ATR Inhibitor M6620 (VX-970) Enhances the Effect of Radiation in Non-Small Cell Lung Cancer Brain Metastasis Patient-Derived Xenografts

Abstract

M6620, a selective ATP-competitive inhibitor of the ATM and RAD3-related (ATR) kinase, is currently under investigation with radiation in patients with non-small cell lung cancer (NSCLC) brain metastases. We evaluated the DNA damage response (DDR) pathway profile of NSCLC and assessed the radiosensitizing effects of M6620 in a preclinical NSCLC brain metastasis model. Mutation analysis and transcriptome profiling of DDR genes and pathways was performed on NSCLC patient samples. NSCLC cell lines were assessed with proliferation, clonogenic survival, apoptosis, cell cycle, and DNA damage signaling and repair assays. NSCLC brain metastasis patient-derived xenograft models were used to assess intracranial response and overall survival. In vivo IHC was performed to confirm in vitro results. A significant portion of NSCLC patient tumors demonstrated enrichment of DDR pathways. DDR pathways correlated with lung squamous cell histology; and mutations in ATR, ATM, BRCA1, BRCA2, CHEK1, and CHEK2 correlated with enrichment of DDR pathways in lung adenocarcinomas. M6620 reduced colony formation after radiotherapy and resulted in inhibition of DNA DSB repair, abrogation of the radiation-induced G2 cell checkpoint, and formation of dysfunctional micronuclei, leading to enhanced radiation-induced mitotic death. The combination of M6620 and radiation resulted in improved overall survival in mice compared with radiation alone. In vivo IHC revealed inhibition of pChk1 in the radiation plus M6620 group. M6620 enhances the effect of radiation in our preclinical NSCLC brain metastasis models, supporting the ongoing clinical trial (NCT02589522) evaluating M6620 in combination with whole brain irradiation in patients with NSCLC brain metastases.

Figure 1.

DNA damage and repair mutation and transcriptional analysis in non-small cell lung cancer patients. A, Percent of DDR mutations in lung adenocarcinoma and squamous cell carcinomas. B, Breakdown of mutations in 53 genes DDR genes. C, Heatmap of pathways and molecular processes (GO terms) involved in DNA maintenance and cell cycle regulation activated in DNA damage response. Age, gender, histological subtype is annotated as well as mutation status of important DDR and clinically relevant oncogenic genes.

Figure 2.

Response to ATR inhibitor M6620 and radiosensitivity effects in three NSCLC cell lines. A, Sensitivity to M6620 determined by proliferation assay in three NSCLC cell lines A549, NCI-H226 and NCI-H520 following varying doses of M6620. B, In vitro radiosensitizing effects of M6620 on A549, NCI-H226, and NCI-H520. Cell cultures were treated with 40 or 80 nM M6620 for 1 h before irradiation and maintained in the medium after irradiation. Colony-forming efficiency was determined 14–21 days later, and survival curves were generated. C, Western blot was used to assess ATR, p-ATR (Thr1989), ATM and p-ATM (Ser1981) in M6620-treated and irradiated cell lines. Cells were pretreated with vehicle (DMSO) or M6620 (40 nM) for 1 h and either collected after (no radiation) or exposed to radiation (10 Gy) at the indicated time points. Points, mean; Bars, SE (n=3).

Figure 3.

Mechanism of cell death after combined radiation and M6620 treatment in A549 NSCLC cells. A, Cell cycle, B, Apoptosis, C, γH2AX foci and D, mitotic cell death analyses. Cells were treated with vehicle (DMSO), M6620 (40 nM or 80 nM), 2 Gy or 10 Gy, or M6620 1 h prior to radiation and analyzed at designated time points. For γH2AX, cells with >10 γH2AX foci were counted and plotted at the time points following 2 Gy radiation. Representative DAPI stained immunofluorescence images and micronuclei quantification are shown. Columns, mean; bars, SE, n=3. ***, P < 0.001, according to t-test (radiation versus M6620 plus radiation).

Figure 4.

The effects of M6620 on radiation-induced patient-derived xenograft tumor growth delay. Tumor growth delay curves of A, UW-Lung-16 and UW-Lung-18 subcutaneous flank xenografts treated with vehicle (5%DMSO+45%PEG300+water), M6620 (60 mg/kg), radiation (2 Gy × 10) or M6620 (60 mg/kg) plus radiation (2 Gy × 10). M6620 was administered daily 1 h before radiation fraction. Points, mean tumor volume in mice after treatment; bars, SE. P < 0.001. IHC analysis of B, UW-Lung-16 and UW-Lung-18 tumors showing H&E, phospho-Chk1 (Ser345), and Cleaved Caspase-3 (CC3) expression and quantification. Tumors were harvested at 8 h following a single dose of vehicle, M6620 (60 mg/kg), radiation (2 Gy) and M6620 plus radiation. The scale bar represents 50 mm and all images are to the same scale. C, In-vivo γH2AX foci analysis after combined radiation and M6620 treatment in UW-Lung-16 and UW-Lung-18. UW-lung-16 and UW-lung-18 tumors were harvested at 8 h following a single dose of vehicle, M6620 (60 mg/kg), radiation (2 Gy) and M6620 plus radiation. γH2AX foci were counted in 50 cells in each section and plotted (n=3). Columns, mean (n=3); bars, SE; ***, P < 0.05 by ANOVA for all graphs (radiation versus M6620 plus radiation).

Figure 5.

The addition of M6620 to radiation improves overall survival in mice bearing NSCLC brain metastases. Mice bearing UW-lung-16 NSCLC brain metastasis tumors were randomized 3 days post implant based on bioluminescence signal to either irradiation (2.5 Gy × 5), or M6620 (60 mg/kg) plus irradiation (2.5 Gy × 5). A single dose of M6620 was delivered as p.o. gavage 1 h before receiving each daily radiation treatment. Each group contained 10 mice. A, Representative bioluminescence imaging of mice post implant, showing pre-treatment day 3 and post-treatment day 11 and day 40. B, Total flux of implanted brain metastases over time. Average total flux between the two groups was statistically significant at day 32 and 40 post implantation (P = 0.01, Students t-test). C, Kaplan-Meier curve showing survival of mice with implanted intracranial UW-lung-16 NSCLC brain metastasis tumors (P = 0.022, Gehan-Breslow-Wilcoxon test). D, Weights between the two treatment groups during and 40 days post-treatment.