Synergistic activity of combining repurposed simvastatin with irinotecan chemotherapy against glioblastoma and its underlying mechanisms

Background and Rationale: Glioblastoma (GBM) is the most common and aggressive primary brain tumor, with a dismal prognosis (approximately 15 months) and inevitable recurrence due to resistance towards standard-of-care therapies. This warrants rapidly translatable therapeutics against GBM and its recurrent subtypes, including glioma stem cells (GSCs) and hypermutated, mismatch-repair deficient (MMRd) sub-phenotypes. In this work, we investigated the synergistic therapeutic potential of a novel combination of the repurposed drug simvastatin with irinotecan chemotherapy towards glioblastoma (GBM) and the underlying molecular mechanisms. We also investigated the efficacy of these agents against models of therapy-resistant GBM, including MMRd phenotypes and GSCs.

Methods: Efficacy of simvastatin and irinotecan alone and in combination against diverse GBM lines (U251MG human GBM line, G34/G88 primary GSCs, SB28 murine GBM line) was assessed using mechanistically distinct cell viability assays. Bulk RNA-Sequencing was performed to uncover the top pathways and genes affected by these drugs, followed by validation of promising pathways (TGF-β signaling and cell death) using targeted phosphoproteomics, genetic manipulation, and functional assays.

Results: We observed synergy between simvastatin and irinotecan across diverse human GBM lines at nanomolar concentrations. A role for multiple cell death pathways, both caspase-dependent (apoptosis) and caspase-independent (autophagy, ferroptosis), was demonstrated. Notably, irinotecan alone and in combination with simvastatin downregulated gene expression of TGF-β family members, as evidenced by RNA-Sequencing. Targeted phosphoproteomics and functional experiments further validated significant inhibition of TGF-β signaling with both treatment types. Additionally, enrichment of immunological (interferons, complement, inflammatory responses, TNF-α) and oncogenic (K-RAS/ERK) signaling pathways was observed with the combination treatment. Furthermore, we observed the efficacy of simvastatin and irinotecan against models of MMR-deficient GBM, including the GBM22TMZ patient-derived xenograft (PDX) line and U251 shMSH2 line (small-hairpin knockdown of MSH2 in U251MG human GBM line) and observed synergy of these agents in combination against these resistant GBM models.

Conclusions: Besides the first detailed demonstration of a robust synergy between simvastatin and irinotecan against conventional and therapy-resistant GBM/GSC lines, this study shows for the first time that both irinotecan and the combination treatment converge on inhibition of TGF-β signaling. This is notable given the lack of TGF-β inhibitors in the clinic. Collectively, this study provides preclinical data suggesting this novel drug combination be tested in patients with GBM and TGF-β driven cancers.