IMMU-47. The impact of ASC expression and NLRP1 inflammasome signaling in glioblastoma

Abstract BACKGROUND The ASC protein (apoptosis-associated speck-like protein containing a CARD (caspase recruitment domain)) was first characterized as a potential tumor suppressor in the context of breast cancer. Subsequently, varying effects of ASC expression were observed across different tumor types. Despite the discovery of ASC in the field of oncology, most recent research has focused on ASC from an immunological perspective, elucidating its function as an adaptor protein essential for NLRP (nucleotide-binding domain and leucine-rich repeat containing protein) inflammasome assembly. In glioblastoma, IDH-wildtype (CNS WHO grade 4) (GBM), the implication of ASC expression and inflammasome functionality remains largely unexplored. METHODS In a translational approach, we first investigated the regulation of ASC expression and its impact on NLRP1 inflammasome functionality in three independent patient-derived GBM cell lines by western blot, ELISA, flow cytometry, qRT-PCR, and live cell imaging. Next, we prospectively analyzed GBM tumor tissue samples of 134 patients to assess ASC expression in the tumor cell compartment and characterized the tumor microenvironment using immunohistochemistry and bulk RNA sequencing. RESULTS In vitro assays revealed that most GBM cell lines express all proteins required for NLRP1 inflammasome cascade execution, except for ASC. Re-expression of ASC via lentiviral transduction of ASC-deficient GBM cells boosted NLRP1 inflammasome signaling. Upon NLRP1 activation, ASC-re-expressing GBM cells presented immune cell-like behaviors, marked by a significant release of the pro-inflammatory cytokine IL-1β, ASC speck formation, and subsequent pyroptotic cell death. In GBM tissue, 69.4% of cases were ASC-deficient, whereas 30.6% of cases possessed the ability of ASC expression by tumor cells. ASC-competent GBM cases were associated with significantly increased intratumoral infiltration and clustering of macrophages and microglia. CONCLUSION Our findings demonstrate that, in the presence of ASC, GBM tumor cells highly engage in inflammasome-mediated immune signaling, thereby substantially modulating the tumor microenvironment and promoting tumor cell clearance through pyroptosis.