Metabolic Inhibition Induces Pyroptosis in Uveal Melanoma

Abstract

Few treatment options are available for patients with metastatic uveal melanoma. Although the bispecific tebentafusp is FDA approved, immunotherapy has largely failed, likely given the poorly immunogenic nature of uveal melanoma. Treatment options that improve the recognition of uveal melanoma by the immune system may be key to reducing disease burden. We investigated whether uveal melanoma has the ability to undergo pyroptosis, a form of immunogenic cell death. Publicly available patient data and cell line analysis showed that uveal melanoma expressed the machinery needed for pyroptosis, including gasdermins D and E (GSDMD and E), caspases 1, 3, 4, and 8, and ninjurin-1. We induced cleavage of GSDMs in uveal melanoma cell lines treated with metabolic inhibitors. In particular, the carnitine palmitoyltransferase 1 (CPT1) inhibitor, etomoxir, induced propidium iodide uptake, caspase 3 cleavage, and the release of HMGB1 and IL-1β, indicating that the observed cleavage of GSDMs led to pyroptosis. Importantly, a gene signature reflecting CPT1A activity correlated with poor prognosis in patients with uveal melanoma and knockdown of CPT1A also induced pyroptosis. Etomoxir-induced pyroptosis was dependent on GSDME but not on GSDMD, and a pyroptosis gene signature correlated with immune infiltration and improved response to immune checkpoint blockade in a set of patients with uveal melanoma. Together, these data show that metabolic inhibitors can induce pyroptosis in uveal melanoma cell lines, potentially offering an approach to enhance inflammation-mediated immune targeting in patients with metastatic uveal melanoma. Implications: Induction of pyroptosis by metabolic inhibition may alter the tumor immune microenvironment and improve the efficacy of immunotherapy in uveal melanoma.

Figure 1.. Pyroptotic gene expression in uveal melanoma.

(A-C) mRNA expression of GSDMD, GSDME, and NINJ1 (A-C, respectively) across TCGA tumor types, ordered by median (Log2 RSEM, Batch normalized from Illumina HiSeq_RNA-SeqV2). (D) Western blots of BAP1, GSDMD, GSDME and caspases CASP1, CASP3, CASP4, CASP8, and loading controls (HSP90, β-actin, or vinculin) in UM cell line lysates.

Figure 2.. Metabolic inhibitors induce gasdermin cleavage in UM cell lines.

(A) UM cell lines MP38, MP46, and MP65 were treated with 100 μM etomoxir (ETO), 50 nM IACS, 40 μM WZB117 (WZB), and 100 μM 6-aminonicotinamide (6AN) for 48 hours. Western blots of GSDME, GSDMD, and loading controls (vinculin and HSP90, n = 3). Bands corresponding to full-length or cleaved N-terminal gasdermin domains are denoted ‘FL’ and ‘NT’ and the GSDMD 43 kDa band is denoted ‘~43 kDa’. (B-C) Quantification of cleaved N-terminal gasdermin domains in the treated UM cell lines from panel A (significant p-values are displayed and non-significant p-values denoted ‘n.s.’ or not shown, one-way ANOVA and Tukey’s HSD, n = 3).

Figure 3.. High CPT1A-activity scores correlate with high-risk indicators and poor prognosis in UM.

Gene set variation analysis was performed on the indicated UM RNA-seq data set with a gene signature representing ‘CPT1A-activity’ (CPT1A-inhib. down signature score, see Table S1) (27). (A) Violin plots of CPT1A-activity scores generated from UM single cell RNA-seq data (GSE139829) grouped by BAP1 status (BAP1-WT = red/pink, BAP1-deficient = blue/green). A Welch’s two sample t-test was performed using the median GSVA score value per patient to determine statistical difference between BAP1-WT and BAP1-Def groups (p-value indicated). (B) Boxplots of GSVA CPT1A-activity scores from (TCGA-UVM) grouped by somatic copy number alteration (SCNA) molecular subtypes (24) 1 and 2 (BAP1-WT = red/pink, n = 38) vs 3 and 4 (BAP1-deficient = blue/green n = 42, two sample t-test with Bonferroni adjustment, p-value indicated). (C) Kaplan-Meier plot and log-rank p value for clinical event of UM metastasis among CPT1A activity clusters with increasing CPT1A ‘activity’ scores (clusters 1 to 3, determined by unsupervised k-means clustering, see Fig. S1 and Table S1). The number of cases and UM metastasis events for each cluster are indicated and tick marks correspond to censoring events (date of last follow-up).

Figure 4.. Etomoxir induces pyroptosis in UM cell lines.

(A+D) Representative phase images merged with propidium iodide staining (PI, red) of UM cell lines MP38 (A) or MP46 (D) taken 72 hours post treatment with vehicle control (DMSO) or 75 μM etomoxir (ETO). (B+E) Line plots representing the portion of PI positive area (PI⁺ area) relative to total confluence (phase area) over time in UM cell lines MP38 (B) or MP46 (E) under treatment with vehicle control (DMSO, blue lines), or increasing doses of ETO (50–100 μM, light to dark red lines) as determined by Incucyte-S3 analysis (mean centered across experiments, n = 5). (C+F) Area under the curve (AUC) of Incucyte-S3 data in B+E representing the sum PI⁺ area relative to total confluence (PI+/Phase) over 72 hours of treatment for MP38 (C) and MP46 (F, p-values indicated, one-way ANOVA and Tukey’s HSD, n = 5). (G) Cleavage of GSDME and GSDMD, PARP, or CASP-3 was assessed by western blots of whole-cell lysates prepared from MP38 and MP46 cells treated with vehicle control (DMSO) or ETO for 72 hours at the indicated concentration. (H). Levels of inflammatory proteins, HMGB1 and IL-1β, in supernatants from cell treatments in G were assessed by western blot. Ponceau-S (Pon-S) staining was used as a loading control (n = 3).

Figure 5.. CPT1A knockdown induces pyroptosis in UM cell lines.

MP38 and MP65 UM cell lines were transfected with non-targeting control (siControl) or CPT1A-targeting siRNAs (siCPT1A #1, siCPT1A #2) for 72 hours prior to culture in serum-free media for duration of the experiment. (A) Representative phase images merged with propidium iodide staining (PI, red) of UM cell lines MP38 (left panel) or MP65 (right panel) after 72 hours. (B) AUC boxplots representing the sum of PI positive area (PI⁺ area) relative to total confluence (PI+/Phase) over 72 hours in UM cell lines MP38 (left) or MP65 (right) as determined by Incucyte-S3 analysis (mean centered across experiments, p-values indicated, one-way ANOVA and Tukey’s HSD, n = 3). (C-D) Cleavage of GSDME and GSDMD, and CPT1A expression were assessed by western blots from whole cell lysates collected from MP38 or MP65 cells after 48 hours. HSP90 serves as loading control. Secreted HMGB1 in was assessed by western blot and quantified by densitometry normalized to Coomassie Blue staining loading control (mean centered across experiments, p-values indicated, one-way ANOVA and Tukey’s HSD, n = 3).

Figure 6.. Etomoxir-induced pyroptotic DAMP release is dependent on GSDME.

UM cell lines were transfected with non-targeting control (siControl), GSDME-targeting or GSDMD targeting siRNA for 72 hours prior to treatment with vehicle control (DMSO) or 75 μM etomoxir (ETO). (A) Representative phase images merged with propidium iodide staining (PI, red) of UM cell lines MP38 (upper panels) or MP46 (lower panels) 48 hours after treatment. (B) Area under the curve (AUC) of Incucyte-S3 data after treatment with etomoxir for MP38 (left plot, first 48 hours, p-values indicated, n = 4) or MP46 (right plot, first 72 hours, p-values indicated, n = 3). Data in ‘B’ were analyzed with a robust two-way ANOVA model with MM-type estimation (see methods). (C-D) Cleavage or knockdown efficiency of GSDME was assessed by western blot of whole-cell lysates (upper blots) in MP38 (left panels) and MP65 (right panels) cell lines. Levels of HMGB1 in supernatants were assessed by western blot using Ponceau-S (Pon-S) staining as a loading control (lower blots) and quantified by densitometry in panel ‘D’ for MP38 and MP65 (p-values indicated, Two-way ANOVA and Tukey’s HSD, n = 3).

Figure 7.. Pyroptosis gene-signature scores correlate with improved response to immune checkpoint blockade in UM.

Gene set variation analysis (GSVA) was performed on RNA-seq data from matched UM patient samples before (pretreatment) and during (on-treatment) a new course of immune checkpoint blockade (ICB, described in detail in Table S2) (29) using immune cell gene sets (28) or pyroptosis gene-signature (‘PScore’) (12). (A) Heatmap of GSVA scores corresponding to PScore and significantly correlated immune signatures are shown in matched UM patient samples (patient number 1–7) grouped by treatment and disease response status (progressive disease = red/pink, stable disease = blue/green). Correlation between PScore and immune signatures are shown (left side, Linear mixed effect model correlation, FDR-adjusted p-values indicated). (B) Boxplots of PScore overlayed with line-slope plots indicating PScore response to ICB treatment in matched patient samples with progressive (red/pink, n = 3) or stable (blue/green, n = 4) disease (p-values indicated), LME modeling was used for statistical analysis (see methods).