The Novel ATR Inhibitor M1774 Induces Replication Protein Overexpression and Broad Synergy with DNA-targeted Anticancer Drugs

Abstract

Ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase inhibitors are in clinical trials. Here we explored the molecular pharmacology and therapeutic combination strategies of the oral ATR inhibitor M1774 (Tuvusertib) with DNA-damaging agents (DDA). As single agent, M1774 suppressed cancer cell viability at nanomolar concentrations, showing greater activity than ceralasertib and berzosertib, but less potency than gartisertib and elimusertib in the small cell lung cancer H146, H82, and DMS114 cell lines. M1774 also efficiently blocked the activation of the ATR-CHK1 checkpoint pathway caused by replication stress induced by TOP1 inhibitors. Combination with non-toxic dose of M1774 enhanced TOP1 inhibitor-induced cancer cell death by enabling unscheduled replication upon replicative damage, thereby increasing genome instability. Tandem mass tag-based quantitative proteomics uncovered that M1774, in the presence of DDA, forces the expression of proteins activating replication (CDC45) and G2-M progression (PLK1 and CCNB1). In particular, the fork protection complex proteins (TIMELESS and TIPIN) were enriched. Low dose of M1774 was found highly synergistic with a broad spectrum of clinical DDAs including TOP1 inhibitors (SN-38/irinotecan, topotecan, exatecan, and exatecan), the TOP2 inhibitor etoposide, cisplatin, the RNA polymerase II inhibitor lurbinectedin, and the PARP inhibitor talazoparib in various models including cancer cell lines, patient-derived organoids, and mouse xenograft models. Furthermore, we demonstrate that M1774 reverses chemoresistance to anticancer DDAs in cancer cells lacking SLFN11 expression, suggesting that SLFN11 can be utilized for patient selection in upcoming clinical trials.

Figure 1.. M1774 is a potent inhibitor of ATR and enhances the activity of TOP1 inhibitors.

A. Comparative analysis of cell viability with ATR inhibitors. Small cell lung cancer cell lines (H146, H82, DMS114) were treated for 72 hours at the indicated drug concentrations. Cell viability was assessed by CellTiter-Glo assay. B. Comparison of IC50 values (μmol/L) of ATR inhibitors and RepStress scores in each cell line. IC50 values were calculated based on panel A. RepStress scores were obtained from CellMinerCDB (https://discover.nci.nih.gov/). C. Inhibition of the ATR/CHK1 pathways by M1774 in comparison with other clinical ATR inhibitors. H146 cells were pretreated with the indicated drug concentrations of ATR inhibitors for 1 hour and then incubated with CPT (100 nmol/L) and the ATR inhibitors for three additional hours. Proteins were examined by Western blotting. D. DNA damage and cell death by M1774. H146 cells were treated with M1774 (500 nmol/L) for the indicated times. Protein levels were assessed by Western blotting. E. Combinatory effects of M1774 and SN-38 on DNA synthesis and cell cycle. H146 cells were incubated with SN-38 (100 nmol/L), M1774 (40 nmol/L), and the combination for 24 hours. Cells were pulse-labeled with EdU (10 μmol/L) 30 min before cell harvest. EdU/DAPI uptake per cell was analyzed by flow cytometry. F. Bar graph showing the percentage of cells in the indicated cycle phases from data generated in E. G, Effects of the combination of SN-38 and M1774. H146 cells were incubated with M1774 (40 nmol/L), SN-38 (100 nmol/L), and the combination for 24 hours. Proteins were assessed by Western blotting.

Figure 2.. Combination with M1774 leads to deleterious unscheduled DNA replication activity.

A. Workflow of TMT-mediated quantitative proteomics in H146 cells treated with SN-38, M1774 and SN-38 + M1774. B. Volcano plot displaying the −log10 (p-value) versus abundance ratio (SN-38/Control) for all quantified peptides. C. Volcano plot displaying the −log10 (p-value) versus abundance ratio (SN38+M1774/Control) for all quantified peptides. D. Canonical pathway analysis identified using the IPA (Ingenuity Pathway Analysis) software. The orange bar reflects a positive z-score indicative for activation of this pathway. E. Network analysis of the differentially expressed proteins between combination and control using IPA. Proteins identified in the dataset are highlighted in red (Up) or in green (Down) when they exhibit a higher or a lower content in the combination compared to the control. Orange (Predicted activation) and blue (Predicted inhibition). F. Validation of the hit proteins detected in the network analysis. H146 cells were treated with SN-38 and M1774, and nuclear fractions were isolated. Protein levels were assessed by Western blotting. G. Effects of Aphidicolin on the unscheduled DNA synthesis caused by M1774. Cells were pulse-labeled with EdU (10 μmol/L) 30 min before cell harvest. EdU/DAPI uptake per cell was analyzed by flow cytometry. H. Detection of deleterious unscheduled DNA replication activity by Confocal immunofluorescence staining. U2OS cells were treated with SN-38 (100 nmol/L) and M1774 (40 nmol/L) for 24 hours. Cells were stained with EdU (green), γH2AX (red), and chromatin with DAPI (blue) (Magnification, 63×).

Figure 3.. Low doses of M1774 are highly synergistic with clinical DNA-damaging agents.

A-D. Top: H146 cells were co-treated with M1774 (40 nmol/L) and the indicated concentrations of SN-38 (A), etoposide (B), cisplatin (C), and talazoparib (D) for 72 hours. Cell viability was measured by CellTiter-Glo assays. Error bars represent standard deviations in the triplicate. Bottom: Combination index (CI) values were calculated with CompuSyn. Additive combination: 0.5<CI<1, and synergistic combinations: 0<CI<0.5. Each CI value was annotated in the plots. E-H. TOP: H82 cells were co-treated with M1774 (20 nmol/L) and the indicated concentrations of SN-38 (E), etoposide (F), cisplatin (G), and talazoparib (H) for 72 hours. Bottom: Combination index (CI) values were calculated with CompuSyn. Additive combination: 0.5<CI<1, and synergistic combinations: 0<CI<0.5. Each CI value was annotated in the plots.

Figure 4.. M1774 reverses the chemoresistance of SLFN11-deficient cells to DNA-damaging agents.

A and B. Top: DMS114 (WT: SLFN11 wild-type) and SLFN11-KO (Knock out) cells were treated with M1774 (40 nmol/L) and the indicated concentrations of SN-38 (A) and cisplatin (B) for 72 hours. Cell viability was measured by CellTiter-Glo assays. Error bars represent standard deviations for triplicate data. Bottom: Combination index values. C and D. DU145 (WT: SLFN11 wild-type) and SLFN11-KO (Knock out) cells were treated with M1774 (40 nmol/L) and the indicated concentrations of exatecan (C) and cisplatin (D) for 72 hours. Bottom: Combination index values. E. Gene expression of SLFN11 and TOP1 are plotted for all the cancer cell line in the GDSC database with CellMinerCDB (http://discover.nci.nih.gov/). Dot colors indicates different cancer types. F. Difference of drug activity between SLFN11-high and SLFN11-low cells in the SCLC NCI-DTP dataset. Plots were generated from data downloaded from CellMinerCDB. * p-value <0.03, ** p-value < 0.005. G. Combination effects of PARP inhibitor talazoparib with SN-38 in SLFN11-WT vs. SLFN11-KO cells. DU145 and paired SLFN11-KO cells were treated with the indicated concentrations of talazoparib and SN-38 (1 nmol/L and 5 nmol/L) for 72 hours. cell viability was measured by CellTiter-Glo assays. Error bars represent standard deviations for triplicate data.

Figure 5.. M1774 synergizes with clinical DNA-damaging agents in SCLC patient-derived organoid models.

A-D. Top: Small cell lung cancer (SCLC) patient-derived organoid (PDO) #1 was co-treated with M1774 (15.6 or 62.5 nmol/L) and the indicated concentrations of topotecan (A), etoposide (B), cisplatin (C), and lurbinectedin (D) for 72 hours. Cell viability was assessed by CellTiter-Glo assays. Error bars represent standard deviations from triplicate data. Bottom: Combination index values calculated with CompuSyn. E-H. Top: SCLC PDO #2 was co-treated with M1774 (15.6 or 62.5 nmol/L) and the indicated concentrations of topotecan (E), etoposide (F), cisplatin (G), and lurbinectedin (H) for 72 hours. Bottom: Combination index values were calculated with CompuSyn.

Figure 6.. M1774 synergistically suppresses tumor growth in combination treatment with irinotecan in H82 small cell lung cancer xenograft models.

A. Tumor volumes were confirmed in H82 SCLC xenografts treated with M1774 (15 mg/kg) at a once-weekly oral dose, Irinotecan (50 mg/kg) at a once-weekly intraperitoneal dose, and the combination of M1774 with irinotecan. Error bars show SEM; *P-value < 0.0001 (Irinotecan, Combination vs Vehicle; Combination vs Irinotecan), two-way ANOVA, n=9/group. B. Changes of body weight for xenografts. C. Progression-free survival by combination treatment compared with control (vehicle) and monotherapy. D~F. Tumor volumes (D), body weight (E), and progression-free survival (F) of N87 gastric carcinoma xenografts treated with M1774 (25 mg/kg) at a once-weekly oral dose, Irinotecan (50 mg/kg) at a once-weekly intraperitoneal dose, and the combination of M1774 with irinotecan. Error bars show SEM; *P-value < 0.0001 (Irinotecan, Combination vs Vehicle; Combination vs Irinotecan), two-way ANOVA, n=10/group. G. Schematic model for how the ATR inhibitor M1774 may be applied in cancer therapy.