Supplementary figures: 1): molecular structure of mibefradil, 2) Mibefradil effects on HIFs, 3) effects of sh-RNA inhibition of Cav3.2 in GSCs, 4) Schematic summary of the findings.

Supplementary methods containing descriptions of PCR primer sequences, Reverse Phase Protein Arrays, RNA-seq, and Rescue Experiments.

Abstract Glioblastoma (GBM) is the most common and most lethal primary malignant brain tumor. The receptor tyrosine kinase MET is frequently upregulated or over activated in GBM. Clinically applicable MET inhibitors have been developed and tested in the lab and in clinical trials. However, resistance to single modality anti-MET drugs frequently occurs, rendering these agents ineffective. This study aimed to determine the mechanisms of MET inhibitor resistance in GBM and to use the acquired information to develop novel therapeutic approaches to overcome resistance. We investigated two clinically applicable MET inhibitors: PF-02341066, an ATP-competitive small molecule inhibitor of MET, and MetMab, a monovalent monoclonal antibody that binds to the extracellular domain of the MET receptor. We generated PF-02341066 and MetMab resistant GBM cell lines and primary cells by subjecting them to increasing concentrations of drug over a period of time. We utilized RNA sequencing (RNA-seq) and reverse phase protein arrays (RPPA) in addition to death and proliferation assays to identify the pathways altered in the resistant GBM cells compared to wild type GBM cells. We discovered many critical proteins that were altered in the resistant cells lines compared to wild type cells. These included FAK, COX-2, p-FGFR1, Vimentin, mTOR and p-STAT3. There was substantial but not complete overlap between the molecules that were altered in cells resistant to the small molecule as compared to molecules that were changed in cells resistant to the antibody. The protein changes in resistant GBM cells were verified by western blotting. Notably, we discovered that both COX-2 and p-FGFR1 were upregulated in GBM resistant cells and thus investigated whether inhibition of these targets could restore MET inhibitor sensitivity. Celecoxib, an FDA-approved drug, acts to inhibit COX-2 and Debio-1347 acts to inhibit the FGFR family, with a higher affinity for FGFR1. Combining Celecoxib or Debio-1347 with PF-02341066 or MetMab led to increased cell death in resistant cells compared to either agent alone. In addition, the drug combination decreased resistance to cell proliferation inhibition compared to either agent alone, indicating restored sensitivity to both MET inhibitors. We are currently undertaking in vivo experiments to establish whether the combination of celecoxib and MET inhibitors can restore sensitivity to MET inhibition in resistant cells as effectively in vivo as seen with in vitro experiments. These data indicate that MET inhibitor resistance can be overcome by targeting the resulting upregulated pathways using FDA-approved drugs and that multi-drug combinations may revert resistance during treatment. Citation Format: Nichola A. Cruickshanks, Ying Zhang, Sarah Hatef, Julia Wulfkuhle, Isela Gallagher, Alexander Koeppel, David Schiff, See Phan, Stephen Turner, Emanuel Petricoin, Roger Abounader. Overcoming MET inhibitor resistance in GBM therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 102. doi:10.1158/1538-7445.AM2017-102

Abstract Glioblastoma stem cells (GSCs) have been implicated in tumor resistance to radio- and chemotherapy. Proliferation of GSCs reportedly requires calcium influx through T-type calcium channels (Cav3.2). In this study we investigated the expression, function, mechanism of action and therapeutic targeting of Cav3.2 with the FDA approved and repurposed drug Mibefradil in glioblastoma (GBM), and GSCs. We found that Cav3.2 is highly expressed in the majority of human GBM specimens and all GCSs. TCGA data analysis demonstrated that approximately 11% of GBM tumors have upregulated Cav3.2 and that overexpression of Cav3.2 is associated with worse prognosis. Furthermore, we demonstrated that Mibefradil inhibits GSC growth and survival and sensitizes GSCs to Temozolomide (TMZ) chemotherapy. Mibefradil inhibited hypoxia inducible factor HIF1a and induced GSC differentiation. To investigate the mechanism of action of Mibefradil, were performed proteomic and transcriptomic screenings of Mibefradil-treated GSCs using reverse phase protein arrays and RNA-seq, followed by functional rescue experiments. Inhibition of Cav3.2 with Mibefradil significantly altered multiple cancer regulatory signaling pathways and molecules as well as the transcription of oncogenes and tumor suppressors. Among other, Mibefradil suppressed GSC growth through inhibition of pro-survival pathways such as AKT/mTOR, whilst simultaneously inducing apoptosis through upregulation of survivin, BAX and cleavage of caspase 9 and PARP. Notably also, RNA-deep sequencing of Mibefradil treated GSCs revealed an increase in expression of tumor suppressors such as TNFRSF14 and HSD17B14 along with a decrease in the expression of several oncogenes such as PDGFA, PDGFB and TGFB1. We also assessed the therapeutic effects of Mibefradil, on established GSC-derived xenografts. Oral administration of Mibefradil significantly inhibited tumor growth, prolonged animal survival and sensitized tumors to inhibition by TMZ and radiation. This study represents the first comprehensive characterization of Cav3.2 in GBM and GSC. The data establish Cav3.2 inhibition by the repurposed FDA-approved drug Mibefradil as a new strategy for GBM therapy. Citation Format: Ying Zhang, Nichola Cruickshanks, Fang Yuan, Baomin Wang, Mary Pahuski, Julia Wulfkuhle, Isela Gallagher, Alexander F. Koeppel, Sarah Hatef, Christopher Papanicolas, Jeongwu Lee, Eli Bar, David Schiff, Stephen D. Turner, Emanuel Petricoin, Lloyd L. Gray, Roger Abouander. Comprehensive characterization of the role of T-type calcium channels in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3147. doi:10.1158/1538-7445.AM2017-3147

Abstract Glioblastoma (GBM) stem-like cells (GSC) promote tumor initiation, progression, and therapeutic resistance. Here, we show how GSCs can be targeted by the FDA-approved drug mibefradil, which inhibits the T-type calcium channel Cav3.2. This calcium channel was highly expressed in human GBM specimens and enriched in GSCs. Analyses of the The Cancer Genome Atlas and REMBRANDT databases confirmed upregulation of Cav3.2 in a subset of tumors and showed that overexpression associated with worse prognosis. Mibefradil treatment or RNAi-mediated attenuation of Cav3.2 was sufficient to inhibit the growth, survival, and stemness of GSCs and also sensitized them to temozolomide chemotherapy. Proteomic and transcriptomic analyses revealed that Cav3.2 inhibition altered cancer signaling pathways and gene transcription. Cav3.2 inhibition suppressed GSC growth in part by inhibiting prosurvival AKT/mTOR pathways and stimulating proapoptotic survivin and BAX pathways. Furthermore, Cav3.2 inhibition decreased expression of oncogenes (PDGFA, PDGFB, and TGFB1) and increased expression of tumor suppressor genes (TNFRSF14 and HSD17B14). Oral administration of mibefradil inhibited growth of GSC-derived GBM murine xenografts, prolonged host survival, and sensitized tumors to temozolomide treatment. Our results offer a comprehensive characterization of Cav3.2 in GBM tumors and GSCs and provide a preclinical proof of concept for repurposing mibefradil as a mechanism-based treatment strategy for GBM. Cancer Res; 77(13); 3479–90. ©2017 AACR.