Robert J. Schumacher

A Preclinical Evaluation of Minnelide as a Therapeutic Agent Against Pancreatic Cancer

Minnelide prevents tumor formation, causes tumor regression, and increases survival in multiple models of pancreatic cancer. Vegetation Is Good for You Your mom always told you to eat your vegetables, but what she probably didn’t tell you is that other plants can be good for you as well. Tripterygium wilfordii, sometimes known as the Thunder God vine, has various uses in traditional Chinese medicine. To better understand and improve upon the healing properties of this vine, the active ingredients have been isolated and characterized. One component of T. wilfordii, triptolide, has shown promising effects against pancreatic cancer cells. New therapies for pancreatic cancer—which is one of the most lethal human malignancies—are desperately needed, but triptolide is poorly soluble in water and thus has limited clinical use. Now, Chugh et al. synthesize a water-soluble form of triptolide, Minnelide, and demonstrate efficacy against pancreatic cancer in multiple animal models. The authors tested Minnelide both in vitro and in multiple preclinical models of pancreatic cancer. Each model has distinct advantages and limitations: Well-studied cancer cell lines and translationally relevant patient tumors were transplanted into mice that lack immune systems, whereas a spontaneous model in immunosufficient mice was, by necessity, a mouse tumor. By combining these approaches, the authors addressed many caveats that frequently plague preclinical studies. Indeed, Minnelide was highly effective in treating pancreatic cancer in all of these complementary models. The next step is to take Minnelide into early clinical trials to see if these results can be reproduced in human patients with pancreatic cancer. Pancreatic cancer is one of the most lethal human malignancies with an all-stage 5-year survival frequency of <5%, which highlights the urgent need for more effective therapeutic strategies. We have previously shown that triptolide, a diterpenoid, is effective against pancreatic cancer cells in vitro as well as in vivo. However, triptolide is poorly soluble in water, limiting its clinical use. We therefore synthesized a water-soluble analog of triptolide, named Minnelide. The efficacy of Minnelide was tested both in vitro and in multiple independent yet complementary in vivo models of pancreatic cancer: an orthotopic model of pancreatic cancer using human pancreatic cancer cell lines in athymic nude mice, a xenograft model where human pancreatic tumors were transplanted into severe combined immunodeficient mice, and a spontaneous pancreatic cancer mouse model (KRasG12D; Trp53R172H; Pdx-1Cre). In these multiple complementary models of pancreatic cancer, Minnelide was highly effective in reducing pancreatic tumor growth and spread, and improving survival. Together, our results suggest that Minnelide shows promise as a potent chemotherapeutic agent against pancreatic cancer, and support the evaluation of Minnelide in clinical trials against this deadly disease.

Phosphonooxymethyl Prodrug of Triptolide: Synthesis, Physicochemical Characterization, and Efficacy in Human Colon Adenocarcinoma and Ovarian Cancer Xenografts.

A disodium phosphonooxymethyl prodrug of the antitumor agent triptolide was prepared from the natural product in three steps (39% yield) and displayed excellent aqueous solubility at pH 7.4 (61 mg/mL) compared to the natural product (17 μg/mL). The estimated shelf life (t90) for hydrolysis of the prodrug at 4 °C and pH 7.4 was found to be two years. In a mouse model of human colon adenocarcinoma (HT-29), the prodrug administered intraperitoneally was effective in reducing or eliminating xenograft tumors at dose levels as low as 0.3 mg/kg when given daily and at 0.9 mg/kg when given less frequently. When given via intraperitoneal and oral routes at daily doses of 0.6 and 0.9 mg/kg, the prodrug was also effective and well tolerated in a mouse model of human ovarian cancer (A2780).

A completely biological “off-the-shelf” arteriovenous graft that recellularizes in baboons

A decellularized tissue tube grown from human skin cells and fibrin becomes repopulated by recipient cells when tested in baboons as a vascular graft for hemodialysis access. Growing grafts for hemodialysis Patients undergoing hemodialysis for renal failure often receive an arteriovenous fistula, a connection between a vein and an artery. These surgical connections fail or cannot be attempted in some patients with compromised vasculature, who instead require vein grafts. As an alternative to autologous or synthetic grafts, Syedain et al. used a tissue engineering approach to generate vascular grafts from sacrificial fibrin scaffolds and human fibroblasts. Decellularized grafts were implanted into baboons and tested as hemodialysis access points. Over the course of 6 months, the grafts were recellularized with host cells and maintained sufficient burst pressure without evidence of immune rejection. Pending additional testing, these grafts represent an additional surgical option for hemodialysis access. Prosthetic arteriovenous grafts (AVGs) conventionally used for hemodialysis are associated with inferior primary patency rates and increased risk of infection compared with autogenous vein grafts. We tissue-engineered an AVG grown from neonatal human dermal fibroblasts entrapped in bovine fibrin gel that is then decellularized. This graft is both “off-the-shelf” (nonliving) and completely biological. Grafts that are 6 mm in diameter and about 15 cm in length were evaluated in a baboon model of hemodialysis access in an axillary-cephalic or axillary-brachial upper arm AVG construction procedure. Daily antiplatelet therapy was given. Grafts underwent both ultrasound assessment and cannulation at 1, 2, 3, and 6 months and were then explanted for analysis. Excluding grafts with cephalic vein outflow that rapidly clotted during development of the model, 3- and 6-month primary patency rates were 83% (5 of 6) and 60% (3 of 5), respectively. At explant, patent grafts were found to be extensively recellularized (including smoothelin-positive smooth muscle cells with a developing endothelium on the luminal surface). We observed no calcifications, loss of burst strength, or outflow stenosis, which are common failure modes of other graft materials. There was no overt immune response. We thus demonstrate the efficacy of an off-the-shelf AVG that is both acellular and completely biological.

Heme Binding Biguanides Target Cytochrome P450-Dependent Cancer Cell Mitochondria

The mechanisms by which cancer cell-intrinsic CYP monooxygenases promote tumor progression are largely unknown. CYP3A4 was unexpectedly associated with breast cancer mitochondria and synthesized arachidonic acid (AA)-derived epoxyeicosatrienoic acids (EETs), which promoted the electron transport chain/respiration and inhibited AMPKα. CYP3A4 knockdown activated AMPKα, promoted autophagy, and prevented mammary tumor formation. The diabetes drug metformin inhibited CYP3A4-mediated EET biosynthesis and depleted cancer cell-intrinsic EETs. Metformin bound to the active-site heme of CYP3A4 in a co-crystal structure, establishing CYP3A4 as a biguanide target. Structure-based design led to discovery of N1-hexyl-N5-benzyl-biguanide (HBB), which bound to the CYP3A4 heme with higher affinity than metformin. HBB potently and specifically inhibited CYP3A4 AA epoxygenase activity. HBB also inhibited growth of established ER+ mammary tumors and suppressed intratumoral mTOR. CYP3A4 AA epoxygenase inhibition by biguanides thus demonstrates convergence between eicosanoid activity in mitochondria and biguanide action in cancer, opening a new avenue for cancer drug discovery.

Engraftment of Reprogrammed Human Bile Duct Cells Transiently Rescues Diabetes in Mice

Differentiation of adult stem/progenitor cells into functional beta cells to provide a successful autologous cell therapy for Type 1 diabetes patients has not yet been achieved. Progenitor cells in the adult pancreas or liver have been considered potential sources of endocrine beta cells based on their ability to repopulate the organ following injury. Here we describe the isolation and reprogramming of a lineage-committed progenitor population of cells in the human bile duct towards a beta cell fate. These cells, which possess SOX9 as a marker, were able to manifest beta cell characteristics upon ectopic expression of pancreatic transcription factors. The beta cells derived from SOX9+ progenitor cells were also able to ameliorate high blood glucose in diabetic mice. The insulin+ islet-like clusters which developed from this progenitor cell type demonstrate the potential of this approach to generate functional, autologous beta cells.

Novel N-1 substituted fluoroquinolones inhibit human topoisomerase I activity and exhibit anti-proliferative activity

SummaryFluoroquinolone-class agents selectively target the bacterial type IIA topoisomerases DNA gyrase and topoisomerase IV, with a few exceptions that target eukaryotic type IIA topoisomerases. Fluoroquinolones bind and stabilize type IIA topoisomerase-DNA covalent complexes that contain a double-strand break. This unique mode of action is referred to as ‘topoisomerase poisoning’. We discovered that two novel fluoroquinolones having aryl functionality at the N-1 position, UITT-3-217 (217) and UITT-3-227 (227), could inhibit the catalytic activity of human topoisomerase II without stabilizing topoisomerase-DNA complexes, i.e., without poisoning it. Surprisingly, these compounds are more effective in inhibiting the catalytic activities of human and bacterial topoisomerase I. The National Cancer Institute’s 60 human tumor cell lines screen revealed significant anti-proliferative activities with 217 and 227 against the majority of 60 cancer cell lines. A proof of concept in vivo efficacy study using an HT-29 xenograft model of human colorectal cancer showed that 217 could inhibit the proliferation of human colorectal cancer cells to a degree comparable to fluorouracil in mice. Although 227 also exhibited anti-proliferative activity, it was not as effective as 217 in this xenograft model. These novel fluoroquinolones may serve as promising lead compounds for the development of new anticancer drugs.

Abstract 44: Hexyl-benzyl-biguanide (HBB) potently and selectively inhibits CYP3A4 epoxygenase activity and inhibits EET stabilization of mitochondrial respiration in ER+HER2- breast cancer cells, inducing glycolysis and pyruvate biosynthesis

Cytochrome P450 3A4 (CYP3A4) promotes ER+HER2- breast cancer cell proliferation and survival, in part, by biosynthesis of epoxyeicosatrienoic acids (EETs). EETs are known to regulate mitochondrial function in non-transformed cells, but the roles of CYP3A4 and EETs in regulation of breast cancer bioenergetics are unknown. Hexyl-benzyl-biguanide (HBB) is useful probe of CYP3A4 epoxygenase activity and selectively inhibits EET biosynthesis (IC50 = 9 uM vs. IC50 = 50 uM for CYP2C8). HBB caused depolarization of mitochondria in MCF-7 cells, while (±)-14,15-EET provided partial protection. The soluble epoxide hydrolase (sEH) inhibitor t-AUCB ameliorated inhibition of oxygen consumption rates (OCR) by HBB (20 uM), while there was no effect on extracellular acidification rate (ECAR), indicating that the primary effect of HBB is on OCR. At 30 minutes, HBB added to MCF-7 cells transiently suppressed phosphorylation of pyruvate kinase muscle isozyme 2 (PKM2) on Tyr-105, which has been reported to favor enzymatically inactive dimer over active tetramer. Suppression of phosphorylated PKM2 correlated with subsequent PKM2 tetramer formation and increase of intracellular pyruvate and extracellular lactate at 1 hour. The (±)-14,15-EET regioisomer reduced the pro-glycolytic PKM2 tetramer at 1 hour, suggesting that HBB may promote PKM2 tetramer, in part, through reduction of EET. Prolonged exposure to HBB (20 uM) in cultured cells activated phosphorylation of PKM2 on Tyr-105, but there was increased cellular necrosis correlating with reduced mitochondrial respiration and reduction of ATP stores, indicating that loss of respiration was the dominant effect. HBB inhibited the ER+HER2- MCF-7 xenograft, similar to CYP3A4 silencing. HBB promoted phosphorylation of intratumoral PKM2 on Tyr-105, consistent with long-term exposure to HBB in cultured MCF-7 cells. Notably, MCF-7 tumor response to HBB did not correlate with phosphorylation of AMPK-alpha on Thr-172, a marker of AMPK activation. Metformin (5 mM) exhibited no effect on PKM2 or its phosphorylation in cultured MCF-7 cells. Together, these results indicate that part of the inhibitory effect of HBB on ER+HER2- breast cancer is mediated through inhibition of respiration. Significance: These results establish HBB as a useful chemical probe of respiration, with indirect effects on PKM2 regulation. HBB may also be useful as a potential therapeutic candidate for ER+HER2- breast cancer. Citation Format: Zhijun Guo, Irina Sevrioukova, Eric Hanse, Xia Zhang, Ilia Denisov, Ting-Lan Chiu, Rebecca Cuellar, Christian Torres, Julia Wulfkuhle, Emanuel Petricoin, Qing Cao, Haitao Chu, Beverly Norris, Robert Schumacher, Ameeta Kelekar, Ian Blair, Jorge Capdevila, John Falck, Thomas Poulos, Steven Sligar, Gunda Georg, Elizabeth Amin, David A. Potter. Hexyl-benzyl-biguanide (HBB) potently and selectively inhibits CYP3A4 epoxygenase activity and inhibits EET stabilization of mitochondrial respiration in ER+HER2- breast cancer cells, inducing glycolysis and pyruvate biosynthesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 44.

Abstract 2689: Breast cancer inhibition by a novel and potent biguanide, N1-hexyl-N5-benzyl-biguanide

Metformin is a widely used biguanide diabetes drug that is associated with decreased breast cancer risk and is currently being studied for treatment and prevention of breast cancer. While metformin and biguanides buformin and phenformin exhibit inhibitory activity against breast cancer in vitro and in vivo, they lack potency (IC50=5-20 mM) and their mechanisms of action remain unclear. More potent biguanides may provide insights into biguanide anti-cancer activity and we therefore studied the novel biguanide N1-hexyl-N5-benzyl-biguanide mesylate (HBB), which potently inhibits the MCF-7 and MDA-MB-231 breast cancer lines (IC50=20 uM for both lines). HBB induces AMPK phosphorylation in both lines at 10 uM concentration, whereas similarly dosed metformin, buformin or phenformin exhibits no activity. HBB also inhibits STAT3 phosphorylation at 10 uM concentration, whereas metformin dosed at 10 uM exhibits no activity. HBB reduced the mitochondrial membrane potential of both lines, but the effect was more prominent in the MDA-MB-231 line. HBB also induced ROS within 2.5 hours of exposure in the MCF-7 and MDA-MB-231 lines and caused rapid necrosis, but not apoptosis. N-acetylcysteine provides partial protection from HBB for MDA-231 line, but not the MCF-7 line. HBB provides proof of principle that highly potent biguanides can be synthesized with at least 250-fold greater potency than metformin, which can provide insights into the cancer inhibitory mechanisms of biguanide drugs. R01 CA113570, Randy Shaver Foundation, CTSI University of Minnesota Citation Format: Zhijun Guo, Kathryn J. Chavez, Juan Alvarez, Xia Zhang, Beverly Norris, Michael Maher, Monique Morgan, Robert J. Schumacher, Rebecca Cuellar, Irina F. Sevrioukova, Thomas L. Poulos, Ilia Denisov, Stephen G. Sligar, Kalpna Gupta, Ian A. Blair, Jorge Capdevila, Ameeta Kelekar, Elizabeth Amin, Gunda Georg, David A. Potter. Breast cancer inhibition by a novel and potent biguanide, N1-hexyl-N5-benzyl-biguanide. [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 2689. doi:10.1158/1538-7445.AM2014-2689

Abstract 82: CYP3A4 promotes mammary carcinoma angiogenesis in a cell iIntrinsic fashion.

Cytochrome P450 3A4 (CYP3A4) has been recently been implicated in breast cancer cell growth and survival, but the role of cell intrinsic CYP3A4 in breast cancer cell engraftment and angiogenesis is unknown. Previously, we have characterized CYP3A4 knock down lines in which CYP3A4 is stably suppressed [J Biol Chem. 2011 May 20;286(20):17543-59]. The CYP3A4 knock down lines exhibit reduced proliferation and clonogenicity in vitro. We therefore tested in an estradiol dependent nude mouse mammary fat pad xenograft model whether the MCF-7 3-18 line (CYP3A4 shRNA), in which CYP3A4 protein levels are suppressed by 60%, exhibits reduced growth compared to a non-target line MCF-7 NT2. Before implantation, the suppression of CYP3A4 mRNA in the shRNA compared to NT2 cells was confirmed by qPCR. After 46 days of monitored tumor growth, the MCF-7 NT2 tumors exhibited a median size of 27±3 mm3 (median±SEM) compared to 2±0.6 mm3 for the MCF-7 CYP3A4 shRNA tumors. Tumor growth was modeled by a Gompertzian method and the difference was statistically significant (P=0.0018). The mammary fat pads were removed after sacrifice at the end of the experiment, inked, oriented and subjected to z-sectioning. All mice exhibited tumors. The CYP3A4 shRNA tumors exhibited necrosis (5 of 6 evaluable mice) whereas the NT2 control tumors did not (0 of 6 evaluable mice) (P=0.0152; two tailed Fisher9s exact test). Angiogenesis was measured by CD31 immunohistochemical staining using an automated microvessel image analysis algorithm (Image Analysis Toolbox, Aperio Technologies) allowing measurement of percentage of area occupied by CD31 staining cells at the cortex of tumor nodules. Percent area occupied by CD31 positive cells was 2.69±0.46 vs. 0.67±0.64 (mean±SEM) (P=0.033). Together, these results suggest that cell intrinsic CYP3A4 is important for mammary carcinoma growth and angiogenesis in a xenograft model. Citation Format: Zhijun Guo, Beverly Norris, Jonathan Henriksen, Monique Morgan, Michael Maher, Robert Schumacher, Robin Bliss, Haitao Chu, Kalpna Gupta, Stephen Schmechel, David Potter. CYP3A4 promotes mammary carcinoma angiogenesis in a cell iIntrinsic fashion. [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 82. doi:10.1158/1538-7445.AM2013-82