Fig 1.
Inhibition of KDM5 demethylases activates interferon-induced genes.
(A) Western blot analysis of histone modifications in MCF7 cells treated with 1 μM KDM5-C70, 10 μM Dong-A-167, 10 μM GDC-50, or 10 μM CPI-48 for 3 days. (B) GSEA of RNA-seq data from MCF7 cells treated with 3 μM KDM5-C70 or CPI-48 for 6 days. (C, D) RT-qPCR (panel C) and western blot (panel D) analyses of MCF7 cells treated with 1 μM KDM5-C70, 10 μM Dong-A-167, 10 μM GDC-50, or 10 μM CPI-48 for 6 days. (E, F) Western blot (panel E) and RT-qPCR (panel F) analyses of MCF7 cells with stable knockout of the indicated genes using the CRISPR/Cas9 system. Control 1, empty vector; control 2, scrambled sequence. KDM5BC, 2 sgRNAs targeting KDM5B and KDM5C. (G) Western blot analysis of MCF7 cells after transfection with indicated plasmids. Representative data from triplicate experiments are shown. Error bar denotes SEM. #p < 0.01 for inhibitors versus DMSO (panel C), and for knockout sgRNA versus average of 2 control sgRNAs (panel F). The numerical values used to generate graphs in panel C and F are available in S1 Data. CRSPR/Cas9, clustered regular interspaced short palindromic repeats/CRISPR-associated protein 9; FDR q, false discovery rate q value; GSEA, gene set enrichment analysis; NES, normalized enrichment score; NOM p, nominal p value; NS, nonspecific band; RNA-seq, RNA sequencing; RT-qPCR, reverse transcription followed by quantitative PCR; sgRNA, single guide RNA.
Fig 2.
Inhibition of KDM5 demethylases primes the innate antiviral immune response.
(A) Representative images of MCF7 cells 24 hours after infection with VSV-GFP viruses at MOI 0.5. Scale bar, 100 μm. (B) Flow cytometry plot (left panel, 24 hours) and quantification of GFP-positive cells (right panel) after infection with VSV-GFP virus for the indicated time at MOI 0.5. (C) qPCR analysis of DNA copy number of vaccinia virus in MCF7 cells at the indicated time after infection at MOI 0.25. (D) Representative crystal violet staining images (left panel) and quantification of relative intensity (right panel) of MCF7 cells 3 days after infection with vaccinia virus at MOI 0.5. (E) qPCR analysis of DNA copy number of vaccinia virus in growth media from the cells shown in panel D. (F) Quantification of flow cytometry analysis for the percentage of GFP-positive cells in control or KDM5B and KDM5C double knockout cells 12 hours after infection with VSV-GFP virus at MOI 0.5. (G) Representative crystal violet staining images (left panel) and quantification of relative intensity (right panel) of the indicated MCF7 knockout cells 3 days after infection with vaccinia virus at MOI 0.25. (H) qPCR analysis of DNA copy number of vaccinia virus in growth media from the cells shown in panel G. Cells were pretreated with DMSO or 1 μM KDM5-C70 for 5 days, followed by no treatment for 1 day, before viral infection in panel A–E. Representative data from triplicate experiments are shown in panel C, E, F, and H. Two or 3 biological replicates are shown in panel B, D, and G. Error bar denotes SEM. #p < 0.01. The numerical values used to generate graphs in B–H are available in S1 Data. MOI, multiplicity of infection; qPCR, quantitative PCR; VSV-GFP, vesicular stomatitis virus carrying a green fluorescent protein reporter.
Fig 3.
Activation of ISGs by KDM5 inhibition is dependent on the cGAS-STING-TBK1-IRF3 signaling pathway.
(A) Schematic of the pattern recognition receptor pathways. (B–D) Western blot (panel B and C) and RT-qPCR (panel D) analyses of MCF7 cells with knockout of the indicated genes after treatment with DMSO or 1 μM KDM5-C70 for 6 days. (E, F) RT-qPCR (panel E) and western blot (panel F) analyses of MCF7 cells with knockout of the indicated genes 5 days after transfection with the indicated siRNAs. (G) RT-qPCR analysis of MCF7 cells with knockout of the indicated genes after treatment with DMSO or 1 μM KDM5-C70 for 6 days. (H) Flow cytometry plots (left panel) and quantification of percentage of GFP-positive cells (right panel) in the indicated MCF7 knockout cells 24 hours after infection with VSV-GFP virus at MOI 0.5. Error bar denotes SEM. Representative data from triplicate experiments are shown in panel D, E, and G. Three biological replicates are shown in panel H. #p < 0.01 for inhibitors versus DMSO (panel D, G, and H), knockdown of KDM5B and KDM5C versus control (panel E). ^p < 0.01 for knockout sgRNA versus control sgRNA (panel D and G). The numerical values used to generate graphs in panel D, E, G, and H are available in S1 Data. cGAS, cyclic GMP-AMP synthase; IRF3, interferon regulatory factor 3; ISG, interferon-stimulated gene; MOI, multiplicity of infection; RT-qPCR, reverse transcription followed by quantitative PCR; sgRNA, single guide RNA; siRNA, small interfering RNA; STING, stimulator of interferon genes; TBK1, TANK-binding kinase 1; VSV-GFP, vesicular stomatitis virus carrying a green fluorescent protein reporter.
Fig 4.
Inhibition of KDM5 demethylases induces STING expression.
(A, B) RT-qPCR analysis (panel A) or western blot (panel B) analysis of the indicated cells after treatment with DMSO or 1 μM KDM5-C70 for 3 days. (C) Western blot analysis of MCF7 cells transfected with the indicated expressing plasmids. (D–F) RT-qPCR (panel D and E) or western blot (panel F) analyses of MCF7 cells with knockout of indicated genes after treatment with DMSO or 1 μM KDM5-C70 for 6 days. (G) Western blot analysis of MCF7 cells 2 days after transfection with different amounts of the indicated plasmid. (H–L) Western blot (panel H) or RT-qPCR (panel I–L) analysis of MCF7, SKBR3, or BT474 cells after treatment with DMSO or 1 μM KDM5-C70 for the indicated length of time. Representative data from triplicate experiments are shown. Error bar denotes SEM. #p < 0.01 for inhibitors versus DMSO (panel A, D, E, and I–L). The numerical values used to generate graphs in panel A, D, E, and I–L are available in S1 Data. RT-qPCR, reverse transcription followed by quantitative PCR; STING, stimulator of interferon genes.
Fig 5.
KDM5B and KDM5C bind to the promoter of STING and directly suppress STING expression.
(A) Western blot (left panel) and RT-qPCR (right panel) analyses of STING in MCF7 cells with knockout of the indicated genes after treatment with DMSO or 1 μM KDM5-C70 for 3 days. (B, C) Western blot analysis of H3K4me3 (upper panel) and RT-qPCR analysis of STING (lower panel) in MCF7 cells (panel B) or in BT474 cells (panel C) after treatment with the indicated compounds for 3 days. The concentration of OICR-9429 was 20 μM. (D) H3K4me3 ChIP-qPCR analysis at the promoter of STING, OAS2, or IFI44L in control or STING knockout MCF7 cells treated with DMSO or 1 μM KDM5-C70 for 1 day or 6 days. (E) KDM5A and KDM5B ChIP-qPCR analysis of MCF7 cells at the STING and NDUFA9 promoters, or downstream of the last STING exon as NC. (F) Analysis of ChIP-seq data for KDM5B binding at the STING genomic region in K562 cells (GSE29611, upper panel) and KDM5C in ZR-75-30 cells (GSE71327, lower panel) [42]. Representative data from triplicate experiments are shown. Error bar denotes SEM. #p < 0.01 for the comparisons shown in panel A–C, for inhibitors versus DMSO (panel D), and for KDM5A or KDM5B ChIP versus IgG ChIP (panel E). The numerical values used to generate graphs in panel A–E are available in S1 Data. ChIP, chromatin immunoprecipitation; ChIP-seq, chromatin immunoprecipitation sequencing; IgG, immunoglobulin G; NC, negative control; RT-qPCR, RT-qPCR, reverse transcription followed by quantitative PCR; STING, stimulator of interferon genes.
Fig 6.
KDM5 inhibition induces a robust interferon response in cancer cells with elevated levels of cytosolic DNA.
(A) Immunostaining of dsDNA and the mitochondrial marker Hsp60 in MCF7 cells. Surface plots of Z-stack images generated with Huygens (left panel). qPCR detecting mtDNA copy number in cells with the indicated treatment (right panel). 10 μM of ddC was used. Scale bar, 10 μm. (B, C) RT-qPCR (panel B) and western blot (panel C) analyses of MCF7 cells with the indicated treatment. MCF7 cells were treated with 10 μM ddC and 1 μM KDM5-C70 for 6 days. Cells were refed every 2 days. (D) dsDNA and DAPI staining of MCF7 cells or SKBR3 cells digested with 50 μg/ml DNase I. Surface plots of Z-stack images generated with Huygens (left panel). Quantification of dsDNA intensity per cell using image J was shown in the right panel. Scale bar, 10 μm. (E) RT-qPCR analyses of SKBR3 cells with the indicated treatment. SKBR3 cells were transfected with 1 μg dsDNA of about 90 bp (dsDNA90) using Lipofectamine 2000 5 hours before treatment with DMSO or 1 μM KDM5-C70 for 3 days. (F) RT-qPCR analysis of SKBR3 cells with knockout of the indicated genes after treatment with 1 μg dsDNA and 1 μM KDM5-C70 for 3 days. Representative data from triplicate experiments are shown. Error bar denotes SEM. #p < 0.01 for panel A, B, and D–F, for KDM5-C70 + dsDNA90 versus DMSO + lipo (panel E), and for knockout sgRNA versus control sgRNA (panel F). The numerical values used to generate graphs in panel A, B, and D–F are available in S1 Data. ddc, dideoxycytidine; dsDNA, double-stranded DNA; lipo, Lipofectamine 2000; mtDNA, mitochondrial DNA; RT-qPCR, RT-qPCR, reverse transcription followed by quantitative PCR; sgRNA, single guide RNA.
Fig 7.
KDM5B is negatively associated with STING expression, CD8+ T-cell infiltration, and clinical outcome.
(A) Anticorrelation between expression of KDM5B and STING in TCGA cancer samples. Normalized STING levels in “KDM5B low” and “KDM5B high” samples of breast invasive carcinoma, bladder urothelial carcinoma, and ovarian serous cystadenocarcinoma from the TCGA datasets. The numerical values used to generate these graphs are available in S1 Data. (B) Correlation between KDM5B and STING in HPV+ head and neck tumors. n = 79. (C) Correlation between KDM5B and CXCL10 (left panel) or STING and CXCL10 (right panel) in HPV+ head and neck tumors. (D) Correlation between KDM5B expression and CD8+ T-cell infiltration in HNSC-HPVpos. (E) Association of CD8+ T-cell infiltration level (left panel) and KDM5B expression (right panel) with survival of HPV+ head and neck cancer patients. Tumors in the top 25th percentile were compared to those in the bottom 25th percentile. (F) A working model for how KDM5i induces innate and adaptive immune responses. HPV, human papilloma virus; HNSC-HPVpos, HPV+ head and neck tumors; ISRE, interferon-stimulated response element; KDM5i, KDM5 inhibitor; STING, stimulator of interferon genes; TCGA, The Cancer Genome Atlas.