Pharmacological activation of STAT1-GSDME pyroptotic circuitry reinforces epigenetic immunotherapy for hepatocellular carcinoma

Abstract

Background: Genomic screening uncovered interferon-gamma (IFNγ) pathway defects in tumours refractory to immune checkpoint blockade (ICB). However, its non-mutational regulation and reversibility for therapeutic development remain less understood.

Objective: We aimed to identify ICB resistance-associated druggable histone deacetylases (HDACs) and develop a readily translatable combination approach for patients with hepatocellular carcinoma (HCC).

Design: We correlated the prognostic outcomes of HCC patients from a pembrolizumab trial (NCT03419481) with tumourous cell expressions of all HDAC isoforms by single-cell RNA sequencing. We investigated the therapeutic efficacy and mechanism of action of selective HDAC inhibition in 4 ICB-resistant orthotopic and spontaneous models using immune profiling, single-cell multiomics and chromatin immunoprecipitation-sequencing and verified by genetic modulations and co-culture systems.

Results: HCC patients showing higher HDAC1/2/3 expressions exhibited deficient IFNγ signalling and poorer survival on ICB therapy. Transient treatment of a selective class-I HDAC inhibitor CXD101 resensitised HDAC1/2/3^high tumours to ICB therapies, resulting in CD8⁺T cell-dependent antitumour and memory T cell responses. Mechanistically, CXD101 synergised with ICB to stimulate STAT1-driven antitumour immunity through enhanced chromatin accessibility and H3K27 hyperacetylation of IFNγ-responsive genes. Intratumoural recruitment of IFNγ⁺GZMB⁺cytotoxic lymphocytes further promoted cleavage of CXD101-induced Gasdermin E (GSDME) to trigger pyroptosis in a STAT1-dependent manner. Notably, deletion of GSDME mimicked STAT1 knockout in abolishing the antitumour efficacy and survival benefit of CXD101-ICB combination therapy by thwarting both pyroptotic and IFNγ responses.

Conclusion: Our immunoepigenetic strategy harnesses IFNγ-mediated network to augment the cancer-immunity cycle, revealing a self-reinforcing STAT1-GSDME pyroptotic circuitry as the mechanistic basis for an ongoing phase-II trial to tackle ICB resistance (NCT05873244).

Keywords: HEPATOCELLULAR CARCINOMA; IMMUNOTHERAPY; INTERFERON.

Figure 1. Tumour cell-intrinsic HDACs correlate with ICB therapy efficacy and survival outcome in patients with HCC. (A) Overview of this study. (B) Dot plot depicting the relationship between the baseline expression levels of tumour cell-intrinsic HDAC isoforms and the survival outcomes of HCC patients treated with pembrolizumab. Each plot represents a p value obtained from the Kaplan-Meier survival analysis of the patients according to their baseline tumourous cell HDAC expression levels. (C) Kaplan-Meier survival analyses of patients with HCC undergone pembrolizumab treatment according to their baseline tumourous cell HDAC1, HDAC2 and HDAC3 expression levels. (D) Expression levels of HDAC1, HDAC2 and HDAC3 in tumour cells of the patients according to pembrolizumab treatment outcomes. (E) GSEA of the genes between the HDAC1/2/3^high and HDAC1/2/3^low tumour cells. Tumour cells were stratified by top (high) and bottom (low) 25% based on the expression levels of HDAC1, HDAC2 or HDAC3. (F, G) TCGA HCC samples with high (n=92) and low (n=92) mRNA levels of HDAC1, HDAC2 or HDAC3 stratified by top and bottom 25% in 369 patients were selected for subsequent analysis. (F) Prediction of potential clinical ICB response in patients between the HDAC1/2/3^high and HDAC1/2/3^low tumour cells using the TIDE signature. R, responders; NR, non-responders. (G) Analysis of TIDE, T cell exclusion and MDSC scores by TIDE algorithm in patients between the HDAC1/2/3^high and HDAC1/2/3^low tumour cells. Statistical significance was assessed by two-sided log-rank (Mantel-Cox) test for (B, C), by Wilcoxon rank sum test for (D), by Kolmogorov-Smirnov test for (E), by two-sided χ² test for (F) or by unpaired two-tailed Student’s t-test for (G). ***p<0.001; ****p<0.0001. HCC, hepatocellular carcinoma; HDAC, histone deacetylase; ICB, immune checkpoint blockade; MDSC, myeloid-derived suppressor cell; NR, non-responders; R, responders; TIDE, Tumour Immune Dysfunction and Exclusion.

Figure 2. A selective class-I HDAC inhibitor overcomes ICB resistance in HCC models. (A) Western blot analysis of HDAC1, HDAC2 and HDAC3 levels in orthotopic Hepa1-6-PD-L1R (left) and RIL-175-PD-1R (right) tumour and non-tumour liver tissues isolated from mice. GAPDH was used as loading control. (B) Treatment schedule of CXD101 and anti-PD-(L)1 antibody in C57BL/6 mice bearing PD-(L)1R tumours. (C, D) Tumour weights and representative images of livers and tumours from indicated groups in Hepa1-6-PD-L1R (C) and RIL-175-PD-1R (D) models (n=8–10). (E) Representative CD45 immunohistochemistry images and statistical analysis of positively stained cells in Hepa1-6-PD-L1R tumours from indicated groups (n=4). Scale bars, 100 µm. Black boxes indicate regions shown in enlarged inset. (F) Proportions (%) of tumour infiltrating CD45⁺cells, CD45⁺CD3⁺CD8⁺T cells, CD45⁺CD3⁺CD4⁺T cells, CD45⁺NK1.1⁺NK cells, CD45⁺CD3⁺NK1.1⁺NKT cells, CD45⁺CD11b⁺Gr1⁺MDSCs, CD45⁺CD11b⁺Gr1⁻CD11c⁺dendritic cells, CD45⁺CD11b⁺Gr1⁻F4/80⁺Ly6C⁻ macrophages of total cells in Hepa1-6-PD-L1R tumours from indicated groups were determined by flow cytometry (n=7–9). (G) Correlation analysis between tumour weights and the proportions of indicated cells in Hepa1-6-PD-L1R tumours. (H) Representative flow cytometry dot plots and proportions of IFNγ⁺ or GZMB⁺ cells in tumour infiltrating CD8⁺T cells from indicated groups in Hepa1-6-PD-L1R model (n=7–9). (I, J) Kaplan-Meier survival analysis of mice from indicated groups in RIL-175-PD-1R (I) and Hepa1-6-PD-L1R (J) ICB-resistant models (n=12–15). (K) Combination therapy cured mice (from J) and naïve mice with same age were challenged with Hepa1-6-PD-L1R cells at 140 days after initial tumour cell inoculation (n=7). Kaplan-Meier survival analysis of mice from indicated groups are shown. (L) Representative flow cytometry dot plots and proportions of CD8⁺CD44⁺CCR7⁺CD62L⁺central memory CD8⁺T cells (CD8⁺T_CM) and CD8⁺CD44⁺CCR7⁻CD62L⁻effector memory CD8⁺T cells (CD8⁺T_EM) in circulating CD8⁺T cells, and the corresponding CD4⁺T_CM and CD4⁺T_EM in circulating CD4⁺T cells from combination therapy cured mice and naive mice (n=7). Data are represented as mean±SD. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparisons correction for (C–F, H), by single-tailed Pearson’s correlation for (G), by two-sided log-rank (Mantel-Cox) test for (I–K) or by unpaired two-tailed Student’s t-test for (L). ns, no significance; *p<0.05; **p<0.01; ***p<0.001. ANOVA, analysis of variance; HCC, hepatocellular carcinoma; HDAC, histone deacetylase; ICB, immune checkpoint blockade.

Figure 3. Single-cell multiomics reveals reactivation of IFNγ/STAT1 signalling by CXD101-ICB combination therapy. (A) Treatment schedule of CXD101 and anti-PD-L1 antibody in mice bearing Hepa1-6-PD-L1R tumours for single-cell multiomics analysis. Fresh tumour tissues from three representative mice per group were employed to avoid the interindividual variability. (B) t-SNE plot showing identified cell clusters within tumour from all merged groups. Cell annotations were derived from RNA analysis according to representative lineage markers. (C) Dot plot showing the RNA expression levels of representative marker genes in annotated cell clusters. (D) Visualisation of the pseudo bulk chromatin accessibility tracks of representative marker gene loci in annotated cell clusters. (E) Distribution fraction of distinct cells in the indicated groups. (F, G) t-SNE plot showing the identified subclusters of tumour cells from RNA (F) and ATAC (G). (H, I) t-SNE plots (H) and proportions (I) of the identified tumour sub-clusters in indicated groups from RNA analysis. (J, K) t-SNE plots (J) and proportions (K) of identified tumour subclusters in indicated groups from ATAC analysis. (L, M) Pathway enrichment of differentially expressed genes for RNA-based cluster 6 (C6 (RNA)) (L) and ATAC-based cluster 6 (C6 (ATAC)) (M), respectively. (N) Overlapping genes between C6 (RNA) and C6 (ATAC). (O) Pathway enrichment of overlapping genes in (N). (P) TRRUST analysis showing the key transcription factors for regulating the overlapping genes. (Q) Violin plots showing the RNA expression levels of Stat1 and Irf1 across the six tumour cell subclusters based on scRNA-seq analysis. (R) GSEA of the genes in combination group versus control group. (S, T) Heatmap for the RNA expression (S) and chromatin accessibility (T) levels of the top 30 IRGs (upregulated in combination therapy-treated group) in tumour cells from indicated groups. (U) Transcription factor-binding motif enrichment analysis showing the key transcription factors in tumour cells of the indicated groups versus control group. ICB, immune checkpoint blockade; TRRUST, Transcriptional Regulatory Relationships Unravelled by Sentence-based Text mining.

Figure 4. CXD101-induced histone hyperacetylation primes IRG activation in response to IFNγ. (A, B) Heatmaps depicting the H3K27ac intensity in genome-wide scale (A) and in IRGs (B) in tumour tissues from the indicated groups as shown in figure 3A. (C, D) Overlapping tracks of ATAC-seq and nanoscale ChIP-seq (H3K27ac, H3K4me1 and H3K4me3) at the Cd74 (C) and Stat1 (D) loci. ATAC-seq data were extracted from the in vivo scATAC-seq of tumour cells. (E) Western blot analysis of STAT1 and p-STAT1 levels in Hepa1-6-PD-L1R cells treated with the indicated concentrations of CXD101 or vehicle control in the presence or absence of IFNγ (10 ng/mL) for 48 hours. GAPDH was used as loading control. (F) RT-qPCR analyses of mRNA levels of the indicated genes in Hepa1-6-PD-L1R cells treated with CXD101 (2 µM) or vehicle control in the presence or absence of IFNγ (10 ng/mL) for 48 hours. Gapdh was used as normalisation control. (G) Western blot analysis of STAT1 levels in Hepa1-6 derived PD-L1R-WT and STAT1-KO cells treated with IFNγ (10 ng/mL) for 48 hours. GAPDH was used as loading control. (H) ChIP-qPCR analyses of H3K27ac occupancy in the enhancer and promoter regions of Stat1 in Hepa1-6 derived PD-L1R-WT or STAT1-KO cells treated with CXD101 (2 µM) or vehicle control in the presence or absence of IFNγ (10 ng/mL) for 48 hours. Stat1 enhancer (site 1) and promoter (site 2) loci for ChIP-qPCR analysis as shown in figure 4D. (I) RT-qPCR analyses of mRNA levels of the indicated genes in Hepa1-6 derived PD-L1R-WT or STAT1-KO cells treated with CXD101 (2 µM) or vehicle control in the presence or absence of IFNγ (10 ng/mL) for 48 hours. Gapdh was used as normalisation control. Data are represented as mean±SEM. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparisons correction. *p<0.05; **p<0.01; ***p<0.001. ANOVA, analysis of variance; ChIP, chromatin immunoprecipitation.

Figure 5. CXD101 and IFNγ/STAT1 signalling coordinate CD8⁺T cell-induced pyroptosis. (A) Schematic illustration of CD8⁺T cell/tumour cell co-culture cytotoxicity system. (B–D) Hepa1-6-PD-L1R cells were pretreated with CXD101 (2 µM) or vehicle control in the presence or absence of IFNγ (10 ng/mL) for 42 hours. CD8⁺T cells at an effector/target ratio (E/T) of 10:1 were co-cultured with drug washed out cells for 6 hours. Cell viabilities (B) morphological changes (C) and LDH release (D) of tumour cells were measured by the corresponding assays. (C) Red arrowheads indicate pyroptotic cells, while blue arrowheads indicate representative CD8⁺T cells binding on pyroptotic tumour cells. Scale bar=20 µm. (E–G) Hepa1-6 derived PD-L1R-WT or STAT1-KO cells were pretreated with CXD101 (2 µM) or vehicle control in the presence or absence of IFNγ (10 ng/mL) for 42 hours, CD8⁺T cells at an E/T of 10:1 were co-cultured with drug washed out cells for 6 hours. Cell viabilities (E) morphological changes (F) and LDH release (G) of tumour cells were measured by the corresponding assays. (F) Red arrowheads indicate pyroptotic cells, scale bar=20 µm. (H) Hepa1-6-PD-L1R cells were pre-treated with CXD101 (2 µM) or vehicle control in the presence or absence of IFNγ (10 ng/mL) for 42 hours. CD8⁺T cells at an E/T of 10:1 were co-cultured with drug washed out cells for 6 hours, followed by Western blot analysis of the indicated proteins. (I) Hepa1-6-PD-L1R cells were treated with DMSO or CXD101 at the indicated concentrations for 48 hours, the Gsdme mRNA and GSDME protein levels were measured by RT-qPCR and Western blot assays, respectively. (J) ChIP-qPCR analysis of H3K27ac occupancy in the enhancer and promoter regions of Gsdme in Hepa1-6-PD-L1R cells treated with CXD101 (2 µM) or vehicle control for 48 hours. Gsdme enhancer and promoter loci for ChIP-qPCR analysis are shown in online supplemental figure 18. (K) Hepa1-6 derived PD-L1R-WT or STAT1-KO cells were pretreated with CXD101 (2 µM) or vehicle control in the presence or absence of IFNγ (10 ng/mL) for 42 hours. CD8⁺T cells at an E/T of 10:1 were co-cultured with drug washed out cells for 6 hours, followed by Western blot analysis of the indicated proteins. Data are represented as mean±SEM. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparisons correction. *p<0.05; **p<0.01; ***p<0.001. ANOVA, analysis of variance; ChIP, chromatin immunoprecipitation; LDH, lactate dehydrogenase.

Figure 6. Epigenetic upregulation of GSDME renders CTL-induced tumour cell pyroptosis. (A) Hepa1-6-PD-L1R cells were pretreated with DMSO or CXD101 at 2 µM for 42 hours. NK92 cells at an E/T of 2:1 were co-cultured with drug washed out cells for 5 hours. The representative images are shown. Red arrowheads indicate pyroptotic cells. Scale bar=20 µm. (B) Hepa1-6-PD-L1R cells were pretreated with DMSO or CXD101 at 2 µM for 42 hours. NK92 cells at indicated E/T were co-cultured with drug washed out cells for 5 hours. The LDH release levels of tumour cells are shown. (C–E) Hepa1-6 derived PD-L1R-empty vector (EV) and GSDME-overexpressed (OE) cells were co-cultured with NK92 cells at an E/T of 2:1 for 5 hours. The indicated protein levels (C) morphological changes (D) and LDH release (E) of tumour cells were measured by the corresponding assays. (D) Red arrowheads indicate pyroptotic cells. Scale bar=20 µm. (F) Hepa1-6-PD-L1R cells were pretreated with DMSO or CXD101 at 2 µM for 42 hours. NK92 cells at an E/T of 2:1 were co-cultured with drug washed out cells for the indicated times, followed by Western blot analysis of the indicated proteins. (G) Hepa1-6 derived PD-L1R-WT or GSDME-KO cells were pretreated with DMSO or CXD101 at 2 µM for 42 hours. NK92 cells at an E/T of 2:1 were co-cultured with drug washed out cells for 5 hours. The indicated protein levels (G) LDH release (H) and morphological changes (I) of tumour cells were measured by the corresponding assays. #, non-specific bands. (I) Red arrowheads indicate pyroptotic cells. Scale bar=20 µm. (J, K) The total and cleaved GSDME levels in Hepa1-6-PD-L1R (J) and RIL-175-PD-1R (K) tumours from indicated groups were measured by Western blot assays. GAPDH was used as loading control. (L, M) The proportions of CD45^-7AAD⁺Annexin-V⁺cells in Hepa1-6-PD-L1R (L) and RIL-175-PD-1R (M) tumours from the indicated groups were detected by flow cytometry assay (n=5–8). (N, O) HMGB1 levels in Hepa1-6-PD-L1R (N) and RIL-175-PD-1R (O) tumours from the indicated groups are shown (n=6–9). Data are represented as mean±SEM (B, E, H) or SD (L–O). Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparisons correction. **p<0.01; ***p<0.001. 7-AAD, 7-aminoactinomycin; ANOVA, analysis of variance; LDH, lactate dehydrogenase.

Figure 7. Deletion of tumourous STAT1 or GSDME abolishes the antitumour effects of CXD101 and anti-PD-L1 combination therapy. (A) Treatment schedule of CXD101 and anti-PD-L1 antibody in C57BL/6 mice bearing the indicated Hepa1-6-PD-L1R tumours. (B) Tumour weights and representative images of livers and tumours from indicated groups are shown (n=9–12). (C) RT-qPCR analyses of mRNA levels of Stat1 and Gsdme in tumours from the indicated groups (n=7–8). (D) The proportions of CD45⁻7AAD⁺Annexin-V⁺cells in tumours from the indicated groups were detected by flow cytometry assay (n=8–9). (E) IFNγ levels in tumours from the indicated groups were measured by ELISA assay (n=6–7). (F) RT-qPCR analyses of mRNA levels of the indicated genes in tumours from indicated groups (n=7–8). (G) Proportions (%) of tumour infiltrating CD45⁺cells, CD45⁺CD3⁺CD8⁺T cells, CD45⁺CD3⁺CD4⁺T cells, CD45⁺NK1.1⁺NK cells and CD45⁺CD3⁺NK1.1⁺NKT cells of total cells in the indicated tumours were determined by flow cytometry (n=8–10). (H) Representative flow cytometry dot plots and proportions of IFNγ⁺ or GZMB⁺ cells in tumour infiltrating CD8⁺T cells from the indicated groups are shown (n=8–10). (I) Kaplan-Meier survival analysis of mice from the indicated groups (n=10–12). Data are represented as mean±SD. Statistical significance was assessed by one-way ANOVA with Holm-Sidak’s multiple comparisons correction. *p<0.05; **p<0.01; ***p<0.001. ANOVA, analysis of variance.

Figure 8. CXD101 synergies with anti-PD-L1 antibody to suppress tumourigenicity in spontaneous HDAC1/2/3^high HCC model. (A) mRNA levels of Hdac1, Hdac2 and Hdac3 in normal livers or the indicated HCC tumours were extracted from the published RNA-seq data (GSE148379). (B) Western blot analysis of HDAC1, HDAC2 and HDAC3 protein levels in MYC^high and CTNNB1^mut tumour and non-tumour liver tissues isolated from mice. GAPDH was used as loading control. (C) Treatment schedule of CXD101 and anti-PD-L1 antibody in C57BL/6 mice bearing MYC^high and CTNNB1^mut tumours by HDTVi of the indicated plasmids. (D) Representative photos (top) and H&E staining images (bottom) of livers and tumours from the indicated groups are shown. Scale bars, 1000 µm. Tumour areas are circled by black dotted lines. (E) Tumour burden in indicated groups was evaluated by liver weight, liver weight vs body weight ratio and tumour area per slide calculated from H&E images. (F) Kaplan-Meier survival analysis of mice from the indicated groups in MYC^high and CTNNB1^mut HCC model (n=13–14). (G) A summary schematic of this study. Data are represented as mean±SD. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparisons correction for (E) and by two-sided log-rank (Mantel-Cox) test for (F). *p<0.05, **p<0.01, ***p<0.001. ANOVA, analysis of variance; HCC, hepatocellular carcinoma; HDTVi, hydrodynamic tail-vein injection.