ATM kinase inhibitor AZD0156 in combination with irinotecan and 5-fluorouracil in preclinical models of colorectal cancer.

BACKGROUND: AZD0156 is an oral inhibitor of ATM, a serine threonine kinase that plays a key role in DNA damage response (DDR) associated with double-strand breaks. Topoisomerase-I inhibitor irinotecan is used clinically to treat colorectal cancer (CRC), often in combination with 5-fluorouracil (5FU). AZD0156 in combination with irinotecan and 5FU was evaluated in preclinical models of CRC to determine whether low doses of AZD0156 enhance the cytotoxicity of irinotecan in chemotherapy regimens used in the clinic. METHODS: Anti-proliferative effects of single-agent AZD0156, the active metabolite of irinotecan (SN38), and combination therapy were evaluated in 12 CRC cell lines. Additional assessment with clonogenic assay, cell cycle analysis, and immunoblotting were performed in 4 selected cell lines. Four colorectal cancer patient derived xenograft (PDX) models were treated with AZD0156, irinotecan, or 5FU alone and in combination for assessment of tumor growth inhibition (TGI). Immunofluorescence was performed on tumor tissues. The DDR mutation profile was compared across in vitro and in vivo models. RESULTS: Enhanced effects on cellular proliferation and regrowth were observed with the combination of AZD0156 and SN38 in select models. In cell cycle analysis of these models, increased G2/M arrest was observed with combination treatment over either single agent. Immunoblotting results suggest an increase in DDR associated with irinotecan therapy, with a reduced effect noted when combined with AZD0156, which is more pronounced in some models. Increased TGI was observed with the combination of AZD0156 and irinotecan as compared to single-agent therapy in some PDX models. The DDR mutation profile was variable across models. CONCLUSIONS: AZD0156 and irinotecan provide a rational and active combination in preclinical colorectal cancer models. Variability across in vivo and in vitro results may be related to the variable DDR mutation profiles of the models evaluated. Further understanding of the implications of individual DDR mutation profiles may help better identify patients more likely to benefit from treatment with the combination of AZD0156 and irinotecan in the clinical setting.

Targeting the DNA Damage Response to Increase Anthracycline-Based Chemotherapy Cytotoxicity in T-Cell Lymphoma.

Mature T-cell lymphomas (MTCLs) represent a heterogeneous group of aggressive non-Hodgkin lymphomas comprising different entities. Anthracycline-based regimens are considered the standard of care in the front-line treatment. However, responses to these approaches have been neither adequate nor durable, and new treatment strategies are urgently needed to improve survival. Genomic instability is a common feature of cancer cells and can be caused by aberrations in the DNA damage response (DDR) and DNA repair mechanisms. Consistently, molecules involved in DDR are being targeted to successfully sensitize cancer cells to chemotherapy. Recent studies showed that some hematological malignancies display constitutive DNA damage and intrinsic DDR activation, but these features have not been investigated yet in MTCLs. In this study, we employed a panel of malignant T cell lines, and we report for the first time the characterization of intrinsic DNA damage and basal DDR activation in preclinical models in T-cell lymphoma. Moreover, we report the efficacy of targeting the apical kinase ATM using the inhibitor AZD0156, in combination with standard chemotherapy to promote apoptotic cell death. These findings suggest that DDR is an attractive pathway to be pharmacologically targeted when developing novel therapies and improving MTCL patients' outcomes.

Epigenetic silencing schlafen-11 sensitizes esophageal cancer to ATM inhibitor.

BACKGROUND: Targeting DNA damage response (DDR) pathway is a cutting-edge strategy. It has been reported that Schlafen-11 (SLFN11) contributes to increase chemosensitivity by participating in DDR. However, the detailed mechanism is unclear. AIM: To investigate the role of SLFN11 in DDR and the application of synthetic lethal in esophageal cancer with SLFN11 defects. METHODS: To reach the purpose, eight esophageal squamous carcinoma cell lines, 142 esophageal dysplasia (ED) and 1007 primary esophageal squamous cell carcinoma (ESCC) samples and various techniques were utilized, including methylation-specific polymerase chain reaction, CRISPR/Cas9 technique, Western blot, colony formation assay, and xenograft mouse model. RESULTS: Methylation of SLFN11 was exhibited in 9.15% of (13/142) ED and 25.62% of primary (258/1007) ESCC cases, and its expression was regulated by promoter region methylation. SLFN11 methylation was significantly associated with tumor differentiation and tumor size (both P < 0.05). However, no significant associations were observed between promoter region methylation and age, gender, smoking, alcohol consumption, TNM stage, or lymph node metastasis. Utilizing DNA damaged model induced by low dose cisplatin, SLFN11 was found to activate non-homologous end-joining and ATR/CHK1 signaling pathways, while inhibiting the ATM/CHK2 signaling pathway. Epigenetic silencing of SLFN11 was found to sensitize the ESCC cells to ATM inhibitor (AZD0156), both in vitro and in vivo. CONCLUSION: SLFN11 is frequently methylated in human ESCC. Methylation of SLFN11 is sensitive marker of ATM inhibitor in ESCC.

Computational Analysis of Non-synonymous SNPs in ATM Kinase: Structural Insights, Functional Implications, and Inhibitor Discovery.

Ataxia telangiectasia-mutated (ATM) protein kinase, a key player in cellular integrity regulation, is known for its role in DNA damage response. This study investigates the broader impact of ATM on cellular processes and potential clinical manifestations arising from mutations, aiming to expand our understanding of ATM's diverse functions beyond conventional roles. The research employs a comprehensive set of computational techniques for a thorough analysis of ATM mutations. The mutation data are curated from dbSNP and HuVarBase databases. A meticulous assessment is conducted, considering factors such as deleterious effects, protein stability, oncogenic potential, and biophysical characteristics of the identified mutations. Conservation analysis, utilizing diverse computational tools, provides insights into the evolutionary significance of these mutations. Molecular docking and dynamic simulation analyses are carried out for selected mutations, investigating their interactions with Y2080D, AZD0156, and quercetin inhibitors to gauge potential therapeutic implications. Among the 419 mutations scrutinized, five (V1913C, Y2080D, L2656P, C2770G, and C2930G) are identified as both disease causing and protein destabilizing. The study reveals the oncogenic potential of these mutations, supported by findings from the COSMIC database. Notably, Y2080D is associated with haematopoietic and lymphoid cancers, while C2770G shows a correlation with squamous cell carcinomas. Molecular docking and dynamic simulation analyses highlight strong binding affinities of quercetin for Y2080D and AZD0156 for C2770G, suggesting potential therapeutic options. In summary, this computational analysis provides a comprehensive understanding of ATM mutations, revealing their potential implications in cellular integrity and cancer development. The study underscores the significance of Y2080D and C2770G mutations, offering valuable insights for future precision medicine targeting-specific ATM. Despite informative computational analyses, a significant research gap exists, necessitating essential in vitro and in vivo studies to validate the predicted effects of ATM mutations on protein structure and function.

The effect of the ATM inhibitor AZD0156 on the radiosensitivity of human breast cancer and lung fibroblast cells.

Aims: To evaluate the effect of the combination of irradiation and AZD0156 on apoptosis, cell cycle progression, and clonogenic survival in human breast cancer and fibroblast cells. Methods and Material: Estrogen receptor-positive breast cancer cell line MCF-7 and healthy lung fibroblast cell line WI-38 were obtained. Following employing proliferation analysis, cytotoxicity analysis was done to calculate the IC50 values of AZD0156 in MCF-7 and WI-38 cell lines. Following the application of AZD0156 and irradiation, flow cytometry analysis was performed for evaluating cell cycle distribution and the extent of apoptosis. Plating efficiency and surviving fraction were calculated for the clonogenic assay. Statistical Analysis Used: SPSS Statistics for Windows, Version 17.0. (SPSS Inc. Chicago) and GraphPad Prism Version 6.0 for Windows (GraphPad Software, San Diego, California USA) softwares were used to analyze data. Results: AZD0156 and irradiation dose of 2-10 Gy had no effect on apoptosis on MCF-7 cells. The combination treatment of AZD0156 and 2 Gy, 4 Gy, 6 Gy, 8 Gy, and 10 Gy irradiation induced G0/G1 phase arrest by 1.79, 1.79, 1.50, 1.25, and 1.52-fold compared to the control group, respectively on MCF-7 cell lines. Combination treatment of AZD0156 and each different irradiation dose affected clonogenic survival owing to increased radiosensitivity (p: 0.02). AZD0156 and irradiation dose of 2 Gy, 4 Gy, 6 Gy, 8 Gy, and 10 Gy decreased the cell viability rate of WI-38 cells by 1.05, 1.18, 1.22, 1.04, and 1.05-fold compared to the control group, respectively. No efficacy was detected on cell cycle analysis, and clonogenic survival was not significantly decreased in WI-38 cells. Conclusion: The combination use of irradiation and AZD0156 has improved efficacy of tumor cell-specific cell cycle arrest and decreasing clonogenic survival.

Simultaneous Delivery of Dual Inhibitors of DNA Damage Repair Sensitizes Pancreatic Cancer Response to Irreversible Electroporation.

Pancreatic ductal adenocarcinoma (PDAC) is an abysmal disease refractory to most standard therapies. Irreversible electroporation (IRE) is a local ablative technique for the clinical treatment of solid tumors, including locally advanced and unresectable PDAC, by intratumorally delivering high-intensity electric pulses to permanently disrupt cell membranes and induce cell death. But the distribution of electric field is uneven within the tumor, and in some regions, tumor cells only experience temporary perturbation to their cell membrane, a phenomenon denoted as reversible electroporation (RE). These tumor cells may survive and therefore are the main culprit of tumor relapse after IRE. We herein showed that RE, although not killing tumor cells, induced DNA double-strand breaks and activated DNA damage repair (DDR) responses. Using reactive oxygen species-sensitive polymeric micelles coloaded with Olaparib, an inhibitor of poly(ADP-ribose) polymerase (PARP), and AZD0156, an inhibitor of ataxia telangiectasia mutated (ATM), the resultant nanoformulation (M-TK-OA) disrupted both homologous recombination and nonhomologous end joining signaling of the DDR response and impaired colony formation in pancreatic cancer cells after RE. The combination of IRE and M-TK-OA significantly prolonged animal survival in both subcutaneous and orthotopic murine PDAC models and elicited CD8+ T cell-mediated antitumor immunity with a sustained antitumor memory. The efficacy of combined IRE and M-TK-OA treatments was partially attributed to the activation of cyclic GMP-AMP synthase-stimulator of interferon genes innate immune responses. Our study suggests that dual inhibition of PARP and ATM with nanomedicine is a promising strategy to enhance the pancreatic cancer response to IRE.

Methylation of TMEM176A, a key ERK signaling regulator, is a novel synthetic lethality marker of ATM inhibitors in human lung cancer.

Aim: The role of TMEM176A methylation in lung cancer and its therapeutic application remains unclear. Materials and methods: Nine lung cancer cell lines and 123 cases of cancer tissue samples were employed. Results:TMEM176A was methylated in 53.66% of primary lung cancer. Restoration of TMEM176A expression induced cell apoptosis and G2/M phase arrest, and inhibited colony formation, cell proliferation, migration and invasion. TMEM176A suppressed H1299 cell xenograft growth in mice. Methylation of TMEM176A activated ERK signaling and sensitized H1299 and H23 cells to AZD0156, an ATM inhibitor. Conclusion: The expression of TMEM176A is regulated by promoter region methylation. Methylation of TMEM176A is a potential lung cancer diagnostic marker and a novel synthetic lethal therapeutic marker for AZD0156.

Lay abstract The TMEM176A gene is often methylated in human lung cancer by addition of a methyl group to the gene promotor region. This regulates the expression of TMEM176A. We found that TMEM176A suppressed lung cancer growth both invitro and invivo by inhibiting ERK signaling. Methylation of TMEM176A sensitized H1299 and H23 cells to AZD0156, an ATM kinase inhibitor used to induce tumor cell death. Re-expression of TMEM176A reduced the sensitivity of these cells to AZD0156. Methylation of TMEM176A is a novel synthetic lethality therapeutic marker of AZD0156 in human lung cancer.