IMMU-43. Immune-fate reprogramming of GBM creates a pyroptosis-fueled immunotherapy platform enhanced by DNA-disrupting agents

Abstract BACKGROUND GBM resists immunotherapy because its “cold” TME excludes T cells. We developed a computationally directed gene therapy that reprograms GBM cells into induced antigen-presenting cells (iAPCs), unexpectedly triggering a striking upregulation of the pyroptotic inflammasome machinery. We hypothesized that coupling iAPCs with DNA-disrupting agents––temozolomide (TMZ) or Tumor-Treating Fields (TTFields)––would provoke pyroptotic immunogenic cell death (ICD) and boost anti-tumor immunity. METHODS Two cell-fate-determinant combinations—murine mPF (mPu.1/Irf8/Id2/Batf3) and human hPI (hPU.1/IKZF1)—were delivered via viral vectors into GBM cells. Pyroptosis-related genes (e.g., GSDMD, AIM2, Caspase-1) were measured by single-cell RNA-seq. Both iAPCs and control GBM cells were then treated with TMZ or TTFields. Pyroptosis was quantified by morphology (cell swelling, “ghost” cells), activated caspase-1 (p10), and live-cell enumeration. In vivo, syngeneic orthotopic GBM mouse models received mPF+TMZ, mPF alone, or TMZ alone with survival as the primary endpoint. RESULTS iAPC conversion instigated a pronounced surge in pyroptosis-related gene expression to levels rivaling or exceeding natural APC controls––AIM2 (3.8-fold vs controls), Caspase-1 (4.4-fold), GSDMD (0.96-fold). Upon TMZ or TTFields exposure, iAPCs displayed classic pyroptotic morphology, 2.5-6fold higher caspase-1 activity (p=0.0292), and 6.5-20fold higher pyroptotic death (p=0.0031) than controls, confirming that DNA-disrupting stress synergizes with iAPCs to drive pyroptosis. In vivo, mPF+TMZ extended survival compared to mPF alone (HR 0.3439; p=0.0058) or TMZ alone (HR 0.2864; p=0.0126). Notably, 30% mPF+TMZ animals died without detectable tumors, indicating excessive CNS swelling. CONCLUSIONS iAPC conversion unleashes a potent pyroptotic program that synergizes with DNA-disrupting therapies to produce in-situ immunization—merging ICD with antigen presentation to elicit durable T cell responses and prolong survival. Excessive neuroinflammation in some animals underscores the need for refined immunomodulation. We are developing a next-generation gene therapy that co-produces iAPCs with PD-1 and VEGF decoys to sustain anti-tumor T-cell responses while mitigating treatment-related cerebral edema.