Novel and Highly Potent ATR Inhibitor M4344 Kills Cancer Cells With Replication Stress, and Enhances the Chemotherapeutic Activity of Widely Used DNA Damaging Agents

Abstract

Although several ATR inhibitors are in development, there are unresolved questions regarding their differential potency, molecular signatures of patients with cancer for predicting activity, and most effective therapeutic combinations. Here, we elucidate how to improve ATR-based chemotherapy with the newly developed ATR inhibitor, M4344 using in vitro and in vivo models. The potency of M4344 was compared with the clinically developed ATR inhibitors BAY1895344, berzosertib, and ceralasertib. The anticancer activity of M4344 was investigated as monotherapy and combination with clinical DNA damaging agents in multiple cancer cell lines, patient-derived tumor organoids, and mouse xenograft models. We also elucidated the anticancer mechanisms and potential biomarkers for M4344. We demonstrate that M4344 is highly potent among the clinically developed ATR inhibitors. Replication stress (RepStress) and neuroendocrine (NE) gene expression signatures are significantly associated with a response to M4344 treatment. M4344 kills cancer cells by inducing cellular catastrophe and DNA damage. M4344 is highly synergistic with a broad range of DNA-targeting anticancer agents. It significantly synergizes with topotecan and irinotecan in patient-derived tumor organoids and xenograft models. Taken together, M4344 is a promising and highly potent ATR inhibitor. It enhances the activity of clinical DNA damaging agents commonly used in cancer treatment including topoisomerase inhibitors, gemcitabine, cisplatin, and talazoparib. RepStress and NE gene expression signatures can be exploited as predictive markers for M4344.

Trial registration: ClinicalTrials.gov NCT02278250 NCT04149145.

©2021 The Authors; Published by the American Association for Cancer Research.

Figures

Figure 1.

Comparison between M4344 and other clinical ATR inhibitors. A, Chemical structures of ATR inhibitors. B, Comparative analysis of cell viability between clinical ATR inhibitors. DU145 cells were treated as indicated for 72 hours. Cell viability was accessed by CellTiter-Glo assay. C–F, Synergistic effects between ATR inhibitors and camptothecin (CPT). Cells were co-incubated with CPT (100 nmol/L) and M4344 (C), BAY1895344 (D), berzosertib (E), and ceralasertib (F) as indicated concentrations for 72 hours. Cell viability was accessed by CellTiter-Glo assay. G, Comparison of combination index values obtained for combination treatments of CPT (6.3 and 12.5 nmol/L) with the indicated ATR inhibitors. H, Inactivation of ATR-mediated CHK1 phosphorylation by ATR inhibitors. DU145 cells were pretreated with the indicated concentrations of ATR inhibitors for 1 hour and then incubated with CPT (100 nmol/L) and the ATR inhibitors for three additional hours. Protein levels were examined by Western blotting (see Supplementary Fig. S1 for effects of M4344 in H82 and U2OS cells).

Figure 2.

M4344 kills cancer cells under replication stress (RepStress) and with NE genomic signatures by replication-mediated DNA damage. A, Cytotoxicity of M4344 as monotherapy in 16 cancer cell lines from different histology. Following incubation with the indicated concentrations of M4344 for 72 hours, cell viability was accessed by CellTiter-Glo assay. B, Correlations between protein levels related to DNA damage response pathways and M4344 sensitivity. Protein levels were mined from the CCLE cell line database. Correlation heatmap between protein levels and IC50 values of M4344 in A were generated by CellMinerCDB (http://discover.nci.nih.gov/cellminercdb). C, Correlation of RepStress signature scores in the GDSC databases with M4344 activity obtained in A. Plots were generated with CellMinerCDB. D, Correlation of NE signature scores in the GDSC databases with M4344 activity in A. Plots were generated by CellMinerCDB. E and F, Gene dependency of M4344 response. Dependency scores of ARID1A and BRG1were mined from the Project Achilles from CCLE. Plots were generated with CellMinerCDB. P values indicate Pearson correlation coefficients. G, Biphasic effect of M4344 on DNA synthesis. H82 cells treated as indicated were pulse-labeled with EdU (10 μmol/L) 30 minutes prior to harvest. Edu incorporation per cell was analyzed by FACS. Numbers indicate percentage of cells in the areas. H, Induction of DNA damage detected by γH2AX Western blotting. Cells were incubated with the indicated concentrations of M4344 for 24 hours.

Figure 3.

M4344 synergizes clinical TOP1 inhibitors. A–D, H82 cells were co-incubated with M4344 (25 nmol/L) and the indicated concentrations of exatecan (A), SN-38 (B), topotecan (C), and indotecan (LMP400; D) for 72 hours. E and F, Conversely, H82 cells were co-incubated with the indicated concentrations of M4344 and exatecan (E, 0.25 nmol/L), SN-38 (F, 1 nmol/L), topotecan (G, 10 nmol/L), and indotecan (LMP400; H, 10 nmol/L) for 72 hours. I–L, Synergy of M4344 with topotecan in four additional cell lines: SK-OV-3 (I), DMS114 (J), U2OS (K), and A549 (L). Cells were cotreated with M4344 (25 nmol/L) and the indicated concentrations of topotecan for 72 hours. Cell viability was accessed by CellTiter-Glo assay. Synergy plots are presented in Supplementary Fig. S3.

Figure 4.

M4344 shows synergy with etoposide, gemcitabine, cisplatin, and talazoparib. A–D, H82 cells were co-incubated with a nontoxic concentration of M4344 (25 nmol/L) and the indicated concentrations of etoposide (A), gemcitabine (B), cisplatin (C), and talazoparib (D) for 72 hours. E–H, H82 cells were coincubated with the indicated concentrations of M4344 and etoposide (E, 1 μmol/L), gemcitabine (F, 1 nmol/L), cisplatin (G, 0.5 μmol/L), and talazoparib (H, 5 nmol/L) for 72 hours. Cell viability was accessed by CellTiter-Glo assay. Synergy plots are presented in Supplementary Fig. S4.

Figure 5.

M4344 synergizes with topotecan in patient derived prostate tumor organoids. A–D, LuCaP 145.2 (A), LuCaP 173.1 (B), MB155 (C), and MB44 (D) organoids were incubated with a noncytotoxic concentration of M4344 (25 nmol/L) and the indicated concentrations of topotecan for 72 hours. Cell viability was quantified using CellTiter-Glo 3D. Data shown are the mean ± SEM (N = 4 for each group). P values indicate statistical difference between groups (topotecan vs. combination; *, P values < 0.0001). E, Bar graph representing normalized AUC values calculated by measuring changes in cell viability of organoids treated as indicated. Data shown are the mean ± SEM (N = 4 for each group; *, P values < 0.0001).

Figure 6.

Efficacy of the combination of M4344 with irinotecan in human small-cell lung cancer tumor xenografts and schematic flow chart summarizing this study. A–D, The in vivo efficacy of M4344 was evaluated in mice transplanted with H82 (A) and H446 (B) cells. Mice received subcutaneous injections in the right flank with cancer cells (in PBS/Matrigel). Mice received M4344 at an oral dose of 10 mg/kg, irinotecan at an intraperitoneal dose of 50 mg/kg or the combination thereof. Both compounds were applied once weekly, the treatment duration was 2 weeks for the H82 and 5 weeks for the H446. For the combination M4344 was applied 24 hours after Irinotecan. Tumor volumes are shown as mean ± SEM (N = 10 mice for each group). C and D, Benefits of progression-free survival by combination treatment, compared with control treatment and either monotherapy. E, M4344 inhibits activation of the ATR signaling pathway, thereby consequently blocking signal transduction from replicative DNA damage. After M43444 treatment, cells undergo replication catastrophe with DNA damage and mitotic defects, leading to cell death. Gene expression signatures including replication stress (RepStress), NE signature, and SWI/SNF inactivation are candidate predictive markers for M4344 in cancer therapy.