The reprogramming impact of SMAC-mimetic on glioblastoma stem cells and the immune tumor microenvironment evolution.

BACKGROUND: Intrinsically resistant glioma stem cells (GSCs) in the setting of a highly immunosuppressive tumor microenvironment (TME) remain the most predominant phenomenon leading to unfavorable therapeutic outcomes in glioblastoma (GBM). Hence there is an unmet need for novel anti-GBM therapeutic paradigms that can effectively target GSCs while simultaneously reprogramming the TME. METHODS: In this study, we leverage evidence from SMAC mimetic screening to evaluate and characterize the anti-tumor and immune TME modulating impacts of the lead SMAC mimetic Xevinapant at the single cell level in GBM. We utilized viability assays and orthotopic human and murine GBM models to assess the survival impacts of Xevinapant on GSCs in vitro and in vivo. Moreover, we employed single-cell RNA sequencing (scRNA-seq) to investigate the modulation impact of Xevinapant on GBM TME. Lastly, we investigated drug combination synergies to address potential mechanisms of tolerance or resistance to Xevinapant. RESULTS: According to our observations, in vitro exposure to Xevinapant induced apoptosis along with significant viability reduction in a dose-dependent manner, in both human and mouse GSCs. Moreover, Xevinapant treatment produced robust anti-tumor effects in vivo and significantly prolonged animal overall survival. Based on single-cell RNA seq analysis, Xevinapant did not only enhance GSCs apoptosis but also activated antitumor effector immune response leading to favorable reprogramming of immunosuppressive TME. Furthermore, we established and queried Xevinapant therapeutic signatures to the LINCS database in an effort to identify small molecules that could reverse treatment-induced tolerance to Xevinapant. We have identified a novel set of candidate small molecules with robust synergy when combined with Xevinapant. CONCLUSIONS: In summary, Xevinapant exhibits robust anti-tumor activity on GSCs and favorable immune modulation of the TME in GBM, hence providing a rationale for further clinical investigation in GBM.