Teresa Fernandes-Alnemri

WNK1 [with no lysine (K)] is a chloride- and potassium-sensitive kinase that regulates cellular ion homeostasis. Although WNK1 negatively regulates the NLRP3 and AIM2 inflammasomes, its role in pyrin inflammasome activation has remained unknown. Here, we identify WNK1 as a critical regulator of pyrin inflammasome assembly. In macrophages, genetic or pharmacologic inhibition of WNK1 suppresses pyrin activation without affecting pyrin dephosphorylation, demonstrating that WNK1 acts downstream of the canonical licensing step. This function occurs largely independently of the canonical SPAK/OXSR1 pathway. Mechanistically, WNK1 regulates microtubule dynamics: Its inhibition stabilizes microtubules, reduces plus-end growth, and impairs pyrin inflammasome assembly. Extracellular ionic conditions bidirectionally regulate this pathway, with hypotonic stress enhancing WNK1 activity and pyrin activation, whereas elevated extracellular potassium chloride suppresses WNK1 activity and inhibits pyrin assembly independently of osmotic stress. Together, these findings define an ion-sensitive WNK1-microtubule axis linking ionic signals to cytoskeletal dynamics to control pyrin inflammasome assembly and explain how WNK1 exerts opposing effects on distinct inflammasome pathways.

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

Figure S4 from Pyroptosis Modulates Multiple Immune Cell Populations in Targeted Therapy–Treated Melanoma

Figure S2 from Pyroptosis Modulates Multiple Immune Cell Populations in Targeted Therapy–Treated Melanoma

Figure S5 from Pyroptosis Modulates Multiple Immune Cell Populations in Targeted Therapy–Treated Melanoma

Uncropped western blots from Pyroptosis Modulates Multiple Immune Cell Populations in Targeted Therapy–Treated Melanoma