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.

Abstract P054: RP-3500: A novel, potent and selective ATR inhibitor that is effective in pre-clinical models as a monotherapy and in combination with PARP inhibitors

Abstract Background: Ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key mediator of cellular DNA damage repair (DDR) and is activated in response to DNA replication stress. ATR is attractive as a drug target in tumors with loss-of-function alterations in complimentary DDR pathways, including ataxia-telangiectasia mutated (ATM) and BRCA. RESULTS: RP-3500 is a novel, orally bioavailable clinical-stage ATR kinase inhibitor. RP-3500 is highly potent with ATR kinase IC50 values of 1.0 and 0.33 nM in biochemical and cell-based assays, respectively. It is highly selective with &amp;gt;30-fold selectivity over mTOR and &amp;gt;2000-fold selectivity over ATM, DNA-PK and PI3Kα kinases. Preclinical tumor xenograft models harboring synthetic lethal (SNIPRx) gene mutations whose loss of function sensitizes to RP-3500 were selected for in vivo studies. RP-3500 treatment resulted in potent single-agent in vivo efficacy and/or tumor regression in multiple models at minimum effective doses (MED) of 5–7 mg/kg once daily. Pharmacodynamic assessments validated target engagement, with a proportional relationship between tumor pCHK1(Ser345) inhibition and circulating RP-3500 plasma levels (IC80 = 18.6 nM). Circulating free plasma levels of RP-3500 at the MED indicate that exposure above the in vivo tumor pCHK1(Ser345) IC80 for 10–12 hours is sufficient for efficacy and dose proportional phosphorylation of DNA damage markers γ-H2AX, pDNA-PKcs and pKAP1. In vitro, tumor cells with ATM loss exhibited increased susceptibility to RP-3500 and a 3-day compound exposure was sufficient to generate a sustained DNA damage response compared to cells with functional ATM expression. In ATM-deficient mouse models, short-duration intermittent (weekly 3 days on/4 days off or 5 days on/2 days off) dosing schedules maximized tumor growth inhibition while minimizing the impact on hematology parameters, including red blood cell depletion. These results emphasize the reversible nature of erythroid toxicity with RP-3500 and the advantage of intermittent dosing schedules to alleviate anemia. The 3 days on/4 days off intermittent treatment schedule also substantially improved the efficacy and tolerability of RP-3500 and PARP inhibitor combinations compared to continuous treatment schedules. Intermittent treatments of RP-3500 given concomitantly with reduced doses of olaparib or niraparib demonstrated synergistic efficacy in Granta-519 (ATM mutn) and SUM149PT (BRCA1mutn) models with minimal hematological adverse effects and superior efficacy to sequential treatment. CONCLUSIONS: These results provide a strong preclinical rationale to support clinical investigation of the novel ATR inhibitor RP-3500 on an intermittent schedule as a monotherapy and in combination with PARP inhibitors as a means of maximizing clinical benefit. RP-3500 is currently evaluated in the ongoing phase 1 TRESR (Treatment Enabled by SNIPRx) study (NCT04497116). Citation Format: Anne Roulston, Michal Zimmerman, Robert Papp, Alex Skeldon, Charles Pellerin, Émilie Dumas-Bérube, Valerie Dumais, Stephane Dorich, Sara Fournier, Li Li, Marie-Ève Leclaire, Shou Yun Yin, Amandine Chefson, Hunain Alam, William Yang, Chloe Fugère-Desjardins, Sabrina Hammond, Kathryn Skorey, Amina Mulani, Victoria Rimkunas, Artur Veloso, Martine Hamel, Rino Stocco, Yael Mamane, Zuomei Li, Jordan Young, Mike Zinda, Cameron Black. RP-3500: A novel, potent and selective ATR inhibitor that is effective in pre-clinical models as a monotherapy and in combination with PARP inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P054.

P–437 The ovaries of transgender men indicate effects of high dose testosterone on the primordial and early growing follicle pool

Abstract Study question Does high-dose testosterone therapy affect the stage distribution, morphological health and DNA damage repair capacity of human ovarian follicles and their survival in vitro? Summary answer Testosterone exposure is associated with reduced follicle growth activation, reduced follicle health and increased DNA damage: these further deteriorate after six days of culture. What is known already: Androgens have diverse actions within the ovary, however, there is a lack of information regarding the long-term effects of high-dose testosterone on ovarian function and reproductive potential. Cumulus-oocyte complexes recovered from transgender men have been successfully matured in-vitro but little is known regarding the impact of this gender affirming endocrine therapy on the primordial follicle pool. Study design, size, duration: Whole ovaries were obtained from four transgender men aged 25–36 years with informed consent at oophorectomy. All patients had received 1000mg testosterone undecanoate intramuscularly at 12–16 week intervals for a minimum of 4 years pre-operatively. Cortical tissues were dissected into small pieces (≈1x1x0.5mm) and either immediately fixed for histological analysis or cultured for 6 days. Testosterone-treated ovaries were compared to cortical biopsies from age-matched healthy women obtained at caesarean section (n = 4, age 26–36). Participants/materials, setting, methods: Follicle number, classification of developmental stage and morphology were evaluated by histological analysis of ovarian cortical tissue from day 0 and 6 days post culture. Immunohistochemical analysis included γH2AX as a marker of DNA damage, and meiotic recombination 11 (MRE11), ataxia telangiectasia mutated (ATM) and Rad51 as DNA repair proteins. A total of 3802 follicles from testosterone exposed and 878 from control ovaries were analysed. Main results and the role of chance: At day 0 (D0), transgender tissue had a higher proportion of non-growing follicles (92.7±1.7%) compared to control (85.4±6.2%, p &amp;lt; 0.05) but a lower proportion of morphologically healthy follicles (non-growing 59%, primary 61%, secondary 36%; vs 83%, 75%, 80% in controls, all p &amp;lt; 0.005). After 6 days in culture, the proportion of growing follicles increased (51.3% vs 46.5%) but follicle health further declined (all stages p &amp;lt; 0.005). DNA damage was assessed by expression of γH2AX. At D0, the proportion of oocytes showing DNA damage was significantly higher in transgender non-growing follicles (48.1±12.5%, vs 12.3±0.25%, p &amp;lt; 0.005). After culture, γH2AX expression increased in both transgender (p &amp;lt; 0.005) and controls (p &amp;lt; 0.005) but remained higher in transgender oocytes (non-growing 72.2%, primary 71.7% vs 27.3%, 46.2%, all p &amp;lt; 0.05). At D0, there was no difference in expression of DNA repair enzymes ATM and RAD51 between transgender and control oocytes, and increased expression of MRE11 in control non-growing follicles (p &amp;lt; 0.05). Post-culture, there was a significant increase in ATM expression in transgender non-growing oocytes compared to control (98.5% vs 77.8%, p &amp;lt; 0.05) and a less marked decline in RAD51 expression(p &amp;lt; 0.05). The expression of MRE–11 in control non-growing oocytes dramatically declined (100% to 58.2%, p &amp;lt; 0.05), unlike in transgender tissue where expression was comparable to D0. Limitations, reasons for caution A large number of follicles have been analysed, but only from a small number of ovaries. DNA damage at D0 and after 6 days of culture may not reflect DNA damage and repair capacity at later stages of follicle growth. The effect of duration of testosterone treatment was not investigated. Wider implications of the findings: These data indicate that high circulating concentrations of testosterone have previously unrecognised effects on the primordial and small-growing follicles of the ovary. These results may have implications for transgender men receiving gender-affirming therapy prior to considering pregnancy or fertility preservation measures. Trial registration number n/a