Enhanced glioblastoma immunotherapy via SMAC mimetic dose escalation and TGFβ blockade.

Background: Glioblastoma (GBM) is the most common primary brain tumor with an overall survival under 21 months. Despite extensive research effort, patient outcomes have improved minimally over the past several decades. The Inhibitor of Apoptosis (IAP) proteins are critical survival factors implicated in both immune regulation and gliomagenesis. Small molecule IAP antagonists called SMAC mimetic compounds (SMCs) are under investigation as cancer therapeutics across multiple malignancies, including GBM. SMCs induce GBM cell death in the presence of inflammatory cytokines, synergize with immune checkpoint inhibitors (ICI), and induce death of microglia and macrophages. Although SMCs show significant efficacy in murine models, complete eradication is not achieved. Here, we aimed to understand the limitations of SMCs in murine GBM and identify strategies to enhance efficacy of combination treatment with ICIs with the goal of informing future translational efforts. Methods: We use animal models, co-culture systems, flow cytometry, and multiplex immunohistochemistry to optimize SMC dosing and delivery, uncovering resistance mechanisms that address key unmet research needs. Results: We demonstrate that although GBM cells are immunologically recognizable, their location within the central nervous system (CNS) limits effective anti-GBM immunity following SMC and ICI combination therapy. Increasing SMC dose potently improves overall survival, which is associated with reduced intratumoral macrophage content, increased microglial involvement, and peripheral immunoactivation. Given the immunosuppressive role of TGFβ, the incorporation of TGFβ blockade further enhances survival outcomes. Conclusion: We comprehensively outline how SMCs can be used in conjunction with ICIs to treat GBM and propose strategies to maximize SMC efficacy.