Timothy J. Purwin

Figure S2. CPT1A ‘activity-signature’ score (GSVA) K-Means clustering and association with SCNA clusters in TCGA-UVM data.

Table S1. PScore and CPT1A ‘activity’ signature gene sets.

Figure S3. Etomoxir-induced, pyroptotic DAMP release does not require GSDMD.

Figure S1. GSDMD cleavage fragments induced by Etomoxir.

Table S2. UM patient ICb treatment groups in figure 7.

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 S3 from Pyroptosis Modulates Multiple Immune Cell Populations in Targeted Therapy–Treated Melanoma

Figure S1 from Pyroptosis Modulates Multiple Immune Cell Populations in Targeted Therapy–Treated Melanoma

Treatment of melanoma with BRAF inhibitors plus MEK inhibitors (BRAFi + MEKi) stimulates an intratumoral immune response, in part through pyroptosis mediated by the pore-forming protein gasdermin E (GSDME/Gsdme). How GSDME mediates effects on tumoral immunity is not well characterized. Using single-cell RNA sequencing and flow cytometry in BRAFi + MEKi–treated melanoma, we show herein that isogenic Gsdme knockout (KO) tumors show decreased infiltration with T cells, natural killer (NK) cells, and regulatory T cells (Treg) compared with control tumors. Infiltrated Tregs in Gsdme KO tumors displayed decreased expression of the IL2 receptor and phenotypic markers associated with suppressive function. Furthermore, intratumoral frequency of phenotypically suppressive Tregs was decreased after BRAFi + MEKi treatment in Gsdme KO tumors engineered to express a pyroptosis-defective mutant form of Gsdme (T6E) compared with Gsdme KO tumors engineered to reexpress wild-type Gsdme. Combining BRAFi + MEKi with a TLR9 agonist limited the regrowth of Gsdme-deficient tumors, and this was associated with a further reduction in intratumoral Tregs. Overall, we show a critical role of GSDME in the modulation of intratumoral immune cells in BRAFi + MEKi–treated melanoma.

Figure S6 from Pyroptosis Modulates Multiple Immune Cell Populations in Targeted Therapy–Treated Melanoma