ARID1A loss enhances sensitivity to c-MET inhibition by dual targeting of GPX4 and iron homeostasis, inducing ferroptosis

Abstract

ARID1A, a subunit of the SWI/SNF chromatin-remodeling complex, functions as a tumor suppressor in various cancer types. Owing to its high frequency of inactivating mutations, ARID1A has emerged as a promising target for the development of anticancer drugs. In this study, we report that ARID1A-deficient colorectal cancer (CRC) cells induce synthetic lethality when treated with inhibitors of c-MET receptor tyrosine kinase. c-MET specific inhibitor PHA-665752 as well as two other FDA-approved drugs, crizotinib and cabozantinib, selectively inhibited the growth of ARID1A-deficient CRC cells in vitro and in xenograft tumor models. Mechanistically, we identified a tripartite functional association among ARID1A, c-MET, and NRF2, where ARID1A and c-MET pathways converge on the NRF2 transcription factor, which regulates the transcription of GPX4, a key regulator of ferroptosis. ARID1A inactivation reduces c-MET expression, decreasing NRF2 nuclear localization and its binding to the GPX4 promoter, resulting in reduced GPX4 transcription. This creates a cellular dependency on the residual c-MET for minimal GPX4 expression to survive the ferroptotic cell death. Additionally, we demonstrate that ARID1A loss leads to increased intracellular labile iron accumulation by downregulating the iron-exporting protein SLC40A1, thereby increasing cellular susceptibility to ferroptosis. Inhibition of c-MET in ARID1A-deficient CRC cells diminishes GPX4 expression, resulting in elevated lipid peroxidation and glutathione depletion, ultimately inducing ferroptosis. This study reveals a novel synthetic lethal relationship between ARID1A and c-MET signaling in promoting ferroptosis and proposes c-MET inhibitors as a potential therapeutic strategy for ARID1A-deficient CRC.

Fig. 1. Screening of Kinase inhibitor library for synthetic lethality in HCT116 cell.

A Immunoblot analysis showing loss of ARID1A expression in the two ARID1A-KO clones. B Schematic illustration of the synthetic lethal kinase inhibitor screening. HCT116 ARID1A-WT and ARID1A-KO cell lines were screened in parallel with 430 kinase inhibitor library in an 8-dose titration format. After incubation with the compound library for 72 h, cell viability was determined by Alamar Blue assay. C A −log2 IC50 plot of the screening results. −log2 scale of IC50 values of the drugs against HCT116 ARID1A-KO and ARID1A-WT cells was plotted. D Dose-response curves of HCT116 ARID1A-WT and two ARID1A-KO clones treated with c-MET kinase inhibitor for 72 h are shown. Error bars represent s.d. (n = 3) from three independent experiments. Dose-response curves of HCT116 ARID1A-isogenic cell pair treated with c-MET kinase inhibitors crizotinib (E) and cabozatinib (F) are shown. HCT116 ARID1A-WT, ARID1A-KO #1, and ARID1A-KO #2 clones were incubated with indicated compounds for 72 h and the cell viability was determined by Alamar Blue assay. Error bars represent s.d. (n = 3) from three independent experiments.

Fig. 2. In vitro and in vivo synthetic lethality in ARID1A-KO HCT116 cells by c-MET kinase inhibitor.

A Silencing of MET expression in HCT116 by siRNA (siMET). GAPDH was used as a control. B Synthetic lethality validation with siMET. ARID1A-WT and ARID1A-KO clone was transfected with 100 μM siMET for 72 h and the cell images were taken. Scale bars, 100 μm. C The cell density was determined with Image J software. ANOVA P value of <0.01. D Schematic illustration of mouse tumor xenograft experiments with HCT116 ARID1A-isogenic cell pair. E Tumor growth curve in nude mice bearing HCT116-WT or HCT116 ARID1A-KO xenografts after intravenous injection of vehicle, 15 or 30 mg kg⁻¹ (mpk) PHA. Error bars represent a P value of <0.01 between vehicle and PHA treatment groups (n = 3). Student’s t-test. F Wet weight measurement of the tumors isolated from mice bearing HCT116-WT or HCT116 ARID1A-KO xenografts at 24 days after injection of vehicle, 15 or 30 mpk PHA. Error bars represent s.d.

Fig. 3. Knocking out ARID1A activates the ferroptosis signaling pathway.

A Schematic illustration of RNA-seq contrasting with HCT116-WT and HCT116 ARID1A-KO cells. B volcano plot depicting the results of the RNA-Seq study. C Top 12 enrichment scores of KEGG pathway enrichment analysis. D GSEA analysis on ARID1A-KO vs. ARID1A-WT samples of the ferroptosis gene set. E The heat map data showed gene expression alteration of ferroptosis-correlated molecules after ARID1A knock out including GPX4, SLC7A11, FTH1, HMOX1, and so on. F Lipid peroxidation level was detected in HCT116-WT and two HCT116 ARID1A-KO clones with C11-BODIPY (581/591). G The ratio of GSH with GSSG in HCT116 and HCT116 ARID1A-KO cells was detected. H GPX4 expression level was detected with western blot analysis in HCT116-WT and two HCT116 ARID1A-KO clones. I Cellular labile iron pool (LIP) was detected by calcein-AM reporter in in HCT116-WT and two HCT116 ARID1A-KO clones. HCT116-WT and two HCT116 ARID1A-KO clones were treated with ferroptosis inducers RSL3 (J) and Erastin (K) for 72 h. The cell viability was determined by Alamar Blue assay. Error bars represent s.d. ANOVA P value < 0.01.

Fig. 4. Synthetic lethality is ferroptosis pathway dependent.

A HCT116-WT and HCT116 ARID1A-KO cells were treated with or without PHA for 72 h. Lipid peroxidation level was conducted with C11-BODIPY assay. B c-MET inhibition reduced the ratio of GSH/GSSG in HCT116 and HCT116 ARID1A-KO cells. C PHA treatment raised iron accumulation, especially in ARID1A-KO cells. D Transfection with 100 μM siMET for 48 h reduced the ratio of GSH with GSSG in HCT116 and HCT116 ARID1A-KO cells. Transfection with 100 μM siMET for 48 h raised ROS intensity (E) lipid peroxidation (F) and iron accumulation (G) in HCT116 and HCT116 ARID1A-KO cells. H, I HCT116 and HCT116 ARID1A-KO cells were treated with or without 4 μM PHA, and 20 μM ferrostatin-1 (Ferr-1) for 72 h, and ROS intensity was analyzed with fluorescence. The fluorescence intensity was calculated with Image J software. Scale bars, 100 μm. J HCT116 and HCT116 ARID1A-KO cells were treated with or without 4 μM PHA, 20 μM Ferr-1 for 72 h. The cell viability was determined by Alamar Blue assay. Error bars represent s.d. from three independent experiments. ANOVA P value of <0.001.

Fig. 5. Synthetic lethality and ferroptosis in ARID1A-deficient RKO cells by PHA treatment.

A Expression level of ARID1A in RKO and two selected ARID1A stable clones were detected with immunoblot analysis. B Dose-response curves of parental RKO and ARID1A-overexpressing (ARID1A^OE) RKO clones treated with PHA. HCT116 ARID1A-WT cells were used as a positive control. Error bars represent s.d. (n = 5) from three independent experiments. C Synthetic lethality with siMET transfection in RKO isogenic cells. RKO and ARID1A^OE clones were transfected with 100 μM siMET for 72 h. Scale bars, 100 μm. D The cell density was determined with Image J software. ANOVA P value of <0.01. E, F RKO and ARID1A^OE clones were treated with or without PHA and Ferr-1. ROS intensity was analyzed with fluorescence. The fluorescence intensity was calculated with Image J software. Scale bars, 100 μm. G RKO and ARID1A^OE clones were treated with or without PHA and Ferr-1. The cell viability was determined by Alamar Blue assay. Error bars represent s.d. from three independent experiments. ANOVA P value of <0.01.

Fig. 6. GPX4 is a target gene for transcription repression by ARID1A.

A Downregulation of c-MET, SLC7A11, and GPX4 level in ARID1A-KO HCT116 cells. B Upregulation of c-MET, SLC7A11, and GPX4 level in ARID1A-overexpressing (ARID1A^OE) RKO clones. C Silencing ARID1A expression decreases c-MET, SLC7A11, and GPX4 levels in HCT116 cells. D Upregulation of c-MET, SLC7A11, and GPX4 level in RKO cells by ARID1A overexpression plasmid transfection. E RT-qPCR analysis of GPX4 mRNA level in HCT116 and ARID1A-KO clones. ANOVA P < 0.01. F Four primers (Primer #1, Primer #2, Primer #3, Primer #4) were designed to cover GPX4 promoter from −1 bp to −1000 bp. G Chip of GPX4 promoter in HCT116 cells using anti-ARID1A antibody. H A representative image showing results of PCR performed on DNA samples precipitated with anti-ARID1A. I ChIP of GPX4 promoter in HCT116 cells using anti-ARID1A antibody. IgG was used as a normalization control. ANOVA P < 0.05. J ChIP of GPX4 promoter in HCT116 and ARID1A-KO cells using anti-Pol II antibody. IgG in each cell line was used as a normalization control. ANOVA P < 0.01.

Fig. 7. c-MET/NRF2/GPX4 axis is a target for synthetic lethality in ARID1A-KO colorectal cancer cells.

A RT-qPCR analysis of GPX4 mRNA level with PHA treatment in HCT116 cells. ANOVA P < 0.01. B Immunoblot analysis showing PHA treatment downregulates GPX4 level in HCT116, Lovo, and SW480 cells. C c-MET silencing induces GPX4 downregulation in HCT116, Lovo, and SW480 cells. Inhibition of GPX4 level by PHA treatment in HCT116 (D) and RKO (E) cells. F, G Overexpression of GPX4 reversed synthetic lethality induced by PHA treatment in HCT116 ARID1A-KO cells. HCT116 ARID1A-KO cells were treated with or without PHA and GPX4 expression plasmid. The cell viability was determined by Alamar Blue assay. ANOVA P value of <0.01. H co-IP showing ARID1A binds with NRF2 in HCT116 cells. I ChIP of GPX4 promoter in HCT116 and ARID1A-KO cells using anti-NRF2 antibody. IgG in each cell line was used as a normalization control. ANOVA P < 0.01. J Immunofluorescences showing that ARID1A loss attenuates NRF2 nuclear localization in HCT116 cells. Scale bars, 20 μm. K c-MET overexpression promotes NRF2 nuclear localization in ARID1A-KO cells. Immunofluorescences show that c-MET overexpression promotes NRF2 nuclear localization in ARID1A-KO cells. Scale bars, 20 μm. L c-MET overexpression promotes the binding between NRF2 and GPX4 promoter. ChIP of GPX4 promoter in ARID1A-KO cells using anti-NRF2 antibody with or without c-MET expression plasmid. IgG in each cell line was used as a normalization control. ANOVA P < 0.01. M ChIP of GPX4 promoter in HCT116 cells using anti-ARID1A antibody with or without PHA treatment. IgG in each cell line was used as a normalization control. ANOVA P < 0.01. N Working model of the synthetic lethality. In ARID1A-WT cells, ARID1A promotes c-MET transcription and cooperatively regulates NRF2 transcription factor functions with c-MET, thereby promoting GPX4 transcription. ARID1A inactivation downregulates c-MET and GPX4 levels, creating a cellular dependency on the residual activity of c-MET and GPX4. ARID1A inactivation also downregulates iron-exporting protein SLC40A1, increasing intracellular iron accumulation and lipid peroxidation. Pharmacological inhibition of c-MET (such as PHA665452) in ARID1A-deficient CRC cells diminishes GPX4 transcription, resulting in increased lipid peroxidation and ultimately inducing ferroptotic cell death. PLOOH a reactive phospholipid hydroperoxide, PLOH non-reactive alcohol, PL• a carbon-centered radical on a phospholipid chain.