Ataxia Telangiectasia Mutated Kinase Activity Research Articles

Effect of PI3-kinase inhibitors on DNA double strand break repair pathways: observations using a site specific DSB induction system

Abstract We established two types of site-specific DNA double strand breaks (DSB) induction systems to elucidate factors which affect the efficiency or quality of DSB repair. For mutation assays, a site specific DSB was generated by the transient expression of a zinc finger nuclease which targets the human HPRT1 gene. A cell line in which time and site specific DSBs can be generated in a HR reporter construct was used for homology-directed repair (HR) analysis. By using these two systems, we investigated the effects of PI3-kinase inhibitors on the efficiency and quality of DSB repair. Ataxia telangiectasia mutated (ATM) kinase inhibition resulted a decrease in mutant frequency and a slight increase in deletion-type mutations accompanied by microhomology. Furthermore, the HR frequency increased significantly when ATM kinase activity was inhibited. Thus, ATM kinase activity might be involved in the suppression of DSB end resection, and this may promote DSB repair through canonical non-homologous end joining.

Abstract LB181: First disclosure of XRD-0394, a novel dual ATM/DNA-PK inhibitor, that potently radiosensitizes and potentiates PARP and topoisomerase inhibitors

Abstract A majority of cancer patients receive radiation therapy (RT) as part of their treatment regimens, whether using external beam therapy or locally-delivered radioisotopes. While often effective, RT may not be efficacious for some tumors while exerting significant short-term and long-term toxicities. Thus, enhancing therapeutic effectiveness of radiation therapy remains an important goal. ATM (ataxia-telangiectasia, mutated) and DNA-PKcs (deoxyribonucleic acid [DNA]-dependent protein kinase, catalytic subunit) are related protein kinases and both are critical regulators of cellular responses to ionizing radiation. We report here the identification and initial characterization of XRD-0394, a potent, specific dual inhibitor of both ATM and DNA-PKcs, whose chemical structure will be disclosed. A dual ATM/DNA-PK inhibitor has practical and theoretical advantages over compounds which inhibit just one of these kinases: 1) blunting both DNA damage response (DDR) pathways enhances radiation-induced tumor cell kill compared to single target inhibition; and 2) simultaneous inhibition of two DDR pathways should overcome intrinsic or acquired resistance of tumor cells to either target alone. Notably, transient inhibition of both kinases by XRD-0394 has little adverse effects on cells in the absence of irradiation. XRD-0394 is orally bioavailable, with excellent pharmacological profiles, demonstrating significant in vitro and in vivo sensitizing activity in combination with ionizing radiation and in combination with immunotherapy and radiation. In addition, a CRISPR screen identified alterations in the Fanconi A/BRCA pathway as an opportunity for synthetic lethality in tumor cells exposed to XRD-0394, an insight that was validated in multiple tumor cell types. In vitro studies demonstrated synergy of XRD-0394 in combination with PARP inhibitors, particularly in BRCA-mutant cells. XRD-0394 also potentiates the effectiveness of topoisomerase inhibitors in vitro. Single doses of XRD-0394 were well-tolerated in combination with palliative RT in subjects with advanced cancer in a Phase Ia clinical trial (NCT05002140). No DLTs were observed. At a dose of 160 mg, plasma concentrations were well above the preclinical target plasma level of 530 ng/mL (1 µM) for up to 15 h and inhibition of ATM kinase activity was demonstrated by inhibition of radiation-induced pKAP1 in tumor tissue. Importantly, this concentration of drug is sufficient to induce potent radiosensitization in numerous preclinical models. The median tmax (2.3 h) and mean terminal t½ (11.1 h) at the 160 mg dose in subjects support coadministration of XRD-0394 and RT within a clinically feasible time frame. These results provide a rationale for future clinical trials with XRD-0394 in combination with RT, RT and immunotherapy, PARP inhibitors and targeted delivery of topoisomerase inhibitors. Citation Format: Tona M. Gilmer, Chun-Hsiang Lai, Kexiao Guo, Katherine Deland, Kathleen A. Ashcraft, Amy E. Stewart, Yaode Wang, Jianmin Fu, Kris C. Wood, Deborah A. Smith, Jonathan T. Yang, Christopher T. Chen, Michael F. Gensheimer, Paul B. Romesser, Steven H. Lin, David G. Kirsch, Michael B. Kastan. First disclosure of XRD-0394, a novel dual ATM/DNA-PK inhibitor, that potently radiosensitizes and potentiates PARP and topoisomerase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB181.

The fructose-bisphosphate, Aldolase A (ALDOA), facilitates DNA-PKcs and ATM kinase activity to regulate DNA double-strand break repair

Glucose metabolism and DNA repair are fundamental cellular processes frequently dysregulated in cancer. In this study, we define a direct role for the glycolytic Aldolase A (ALDOA) protein in DNA double-strand break (DSB) repair. ALDOA is a fructose biphosphate Aldolase that catalyses fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), during glycolysis. Here, we show that upon DNA damage induced by ionising radiation (IR), ALDOA translocates from the cytoplasm into the nucleus, where it partially co-localises with the DNA DSB marker γ-H2AX. DNA damage was shown to be elevated in ALDOA-depleted cells prior to IR and following IR the damage was repaired more slowly. Consistent with this, cells depleted of ALDOA exhibited decreased DNA DSB repair via non-homologous end-joining and homologous recombination. In support of the defective repair observed in its absence, ALDOA was found to associate with the major DSB repair effector kinases, DNA-dependent Protein Kinase (DNA-PK) and Ataxia Telangiectasia Mutated (ATM) and their autophosphorylation was decreased when ALDOA was depleted. Together, these data establish a role for an essential metabolic protein, ALDOA in DNA DSB repair and suggests that targeting ALDOA may enable the concurrent targeting of cancer metabolism and DNA repair to induce tumour cell death.

Abstract 2405: Delineating molecular vulnerabilities of ATM mutant prostate cancers

Abstract Background: Mutations in DNA Damage Response (DDR) genes, including Ataxia, Telangiectasia, Mutated (ATM), are common in advanced castration-resistant prostate cancers (CRPCs). Poly (ADP-ribose) polymerase (PARP) inhibitors are approved in DDR mutant CRPCs, but demonstrate limited clinical efficacy in CRPCs with ATM mutations. In this project, we sought to specifically define the impact of ATM loss on DDR pathways in CRPC, with the goal of identifying alternate therapeutic vulnerabilities. Methods: ATM-KO CRPC cell lines were generated via CRISPR-Cas9 mediated knockout. ATM loss and abolishment of downstream ATM kinase activity was confirmed via western blot. We performed an unbiased phospho-proteomic evaluation of DDR pathways in parental and ATM-KO cells after ionizing radiation (IR). Clonogenic survival assays were performed with either an ATR inhibitor (VX970), the selective DNA-PKcs inhibitor (M3814), or combination therapy. Kinetics of DDR protein recruitment and resolution were interrogated with immunofluorescence (IF) staining for γH2ax, 53BP1, MDC1, and Rad51 foci. Results: ATM-KO cells were able to effectively repair DNA damage following IR, as measured by recruitment and resolution of γH2ax, 53BP1, MDC1, and Rad51 foci. Phospho-proteomic studies demonstrated that ATM-KO cells maintain canonical DDR pathways through ATR and DNA-PKcs kinase activation. Treatment of ATM-KO cells with either VX-970 or M3814 only incrementally affected DDR in ATM-KO cells compared to parental controls, as evidenced by clonogenic survival assays and maintenance of DDR foci. Importantly, combination treatment with VX-970 and M3814 prevented downstream DDR foci recruitment and radio-sensitized ATM-KO CRPC to a greater extent than parental controls. This suggested that activity of any of the trinity of kinases is sufficient to mediate DDR, and that blockade of both ATR and DNA-PKcs is required to effectively prevent DDR in ATM-KO CRPC. We then leveraged a RUVBL1 ATPase inhibitor Compound B, which significantly attenuates levels of these kinases in lung cancer cells. We confirmed that Compound B treatment attenuated ATR and DNA-PKcs protein expression and kinase activity in ATM-KO CRPC cells, and demonstrated sensitivity of ATM-KO cells to Compound B. Conclusions: Our data demonstrates that dual targeting of ATR and DNA-PKcs is necessary in ATM-KO CRPC, as either kinase is independently capable of mediating DDR following IR. Our initial studies indicate that the RUVBL1 ATPase inhibitor Compound B may effectively block DDR in ATM-mutant CRPC, and could be utilized as a novel therapeutic strategy in this molecular subtype. Citation Format: Mia Hofstad, Lan Yu, Andrea Woods, Zoi Sychev, Collin Gilbreath, Xiaofang Huo, Ralf Kittler, Justin M. Drake, Ganesh V. Raj. Delineating molecular vulnerabilities of ATM mutant prostate cancers [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 2405.

Characteristics of Rare Germline ATM Variants in Chronic Lymphocytic Leukemia (CLL)

Background The use of next-generation sequencing in the clinic has increasingly uncovered new variants of unknown significance (VUS), which are often ignored clinically. Ataxia telangiectasia mutated (ATM) is a tumor-suppressor gene that has a critical role in DNA double-strand break repair and has been implicated in the pathogenesis of CLL. In this study, we assessed rare germline ATM variants including VUS using a novel variant calling algorithm, evaluated their impact on the clinical characteristics of CLL and functionally assessed their effect on ATM kinase activity. Methods We evaluated the entire ATM coding region via germline whole exome sequencing from 885 CLL patients representing three different previously reported cohorts (327 DFCI; 276 German CLL Study Group, and 282 ICGC), along with a cohort of ancestry matched controls. We utilized DeepVariant (Poplin et al Nature Biotech 2018) to detect germline variants, followed by a standardized quality control workflow (AlDubayan et al JAMA 2020). All individuals harbored ATM germline variants. Variants were annotated using dbSNP, Clinvar and Varsome, and variants with GnomAD population allele frequency (AF) < 1% were defined as rare. For clinical characterization of CLL patients with rare germline ATM missense variants, we focused on the DFCI cohort. For functional analysis, we evaluated ATM kinase activity by ATM and KAP1 phosphorylation in response to 1 Gy gamma-irradiation using flow cytometry in primary CLL samples with/without rare germline ATM missense variants. We also generated CRISPR/Cas9 engineered cell lines to evaluate the function of ATM p.L2307F which was frequent in our CLL cohort and which we previously demonstrated as enriched in CLL, yet generally annotated as a VUS. Results We identified a total of 130 ATM germline variants among the CLL cases, including 87 missense, and 9 splice site alterations. 80 of 87 missense variants were rare and 59 were annotated as VUS. The top 5 most frequent rare germline missense variants with high odds ratio for association with CLL were ATM p.L2332P (AF cases vs controls, 0.328% vs 0.0188%, OR 17.46), p.L2307F (OR 16.88), p.F763L (OR 18.89), p.Y1442H (OR 35.39) and p.S99G (OR 16.28). In 63 DFCI patients with rare missense variants, the median age at diagnosis was 55 (31-82), 21.7% had a familial history of CLL and 29% had del(11q). Phosphorylation of ATM and KAP1 (pATM, pKAP1) in response to gamma-irradiation was reduced in 12 patients assessed (8 rare and 4 non-rare) with germline ATM variants (ATM variant alone group) compared to those without any somatic or germline ATM aberrancy (WT group) (P<0.01). Similar results were seen when the analysis was restricted to the 8 CLL patients with rare ATM variants. Phosphorylation levels were also significantly lower in 17 patients with rare germline ATM variants with concurrent del(11q) (del 11q + ATM variant group) compared to 13 of those with del(11q) alone (del 11q group) (P<0.01) (Figure 1). Altogether, these results suggest that germline ATM variants have a functional impact on DNA damage response in primary CLL cells. We previously identified the VUS variant ATM p.L2307F as particularly enriched among CLL patients (Tiao G et al., Leukemia. 2017). We therefore generated ATM L2307F knock-in NALM-6 cell lines via CRISPR/Cas9. We generated 7 cell lines including ATM wild type (+/+), ATM monoallelic mutated (+/L2307F), ATM monoallelic knocked out (+/-), ATM monoallelic mutated with knock out of the other allele (-/L2307F), and ATM biallelic knockout (-/-). The induction of both pATM and pKAP1 levels by gamma irradiation was reduced in proportion to ATM mutational status as shown in Figure 2. Conclusions We demonstrate that rare ATM germline variants are common in CLL and affect the DNA damage response in primary CLL cells when controlling for somatic 11q deletion. We further characterize the p.L2307F variant using CRISPR and demonstrate that this allele represents a functional hypomorph in cell-based assays. Further studies are needed to assess the impact of each individual rare germline variant on the function of ATM, CLL pathogenesis and the inherited risk of CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MRN-dependent and independent pathways for recruitment of TOPBP1 to DNA double-strand breaks.

Ataxia Telangiectasia mutated and RAD3-related (ATR) kinase is activated by DNA replication stress and also by various forms of DNA damage, including DNA double-strand breaks (DSBs). Recruitment to sites of damage is insufficient for ATR activation as one of two known ATR activators, either topoisomerase II-binding protein (TOPBP1) or Ewing’s tumor-associated antigen 1, must also be present for signaling to initiate. Here, we employ our recently established DSB-mediated ATR activation in Xenopus egg extract (DMAX) system to examine how TOPBP1 is recruited to DSBs, so that it may activate ATR. We report that TOPBP1 is only transiently present at DSBs, with a half-life of less than 10 minutes. We also examined the relationship between TOPBP1 and the MRE11-RAD50-NBS1 (MRN), CtBP interacting protein (CtIP), and Ataxia Telangiectasia mutated (ATM) network of proteins. Loss of MRN prevents CtIP recruitment to DSBs, and partially inhibits TOPBP1 recruitment. Loss of CtIP has no impact on either MRN or TOPBP1 recruitment. Loss of ATM kinase activity prevents CtIP recruitment and enhances MRN and TOPBP1 recruitment. These findings demonstrate that there are MRN-dependent and independent pathways that recruit TOPBP1 to DSBs for ATR activation. Lastly, we find that both the 9-1-1 complex and MDC1 are dispensable for TOPBP1 recruitment to DSBs.

A New Class of Selective ATM Inhibitors as Combination Partners of DNA Double-Strand Break Inducing Cancer Therapies.

Radiotherapy and chemical DNA-damaging agents are among the most widely used classes of cancer therapeutics today. Double-strand breaks (DSB) induced by many of these treatments are lethal to cancer cells if left unrepaired. Ataxia telangiectasia-mutated (ATM) kinase plays a key role in the DNA damage response by driving DSB repair and cell-cycle checkpoints to protect cancer cells. Inhibitors of ATM catalytic activity have been shown to suppress DSB DNA repair, block checkpoint controls and enhance the therapeutic effect of radiotherapy and other DSB-inducing modalities. Here, we describe the pharmacological activities of two highly potent and selective ATM inhibitors from a new chemical class, M3541 and M4076. In biochemical assays, they inhibited ATM kinase activity with a sub-nanomolar potency and showed remarkable selectivity against other protein kinases. In cancer cells, the ATM inhibitors suppressed DSB repair, clonogenic cancer cell growth, and potentiated antitumor activity of ionizing radiation in cancer cell lines. Oral administration of M3541 and M4076 to immunodeficient mice bearing human tumor xenografts with a clinically relevant radiotherapy regimen strongly enhanced the antitumor activity, leading to complete tumor regressions. The efficacy correlated with the inhibition of ATM activity and modulation of its downstream targets in the xenograft tissues. In vitro and in vivo experiments demonstrated strong combination potential with PARP and topoisomerase I inhibitors. M4076 is currently under clinical investigation.

Implication of Ataxia-Telangiectasia-mutated kinase in epithelium-mesenchyme transition.

Impairment of genome instability drives the development of cancer by disrupting anti-cancer barriers. Upon genotoxic insults, DNA damage responsive factors, notably ATM kinase, is crucial to protect genomic integrity while promoting cell death. Meanwhile, cytotoxic therapy-inducing DNA lesions is double-edged sword by causing cancer metastasis based on animal models and clinical observations. The underlying mechanisms for the procancer effect of cytotoxic therapies are poorly understood. Here, we report that cancer cells subjected to cytotoxic treatments elicit dramatic alteration of gene expression controlling the potential of epithelium-mesenchyme transition (EMT). Resultantly, EMT-dependent cell mobility is potently induced upon DNA damage. This stimulation of EMT is mainly Ataxia-Telangiectasia-mutated (ATM)-dependent, as the chemical inhibitor specifically inhibiting ATM kinase activity can suppress the EMT gene expression and thus cell mobility. At last, we show that cancer cells with ATM activation display increased metastatic potential in ovarian cancer tissues. Taken together, we reveal a novel role of ATM in promoting metastatic potential of cancer cells by favoring EMT gene expression.

ATM inhibition enhances Auranofin-induced oxidative stress and cell death in lung cell lines.

Ataxia-Telangiectasia (A-T), a pleiotropic chromosomal breakage syndrome, is caused by the loss of the kinase Ataxia-telangiectasia mutated (ATM). ATM is not only involved in the response to DNA damage, but also in sensing and counteracting oxidative stress. Since a disturbed redox balance has been implicated in the pathophysiology of A-T lung disease, we aimed to further explore the interplay between ATM and oxidative stress in lung cells. Using a kinetic trapping approach, we could demonstrate an interaction between the trapping mutant TRX1-CS and ATM upon oxidative stress. We could further show that combined inhibition of thioredoxin reductase (TrxR) and ATM kinase activity, using Auranofin and KU55933 respectively, induced an increase in cellular reactive oxygen species (ROS) levels and protein oxidation in lung cells. Furthermore, ATM inhibition sensitized lung cells to Auranofin-induced cell death that could be rescued by ROS scavengers. As a consequence, targeted reduction of ATM by TRX1 could serve as a regulator of oxidative ATM activation and contribute to the maintenance of the cellular redox homeostasis. These results highlight the importance of the redox-active function of ATM in preventing ROS accumulation and cell death in lung cells.

Activation of ATM kinase by ROS generated during ionophore-induced mitophagy in human T and B cell malignancies.

Ataxia telangiectasia mutated (ATM), a critical DNA damage sensor, also possesses non-nuclear functions owing to its presence in extra-nuclear compartments, including peroxisomes, lysosomes, and mitochondria. ATM is frequently altered in several human cancers. Recently, we and others have shown that loss of ATM is associated with defective mitochondrial autophagy (mitophagy) in ataxia-telangiectasia (A-T) fibroblasts and B-cell lymphomas. Further, we reported that ATM protein but not ATM kinase activity is required for mitophagy. However, the mechanism of ATM kinase activation during ionophore-induced mitophagy is unknown. In the work reported here, using several ionophores in A-T and multiple T-cell and B-cell lymphoma cell lines, we show that ionophore-induced mitophagy triggers oxidative stress-induced ATMSer1981 phosphorylation through ROS activation, which is different from neocarzinostatin-induced activation of ATMSer1981, Smc1Ser966, and Kap1Ser824. We used A-T cells overexpressed with WT or S1981A (auto-phosphorylation dead) ATM plasmids and show that ATM is activated by ROS-induced oxidative stress emanating from ionophore-induced mitochondrial damage and mitophagy. The antioxidants N-acetylcysteine and glutathione significantly inhibited ROS production and ATMSer1981 phosphorylation but failed to inhibit mitophagy as determined by retroviral infection with mt-mKeima construct followed by lysosomal dual-excitation ratiometric pH measurements. Our data suggest that while ATM kinase does not participate in mitophagy, it is activated via elevated ROS.

ATM-mediated DNA double-strand break response facilitated oncolytic Newcastle disease virus replication and promoted syncytium formation in tumor cells.

Deoxyribonucleic acid (DNA) damage response (DDR) is the fundamental cellular response for maintaining genomic integrity and suppressing tumorigenesis. The activation of ataxia telangiectasia-mutated (ATM) kinase is central to DNA double-strand break (DSB) for maintaining host-genome integrity in mammalian cells. Oncolytic Newcastle disease virus (NDV) can selectively replicate in tumor cells; however, its influence on the genome integrity of tumor cells is not well-elucidated. Here, we found that membrane fusion and NDV infection triggered DSBs in tumor cells. The late replication and membrane fusion of NDV mechanistically activated the ATM-mediated DSB pathway via the ATM-Chk2 axis, as evidenced by the hallmarks of DSBs, i.e., auto-phosphorylated ATM and phosphorylated H2AX and Chk2. Immunofluorescence data showed that multifaceted ATM-controlled phosphorylation markedly induced the formation of pan-nuclear punctum foci in response to NDV infection and F-HN co-expression. Specific drug-inhibitory experiments on ATM kinase activity further suggested that ATM-mediated DSBs facilitated NDV replication and membrane fusion. We confirmed that the Mre11-RAD50-NBS1 (MRN) complex sensed the DSB signal activation triggered by NDV infection and membrane fusion. The pharmacological inhibition of MRN activity also significantly inhibited intracellular and extracellular NDV replication and syncytia formation. Collectively, these data identified for the first time a direct link between the membrane fusion induced by virus infection and DDR pathways, thereby providing new insights into the efficient replication of oncolytic NDV in tumor cells.

Abstract P5-03-02: Cancer risks associated with pathogenic variants in the ataxia telangiectasia mutated (ATM) gene

Abstract Background: Germ-line pathogenic variants (PV) in ATM are often considered low-to-moderate penetrance risk factors for breast cancer (BC), conferring a two-fold increased BC risk among heterozygous PV carriers. Several other cancers have been associated with ATM PV such as melanoma, thyroid cancer, prostate cancer, pancreatic cancer, gastro-esophageal cancers, colorectal cancer, and ovarian cancer. Estimating the diverse cancer risks attributable to ATM PVs may present challenges due to the low-to-moderate penetrance of ATM PV for some cancers, requiring large samples of tested patients to estimate risk with precision, and the potential for substantial variability in penetrance of particular ATM PV such as the c.7271T&amp;gt;G PV reported by previous studies. We investigated cancer risks with ATM PV in a large clinical testing dataset. Methods: Results of 614,344 individuals undergoing clinical analysis of ATM as part of a multi-gene hereditary cancer gene panel between 09/2013 and 06/2019 were reviewed. Patient age, sex, ancestry, personal and family cancer history were extracted from a test requisition form. PV included variants classified as suspected deleterious or deleterious (hereafter referred to as pathogenic). Risk estimates are estimated using multivariate logistic regression and are reported as odds ratios (OR) with 95% confidence intervals (CI). Results: PV in ATM were identified in 4,372/614,344 (0.7%) individuals: 4,155/592,881 (0.7%) females and 217/21,463 (1.0%) males. 1,603/4,155 (38.6%) of female ATM PV carriers had invasive BC or DCIS. Overall, ATM PV were associated with a 2-fold increased risk for invasive BC [OR 2.01, 95% CI 1.86-2.16] and DCIS [OR 1.82, 95% CI 1.62-2.04]. A prevalent missense PV c.7271T&amp;gt;G, predicted to lead to inactivation of ATM kinase activity and that has previously been described as high penetrance (Goldgar DE et al, Breast Cancer Res 2011), represented 5.1% (n=223) of all ATM PV, and was associated with a particularly high risk of invasive BC [OR 3.80, 95% CI 2.78-5.19] but no increased risk of DCIS. Significantly elevated risks of pancreatic cancer, gastric cancer, prostate cancer, ovarian cancer, colon cancer and melanoma were also observed among ATM PV carriers (see Table 1). Median age of invasive BC diagnosis was similar in carriers and non-carriers of ATM PV. Conclusions: ATM PV are associated with 1.5-fold to &amp;gt;4-fold increased cancer risks across a variety of disease sites, including invasive BC (2-fold increased risk). Cancer risks also demonstrate mutation-specific variability, as suggested by the 4-fold increased risk of invasive BC in carriers of the ATM PV c.7271T&amp;gt;G. Larger and/or pooled studies are critical to further refine the breadth and magnitude of ATM-associated cancer risk and to improve clinical management guidelines for ATM PV carriers. Table 1: Odds ratios for cancer risks in c.7271T&amp;gt;G and all other ATM PV carriersAll ATM PVs (excluding c.7271T&amp;gt;G)c.7271T&amp;gt;GCancer OR (95% CI)p-valueOR (95% CI)p-valueBreast, Invasive1.94 (1.79, 2.09)&amp;lt;0.00013.80 (2.78, 5.19)&amp;lt;0.0001Breast, DCIS1.83 (1.62, 2.05)&amp;lt;0.00011.63 (0.97, 2.74)0.0624Breast, Lobular invasive0.94 (0.73, 1.21)0.62330.84 (0.27, 2.64)0.7671Breast, Male1.44 (0.86, 2.42)0.16884.24 (0.46, 39.15)0.2032Ovarian1.55 (1.32, 1.82)&amp;lt;0.00011.99 (0.99, 4.02)0.0532Colorectal1.51 (1.25, 1.82)&amp;lt;0.00011.67 (0.67, 4.18)0.2693Endometrial1.10 (0.88, 1.39)0.39610.89 (0.28, 2.81)0.8401Melanoma1.44 (1.14, 1.81)0.00182.16 (1.00, 4.64)0.0491Prostate2.61 (1.93, 3.52)&amp;lt;0.00014.37 (0.70, 27.26)0.1146Pancreatic4.18 (3.17, 5.49)&amp;lt;0.00012.50 (0.34, 18.21)0.3660Gastric3.09 (1.73, 5.53)0.0001—— Citation Format: Michael J Hall, Katie Larson, Ryan Bernhisel, Elisha Hughes, Eric Rosenthal, Nanda Singh, Johnathan M Lancaster, Allison W Kurian. Cancer risks associated with pathogenic variants in the ataxia telangiectasia mutated (ATM) gene [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-03-02.

Ataxia-telangiectasia mutated interacts with Parkin and induces mitophagy independent of kinase activity. Evidence from mantle cell lymphoma.

Ataxia telangiectasia mutated (ATM), a critical DNA damage sensor with protein kinase activity, is frequently altered in human cancers including mantle cell lymphoma. Loss of ATM protein is linked to accumulation of nonfunctional mitochondria and defective mitophagy in both murine thymocytes and in ataxia-telangiectasia cells. However, the mechanistic role of ATM kinase in cancer cell mitophagy is unknown. Here, we provide evidence that FCCP-induced mitophagy in mantle cell lymphoma and other cancer cell lines is dependent on ATM but independent of its kinase function. While Granta-519 mantle cell lymphoma cells possess single copy kinase-dead ATM and are resistant to FCCP-induced mitophagy, both Jeko-1 and Mino cells are ATMproficient and induce mitophagy. Stable knockdown of ATM in Jeko-1 and Mino cells conferred resistance to mitophagy and was associated with reduced ATP production, oxygen consumption, and increased mitochondrial reactive oxygen species. ATM interacts with the E3 ubiquitin ligase Parkin in a kinase-independent manner. Knockdown of ATM in HeLa cells resulted in proteasomal degradation of GFP-Parkin which was rescued by the proteasome inhibitor, MG132, suggesting that the ATMParkin interaction is important for Parkin stability. Neither loss of ATM kinase activity in primary B-cell lymphomas nor inhibition of ATM kinase in mantle cell lymphoma, ataxia-telangiectasia and HeLa cell lines mitigated FCCP- or CCCP-induced mitophagy suggesting that ATM kinase activity is dispensable for mitophagy. Malignant B-cell lymphomas without detectable ATM, Parkin, Pink1, and Parkin-UbSer65 phosphorylation were resistant to mitophagy, providing the first molecular evidence of the role of ATM in mitophagy in mantle cell lymphoma and other B-cell lymphomas.

Ataxia telangiectasia mutated pathway disruption affects hepatic DNA and tissue damage in nonalcoholic fatty liver disease

To overcome the rising burdens of nonalcoholic fatty liver disease, mechanistic linkages in mitochondrial dysfunction, inflammation and hepatic injury are critical. As ataxia telangiectasia mutated (ATM) gene oversees DNA integrity and mitochondrial homeostasis, we analyzed mRNAs and total proteins or phosphoproteins related to ATM gene by arrays in subjects with healthy liver, fatty liver or nonalcoholic steatohepatitis. Functional genomics approaches were used to query DNA damage or cell growth events. The effects of fatty acid-induced toxicity in mitochondrial health, DNA integrity and cell proliferation were validated in HuH-7 cells, including by inhibiting ATM kinase activity or knckdown of its mRNA. In fatty livers, DNA damage and ATM pathway activation was observed. During induced steatosis in HuH-7 cells, lowering of ATM activity produced mitochondrial dysregulation, DNA damage and cell growth inhibition. In livers undergoing steatohepatitis, ATM was depleted with increased hepatic DNA damage and growth-arrest due to cell cycle checkpoint activations. Moreover, molecular signatures of oncogenesis were associated with upstream mechanistic networks directing cell metabolism, inflammation or growth that were either activated (in fatty liver) or inactivated (in steatohepatitis). To compensate for hepatic growth arrest, preoncogenic oval cell populations expressing connexin-43 and/or albumin emerged. These oval cells avoided DNA damage and proliferated actively. We concluded that ATM is a major contributor to the onset and progression of nonalcoholic fatty liver disease. Therefore, specific markers for ATM pathway dysregulation will allow prospective segregation of cohorts for disease susceptibility and progression from steatosis to steatohepatitis. This will offer superior design and evaluation parameters for clinical trials. Restoration of ATM activity with targeted therapies should be appropriate for nonalcoholic fatty liver disease.

Homozygous pathogenic variant in BRAT1 associated with nonprogressive cerebellar ataxia.

ObjectiveTo investigate the pathogenicity of a novel homozygous BRAT1 variant in 2 siblings with nonprogressive cerebellar ataxia (NPCA) through functional studies on primary and immortalized patient cell lines.MethodsBRAT1 protein levels and ataxia-telangiectasia mutated (ATM) kinase activity in patient-derived and control cell lines were assessed by Western blotting. The impact of the novel BRAT1 variants on mitochondrial function was also assessed, by comparing patient and control cell lines for rates of oxygen consumption and for phosphorylation (S293) of the E1⍺ subunit of pyruvate dehydrogenase (PDH).ResultsTwo male siblings with NPCA, mild intellectual disability, and isolated cerebellar atrophy were found to be homozygous for a c.185T>A (p.Val62Glu) variant in BRAT1 by whole exome sequencing. Western blotting revealed markedly decreased BRAT1 protein levels in lymphocytes and/or fibroblast cells from both affected siblings compared to control cell lines. There were no differences between the patient and control cells in ATM kinase activation, following ionizing radiation. Mitochondrial studies were initially suggestive of a defect in regulation of PDH activity, but there was no evidence of increased phosphorylation of the E1⍺ subunit of the PDH complex. Measurement of oxygen consumption rates similarly failed to identify differences between patient and control cells.ConclusionsBiallelic pathogenic variants in BRAT1 can be associated with NPCA, a phenotype considerably milder than previously reported. Surprisingly, despite the molecular role currently proposed for BRAT1 in ATM regulation, this disorder is unlikely to result from defective ATM kinase or mitochondrial dysfunction.

Abstract 4868: A preclinical PK/PD model based on a mouse glioblastoma survival model for AZD1390, a novel, brain-penetrant ATM kinase inhibitor, to predict the inhibition of DNA damage response induced by radiation and the human efficacious dose

Abstract AZD1390 is a novel, highly selective, brain-penetrant, potent inhibitor of the Ataxia Telangiectasia Mutated (ATM) kinase. ATM is activated in response to double-strand breaks (DSBs) and coordinates cellular responses to ionising radiation and other insults. Radiation is the mainstay of treatment for patients with brain tumors, and glioma cells are exquisitely sensitive to ATM inhibition. Therefore, AZD1390 is in clinical development in combination with radiation therapy for the treatment of patients with Glioblastoma Multiforme (GBM) and brain metastases from solid tumours (NCT03423628). [1] The aim of this work was to develop a translational PK/PD-efficacy model for AZD1390 that would enable the project team to assess the extent/duration of inhibition of target engagement required and help to predict the optimal dose and regimen of AZD1390 to treat patients with brain malignancies in combination with radiation. Time-course data of pharmacokinetics (PK), pharmacodynamics (PD) and tumor growth inhibition from mouse NCI-H2228 brain tumor orthotopic model studies were used to build a PK-PD/Efficacy or survival model. ATM activation is induced by IR treatment, becoming transiently phosphorylated within minutes of IR exposure and dissipates over a 24-hour period. This model quantitatively and dynamically integrates AZD1390 brain PK to the rate and extent of inhibition of phosphorylation of ATM, a proximal PD marker of ATM kinase activation caused by radiation induced DNA damage in the tumor and rate of induction of cell death (determined by bioluminescence signalling as a measure of tumor growth). In parallel, we also modelled cell cycle profiles in GBM cell lines to complement the in-vivo modelling work. The pATM time-course relative to IR radiation (2 Gy) was modelled using AZD1390 concentrations in the brain tumor and the corresponding pATM inhibition, which was then linked to efficacy by estimating tumor cell kill rate via exponential tumor growth model. The free brain concentration of AZD1390 that resulted in half-maximal inhibition (EC50) of pATM after radiation was estimated to be 0.8 nM (range 0.4 to 1.6 nM). Significant tumor regression in orthotopic tumor model was observed at doses &amp;gt;5 mg/kg. An average pATM inhibition over 24h in the mouse GBM survival model was in the range of 44% (5 mg/kg QD) to 88% (20 mg/kg BD). The % overall survival at 5 mg/kg QD and 20 mg/kg BD dose were 56% and 100%, respectively. Conclusion: The translation of mouse survival data to clinical schedules is unknown, thus efficacious dose was anchored to pATM inhibition required to deliver significant tumor regression.

Abstract 3500: Highly potent and selective ATM kinase inhibitor M4076: A clinical candidate drug with strong anti-tumor activity in combination therapies

Abstract M4076 is an ATP-competitive inhibitor of the Ataxia telangiectasia mutated (ATM) kinase (IC50 &amp;lt; 1 nM), which targets tumor cell survival and growth by inhibiting double-strand break (DSB) repair as well as checkpoint control. DSB repair is crucial for survival of malignant tumor cells, especially under treatment with DNA damaging chemo- and radiotherapy. As such, the rationale of pharmacological inhibition of ATM is to increase and maintain the extent of unrepaired DNA damage generated by radio-, chemotherapy, and targeted therapies to drive tumor cells into cell death. We have developed an orally administered, sub-nanomolar potent &amp; selective kinase inhibitor of ATM, M4076, for cancer therapy in combination with DNA damaging modalities. Here, we present its structure-activity relationship, its chemical structure (first-time disclosure) &amp; physicochemical profile as well as its preclinical characterization using biochemical, cellular &amp; human tumor xenograft models. M4076 sensitizes tumor cell lines to radiation therapy in vitro and strongly enhances the anti-tumor activity of ionizing radiation in vivo. These effects are due to the inhibition of ATM kinase activity as demonstrated by the inhibition of radiotherapy induced ATM autophosphorylation and CHK2 phosphorylation in xenograft lysates. Citation Format: Thomas Fuchss, Ulrich Graedler, Kai Schiemann, Daniel Kuhn, Holger Kubas, Heike Dahmen, Astrid Zimmermann, Frank Zenke, Andree Blaukat. Highly potent and selective ATM kinase inhibitor M4076: A clinical candidate drug with strong anti-tumor activity in combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3500.

NMR– and MD simulation–based structural characterization of the membrane-associating FATC domain of ataxia telangiectasia mutated

The Ser/Thr protein kinase ataxia telangiectasia mutated (ATM) plays an important role in the DNA damage response, signaling in response to redox signals, the control of metabolic processes, and mitochondrial homeostasis. ATM localizes to the nucleus and at the plasma membrane, mitochondria, peroxisomes, and other cytoplasmic vesicular structures. It has been shown that the C-terminal FATC domain of human ATM (hATMfatc) can interact with a range of membrane mimetics and may thereby act as a membrane-anchoring unit. Here, NMR structural and 15N relaxation data, NMR data using spin-labeled micelles, and MD simulations of micelle-associated hATMfatc revealed that it binds the micelle by a dynamic assembly of three helices with many residues of hATMfatc located in the headgroup region. We observed that none of the three helices penetrates the micelle deeply or makes significant tertiary contacts to the other helices. NMR-monitored interaction experiments with hATMfatc variants in which two conserved aromatic residues (Phe3049 and Trp3052) were either individually or both replaced by alanine disclosed that the double substitution does not abrogate the interaction with micelles and bicelles at the high concentrations at which these aggregates are typically used, but impairs interactions with small unilamellar vesicles, usually used at much lower lipid concentrations and considered a better mimetic for natural membranes. We conclude that the observed dynamic structure of micelle-associated hATMfatc may enable it to interact with differently composed membranes or membrane-associated interaction partners and thereby regulate ATM's kinase activity. Moreover, the FATC domain of ATM may function as a membrane-anchoring unit for other biomolecules.

Nucleolar protein nucleolin functions in replication stress-induced DNA damage responses.

The nucleolus contains multiple copies of ribosomal (r)DNA, which indicate sites of frequent replication stress and suggest the existence of a mechanism to prevent replication stress–related rDNA instability and the possibility that such a mechanism contributes to the whole genomic stability against replication stress. We have previously reported that nucleolin, a major nucleolar protein, is involved in ionizing radiation–induced DNA damage responses (DDRs) such as ataxia telangiectasia mutated (ATM)-dependent cell cycle checkpoints and homologous recombination (HR) repair. Here, we investigated the role of nucleolin in DDR due to replication stress. The results indicate that following replication stress, nucleolin interacted with the histone γH2AX, proliferating cell nuclear antigen (PCNA), and replication protein A (RPA)32, suggesting that it may be recruited to DNA damage sites on the replication fork. Furthermore, the knockdown of nucleolin by siRNA reduced the activation of ATM and RAD3-related (ATR) kinase and the formation of RAD51 and RPA32 foci after replication stress due to UV or camptothecin exposure, whereas nucleolin overexpression augmented ATR-dependent phosphorylation and RAD51 and RPA accumulation on chromatin. Moreover, these overexpressing cells seemed to increase repair activity and resistance to replication stress. Our results indicate that nucleolin plays an important role in replication stress–induced DDRs such as ATR activation and HR repair. Given that nucleolin overexpression is often observed in many types of cancer cells, our findings suggest that nucleolin is involved in the regulation of resistance to replication stress that may otherwise lead to tumorigenesis and it could be a possible target for chemotherapy and radiotherapy.

Genotype–phenotype correlations in ataxia telangiectasia patients with ATM c.3576G>A and c.8147T>C mutations

BackgroundAtaxia telangiectasia (A-T) is a neurodegenerative disorder. While patients with classic A-T generally die in their 20s, some patients with variant A-T, who have residual ataxia-telangiectasia mutated (ATM) kinase activity,...