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.

In vivo randomization/matching data

Abstract Purpose: Glioblastoma (GBM) is the most common and most lethal primary malignant brain tumor. The receptor tyrosine kinase MET is frequently upregulated or overactivated in GBM. Although clinically applicable MET inhibitors have been developed, resistance to single modality anti-MET drugs frequently occurs, rendering these agents ineffective. We aimed to determine the mechanisms of MET inhibitor resistance in GBM and use the acquired information to develop novel therapeutic approaches to overcome resistance. Experimental Design: We investigated two clinically applicable MET inhibitors: crizotinib, an ATP-competitive small molecule inhibitor of MET, and onartuzumab, a monovalent monoclonal antibody that binds to the extracellular domain of the MET receptor. We developed new MET inhibitor–resistant cells lines and animal models and used reverse phase protein arrays (RPPA) and functional assays to uncover the compensatory pathways in MET inhibitor–resistant GBM. Results: We identified critical proteins that were altered in MET inhibitor–resistant GBM including mTOR, FGFR1, EGFR, STAT3, and COX-2. Simultaneous inhibition of MET and one of these upregulated proteins led to increased cell death and inhibition of cell proliferation in resistant cells compared with either agent alone. In addition, in vivo treatment of mice bearing MET-resistant orthotopic xenografts with COX-2 or FGFR pharmacological inhibitors in combination with MET inhibitor restored sensitivity to MET inhibition and significantly inhibited tumor growth. Conclusions: These data uncover the molecular basis of adaptive resistance to MET inhibitors and identify new FDA-approved multidrug therapeutic combinations that can overcome resistance.

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.

Abstract The androgen receptor (AR) is widely expressed in breast cancer, and evidence suggests dependence on AR signaling for growth and survival. AR antagonists such as enzalutamide and seviteronel have shown success in preclinical models and clinical trials of prostate cancer and are currently being evaluated in breast cancer. Reciprocal regulation between AR and the HER2/PI3K/mTOR pathway may contribute to resistance to HER2- and mTOR-targeted therapies; thus, dual inhibition of these pathways may synergistically inhibit breast cancer growth. HER2+ and triple-negative breast cancer cell lines were treated with AR antagonist plus anti-HER2 mAb trastuzumab or mTOR inhibitor everolimus. Apoptosis, cell proliferation, and drug synergy were measured in vitro. Pathway component genes and proteins were measured by qRT-PCR, Western blot, and reverse phase protein array. In vivo, HER2+ breast cancer xenografts were treated with enzalutamide, everolimus, trastuzumab, and combinations of these drugs. AR antagonists inhibited proliferation of both HER2+ and TNBC cell lines. Combining AR antagonist and either everolimus or trastuzumab resulted in synergistic inhibition of proliferation. Dihydrotestosterone caused increased phosphorylation of HER2 and/or HER3 that was attenuated by AR inhibition. Everolimus caused an increase in total AR, phosphorylation of HER2 and/or HER3, and these effects were abrogated by enzalutamide. Growth of trastuzumab-resistant HER2+ xenograft tumors was inhibited by enzalutamide, and combining enzalutamide with everolimus decreased tumor viability more than either single agent. AR antagonists synergize with FDA-approved breast cancer therapies such as everolimus and trastuzumab through distinct mechanisms. Treatment combinations are effective in trastuzumab-resistant HER2+ breast cancer cells in vivo. Mol Cancer Ther; 16(7); 1389–400. ©2017 AACR.

Glioblastoma stem cells (GSC) have been implicated in tumor resistance to radio- and chemotherapy. T type calcium channels (Cav3.2) regulate cell cycle progression by mediating the necessary influx of calcium for transit past the G1/S cell cycle checkpoint. We hypothesized that treating GSCs and GSC-derived xenografts with the FDA-approved Cav3.2 channel blocker mibefradil would synchronize GSCs to enter the S phase, and consequently sensitize them to cytotoxic therapies. We demonstrated that Cav3.2 is highly expressed in the majority of human GBM specimens and all GCSs, compared to normal brain tissue or glioma cell lines, respectively. Mibefradil treatment inhibited GSC proliferation and induced cell death. Furthermore, mibefradil sensitized GSCs to temozolomide treatment (TMZ) and increased TMZ-induced cell death by 25-63% (p < 0.05) in vitro. To determine the effect of mibefradil on glioblastoma xenograft growth, we implanted GSCs in the brains of immunodeficient mice and treated the mice with mibefradil and/or TMZ and monitored tumor growth by MRI. We found that mibefradil increased TMZ-induced tumor growth inhibition by 60% (p < 0.05). Mibefradil also significantly improved the survival of TMZ-treated mice bearing GSC-derived xenografts. To further investigate the mechanism of action of mibefradil, we performed reverse phase protein arrays on GSCs treated with mibefradil. We found that mibefradil strongly regulated GSC apoptosis by regulating BCL2, PUMA and BAX expressions/activations as well as caspase cleavage. Mibefradil also altered proteins involved in autophagy and invasion including LC3, FAK and other. We are currently performing RNA-seq to determine the transcriptome wide changes that are induced by Cav3.2 inhibition. Altogether, the data provide mechanistic and functional rationales for the use of Cav3.2 inhibitors such as mibefradil as a new adjuvant therapy that enhances the efficacy of cytotoxic therapies in glioblastoma by targeting glioblastoma stem cells.

Previously, it has been shown that pancreatic ductal adenocarcinoma (PDA) tumors exhibit high levels of hypoxia, characterized by low oxygen pressure (pO2) and decreased O2 intracellular perfusion. Chronic hypoxia is strongly associated with resistance to cytotoxic chemotherapy and chemoradiation in an understudied phenomenon known as hypoxia-induced chemoresistance. The hypoxia-inducible, pro-oncogenic, serine-threonine kinase PIM1 (Proviral Integration site for Moloney murine leukemia virus 1) has emerged as a key regulator of hypoxia-induced chemoresistance in PDA and other cancers. Although its role in therapeutic resistance has been described previously, the molecular mechanism behind PIM1 overexpression in PDA is unknown. Here, we demonstrate that cis-acting AU-rich elements (ARE) present within a 38-base pair region of the PIM1 mRNA 3'-untranslated region mediate a regulatory interaction with the mRNA stability factor HuR (Hu antigen R) in the context of tumor hypoxia. Predominantly expressed in the nucleus in PDA cells, HuR translocates to the cytoplasm in response to hypoxic stress and stabilizes the PIM1 mRNA transcript, resulting in PIM1 protein overexpression. A reverse-phase protein array revealed that HuR-mediated regulation of PIM1 protects cells from hypoxic stress through phosphorylation and inactivation of the apoptotic effector BAD and activation of MEK1/2. Importantly, pharmacological inhibition of HuR by MS-444 inhibits HuR homodimerization and its cytoplasmic translocation, abrogates hypoxia-induced PIM1 overexpression and markedly enhances PDA cell sensitivity to oxaliplatin and 5-fluorouracil under physiologic low oxygen conditions. Taken together, these results support the notion that HuR has prosurvival properties in PDA cells by enabling them with growth advantages in stressful tumor microenvironment niches. Accordingly, these studies provide evidence that therapeutic disruption of HuR's regulation of PIM1 may be a key strategy in breaking an elusive chemotherapeutic resistance mechanism acquired by PDA cells that reside in hypoxic PDA microenvironments.