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Inhibition of a G9a/DNMT network triggers immune-mediated bladder cancer regression

Abstract

Bladder cancer is lethal in its advanced, muscle-invasive phase with very limited therapeutic advances1,2. Recent molecular characterization has defined new (epi)genetic drivers and potential targets for bladder cancer3,4. The immune checkpoint inhibitors have shown remarkable efficacy but only in a limited fraction of bladder cancer patients5,6,7,8. Here, we show that high G9a (EHMT2) expression is associated with poor clinical outcome in bladder cancer and that targeting G9a/DNMT methyltransferase activity with a novel inhibitor (CM-272) induces apoptosis and immunogenic cell death. Using an immunocompetent quadruple-knockout (PtenloxP/loxP; Trp53loxP/loxP; Rb1loxP/loxP; Rbl1−/−) transgenic mouse model of aggressive metastatic, muscle-invasive bladder cancer, we demonstrate that CM-272 + cisplatin treatment results in statistically significant regression of established tumors and metastases. The antitumor effect is significantly improved when CM-272 is combined with anti-programmed cell death ligand 1, even in the absence of cisplatin. These effects are associated with an endogenous antitumor immune response and immunogenic cell death with the conversion of a cold immune tumor into a hot tumor. Finally, increased G9a expression was associated with resistance to programmed cell death protein 1 inhibition in a cohort of patients with bladder cancer. In summary, these findings support new and promising opportunities for the treatment of bladder cancer using a combination of epigenetic inhibitors and immune checkpoint blockade.

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Fig. 1: Relevance of G9a in human BC and effect of G9a/DNMT dual inhibitor in BC cells.
Fig. 2: Combination of CM-272 and CDDP induces activation of immune-related pathways in a QKO transgenic model of BC.
Fig. 3: G9a/DNMT inhibition enhances responses to PD-L1 blockade and induces tumor regression.
Fig. 4: Levels of G9a, EZH2, H3K9me2 and H3K27me3 histone marks associated with clinical responses to anti-PD-1 immunotherapy in patients with BC.

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Data availability

Datasets have been deposited in the Gene Expression Omnibus with accession nos. GSE115544, GSE115485 and GSE111636. Full uncropped blots are available as Source data.

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Acknowledgements

We acknowledge the patients and their families. We particularly thank all the people from the Paramio and Prosper laboratories for discussion and suggestions. We express our acknowledgement to F.X. Real (CNIO) for his critical reading of the manuscript and helpful comments. This study was cofunded by European Regional Development Fund (FEDER) grants from MINECO (no. SAF2015-66015-R to J.M.P. and no. SAF2016-78568-R to J.J.L.), Instituto de Salud Carlos III (RETIC no. RD12/0036/0009 to J.M.P., RTICC no. RD12/0036/0068 to F.P., no. PI14/01867 to F.P., no. PI16/02024 to X.A., no. PI17/00701 to F.P., CIBERONC no. CB16/12/00489 to F.P., CIBERONC no. CB16/12/00228 to J.M.P. and ERANET-TRANSCAN-2 EPICA to F.P.), Departamento de Salud del Gobierno de Navarra no. 40/2016 to X.A. and Gobierno de Navarra Industria (0011-1411-2017-000028; 0011-1411-2017-000029; 0011-1411-2017-000030; Proyecto DIANA to F.P.). We also thank the Fundación Fuentes Dutor (to J.O.) and Fundación La Caixa Hepacare Project (to J.J.L.) for financial support.

Author information

Authors and Affiliations

Authors

Contributions

C.Segovia, E.S.J.-E., E.M.-M., X.A., F.P. and J.M.P. conceived and designed the study. C.Segovia, E.S.J.-E., E.M.-M., M.M.-F., P.F., J.A.C., M.D. and J.O. developed the methodology. L.G., E.M., A.V.-Z., I.L., C.R., C.Segrelles, L.V.V., O.R., N.C., A.B., C.S.-C., F.F.L.-C., P.F., J.A.C., M.D., F.V., F.G.-R., G.d.V. and D.C. acquired the data and assisted with the experiments. C.Segovia, E.S.J.-E., E.M.-M., P.F., J.A.C., M.D., J.J.L., X.A., F.P. and J.M.P. analyzed and interpreted the data. C.Segovia, E.S.J.-E., E.M.-M., J.J.L., J.O., X.A., F.P. and J.M.P. wrote, reviewed and/or revised the manuscript. L.V.V. and J.M.P. provided administrative, technical and material support (that is, reported or organized the data, built the databases). X.A., F.P. and J.M.P. supervised the study.

Corresponding authors

Correspondence to Xabier Agirre, Felipe Prósper or Jesús M. Paramio.

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The authors declare no competing interests.

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Peer review information: Javier Carmona was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Alterations in G9a gene in the TCGA BC database.

a, Summary of G9a gene alterations observed in the TCGA database. Classification was made according to z-scores of mRNA expression (RNA-Seq V2 RSEM) with a threshold ± 2.0. b, Summary of G9a gene mutations localization reported in the TCGA database. c, Heatmap showing the supervised classification of genes bound by G9a according to the different TCGA BC molecular subtypes. d, Summary of G9a gene expression in the different TCGA molecular subtypes according to z-scores of mRNA expression (RNA-Seq V2 RSEM). e, Summary of G9a gene expression at different tumor stages from the TCGA database. f, Summary of EZH2 gene expression in the different TCGA molecular subtypes according to z-scores of mRNA expression (RNA-Seq V2 RSEM). g, Correlation analyses between G9a and EZH2 gene expression from TCGA database. n = 404 patients. h, Representative immunohistochemistry images of G9a expression in NMIBC samples showing a negative (left panel) and positive (right panel) example. Histological analyses and staining were performed in all clinical samples (patient), scoring at least two different sections from each tumor. Bar, 150 µm. i, Kaplan–Meier graph showing recurrence according to G9a protein staining in human NMIBC samples. The P value was determined by the log-rank test. df, n = 404 patients. Data are represented as the mean ± s.e.m. The P value was estimated using a two-sided t-test.

Extended Data Fig. 2 Effect of inhibition of G9a and DNMT1 in BC cells.

a, Summary of gene mutations and GI50 values for CM-272 in BC cell lines. b, Proliferation of RT112 and 5637 cell lines after treatment with A-366 or decitabine alone or in combination at different doses. A combination index lower than 1 indicates a synergistic effect as defined by Chou and Talalay36 (combination index > 0.85, slight or no synergism; combination index between 0.7 and 0.85, moderate synergism; combination index between 0.3 and 0.7, synergism; combination index between 0.1 and 0.3, strong synergism). Combination doses were based on GI50 values for A-366 and decitabine against the tested cell line (the GI50is >50 µM in both cases). *P < 0.05. One-tailed Mann–Whitney U-test. n = 3 independent experiments. Data are represented as the mean ± s.d. c, Heatmaps of cell proliferation of the 5637 and RT112 cell lines after treatment with A-366 or decitabine alone or in combination at different doses (right). Heatmaps of combination indexes (left). d, Proliferation of RT112 and 5637 cells after treatment with specific siRNAs against G9a and DNMT1 alone or in combination. Data are represented as the mean ± s.d. n = 2 independent experiments.

Extended Data Fig. 3 Effect of EZH2 inhibition in BC cells.

a, Effect of CM-272 on the enzymatic activities of distinct human EZH2 mutants, EZH2–EED binding activity, G9a and DNMT using in vitro assays. In this study, we included the recombinant human EZH2 A677G, A738T, Y641S, P132S, Y641C, Y641F, Y641H and Y641N. b, Immunoblot showing the expression of the cited proteins or histone marks in parental 5637 cells or derivatives overexpressing 5637 EZH2 or on EZH2 knockdown (two different shRNAs denoting shEzh2#1 and shEzh2#2 were used) (left panel). A representative example of two independent experiments is shown. Full uncropped blots are available as Source data. Proliferation of parental RT112 cells or derivatives (overexpression or knockdown of EZH2) after treatment with CM-272 (right panel). Results are the mean ± s.e.m. of three independent experiments. c, Correlation between G9a and EZH2 RNA (upper panel) and protein expression (lower panel) in BC patients. Correlation was calculated using Spearman correlation analysis. n = 55 patients. dg, Proliferation of RT112 (d,e) or 5637 cells (f,g) after treatment with GSK-126 (EZH2 inhibitor) alone or in combination with A-366 (d,f) or decitabine (e,g). At several tested concentrations, the combination index is >1 according to the range of combinations indexes defined by Chou and Talalay40. The combination study set-up was based on GI50 values for the assayed molecules. The asterisks show the P values of one-tailed Mann–Whitney U-tests (*P < 0.05). The results represent the mean ± s.d. of three independent experiments.

Source data

Extended Data Fig. 4 In vitro and in vivo activity of CM-272 and CDDP in BC cells.

a, Cell proliferation of BC cells (RT112, 5637 and UMUC1) after treatment with CM-272 alone or in combination with CDDP, showing a synergistic effect (combination index <1 as defined by Chou and Talalay36). The asterisks show the P values of a one-tailed Mann–Whitney U-test (*P < 0.05). n = 3 independent experiments. The error bars indicate the s.d. b, RT112 cells (5 × 106) were subcutaneously implanted in the flanks of nude mice (5 per group). When tumors reached 150–250 mm3, mice were treated intraperitoneally with CM-272 (5 mg kg−1 5 d per week), CDDP (6 mg kg−1 once a week) or both compounds for 2 weeks. Tumor volume was related to baseline volume before treatment. Data are represented as the mean ± s.e.m. P values were determined by a one-sided Mann–Whitney U-test. c, H&E-stained and immunohistochemistry images showing the expression of the cited proteins or histone marks in tumor xenografts corresponding to the cited treatment groups. The representative images of at least 6 independent samples obtained from 3–6 different animals are shown. Scale bars, 150 µm. d, Immunoblot showing the expression of the cited proteins or histone marks in tumor xenografts corresponding to the cited treatment groups. Tubulin and total H3 levels were used to normalize loading. A representative example of two independent experiments including three independent samples from each group of mice is shown. Full uncropped blots are available as.

Source data

Extended Data Fig. 5 Genomic characterization of PtenloxP/loxP, Trp53loxP/loxP, Rb1loxP/loxPand Rbl1−/− mice.

a, Heatmap of normal bladder, tumors at early and advanced stages and visceral metastasis showing the expression of various genes associated with basal and luminal human BC molecular subtypes. b, GSEA data show the positive correlation of genes upregulated in QKO mouse tumors with upregulated genes in the human basal/squamous molecular subtype from the TCGA database, and the negative correlation of mouse upregulated genes with those upregulated in human luminal-papillary and luminal molecular subtypes. n = 5 mouse samples. c, Heatmap showing the augmented activity of PKB/Akt, ERK and S6K, together with reduced PTEN expression in specific groups of human tumors (clusters 6 and 9 according to self-organizing tree algorithm (SOTA)). d, Clusters 6 and 9 of human tumors are enriched in basal/squamous, luminal-infiltrated and neuronal subtypes. The P value was obtained using a two-sided Fisher exact test. e, Human tumors included in clusters 6 and 9 display predominant co-occurring mutations in RB1 and TP53 tumor suppressor genes. f, Human tumors included in clusters 6 and 9 display increased distant metastasis. g,h, Human tumors included in clusters 6 and 9 display increased expression of the G9a (g) and EZH2 (h) genes. Data are represented as the mean ± s.e.m. The P value was obtained using a two-sided Mann–Whitney U-test. n = 339 patients. i, Kaplan–Meyer graph showing that human patients harboring tumors included in clusters 6 and 9 display decreased survival. The P value was obtained by log-rank test. j, GSEA between bladder tumors from the transgenic mouse model and tumors treated with a combination of CM-272 and CDDP, showing reduced enrichment of the E2F and MYC target genes and in genes involved in epithelial–mesenchymal transition in treated tumors. n = 5 mouse samples. k, Unsupervised heatmap using the previously identified differentially expressed transcripts between tumors and normal bladder. l, GSEA between tumors from the transgenic mouse model (Tumor) and tumors treated with a combination of CM-272 and CDDP show increased expression in treated tumors of genes repressed by EZH2 and decreased expression in treated tumors of genes induced by epidermal growth factor receptor in mice BC tumors. n = 5 mouse samples.

Extended Data Fig. 6 CM-272 induces an immune-mediated antitumor effect in vitro in BC.

a, Gene Ontology categories upregulated in RT112 and 5637 cells after treatment with CM-272. A two-sided t-test was used. n = 6 samples. b, GSEA of 5637 cells treated with CM-272 versus untreated cells (Control) showing enrichment in the genes corresponding to interferon-α, interferon-γ and tumor necrosis factor-α via nuclear factor kappa-light-chain-enhancer of activated B cells response. A two-sided t-test was used. n = 6 samples. c, Heatmap showing gene expression changes in RT112 and 5637 BC cell lines after CM-272 treatment. d, Venn diagrams showing the overlapping transcripts down- (upper panel) or upregulated (lower panel) by CM-272 treatment in the RT112 and 5637 BC cell lines. A one-sided Fisher’s exact test was used. e, RT–qPCR of interferon-response genes in RT112 and 5637 cell lines after treatment with CM-272 for 48 h. n = 2 independent experiments. The data represent the mean value. f, ChIP with qPCR analysis of interferon-response genes in RT112 and 5637 cells treated for 48 h (700 and 880 nM, respectively). n = 2 independent experiments. The data represent the mean value. g, Immunofluorescence of dsRNAs after treatment of 5637 and RT112 cells with CM-272. A representative example of three independent experiments is shown. h, RT–qPCR analysis of endogenous retroviruses after treatment of 5637 and RT112 cells with CM-272. n = 3 independent experiments. The data represent the mean ± s.d. i, Calreticulin exposure determined by flow cytometry in RT112 cells after 48 h of treatment with 250, 500 and 1000 nM of CM-272. MFI ratio of calreticulin expression: \({\mathrm{MFI}}\,{\mathrm{ratio}}\,{\mathrm{of}}\,{\mathrm{calreticulin}}\,{\mathrm{expression}} = \frac{{{\mathrm{MFI}}\,{\mathrm{for}}\,{\mathrm{calreticulin}}\,{\mathrm{after}}\,{\mathrm{CM - 272}}\,{\mathrm{treatment}}}}{{{\mathrm{MFI}}\,{\mathrm{for}}\,{\mathrm{calreticulin}}\,{\mathrm{of}}\,{\mathrm{untreated}}\,{\mathrm{cells}}}}\). n = 4 independent experiments. The data represent the mean ± s.d. The P value was estimated using a two-tailed Mann–Whitney U-test. *P < 0.05. j, HMBG1 secretion determined by ELISA analysis in the supernatants of RT112 cells after treatment with CM-272 for 48 h. n = 4 independent experiments. The data represent the mean ± s.d. The P value was estimated using a two-tailed Mann–Whitney U-test. *P < 0.05. k, Expression of major histocompatibility genes in the RT112 and 5367 cell lines before and after treatment with CM-272. The data represent the mean ± s.e.m. The P value was obtained using a one-sided Mann–Whitney U-test. n = 3 samples. l, Expression of cited natural killer cell ligands in RT112 and 5637 cells after treatment with CM-272 for 48 h. The data represent the mean ± s.e.m. n = 6 samples.

Extended Data Fig. 7 CM-272 induces an immune-mediated antitumor effect in vivo in BC.

a, GSEA between normal bladder (Control) and CM-272 + CDDP-treated BC tumors from the transgenic mouse model showing enrichment in the genes corresponding to interferon-γ, interferon-α, inflammatory response and allograft rejection in the treated tumors. n = 4 samples. b, Expression of negative immunoregulatory cytokines and chemoattractants in BC tumors from transgenic mice treated with CM-272 + CDDP. The P value was obtained using a one-sided Mann–Whitney U-test. n = 3. c, Expression of interferon regulatory factors in BC tumors from animals treated with CM-272 and CDDP. P value was obtained using a one-sided Mann–Whitney U-test. n = 3. d, Expression of T cell activators in BC tumors from animals treated with CM-272 and CDDP. P value was obtained using a one-sided Mann–Whitney U-test. n = 3. e, Expression of NKG2D ligands and receptors in BC tumors from animals treated with CM-272 + CDDP. The P value was obtained using a one-sided Mann–Whitney U-test. n = 3. be, Results are shown as the mean ± s.e.m. of three independent experiments. f, Expression of major histocompatibility genes in BC tumors from animals treated with CM-272 + CDDP. The data represent the mean ± s.e.m. The P value was obtained using a one-sided Mann–Whitney U-test. n = 3.

Extended Data Fig. 8 Expression and epigenetic regulation analysis of PD-L1, PD-1 and CTLA4 in the RT112 cell line.

a, RT–qPCR analysis of PD-L1, PD-1 and CTLA4 in the RT112 cell line after treatment with CM-272 for 48 h. n = 3 independent experiments. The data represent the mean with regard to untreated cells. b, Flow cytometry analysis of PD-L1, PD1 and CTLA4 levels after treatment with CM-272 for 48 h. The MFI ratio is represented as: \({\mathrm{MFI}}\,{\mathrm{ratio}} = \frac{{\left( {{\mathrm{MFI}}\,{\mathrm{for}}\,{\mathrm{immune}}\,{\mathrm{checkpoint}}/{\mathrm{MFI}}\,{\mathrm{for}}\,{\mathrm{isotype}}\,{\mathrm{control}}\,{\mathrm{after}}\,{\mathrm{CM - 272}}\,{\mathrm{treatment}}} \right)}}{{\left( {{\mathrm{MFI}}\,{\mathrm{for}}\,{\mathrm{immune}}\,{\mathrm{checkpoint}}/{\mathrm{MFI}}\,{\mathrm{for}}\,{\mathrm{isotype}}\,{\mathrm{control}}\,{\mathrm{of}}\,{\mathrm{untreated}}\,{\mathrm{cells}}} \right)}}\). n = 2 independent experiments. c, Quantitative ChIP–PCR analysis of H3K9me2 levels in the PD-L1 promoter region in the RT112 cell line after treatment with CM-272 for 48 h. n = 2 independent experiments. d, DNA methylation analysis by pyrosequencing of the promoter region of PD-L1 after CM-272 treatment. The data shown are representative of two independent experiments. Control: untreated RT112 cells; CM-272: RT112 cells after CM-272 treatment; Methylation-positive control: methylation-positive control is a universally methylated DNA.

Extended Data Fig. 9 Appearance of a tumor and a metastasis in QKO mice.

a, Histological appearance of a tumor 1 month after the end of treatment with CM-272 and anti-PD-L1. a’, a’’ and a’’’ show three areas of a at high magnification. A representative example of three independent lesions is shown. b, Histological appearance of a metastatic lesion 1 month after the end of treatment with CM-272 and anti-PD-L1. b’, b’’ and b’’’ show three areas of b at high magnification. Note the presence of necrotic and highly inflamed areas close to those showing normal tumor cell appearance. a,b, scale bar, 1 mm; a’, a’’, a’’ and b’, b’’, b’’, scale bar, 200 µm. A representative example of three independent lesions is shown.

Extended Data Fig. 10 Increased expression of G9a and EZH2 in non-responders treated with anti-PD-1 immunotherapy.

Heatmap showing the supervised classification of genes bound by G9 and EZH2 in advanced urothelial carcinoma patients that responded or progressed to PD-1 treatment.

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Segovia, C., San José-Enériz, E., Munera-Maravilla, E. et al. Inhibition of a G9a/DNMT network triggers immune-mediated bladder cancer regression. Nat Med 25, 1073–1081 (2019). https://doi.org/10.1038/s41591-019-0499-y

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