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.

AbstractOvarian cancer is the deadliest gynecologic cancer, due in large part to the diagnosis of advanced stage disease, the development of platinum resistance, and inadequate treatment alternatives. Recent studies by our group and others have shown that T-type calcium (Ca2+) channels play a reinforcing role in cancer cell proliferation, cell-cycle progression, and apoptosis evasion. Therefore, we investigated whether T-type Ca2+ channels affect ovarian tumor growth and response to platinum agents. Inhibition of T-type Ca2+ channels with mibefradil or by silencing expression resulted in growth suppression in ovarian cancer cells with a simultaneous increase in apoptosis, which was accompanied by decreased expression of the antiapoptotic gene survivin (BIRC5). Analysis of intracellular signaling revealed mibefradil reduced AKT phosphorylation, increased the levels and nuclear retention of FOXO transcription factors that repress BIRC5 expression, and decreased the expression of FOXM1, which promotes BIRC5 expression. Combining carboplatin with mibefradil synergistically increased apoptosis in vitro. Importantly, mibefradil rendered platinum-resistant ovarian tumors sensitive to carboplatin in a mouse model of peritoneal metastasis. Together, the data provide rationale for future use of T-type channel antagonists together with platinum agents for the treatment of ovarian cancer. Mol Cancer Ther; 15(3); 460–70. ©2016 AACR.

AbstractOvarian cancer is the deadliest gynecologic cancer, due in large part to the diagnosis of advanced stage disease, the development of platinum resistance, and inadequate treatment alternatives. Recent studies by our group and others have shown that T-type calcium (Ca2+) channels play a reinforcing role in cancer cell proliferation, cell-cycle progression, and apoptosis evasion. Therefore, we investigated whether T-type Ca2+ channels affect ovarian tumor growth and response to platinum agents. Inhibition of T-type Ca2+ channels with mibefradil or by silencing expression resulted in growth suppression in ovarian cancer cells with a simultaneous increase in apoptosis, which was accompanied by decreased expression of the antiapoptotic gene survivin (BIRC5). Analysis of intracellular signaling revealed mibefradil reduced AKT phosphorylation, increased the levels and nuclear retention of FOXO transcription factors that repress BIRC5 expression, and decreased the expression of FOXM1, which promotes BIRC5 expression. Combining carboplatin with mibefradil synergistically increased apoptosis in vitro. Importantly, mibefradil rendered platinum-resistant ovarian tumors sensitive to carboplatin in a mouse model of peritoneal metastasis. Together, the data provide rationale for future use of T-type channel antagonists together with platinum agents for the treatment of ovarian cancer. Mol Cancer Ther; 15(3); 460–70. ©2016 AACR.

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

Supplementary Figure 1: Relative T-type Ca2+ channel gene expression; Supplementary Figure 2: Down regulation of T-type Ca2+ channel gene expression decreases survivin protein level; Supplementary Figure 3: Effects of Mibefradil on AKT activity and its role in survivin stabilization; Supplementary Figure 4: Effects of carboplatin and down regulation of T-type Ca2+ channel expression on survivin expression; Supplementary Table 1: Sequence of siRNA targeted against T-type channel subunit; Supplementary Table 2: Sequence of primers designed for RT-qPCR amplification for gene expression; Supplementary Table 3: Sequence of primers designed for amplification of genomic DNA at the BIRC5 promoter containing and in close proximity to FoxO binding sequence (BS, Fig 3D); Supplementary Table 4: Combination indexes calculated for sequential treatment with the indicated concentrations of Mibefradil and carboPt in selected ovarian cancer cell lines (Fig 4A).

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.

Ovarian cancer is the deadliest gynecologic cancer, due in large part to the diagnosis of advanced stage disease, the development of platinum resistance, and inadequate treatment alternatives. Recent studies by our group and others have shown that T-type calcium (Ca2+) channels play a reinforcing role in cancer cell proliferation, cell-cycle progression, and apoptosis evasion. Therefore, we investigated whether T-type Ca2+ channels affect ovarian tumor growth and response to platinum agents. Inhibition of T-type Ca2+ channels with mibefradil or by silencing expression resulted in growth suppression in ovarian cancer cells with a simultaneous increase in apoptosis, which was accompanied by decreased expression of the antiapoptotic gene survivin (BIRC5). Analysis of intracellular signaling revealed mibefradil reduced AKT phosphorylation, increased the levels and nuclear retention of FOXO transcription factors that repress BIRC5 expression, and decreased the expression of FOXM1, which promotes BIRC5 expression. Combining carboplatin with mibefradil synergistically increased apoptosis in vitro. Importantly, mibefradil rendered platinum-resistant ovarian tumors sensitive to carboplatin in a mouse model of peritoneal metastasis. Together, the data provide rationale for future use of T-type channel antagonists together with platinum agents for the treatment of ovarian cancer. Mol Cancer Ther; 15(3); 460–70. ©2016 AACR.

Abstract This study's aim was to define a novel molecular target and to develop an effective agent capable of overcoming intrinsic and acquired resistance to anti-EGFR therapy. Despite introduction of new therapies, lung cancer is a major cause of cancer death. One of its characteristics is the expression and activation of epidermal growth factor receptor (EGFR). Activating mutations in EGFR are present in approximately 20% of patients with non-small cell lung cancer (NSCLC), mostly in never-smokers. While most patients initially respond to treatment with anti-EGFR therapy, essentially all patients develop acquired drug resistance. T-type calcium channels, are present in lung cancer and support cell proliferation. Importantly, their expression often coincides with abnormal expression or mutations in EGFR. In this study, we investigated the effects of T-type channel inhibition on NSCLC survival and resistance to targeted therapy in vitro. We used a panel of cell lines differing in EGFR mutation status and sensitivity to EGFR tyrosine kinase inhibitors (TKIs). Our results show that T-type channel inhibitors alone arrest cells in the G1/G0 phase of the cell cycle and induce cell death. The treatment reduces activity of both PI3K/AKT/mTOR and JAK2/STAT5 pathways, often upregulated as a mechanism of acquired resistance to TKIs. Importantly, combined treatment with TKI, gefitinib, and T-type channels inhibitor, mibefradil, resulted in synergistic growth inhibition. Our results demonstrate a connection between T-type Ca2+ channels and aberrant EGFR activity in NSCLC, and provide the rationale for future use of two targeted therapies together. Since T-type calcium channel inhibitors are already undergoing clinical trials, our discoveries could be quickly translated into clinical practice. Citation Format: Barbara Dziegielewska, Lloyd S. Gray, Jaroslaw Dziegielewski. Overcoming resistance to anti-EGFR therapy in non-small cell lung cancer with a T-type Ca2+ channels inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 750. doi:10.1158/1538-7445.AM2015-750

Abstract Glioblastoma multiforme (GBM) is the most common and lethal primary brain tumor. Although introduction of the Stupp regimen combining radiation (IR) with concomitant and adjuvant temozolomide (TMZ) led to increased patient survival, the cure rate of GBM is disappointing. We postulated that sensitization of cancer cells to therapy can be achieved by targeting intracellular pathways and processes. Recently, T-type Ca2+ channels have been proposed as a molecular target for GBM sensitization to chemo- and radiotherapy (Keir et al., 2013; Sheehan et al., 2013). In the present studies, we investigated the effects of T-type channel inhibition on GBM cells’ responses to the Stupp regimen. GBM cells differing in their resistance to TMZ and IR were treated with theT-type channel antagonist, mibefradil, either concomitantly with TMZ or sequentially (Interlaced TherapyTM). Cell viability and clonogenic survival were determined, demonstrating significant enhancement of the Stupp regimen's anticancer effects with co-treatment with T-type channels inhibitors. Importantly for DNA-targeted therapies, we observed a decrease in cells’ ability to repair DNA damage in the presence of mibefradil. The molecular mechanism of this decrease was investigated by assessing expression levels, localization and phosphorylation status of ATM, DNA-PK, γH2AX, Chk1/2, Kap1, p53, Rad51. These mechanistic studies demonstrated that inhibiting T-type Ca2+ channels in GBM affects DNA damage signaling and repair through reduction of repair protein activation and changes their cellular localization. The observed effects are specific for T-type channels inhibition, since L-type channels inhibitors had no effect. Our study provide a rational molecular mechanism for sensitization of GBM to the Stupp regimen by T-type Ca2+ channel inhibition, which will facilitate current and future clinical trials and potentially lead to new treatment options for this deadly brain tumor. Citation Format: Jaroslaw Dziegielewski, Barbara Dziegielewska, Lloyd S. Gray. Enhancing the Stupp regimen in glioblastoma cancer cells with a T-type calcium channels inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1808. doi:10.1158/1538-7445.AM2015-1808

The expanding "valley of death" in drug development is leaving potentially life-saving new chemical entities and molecular targets fallow. This situation is forcing early-stage companies to think creatively about moving their technologies forward, especially as institutional investors show more interest in later stages of development. Drug repurposing, a strategy to examine existing drugs for therapeutic value against different diseases, is an emerging method to bring an off-market drug back onto the market. Tau Therapeutics LLC identified the role of T-type calcium channel blockers (Cav3) in cancer proliferation, but the company was unable to attract funding while having both a nonvalidated drug target and new chemical entities. To change the risk profile of the company, Tau set out to repurpose the known Cav3 drug mibefradil as a proof of concept for the treatment of cancer. Mibefradil was launched for hypertension in the 1990s but withdrawn because of drug-drug interactions. A new sequential combination treatment, termed Interlaced Therapy™, uses short-term administration of mibefradil to enhance the overall therapeutic potential of conventional anticancer agents. Mibefradil is currently in a phase Ib clinical trial with the National Cancer Institute (NCI) Adult Brain Tumor Consortium. Mibefradil has been repurposed from an abandoned antihypertensive to a targeted solid tumor treatment, and it has been rescued from drug-drug interactions by using short-term dose exposure. Tau is using the early success of mibefradil as a proof of concept to build a platform technology of Cav3 blockers for broad antitumor applications in combination with new targeted cancer therapies, well-established chemotherapies, and radiation.

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.

T-type calcium channels are involved in a multitude of cellular processes, both physiological and pathological, including cancer. T-type channels are also often aberrantly expressed in different human cancers and participate in the regulation of cell cycle progression, proliferation, migration, and survival. Here, we review the recent literature and discuss the controversies, supporting the role of T-type Ca(2+) channels in cancer cells and the proposed use of channels blockers as anticancer agents. A growing number of reports show that pharmacological inhibition or RNAi-mediated downregulation of T-type channels leads to inhibition of cancer cell proliferation and increased cancer cell death. In addition to a single agent activity, experimental results demonstrate that T-type channel blockers enhance the anticancer effects of conventional radio- and chemotherapy. At present, the detailed biological mechanism(s) underlying the anticancer activity of these channel blockers is not fully understood. Recent findings and ideas summarized here identify T-type Ca(2+) channels as a molecular target for anticancer therapy and offer new directions for the design of novel therapeutic strategies employing channels blockers. Physiological relevance: T-type calcium channels are often aberrantly expressed or deregulated in cancer cells, supporting their proliferation, survival, and resistance to treatment; therefore, T-type Ca(2+) channels could be attractive molecular targets for anticancer therapy.

Abstract Introduction: Survival for patients with pancreatic ductal adenocarcinoma (PDAC) remains dismal and novel therapeutic strategies are needed. Mutations in the KRAS oncogene are important drivers of PDAC progression; however, monotherapies targeting the RAS pathway have shown only modest efficacy. The RAS pathway is an activator of calcium (Ca2+) signaling, which has been implicated in PDAC progression. We hypothesized that MEK inhibition combined with the T-type calcium channel inhibitor mibefradil would result in enhanced growth inhibition of PDAC tumors. Methods and Results: Gene expression profiling of patient-derived PDAC tumors (relative to normal pancreas) and tumors chronically treated with the MEK inhibitor trametinib in vivo revealed an upregulation of Ca2+-signaling related genes including members of the calpain pathway and the calmodulin pathway, both implicated in PDAC progression. To assess the in vivo efficacy of combined T-type calcium channel inhibition and MEK inhibition, mice were engrafted orthotopically with patient-derived KRAS-mutant (Tumor 366) and KRAS-wild type (Tumor 738) PDAC tumors and treated with control, the MEK inhibitor trametinib, the T-type calcium channel inhibitor mibefradil, or combination. Combination therapy with trametinib plus mibefradil was highly effective with greater inhibition of PDAC growth than either therapy alone in the KRAS-mutant Tumor 366, however mibefradil did not augment the level of growth inhibition achieved by trametinib alone in the KRAS-wild type Tumor 738. To evaluate combination trametinib plus mibefradil therapy in patient-derived PDAC tumors following chronic treatment with trametinib, mice were engrafted orthotopically with patient-derived PDAC tumors, allowed to grow to 300-500mm3, and treated with trametinib for 4-6 weeks. Following initial treatment, tumors were harvested, re-implanted, and again exposed to therapy over 4-6 weeks. The cycles of treatment and reimplantation were continued until tumor growth was not significantly reduced by trametinib treatment. Therapy with trametinib plus mibefradil nearly completely inhibited growth in KRAS-mutant PDAC tumors (Tumors 608 and 366) previously resistant to chronic trametinib therapy. Conclusions: Combination therapy with the MEK inhibitor trametinib plus the T-type calcium channel inhibitor mibefradil results in significant growth inhibition of KRAS-mutant patient-derived PDAC tumors. Moreover, this combination therapy results in near complete growth inhibition of PDAC tumors that have acquired resistance to trametinib. The combination therapy of mibefradil plus trametinib should be further evaluated in clinical trials for patients with PDAC. Citation Format: Timothy Eric Newhook, James M. Lindberg, Sara J. Adair, Edik Blais, Jason Papin, Lloyd Gray, J. Thomas Parsons, Todd W. Bauer. Combination therapy with a MEK inhibitor plus T-type calcium channel inhibitor is highly effective in patient-derived pancreatic ductal adenocarcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 808. doi:10.1158/1538-7445.AM2014-808

Abstract The inability to successfully treat women with ovarian cancer is due in large part to the advanced stage of disease at diagnosis and the development of platinum resistance. T-type calcium channels have recently garnered interest as potential targets for the treatment of many cancers. We investigated the hypothesis that inhibiting T-type calcium channels would sensitize ovarian cancer cells to platinum based chemotherapy using cell culture and mouse models. Sequential treatment of cultured ovarian cancer cell lines or primary cells obtained from patients with mibefradil followed by carboplatin showed a dose-dependent decrease in cell number that was greater than observed with either drug alone. Importantly, treatment of platinum-resistant tumor cells with mibefradil followed by carboplatin also decreased cell number. Moreover, either simultaneous administration of both drugs or treatment with carboplatin followed by mibefradil did not alter cellular response to carboplatin. Based on these findings, we tested the effect of mibefradil and carboplatin in a mouse model of peritoneal ovarian cancer metastasis with platinum-resistant ovarian cancer cells. Tumor-bearing mice were subjected to 3 cycles of Interlaced TherapyTM, which consists of 5 days of mibefradil treatment followed by one dose of carboplatin and 2 days off. We found that while neither drug alone significantly affected tumor growth compared to control, the mice that received the dual treatment had a dramatic reduction in tumor burden. Together, the data provide pre-clinical evidence supporting the use of T-type calcium channel blockers with platinum-based chemotherapy to treat ovarian cancer. Citation Format: Eli V. Casarez, Lloyd Gray, Amir A. Jazaeri, Jill K. Slack-Davis. Inhibition of T-type calcium channels sensitizes ovarian cancer cell growth and metastasis to platinum-based chemotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 945. doi:10.1158/1538-7445.AM2013-945

The purpose of this study is to demonstrate MCF-7 cells' dependence on calcium for growth and to exploit that dependence to improve chemotherapy efficacy.Fura-2 fluorescence imaging shows that MCF-7 cells maintain a higher basal intracellular calcium concentration than non-tumorigenic MCF-10A cells.Blocking T-type calcium channels with mibefradil reduced MCF-7 intracellular calcium concentration.Flow cytometry shows that knocking down T-type calcium channel expression with siRNA caused an increase in MCF-7 cells in G1 phase and a decrease in cells in S phase.Proliferation assays of MCF-7 cells treated with EGTA and thapsigargin reveal the dependence of MCF-7 cell growth on extracellular and intracellular calcium sources, respectively.In vitro, interlaced treatment that alternated the T-type calcium channel blocker NNC-55-0396 with paclitaxel more effectively reduced MCF-7 cell number than chemotherapy alone.In a mouse in vivo model, interlaced mibefradil and paclitaxel more effectively reduced MCF-7 xenograft size than chemotherapy alone.These findings indicate that MCF-7 cells are dependent on calcium for proliferation, particularly in passing the G1/S cell cycle checkpoint.Further, this dependence on calcium can be exploited by alternating treatment with T-type calcium channel blockers with paclitaxel in an interlaced therapy scheme that increases the efficacy of the chemotherapy.

T-type Ca2+ channels are a group of low voltage-gated calcium channels which seem to be crucial for embryonic or stem cell proliferation and differentiation; however, they could be also aberrantly expressed in several human tumors. Since calcium entry is required for smooth transition through the cell cycle, it has been hypothesized that the inhibition of T-type Ca2+ channels blocks cells in G0/G1 phase of cell cycle, while a release from inhibition leads to increased number of cells entering cell cycle and thus increased susceptibility to conventional antitumor therapy. In this study we investigated the effects of T-type channel inactivation, or downregulation, on cell cycle progression, cell death and survival, and resistance to radiotherapy (RT). The experiments were conducted using several cancer cell lines expressing T-type Ca2+ channels (glioblastoma, colon and prostate) and selective and structurally unrelated calcium channels antagonists, including Mibefradil, a drug that is currently undergoing clinical trial for its antitumor properties, and is planned to be tested as radiosensitizing agent in recurrent gliobastoma tumors. The effects of T-type Ca2+ channel inhibition on cell cycle progression, cell death and the expression/activation of cell cycle regulated proteins were assessed. Finally, the combination treatments including T-type channel antagonist and radiotherapy were assayed. We show that T-type Ca2+ channels are important for cell cycle progression and resistance to RT, and that their inhibition leads to enhanced cell death and reduced survival. Our study demonstrates that one of the earliest events evoked by T-type channel inhibition is a deregulation of pro-survival pathway PI3K/Akt/mTOR and activation of pro-apoptotic stress-activated p38MAPK pathway. The results support the idea of T-type Ca2+ channel as a novel molecular target for antitumor therapy and provide a rational and plausible biological mechanism responsible for the effects induced by T-type Ca2+ channel antagonists in cancer cells. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A239. Citation Format: Barbara Dziegielewska, Nicholas C.K. Valerie, Amol S. Hosing, James M. Larner, David L. Brautigan, Lloyd S. Gray, Jaroslaw Dziegielewski. T-type calcium channels as a novel molecular target for tumor therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A239.

Ca2+ influx at critical points in the cell cycle is required for proliferation. This requirement is so ubiquitous that its occurrence is often treated as background noise. Yet without it, cells stop dividing, suggesting an obvious and potentially effective way to treat cancer. To control proliferation by controlling Ca2+ influx requires that the mechanism be elucidated, but this field of study has been filled with controversy and devoid of therapeutic utility. In this study, the authors present a model for the regulation of Ca2+ influx at the G1/S restriction point in cancer and stem cells that is simple, cohesive and, we believe, reasonably complete. The model illustrates the essential role of T-type Ca2+ channels in mediating influx and points clearly to the therapeutic strategies that have recently entered clinical trials.