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 (scRNA-seq) and flow cytometry, we show that Gsdme knockout (KO) tumors have decreased infiltration of T cells, natural killer (NK) cells and regulatory T cells (Tregs) following BRAFi + MEKi-treatment compared to control tumors. Infiltrated Tregs in Gsdme KO tumors displayed decreased expression of the interleukin 2 receptor and phenotypic markers that are associated with a suppressive function of Tregs. Furthermore, intratumoral, phenotypically suppressive Tregs were decreased after BRAFi + MEKi treatment in Gsdme KO tumors expressing a pyroptosis-defective mutant form of Gsdme (T6E) compared to tumors expressing wild-type Gsdme. Use of a TLR9 agonist, which alters the tumor immune microenvironment, decreased the regrowth of Gsdme KO tumors on BRAFi + MEKi treatment, correlating to a further decrease in intratumoral Tregs. Overall, we show a critical role of GSDME in the modulation of intratumoral immune cells, particularly Tregs, in BRAFi + MEKi-treated melanoma. Overall design: YUMM1.7 CTL and Gsdme KO cells were injected intradermally into the shaved right flank of C57BL/6J mice. When tumors reached approximately 100-200 mm3, animals were administered BRAFi + MEKi or vehicle control chow. On day 4, BRAFi + MEKi-treated tumors (n=5) and control-treated tumors (n=3) were harvested and pooled into single samples.