Shrey Modi

Impaired synthesis of stromal components in response to Minnelide improves vascular function, drug delivery and survival in pancreatic cancer

Purpose: Pancreatic cancer stromal microenvironment is considered to be the major reason for failure of conventional and targeted therapy for this disease. The desmoplastic stroma, comprising mainly collagen and glycosaminoglycans like hyaluronan (HA), is responsible for compression of vasculature in the tumor resulting in impaired drug delivery and poor prognosis. Minnelide, a water-soluble prodrug of triptolide currently in phase I clinical trial, has been very effective in multiple animal models of pancreatic cancer. However, whether Minnelide will have efficacious delivery into the tumor despite the desmoplastic stroma has not been evaluated before. Experiment Design: Patient tumor-derived xenografts (PDX) and spontaneous pancreatic cancer mice were treated with 0.42 and 0.21 mg/kg body weight for 30 days. Stromal components were determined by IHC and ELISA-based assays. Vascular functionality and drug delivery to the tumor were assessed following treatment with Minnelide. Result: Our current study shows that treatment with Minnelide resulted in reduction of ECM components like HA and collagen in the pancreatic cancer stroma of both the spontaneous KPC mice as well as in patient tumor xenografts. Furthermore, treatment with Minnelide improved functional vasculature in the tumors resulting in four times more functional vessels in the treated animals compared with untreated animals. Consistent with this observation, Minnelide also resulted in increased drug delivery into the tumor compared with untreated animals. Along with this, Minnelide also decreased viability of the stromal cells along with the tumor cells in pancreatic adenocarcinoma. Conclusions: In conclusion, these results are extremely promising as they indicate that Minnelide, along with having anticancer effects is also able to deplete stroma in pancreatic tumors, which makes it an effective therapy for pancreatic cancer. Clin Cancer Res; 22(2); 415–25. ©2015 AACR.

Downregulation of Sp1 by Minnelide leads to decrease in HSP70 and decrease in tumor burden of Gastric cancer

Background Gastric cancer is the third leading cause of cancer related mortality worldwide with poor survival rates. Even though a number of chemotherapeutic compounds have been used against this disease, stomach cancer has not been particularly sensitive to these drugs. In this study we have evaluated the effect of triptolide, a naturally derived diterpene triepoxide and its water soluble pro-drug Minnelide on several gastric adenocarcinoma cell lines both as monotherapy and in combination with CPT-11. Methods Gastric cancer cell lines MKN28 and MKN45 were treated with varying doses of triptolide in vitro. Cell viability was measured using MTT based assay kit. Apoptotic cell death was assayed by measuring caspase activity. Effect of the triptolide pro-drug, Minnelide, was evaluated by implanting the gastric cancer cells subcutaneously in athymic nude mice. Results Gastric cancer cell lines MKN28 and MKN45 cells exhibited decreased cell viability and increased apoptosis when treated with varying doses of triptolide in vitro. When implanted in athymic nude mice, treatment with Minnelide reduced tumor burden in both MKN28 derived tumors as well as MKN45 derived tumors. Additionally, we also evaluated Minnelide as a single agent and in combination with CPT-11 in the NCI-N87 human gastric tumor xenograft model. Conclusion Our results indicated that the combination of Minnelide with CPT-11 resulted in significantly smaller tumors compared to control. These studies are extremely encouraging as Minnelide is currently undergoing phase 1 clinical trials for gastrointestinal cancers.

Minnelide Inhibits Androgen Dependent, Castration Resistant Prostate Cancer Growth by Decreasing Expression of Androgen Receptor Full Length and Splice Variants

With almost 30,000 deaths per year, prostate cancer is the second‐leading cause of cancer‐related death in men. Androgen Deprivation Therapy (ADT) has been the corner stone of prostate cancer treatment for decades. However, despite an initial response of prostate cancer to ADT, this eventually fails and the tumors recur, resulting in Castration Resistant Prostate Cancer (CRPC). Triptolide, a diterpene triepoxide, has been tested for its anti‐tumor properties in a number of cancers for over a decade. Owing to its poor solubility in aqueous medium, its clinical application had been limited. To circumvent this problem, we have synthesized a water‐soluble pro‐drug of triptolide, Minnelide, that is currently being evaluated in a Phase 1 clinical trial against gastrointestinal tumors. In the current study, we assessed the therapeutic potential of Minnelide and its active compound triptolide against androgen dependent prostate cancer both in vitro as well as in vivo.

A Novel Immunocompetent Mouse Model of Pancreatic Cancer with Robust Stroma: a Valuable Tool for Preclinical Evaluation of New Therapies

A valid preclinical tumor model should recapitulate the tumor microenvironment. Immune and stromal components are absent in immunodeficient models of pancreatic cancer. While these components are present in genetically engineered models such as KrasG12D; Trp53R172H; Pdx-1Cre (KPC), immense variability in development of invasive disease makes them unsuitable for evaluation of novel therapies. We have generated a novel mouse model of pancreatic cancer by implanting tumor fragments from KPC mice into the pancreas of wild type mice. Three-millimeter tumor pieces from KPC mice were implanted into the pancreas of C57BL/6J mice. Four to eight weeks later, tumors were harvested, and stromal and immune components were evaluated. The efficacy of Minnelide, a novel compound which has been shown to be effective against pancreatic cancer in a number of preclinical murine models, was evaluated. In our model, consistent tumor growth and metastases were observed. Tumors demonstrated intense desmoplasia and leukocytic infiltration which was comparable to that in the genetically engineered KPC model and significantly more than that observed in KPC tumor-derived cell line implantation model. Minnelide treatment resulted in a significant decrease in the tumor weight and volume. This novel model demonstrates a consistent growth rate and tumor-associated mortality and recapitulates the tumor microenvironment. This convenient model is a valuable tool to evaluate novel therapies.

Control of Apoptosis in Treatment and Biology of Pancreatic Cancer

Pancreatic cancer is estimated to be the 12th most common cancer in the United States in 2014 and yet this malignancy is the fourth leading cause of cancer‐related death in the United States. Late detection and resistance to therapy are the major causes for its dismal prognosis. Apoptosis is an actively orchestrated cell death mechanism that serves to maintain tissue homoeostasis. Cancer develops from normal cells by accruing significant changes through one or more mechanisms, leading to DNA damage and mutations, which in a normal cell would induce this programmed cell death pathway. As a result, evasion of apoptosis is one of the hallmarks of cancer cells. PDAC is notoriously resistant to apoptosis, thereby explaining its aggressive nature and resistance to conventional treatment modalities. The current review is focus on understanding different intrinsic and extrinsic pathways in pancreatic cancer that may affect apoptosis in this disease. J. Cell. Biochem. 117: 279–288, 2016.

“Heat shock protein 70 in pancreatic diseases: Friend or foe”

The heat shock response in pancreatitis that is activated via HSP70 protects acinar cells through multiple simultaneous mechanisms. It inhibits trypsinogen activation and modulates NF‐κB signaling to limit acinar cell injury. On the other hand, HSP70 is overexpressed in pancreatic cancer and is hijacked by the cellular machinery to inhibit apoptosis. Inhibition of HSP70 in pancreatic cancer by a novel compound, Minnelide, has shown considerable clinical promise.

Regulation of angiogenesis by microRNAs in cardiovascular diseases

Non-coding RNAs are functional RNA molecules comprising the majority of human transcriptome. Only about 1.5% of the human genome is transcribed into messenger RNAs (mRNA) that are translated into proteins. Among the non-coding RNAs, miRNAs are extensively studied and miR targets in endothelial cells, perivascular cells, and angiogenic signaling are relatively well defined. MicroRNAs not only regulate transcripts in situ but also function as paracrine mediators in affecting angiogenesis at distant sites. Exosomal miRs are implicated in modulating endothelial cell function and angiogenesis. Thus miRs have been shown to affect tissue microenvironment in a multitude of ways. A comprehensive analysis of the role of miRs in modulation of angiogenesis and their impact on cardiovascular diseases is presented in this review.

Cell-permeable iron inhibits vascular endothelial growth factor receptor-2 signaling and tumor angiogenesis

Angiogenesis is important for tumor growth and metastasis. Hypoxia in tumors drives this angiogenic response by stabilizing Hypoxia Inducible Factors (HIF) and target genes like Vascular Endothelial Growth Factor (VEGF). HIF stability is regulated by Prolylhydroxylases (PHD)-mediated modification. Iron is an important cofactor in regulating the enzymatic activity of PHDs. Reducing intracellular iron, for instance, mimics hypoxia and induces a pro-angiogenic response. It is hypothesized that increasing the intracellular iron levels will have an opposite, anti-angiogenic effect. We tested this hypothesis by perturbing iron homeostasis in endothelial cells using a unique form of iron, Ferric Ammonium Citrate (FAC). FAC is a cell-permeable form of iron, which can passively enter into cells bypassing the transferrin receptor mediated uptake of transferrin-bound iron. Our studies show that FAC does not decrease the levels of HIF-1α and HIF-2α in endothelial cells but inhibits the autocrine stimulation of VEGF-Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) system by blocking receptor tyrosine kinase phosphorylation. FAC inhibits VEGF-induced endothelial cell proliferation, migration, tube formation and sprouting. Finally, systemic administration of FAC inhibits VEGF and tumor cell-induced angiogenesis in vivo. In conclusion, our studies show that cell-permeable iron attenuates VEGFR-2 mediated signaling and inhibits tumor angiogenesis.

Synergy of water soluble prodrug triptolide (minnelide) with gemcitabine and nab-paclitaxel in pancreatic cancer.

259 Background: Gemcitabine with nab-paclitaxel is the standard of care for metastatic Pancreatic Ductal Adenocarcinoma (PDAC). Addition of nab-paclitaxel to gemcitabine improves the survival of patients with metastatic pancreatic cancer by only 6 weeks while increasing toxicity. Meaningful combinations that reduce the doses while improving efficacy are required. Water soluble prodrug of triptolide (Minnelide) is effective against PDAC in preclinical studies and has demonstrated activity in the ongoing phase I trial. We have evaluated the combination of low dose triptolide prodrug with lowered doses of Abraxane and gemcitabine in PDAC. Methods: Pancreatic cancer cell lines S2-013 and S2-VP10 were xenografted both subcutaneously as well as orthotopically in athymic nude mice. Efficacy of a combination of triptolide prodrug (Minnelide: 0.21mg/kg) with reduced dose of gemcitabine 100mg/kg + Abraxane 10mg/kg (~40% of the clinically effective dose) was evaluated and compared with both standard of care gemcitab...

Abstract 3499: Triptolide pro-drug decreases tumor burden and halts tumor progression in a murine model of acute myeloid leukemia

Introduction Standard treatment for acute myelogenous leukemia (AML) has not changed over the past few decades and relies primarily on the traditional “7 + 3″ regimen of daunorubicin, administered over 3 days and Cytarabine, administered over 7 days. This chemo-intensive regimen is poorly tolerated by patients and is characterized by a high rate of relapse and treatment failure. Triptolide is a diterpenoid tri-epoxide compound isolated from the Chinese herb Tripterygium wilfordii. The water-soluble pro-drug of triptolide, Minnelide has been shown by our group to be highly effective in a number of pre-clinical models of solid tumors and is currently undergoing Phase I Clinical trial for gastrointestinal tumors. Interim analysis of the Phase I data has shown that the equivalent mouse dose of 0.2 mg/kg/day is well tolerated by patients with no reported dose limiting toxicity. Methods Primary AML apheresis samples from patients with acute myeloid leukemia and multiple AML cell Lines (THP1, KG1, Kasumi1, HL-60) were treated with Triptolide at doses from of 2.5 nM to 50nM and cell viability was measured using a formazan based colorimetric assay. Apoptosis was measured using Annexin V via flowcytometry and colony forming ability of AML cell lines was measured using a methylcellulose based assay. To generate an engraftment model of AML, NOD.Rag1-/-;γcnull (NRG) animals expressing human interleukin-3 (IL-3) and human GM-CSF (NRGS) were injected luciferase expressing THP1 cells after sublethal irradiation of 250 cGy. These animals were then serially evaluated using In Vivo Imaging System (IVIS) and leukemic burden was calculated the total bioluminescent signal after intra-periotneal injection of luciferin. Treatment with vehicle or Minnelide at a dose of 0.1mg/kg/day and 0.15 mg/kg/day was started on the 10th day after confirming engraftment using IVIS. Results Triptolide at a dose range of 2.5 nM to 50 nM produced a dose and time dependent killing of leukemic cells in both cell lines and primary AML patient samples. The IC-50 for THP1, KG1, Kasumi1 and HL-60 cell lines with triptolide treatment at 48 hours were 5 ± 0.8 nM, 8 ± 0.7 nM, 7.2 ± 0.6 nM, 10 nM ± 2 nM respectively. Treatment with triptolide at a dose of 2.5 nM induced cell death and apoptosis as measured by Annexin V posiitvity in primary AML apheresis samples. Colony formation by THP1 cells and KG1 cells was completely abrogated by treatment with Triptolide at 2.5 nM and 25 nM respectively. Vehicle treated mice showed a rapid progression of disease burden when compared to Minnelide treated mice. At a dose of 0.1 mg/kg/day and 0.15 mg/kg/day, these mice had no appreciable increase in leukemic burden over normal mice when assessed by IVIS. Conclusion We show that Minnelide induces cell death at therapeutically relevant concentrations in vitro and decreased leukemic burden in a murine model of AML. Minnelide may emerge as a novel therapeutic strategy in treating patients with AML. Citation Format: Bhuwan Giri, Sulagna Banerjee, John George, Shrey Modi, Vineet Kumar Gupta, Mahendra K. Singh, Vikas Dudeja, Ashok K. Saluja. Triptolide pro-drug decreases tumor burden and halts tumor progression in a murine model of acute myeloid leukemia. [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 3499.