Ataxia Telangiectasia Mutated Kinase Inhibitor Research Articles

CTIM-16. SAFETY AND PRELIMINARY EFFICACY OF AZD1390 + RADIATION THERAPY FOR GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma is an aggressive cancer; intensity-modulated radiation therapy (IMRT) with concomitant and adjuvant temozolomide is the first-line standard of care. AZD1390, an ataxia telangiectasia mutated kinase inhibitor, aims to augment the efficacy of IMRT without exacerbating neurotoxicity. AZD1390 is optimized for blood–brain barrier penetration, confirmed in healthy volunteers (NCT03215381) and patients with glioblastoma (Phase 0; NCT05182905). This Phase 1, open-label study (NCT03423628) evaluated safety and efficacy of AZD1390 and IMRT in patients with glioblastoma. METHODS Adult patients received escalating once-daily AZD1390 doses with IMRT 35 Gy in 10 fractions over 2 weeks (Arm A, recurrent glioblastoma) or IMRT 60 Gy in 30 fractions over 6 weeks (Arm C, newly-diagnosed O-6-methylguanine-DNA methyltransferase unmethylated glioblastoma). Patients received 2 additional weeks of adjuvant AZD1390 post-IMRT. Primary objective was safety; secondary objectives included efficacy and pharmacokinetics. RESULTS As of Feb 6, 2024, 115 patients (Arm A=75; Arm C=40) received AZD1390 10–900 mg/day. Pharmacokinetics were linear, with a slightly more-than dose-proportional increase and mean elimination half-life ~9–11 hours. Most patients had ≥1 treatment-emergent adverse event (AE), most commonly fatigue (51.3%), nausea (39.1%) and headache (38.3%). Grade ≥3 AZD1390-related AEs occurred in 18/115 patients. The maximum tolerated dose was 400 mg (Arm A) and 300 mg (Arm C). Dose-limiting toxicities included creatinine kinase elevation (Arm A) and radiation skin injury (Arm C). AEs led to AZD1390 discontinuation in 15/115 patients. Safety profiles were mostly consistent across Arms despite different radiation schedules. At target-engaging doses, median overall survival was 12.7 months (95% CI: 10.7–18.9; Arm A) and still maturing (Arm C). CONCLUSIONS Concurrent AZD1390 and IMRT demonstrated manageable safety at doses with known target engagement. Preliminary efficacy is encouraging (Arm A). AZD1390 can potentially act as a radiosensitizer for glioblastoma treatment. Clinical investigation is ongoing.

Early inhibition of the ATM/p53 pathway reduces the susceptibility to atrial fibrillation and atrial remodeling following acute myocardial infarction

Atrial fibrillation (AF) emerges as a critical complication following acute myocardial infarction (AMI) and is associated with a significant increased risk of heart failure, stroke and mortality. Ataxia telangiectasia mutated (ATM), a key player in DNA damage repair (DDR), has been implicated in multiple cardiovascular conditions, however, its involvement in the development of AF following AMI remains unexplored. This study seeks to clarify the contribution of the ATM/p53 pathway in the onset of AF post-AMI and to investigate the underlying mechanisms. The rat model of AMI was established by ligating left anterior descending coronary artery in the presence or absence of Ku55933 (an ATM kinase inhibitor, 5 mg/kg/d) treatment. Rats receiving Ku55933 were further divided into the early administration group (administered on days 1, 2, 4, and 7 post-AMI) and the late administration group (administered on days 8, 9, 11 and 14 post-AMI). RNA-sequencing was performed 14 days post-operation. In vitro, H2O2-challenged HL-1 atrial muscle cells were utilized to evaluate the potential effects of different ATM inhibition schemes, including earlier, middle, and late periods of intervention. Fourteen days post-AMI injury, the animals exhibited significantly increased AF inducibility, exacerbated atrial electrical/structural remodeling, reduced ventricular function and exacerbated atrial DNA damage, as evidenced by enhanced ATM/p53 signaling as well as γH2AX level. These effects were partially consistent with the enrichment results of bioinformatics analysis. Notably, the deleterious effects were ameliorated by early, but not late, administration of Ku55933. Mechanistically, inhibition of ATM signaling successfully suppressed atrial NLRP3 inflammasome-mediated pyroptotic pathway. Additionally, the results were validated in the in vitro experiments demonstrating that early inhibition of Ku55933 not only attenuated cellular ATM/p53 signaling, but also mitigated inflammatory response by reducing NLRP3 activation. Collectively, hyperactivation of ATM/p53 contributed to the pathogenesis of AF following AMI. Early intervention with ATM inhibitors substantially mitigated AF susceptibility and atrial electrical/structural remodeling, highlighting a novel therapeutic avenue against cardiac arrhythmia following AMI.

Discovery of [1,2,3]Triazolo[4,5-c]quinoline Derivatives as a New Class of Ataxia-Telangiectasia Mutated Kinase Inhibitors.

Ataxia-telangiectasia mutated (ATM) is an atypical serine/threonine protein kinase which is implicated in the repair of DNA double-strand breaks. Numerous reports have shown that ATM inhibition is an attractive target for radiotherapy and chemotherapy sensitization. Herein we report a new series of ATM kinase inhibitors containing the 1H-[1,2,3]triazolo[4,5-c]quinoline scaffold, which was obtained by virtual screening, structural optimization, and structure-activity relationship studies. Among the inhibitors, A011 was one of the most potent, with an IC50 value of 1.0 nM against ATM. In colorectal cancer cells (SW620 and HCT116), A011 was able to inhibit activation of ATM signaling induced by irinotecan (CPT-11) and ionizing radiation and then increased the sensitivity of colorectal cancer cells to irinotecan and ionizing radiation through increasing G2/M arrest and inducing apoptosis. In the SW620 human colorectal adenocarcinoma tumor xenograft model, A011 sensitized SW620 to CPT-11 by inhibiting ATM activity. Collectively, this work has identified a promising lead in the discovery of potent inhibitors against ATM.

Inhibition of ATM kinase rescues planarian regeneration after lethal radiation

As stem cells divide, they acquire mutations that can be passed on to daughter cells. To mitigate potentially deleterious outcomes, cells activate the DNA damage response (DDR) network, which governs several cellular outcomes following DNA damage, including repairing DNA or undergoing apoptosis. At the helm of the DDR are three PI3‐like kinases including Ataxia‐Telangiectasia Mutated (ATM). We report here that knockdown of ATM in planarian flatworms enables stem cells to withstand lethal doses of radiation which would otherwise induce cell death. In this context, stem cells circumvent apoptosis, replicate their DNA, and recover function using homologous recombination‐mediated DNA repair. Despite radiation exposure, atm knockdown animals survive long‐term and regenerate new tissues. These effects occur independently of ATM's canonical downstream effector p53. Together, our results demonstrate that in planarians, ATM promotes radiation‐induced apoptosis. This acute, ATM‐dependent apoptosis is a key determinant of long‐term animal survival. Our results suggest that inhibition of ATM in these organisms could, therefore, potentially favor cell survival after radiation without obvious effects on stem cell behavior.

A Triphenylphosphonium-Functionalized Delivery System for an ATM Kinase Inhibitor That Ameliorates Doxorubicin Resistance in Breast Carcinoma Mammospheres

The enzyme ataxia-telangiectasia mutated (ATM) kinase is a pluripotent signaling mediator which activates cellular responses to genotoxic and metabolic stress. It has been shown that ATM enables the growth of mammalian adenocarcinoma stem cells, and therefore the potential benefits in cancer chemotherapy of a number of ATM inhibitors, such as KU-55933 (KU), are currently being investigated. We assayed the effects of utilizing a triphenylphosphonium-functionalized nanocarrier delivery system for KU on breast cancer cells grown either as a monolayer or in three-dimensional mammospheres. We observed that the encapsulated KU was effective against chemotherapy-resistant mammospheres of breast cancer cells, while having comparably lower cytotoxicity against adherent cells grown as monolayers. We also noted that the encapsulated KU sensitized the mammospheres to the anthracycline drug doxorubicin significantly, while having only a weak effect on adherent breast cancer cells. Our results suggest that triphenylphosphonium-functionalized drug delivery systems that contain encapsulated KU, or compounds with a similar impact, are a useful addition to chemotherapeutic treatment schemes that target proliferating cancers.

Ataxia telangiectasia mutated kinaseinhibition promotes irradiation-induced PD-L1 expression in tumour-associated macrophages through IFN-I/JAK signalling pathway.

Tumour-associated macrophages (TAMs) are one of the primary sources of PD-L1 expression in the tumour microenvironment (TME). Ionizing radiation (IR) promotes PD-L1 expression in tumour cells. However, the effect of IR on macrophage PD-L1 expression and the underlying mechanisms remain unclear. ATM kinase, as the key kinase for initiating DNA damage repair (DDR) process, is associated with innate immune STING axis activation. Here, we explored the molecular mechanism implicated in macrophage PD-L1 expression regulated by IR as well as the role of ATM kinase in this process. IR-regulated PD-L1 expression in macrophages and associated signalling pathways were explored by in vitro studies using murine and human macrophage cell lines. A colorectal xenograft murine model was employed to demonstrate the impact of targeting ATM and PD-L1 expression in TAMs following IR on growth of tumour in vivo. IR up-regulated PD-L1 expression in macrophages, which was further augmented by ATM kinase inhibition. ATM inhibition increased IR-induced DNA damage, which activated STING/interferon regulatory factor 3 (IRF3) signalling pathway and up-regulated type I interferon (IFN-I) expression in macrophages. IFN-I bound to the IFN α receptor 1 on macrophages, activated the downstream JAK1 and STAT1/3 signalling and eventually led to PD-L1 up-expression. ATM inhibition augmented IR-induced PD-L1 expression in macrophages and CD8+ T cell infiltration, and promoted anti-tumour efficacy in vivo. These results suggested that ATM inhibition promoted IR-induced PD-L1 expression through the activation of innate immunity in TAMs, which provided a novel approach to enhance the anti-tumour efficacy of RT.

Small-molecule targeted therapies induce dependence on DNA double-strand break repair in residual tumor cells.

Residual cancer cells that survive upfront drug treatments act as a reservoir from which eventual resistant disease emerges. Although there is great interest in therapeutically targeting residual cells, efforts to do so are hampered by our limited knowledge of the vulnerabilities existing in this cell state. Here, we report that diverse oncogene-targeted therapies, including inhibitors of epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), KRAS, and BRAF, induce DNA double strand breaks and consequently, ataxia-telangiectasia mutated (ATM)-dependent DNA repair in oncogene-matched residual tumor cells. This DNA damage response, observed in cell lines, mouse xenograft models, and human patients, is driven by a pathway involving the activation of caspases 3 and 7 and the downstream caspase-activated deoxyribonuclease (CAD). CAD is, in turn, activated through caspase-mediated degradation of its endogenous inhibitor, ICAD. In models of EGFR mutant non-small cell lung cancer (NSCLC), tumor cells that survive treatment with small-molecule EGFR-targeted therapies are thus synthetically dependent on ATM, and combined treatment with an ATM kinase inhibitor eradicates these cells in vivo. This led to more penetrant and durable responses in EGFR mutant NSCLC mouse xenograft models, including those derived from both established cell lines and patient tumors. Last, we found that rare patients with EGFR mutant NSCLC harboring co-occurring, loss-of-function mutations in ATM exhibit extended progression-free survival on first generation EGFR inhibitor therapy relative to patients with EGFR mutant NSCLC lacking deleterious ATM mutations. Together, these findings establish a rationale for the mechanism-based integration of ATM inhibitors alongside existing targeted therapies.

Molecular basis of human ATM kinase inhibition.

Human checkpoint kinase ataxia telangiectasia-mutated (ATM) plays a key role in initiation of the DNA damage response following DNA double-strand breaks. ATM inhibition is a promising approach in cancer therapy, but, so far, detailed insights into the binding modes of known ATM inhibitors have been hampered due to the lack of high-resolution ATM structures. Using cryo-EM, we have determined the structure of human ATM to an overall resolution sufficient to build a near-complete atomic model and identify two hitherto unknown zinc-binding motifs. We determined the structure of the kinase domain bound to ATPγS and to the ATM inhibitors KU-55933 and M4076 at 2.8 Å, 2.8 Å and 3.0 Å resolution, respectively. The mode of action and selectivity of the ATM inhibitors can be explained by structural comparison and provide a framework for structure-based drug design.

Abstract PO-088: Bintrafusp alfa enhances anti-tumor efficacy in combination with radiation therapy plus the ataxia telangiectasia mutated (ATM) inhibitor, M4076

Abstract Ionizing radiation therapy (RT) is a standard of care for many cancer indications. RT promotes the activation of transforming growth factor (TGF)-β, a pleotropic cytokine that mediates RT-induced fibrosis and suppresses anti-tumor immunity. Additionally, RT induces the expression of the immunosuppressive checkpoint PD-L1 on tumor cells. Bintrafusp alfa (M7824) is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor, functioning as a TGF-β “trap”, fused to a human IgG1 antibody blocking PD-L1. Bintrafusp alfa enhances anti-tumor immunity elicited by RT and reverses RT-induced fibrosis in preclinical models. Inhibition of ataxia telangiectasia mutated (ATM) increases the cancer cell's sensitivity to radiotherapy by blocking cellular pathways which facilitate the response to, and repair of, RT induced DNA double strand breaks. We hypothesize that a combination therapy consisting of bintrafusp alfa, RT and the ATM kinase inhibitor M4076 will show greater anti-tumor efficacy than the respective mono- or dual-combination therapies in immune competent preclinical solid tumor models. The anti-tumor efficacy of bintrafusp alfa, RT, and M4076 triple combination therapy was explored in the immune therapy resistant 4T1 murine syngeneic breast carcinoma model. This triple combination therapy significantly increased anti-tumor efficacy and prolonged overall survival relative to the bintrafusp alfa + RT or M4076 + RT dual combination therapies. The triple combination was further evaluated in the more immunogenic MC38 colon carcinoma model. MC38 tumor bearing mice treated with the triple combination therapy showed a significantly greater tumor growth inhibition and long term tumor free survival when compared with the bintrafusp alfa + RT or M4076 + RT treatment groups. Finally, the bintrafusp alfa + RT + M4076 combination therapy also showed significantly greater tumor growth inhibition and long term tumor free survival when compared to the combination of anti-PD-L1 + RT + M4076. The bintrafusp alfa triple combination resulted in 8 of 10 mice showing long term tumor free survival, whereas the anti-PD-L1 triple combination failed to elicit tumor free survival (0 of 10 mice tumor free). These studies demonstrate the superior anti-tumor efficacy of the Bintrafusp alfa + RT + M4076 triple combination therapy over that of the dual combination therapies of RT + bintrafusp alfa or RT + M4076. These data in the MC38 model also suggest that bintrafusp alfa may promote a superior anti-tumor immune response relative to that of anti-PD-L1 when in combination with RT and M4076. Together, these data support the rational to develop a bintrafusp alfa + RT + M4076 combination therapy for the treatment of solid tumors. Citation Format: Adam Lazorchak, Huakui Yu, Guozhong Qin, Jin Qi, Bo Marelli, Yan Lan. Bintrafusp alfa enhances anti-tumor efficacy in combination with radiation therapy plus the ataxia telangiectasia mutated (ATM) inhibitor, M4076 [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-088.

The DNA repair protein ATM as a target in autism spectrum disorder.

Impairment of the GABAergic system has been reported in epilepsy, autism, attention deficit hyperactivity disorder, and schizophrenia. We recently demonstrated that ataxia telangiectasia mutated (ATM) directly shapes the development of the GABAergic system. Here, we show for the first time to our knowledge how the abnormal expression of ATM affects the pathological condition of autism. We exploited 2 different animal models of autism, the methyl CpG binding protein 2–null (Mecp2y/–) mouse model of Rett syndrome and mice prenatally exposed to valproic acid, and found increased ATM levels. Accordingly, treatment with the specific ATM kinase inhibitor KU55933 (KU) normalized molecular, functional, and behavioral defects in these mouse models, such as (a) delayed GABAergic development, (b) hippocampal hyperexcitability, (c) low cognitive performances, and (d) social impairments. Mechanistically, we demonstrate that KU administration to WT hippocampal neurons leads to (a) higher early growth response 4 activity on Kcc2b promoter, (b) increased expression of Mecp2, and (c) potentiated GABA transmission. These results provide evidence and molecular substrates for the pharmacological development of ATM inhibition in autism spectrum disorders.

MAD2B-mediated cell cycle reentry of podocytes is involved in the pathogenesis of FSGS.

Rationale: Focal segmental glomerulosclerosis (FSGS) is characterized by the dysfunction of “post-mitotic” podocytes. The reentry of podocytes in the cell cycle will ultimately result in cell death. Mitotic arrest deficient 2-like protein 2 (MAD2B), an inhibitor of anaphase-promoting complex (APC)/cyclosome, precisely controls the metaphase to anaphase transition and ordered cell cycle progression. However, the role of MAD2B in FSGS podocyte injury remains unknown.Methods: To explore MAD2B function in podocyte cell cycle reentry, we used conditional mutant mice lacking MAD2B selectively in podocytes in ADR-induced FSGS murine model. Additionally, KU-55933, a specific inhibitor of ataxia-telangiectasia mutated (ATM) was utilized in vivo and in vitro to explore the role of ATM in regulating MAD2B.Results: The expression of MAD2B in podocytes was dramatically increased in patients with FSGS and ADR-treated mice along with podocyte cell cycle reentry. Podocyte-specific knockout of MAD2B effectively attenuated proteinuria, podocyte injury, and prevented the aberrant cell cycle reentry. By bioinformatics analysis we revealed that ATM kinase is a key upstream regulator of MAD2B. Furthermore, inhibition of ATM kinase abolished MAD2B-driven cell cycle reentry and alleviated podocyte impairment in FSGS murine model. In vitro studies by site-directed mutagenesis and immunoprecipitation we revealed ATM phosphorylated MAD2B and consequently hampered the ubiquitination of MAD2B in a phosphorylation-dependent manner.Conclusions: ATM kinase-MAD2B axis importantly contributes to the cell cycle reentry of podocytes, which is a novel pathogenic mechanism of FSGS, and may shed light on the development of its therapeutic approaches.

Abstract 4130: Improvement of drug efficacy of polo-like kinase 4 inhibitor in the treatment of uterine leiomyosarcoma

Abstract Introduction: Uterine leiomyosarcoma (Ut-LMS) is aggressive. Recurrence usually develops in 50 - 70% of patients and effective chemotherapy regime are limited. Polo-like kinase 4 (PLK4) is vital modulator of centriole duplication and cell mitotic progression. Inhibition of PLK4 disrupts mitosis, which leads to polyploidy, further genomic instability, mitotic catastrophe, and cell death. Recently, CFI-400945, a PLK4 inhibitor, has been found effective in preclinical studies in the treatment of several malignancies but its effectiveness on Ut-LMS has not been investigated. In phase I studies, CFI-400945 was well tolerated although toxicity, primarily reversible neutropenia, was observed at higher doses. Ataxia-telangiectasia mutated (ATM) is a key kinase in the DNA damage response pathway. AZD0156, an ATM kinase inhibitor, has recently been proposed to be used with other DNA damaging agents in cancer treatments. We explored the effectiveness of a combined approach with CFI-400945 and AZD0156 in Ut-LMS. Methods: Ut-LMS cell lines SK-UT-1, SKN and SK-LMS-1 cultured in-vitro were treated with CFI-400945 and AZD0156. PLK4 gene expression in these cell lines was also determined by RT-qPCR assays. Cell proliferation was examined using the sulforhodamine B (SRB) assay while apoptosis was examined by Annexin V-PI and caspase 3/7 assays. Immunofluorescence confocal centrosome quantification and flow cytometry PI cell cycle analysis were used to detect centrosome overduplication and generation of aneuploidy cells. The effect of gene expression in normal uterine smooth muscle cells and Ut-LMS cell lines treated with CFI-400945 was performed by RNA-Seq. DNA damage was detected using neutral comet assay. Results: Ut-LMS cell lines expressed high levels of PLK4 and were highly sensitive (IC50 in nM range) to CFI-400945. Centrosome overduplication, aneuploidy cells, and cell growth inhibition were observed in a dose-dependent manner, and prominent apoptosis was observed at 48 hours after drug treatment. Gene set enrichment analysis on RNA-Seq data indicated that cells were under genotoxic stress after CFI-400945 treatment while comet assay verified DNA damage. Cell arrest and apoptosis with CFI-400945 was further enhanced by the addition of AZD0156. Combined treatment of CFI-400945 and AZD0156 produced synergistic antineoplastic effects in all three Ut-LMS cell lines, with combination indices <1. DNA damage was also enhanced in the combined treatment group versus the single treatment group. Conclusion: The PLK4 inhibitor CFI-400945 was a potent treatment for Ut-LMS in vitro. This effect was further augmented when combined with the DNA damage response pathway inhibitor AZD0156. The synergistic effect of these agents is promising, and should be investigated further in the treatment of Ut-LMS Citation Format: Hok Yeung Horace Lee, Kin Long Chow, Ho Shing Wong, Tsz Yan Chong, Ka Yu Tse, Mark R. Bray, Tak Wah Mak, Philip PC Ip. Improvement of drug efficacy of polo-like kinase 4 inhibitor in the treatment of uterine leiomyosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4130.

Adefovir dipivoxil induces DNA replication stress and augments ATR inhibitor-related cytotoxicity.

Replication stress is a common feature of cancer cells. Ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) signalling, a DNA damage repair (DDR) pathway, is activated by regions of single-stranded DNA (ssDNA) that can arise during replication stress. ATR delays cell cycle progression and prevents DNA replication fork collapse, which prohibits cell death and promotes proliferation. Several ATR inhibitors have been developed in order to restrain this protective mechanism in tumours. It is known, however, that despite other effective anticancer chemotherapy treatments targeting DDR pathways, resistance occurs. This begets the need to identify combination treatments to overcome resistance and prevent tumour cell growth. We conducted a drug screen to identify potential synergistic combination treatments by screening an ATR inhibitor (VE822) together with compounds from a bioactive small molecule library. The screen identified adefovir dipivoxil, a reverse transcriptase inhibitor and nucleoside analogue, as a compound that has increased cytotoxicity in the presence of ATR, but not ATM or DNA-dependant protein kinase (DNA-PK) inhibition. Here we demonstrate that adefovir dipivoxil induces DNA replication stress, activates ATR signalling and stalls cells in S phase. This simultaneous induction of replication stress and inhibition of ATR signalling lead to a marked increase in pan-nuclear γH2AX-positive cells, ssDNA accumulation and cell death, indicative of replication catastrophe.

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.

Regulation of the Notch-ATM-abl axis by geranylgeranyl diphosphate synthase inhibition

Notch proteins drive oncogenesis of many cancers, most prominently T-cell acute lymphoblastic leukemia (T-ALL). Because geranylgeranylated Rab proteins regulate Notch processing, we hypothesized that inhibition of geranylgeranyl diphosphate synthase (GGDPS) would impair Notch processing and reduce viability of T-ALL cells that express Notch. Here, we show that GGDPS inhibition reduces Notch1 expression and impairs the proliferation of T-ALL cells. GGDPS inhibition also reduces Rab7 membrane association and depletes Notch1 mRNA. GGDPS inhibition increases phosphorylation of histone H2A.X, and inhibitors of ataxia telangiectasia-mutated kinase (ATM) mitigate GGDPS inhibitor-induced apoptosis. GGDPS inhibition also influences c-abl activity downstream of caspases, and inhibitors of these enzymes prevent GGDPS inhibitor-induced apoptosis. Surprisingly, induction of apoptosis by GGDPS inhibition is reduced by co-treatment with γ-secretase inhibitors. While inhibitors of γ-secretase deplete one specific form of the Notch1 intracellular domain (NICD), they also increase Notch1 mRNA expression and increase alternate forms of Notch1 protein expression in cells treated with a GGDPS inhibitor. Furthermore, inhibitors of γ-secretase and ATM increase Notch1 mRNA stability independent of GGDPS inhibition. These results provide a model by which T-ALL cells use Notch1 to avoid DNA-damage-induced apoptosis, and can be overcome by inhibition of GGDPS through effects on Notch1 expression and its subsequent response.

ATM in DNA repair in cancer.

Alterations in DNA damage response (DDR) pathways are hallmarks of cancer. Incorrect repair of DNA lesions often leads to genomic instability. Ataxia telangiectasia mutated (ATM), a core component of the DNA repair system, is activated to enhance the homologous recombination (HR) repair pathway upon DNA double-strand breaks. Although ATM signaling has been widely studied in different types of cancer, its research is still lacking compared with other DDR-involved molecules such as PARP and ATR. There is still a vast research opportunity for the development of ATM inhibitors as anticancer agents. Here, we focus on the recent findings of ATM signaling in DNA repair of cancer. Previous studies have identified several partners of ATM, some of which promote ATM signaling, while others have the opposite effect. ATM inhibitors, including KU-55933, KU-60019, KU-59403, CP-466722, AZ31, AZ32, AZD0156, and AZD1390, have been evaluated for their antitumor effects. It has been revealed that ATM inhibition increases a cancer cell's sensitivity to radiotherapy. Moreover, the combination with PARP or ATR inhibitors has synergistic lethality in some cancers. Of note, among these ATM inhibitors, AZD0156 and AZD1390 achieve potent and highly selective ATM kinase inhibition and have an excellent ability to penetrate the blood-brain barrier. Currently, AZD0156 and AZD1390 are under investigation in phase I clinical trials. Taken together, targeting ATM may be a promising strategy for cancer treatment. Hence, further development of ATM inhibitors is urgently needed in cancer research.

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 >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 < 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 & 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) & physicochemical profile as well as its preclinical characterization using biochemical, cellular & 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.

Optimization of Potent and Selective Ataxia Telangiectasia-Mutated Inhibitors Suitable for a Proof-of-Concept Study in Huntington's Disease Models.

Genetic and pharmacological evidence indicates that the reduction of ataxia telangiectasia-mutated (ATM) kinase activity can ameliorate mutant huntingtin (mHTT) toxicity in cellular and animal models of Huntington's disease (HD), suggesting that selective inhibition of ATM could provide a novel clinical intervention to treat HD. Here, we describe the development and characterization of ATM inhibitor molecules to enable in vivo proof-of-concept studies in HD animal models. Starting from previously reported ATM inhibitors, we aimed with few modifications to increase brain exposure by decreasing P-glycoprotein liability while maintaining potency and selectivity. Here, we report brain-penetrant ATM inhibitors that have robust pharmacodynamic (PD) effects consistent with ATM kinase inhibition in the mouse brain and an understandable pharmacokinetic/PD (PK/PD) relationship. Compound 17 engages ATM kinase and shows robust dose-dependent inhibition of X-ray irradiation-induced KAP1 phosphorylation in the mouse brain. Furthermore, compound 17 protects against mHTT (Q73)-induced cytotoxicity in a cortical-striatal cell model of HD.

Thymidylate kinase is critical for DNA repair via ATM-dependent Tip60 complex formation.

Cellular supply of deoxythymidine triphosphate (dTTP) is crucial for DNA replication and repair. Thymidylate kinase (TMPK) catalyzes the conversion of thymidine monophosphate to thymidine diphosphate, which is an essential step for dTTP synthesis. Despite their major cellular localization in cytosol, TMPK and ribonucleotide reductase (RNR) are detected at DNA damage sites for local dNDP formation. Because deoxyuridine diphosphate is synthesized by RNR, the simultaneous recruitment of TMPK and RNR to DNA damage sites is critical for preventing deoxyuridine triphosphate-mediated toxic repair. This study investigates the mechanism responsible for the recruitment of TMPK to DNA damage sites. Our data demonstrate the requirement of ataxia telangiectasia mutated (ATM) kinase activity for TMPK recruitment to DNA lesion sites. Moreover, we find that TMPK is able to form the complex with histone acetyltransferase Tip60 and RNR. Inhibition of ATM kinase reduces the complex formation and TMPK phosphorylation. Our analysis further shows the presence of TMPK phosphorylation at serine 88, which is an ATM kinase consensus site. A phosphorylation-defective mutation at this site suppresses TMPK recruitment to DNA damage sites and the complex formation with Tip60. Finally, we provide evidence that this site is critical for the function of TMPK in DNA repair but not for catalytic activity. Together, these findings suggest that Tip60-ATM signaling has a functional contribution to the recruitment of TMPK to DNA damage sites, thereby increasing local dTTP synthesis for DNA repair.-Hu, C.-M., Tsao, N., Wang, Y.-T., Chen, Y.-J., Chang, Z.-F. Thymidylate kinase is critical for DNA repair via ATM-dependent Tip60 complex formation.