Pancreatic ductal adenocarcinoma (PDA) remains a lethal human malignancy with historically limited success in treatment. The role of aberrant Notch signaling, which requires the constitutive activation of γ-secretase, in the initiation and progression of PDA is well defined and inhibitors of this pathway are currently in clinical trials. Here we investigated the in vivo therapeutic effect of PF-03084014, a selective γ-secretase inhibitor, alone and in combination with gemcitabine in pancreatic cancer xenografts. PF-03084014 treatment inhibited the cleavage of nuclear Notch 1 intracellular domain and Notch targets Hes-1 and Hey-1. Gemcitabine treatment showed good response but not capable of inducing tumor regressions and targeting the tumor-resident cancer stem cells (CD24+CD44+ and ALDH+ tumor cells). A combination of PF-03084014 and gemcitabine treatment resulted tumor regression in 3 of 4 subcutaneously implanted xenograft models. PF-03084014, and in combination with gemcitabine reduced putative cancer stem cells, indicating that PF-03084014 target the especially dangerous and resilient cancer stem cells within pancreatic tumors. Tumor re-growth curves plotted after drug treatments demonstrated that the effect of the combination therapy was sustainable than that of gemcitabine. Notably, in a highly aggressive orthotopic model, PF-03084014 and gemcitabine combination was effective in inducing apoptosis, inhibition of tumor cell proliferation and angiogenesis, resulting in the attenuation of primary tumor growth as well as controlling metastatic dissemination, compared to gemcitabine treatment. In summary, our preclinical data suggest that PF-03084014 has greater anti-tumor activity in combination with gemcitabine in PDA and provides rationale for the further investigation of this combination in PDA.

PURPOSE Recent microarray and RNA-sequencing studies have uncovered aberrantly expressed microRNAs (miRs)in Barrett’s esophagus (BE)-associated esophageal adenocarcinoma (EAC). The functional significance of these miRs in EAC initiation and progression is largely unknown. EXPERIMENTAL DESIGN Expression levels of miR-199a/b-3p, −199a-5p, −199b-5p, −200b, −200c, −223, and −375 were determined in microdissected tissues from cardiac mucosa, BE, dysplastic BE, and EAC using quantitative real time PCR. MiR-223 expression was validated in precursors and EACs from 95 EAC patients by in situ hybridization (ISH). MiR-223 was transfected into two EAC cell lines, and in vitro assays were performed. Target genes were identified using Illumina microarray, and results were validated in cell lines and human specimens. RESULTS MiR-199 family members and miR-223 were significantly over-expressed in EAC, however only miR-223 showed a stepwise increase during EAC carcinogenesis. A similar trend was observed by ISH, which additionally showed that miR-223 is exclusively expressed by the epithelial compartment.MiR-223 over-expressing cells had statistically significantly more migratory and invasive potential than scramble sequence transfected cells. PARP1was identified as a direct target gene of miR-223 in EAC cells. Increased sensitivity to chemotherapy was observed in cells with enforced miR-223 expression and reduced PARP1. CONCLUSIONS MiR-223 is significantly up-regulated during the BE-dysplasia-EAC sequence. Although high miR-223 levels might contribute to an aggressive phenotype, our results also suggest that EAC patients with high miR-223 levels might benefit from treatment with DNA-damaging agents.

To identify effective metabolic inhibitors to suppress the aggressive growth of pancreatic ductal adenocarcinoma (PDAC), we explored the antitumor efficacy of metabolic inhibitors, as single agents, in a panel of patient-derived PDAC xenograft models (PDX) and investigated whether genomic alterations of tumors correlate with the sensitivity to metabolic inhibitors. Mice with established PDAC tumors from 6 to 13 individual PDXs were randomized and treated, once daily for 4 weeks, with either sterile PBS (vehicle) or the glutaminase inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), transaminase inhibitor aminooxyacetate (AOA), pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA), autophagy inhibitor chloroquine (CQ), and mitochondrial complex I inhibitor phenformin/metformin. Among the agents tested, phenformin showed significant tumor growth inhibition (>30% compared with vehicle) in 5 of 12 individual PDXs. Metformin, at a fivefold higher dose, displayed significant tumor growth inhibition in 3 of 12 PDXs similar to BPTES (2/8 PDXs) and DCA (2/6 PDXs). AOA and CQ had the lowest response rates. Gene set enrichment analysis conducted using the baseline gene expression profile of pancreatic tumors identified a gene expression signature that inversely correlated with phenformin sensitivity, which is in agreement with the phenformin gene expression signature of NIH Library of Integrated Network-based Cellular Signatures (LINCS). The PDXs that were more sensitive to phenformin showed a baseline reduction in amino acids and elevation in oxidized glutathione. There was no correlation between phenformin response and genetic alterations in , or Phenformin treatment showed relatively higher antitumor efficacy against established PDAC tumors, compared with the efficacy of other metabolic inhibitors and metformin. Phenformin treatment significantly diminished PDAC tumor progression and prolonged tumor doubling time. Overall, our results serve as a foundation for further evaluation of phenformin as a therapeutic agent in pancreatic cancer. .

Background:Albumin-bound paclitaxel (nab-paclitaxel, nab-PTX) plus gemcitabine (GEM) combination has demonstrated efficient antitumour activity and statistically significant overall survival of patients with metastatic pancreatic ductal adenocarcinoma (PDAC) compared with GEM monotherapy. This regimen is currently approved as a standard of care treatment option for patients with metastatic PDAC. It is unclear whether cremophor-based PTX combined with GEM provide a similar level of therapeutic efficacy in PDAC.Methods:We comprehensively explored the antitumour efficacy, effect on metastatic dissemination, tumour stroma and survival advantage following GEM, PTX and nab-PTX as monotherapy or in combination with GEM in a locally advanced, and a highly metastatic orthotopic model of human PDAC.Results:Nab-PTX treatment resulted in significantly higher paclitaxel tumour plasma ratio (1.98-fold), robust stromal depletion, antitumour efficacy (3.79-fold) and survival benefit compared with PTX treatment. PTX plus GEM treatment showed no survival gain over GEM monotherapy. However, nab-PTX in combination with GEM decreased primary tumour burden, metastatic dissemination and significantly increased median survival of animals compared with either agents alone. These therapeutic effects were accompanied by depletion of dense fibrotic tumour stroma and decreased proliferation of carcinoma cells. Notably, nab-PTX monotherapy was equivalent to nab-PTX plus GEM in providing survival advantage to mice in a highly aggressive metastatic PDAC model, indicating that nab-PTX could potentially stop the progression of late-stage pancreatic cancer.Conclusions:Our data confirmed that therapeutic efficacy of PTX and nab-PTX vary widely, and the contention that these agents elicit similar antitumour response was not supported. The addition of PTX to GEM showed no survival advantage, concluding that a clinical combination of PTX and GEM may unlikely to provide significant survival advantage over GEM monotherapy and may not be a viable alternative to the current standard-of-care nab-PTX plus GEM regimen for the treatment of PDAC patients.

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Background and Aim: Pancreatic ductal adenocarcinoma remains a lethal malignancy with historically limited success in treatment. This study aims to (i) develop a Highly Active Anti-Tumor Therapy (HAATT) by specifically targeting multiple components of the mammalian target of rapamycin (mTOR) signaling and (ii) attempt to tailor metabolism-targeted therapy by analyzing the global metaboloic profile of tumors. Methods: mTOR plays a central role in regulating cell growth, proliferation and survival. Here we sought to develop a therapy intend to severely harness mTOR signaling. We formulated a multi-drug regimen comprising Metformin, Rapamycin, and PP242. We investigated the effect of individual agents or in combinations, both in vitro as well as in vivo using human pancreatic cancer cell lines and patient-derived pancreatic cancer xenografts. Mice with established tumors (both s.c and orthotopically implanted) were pre-treated with 2 cycles of gemcitabine, randomized and treated with individual agents or in combinations. Tumors were harvested at various time-points and used for PD assays and functional screens including global metabolic profiles. Results: Our studies demonstrate that the triple combination therapy specifically inactivates mTOR targets, resulting in durable suppression of tumor cell proliferation. Although, gemcitabine therapy was initially effective, but tumors resumed growth upon cessation of gemcitabine treatment. Responses to single agents or doublets were temporary and tumors grew progressively. Conversely, addition of triple combination therapy to post-gemcitabine treatment not only resulted in rapid regression of gemcitabine refractory tumors, which lasted up to 2 months, but also remarkably delayed tumor recurrence. Notably in gemcitabine refractory orthotopic model, the triple combination therapy was effective in inhibiting tumor angiogenesis resulting in the suppression of primary tumor growth. Analysis of tumor samples revealed that combination treatment remarkably suppressed tumor cell proliferation and reversed several key metabolic alterations found in gemcitabine resistant tumors. Conclusion: Our results demonstrate that the triple combination therapy is remarkably effective in inhibiting mTOR targets, resulting in sustained tumor growth inhibition and remarkably delaying tumor recurrence in clinically relevant models established from the tumors resected from pancreatic cancer patients. Our findings have translational relevance and lay framework towards the establishment of a highly effective therapeutic strategy to substantially improve pancreatic cancer therapy. Acknowledgement: This study is supported by a Stand Up To Cancer Dream Team Translational Cancer Research Grant (SU2C-AACR-DT0509) and funding from Commonwealth Foundation for Cancer Research. Citation Format: N.V Rajeshkumar, Shinichi Yabuuchi, Chirag Patel, Shweta G. Pai, Dung T. Le, Daniel D. Von Hoff, Chi V. Dang, Anirban Maitra, Jonathan Powell. Targeting multiple components of the mammalian target of rapamycin (mTOR) signaling: A novel combination therapy to improve pancreatic cancer treatment. [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 5396. doi:10.1158/1538-7445.AM2013-5396

Background and Aim: Advanced pancreatic cancer is both deadly and difficult to treat with success. Here, we disseminate the results of comprehensive preclinical studies which laid a strong foundation for the rapid bench to bedside development of nab-paclitaxel, an albumin-bound formulation of paclitaxel, in combination with gemcitabine, a regimen recently approved by U.S. Food and Drug Administration as a first-line treatment for patients with metastatic pancreatic cancer. Materials and Methods: We enrolled a total of 650 mice with established pancreatic tumors originated from a collection of patient-derived pancreatic cancer xenografts. The present study investigated the anti-tumor activity, survival advantage and mechanism of action of nab-paclitaxel or Cremophor EL™ (CreEL)-based paclitaxel monotherapy and in combination with gemcitabine. Results: When tested in mice with subcutaneous tumors originating from 11 separate individual patient xenografts, nab-paclitaxel plus gemcitabine treatment demonstrated superior tumor regression response, robustly depleted the tumor desmoplatic stroma, leading to enhanced gemcitabine uptake (2.8-fold) in the tumor compared to tumors in the gemcitabine alone treated mice. In orthotopic models, nab-paclitaxel treatment leads to an average of 3.64-fold decrease in primary tumor volumes compared to CreEL-based paclitaxel. Intra-tumor stromal collapse combined with decreased tumor cell proliferation was clearly evident in the primary tumors of nab-paclitaxel treated mice compared to CreEL-based paclitaxel, when the mice were sacrificed immediately after five consecutive day's treatment or three weeks after the final dose of the agents. In a highly aggressive orthotopic model, nab-paclitaxel plus gemcitabine treatment prevented primary tumor progression, and metastatic spread to liver, lymph nodes and diaphragm. While CreEL-based paclitaxel plus gemcitabine treatment failed to enhance mouse survival compared to gemcitabine monotherapy, the nab-paclitaxel plus gemcitabine combination proved statistically significant (p=0.0133) in enhancing survival. nab-paclitaxel monotherapy demonstrated statistically significant survival advantage compared to CreEL-based paclitaxel monotherapy (p=0.0072). Remarkably, nab-paclitaxel monotherapy was equivalent to nab-paclitaxel plus gemcitabine in providing survival advantage in a highly aggressive metastatic model of pancreatic cancer. Conclusion: Our results demonstrated that co-treatment with nab-paclitaxel and gemcitabine resulted in superior tumor regression response, stromal depletion and enhanced intra-tumoral gemcitabine uptake compared with either single agent alone. nab-paclitaxel demonstrated superior anti-tumor activity and provided a statistically significant survival advantage compared to CreEL-based paclitaxel. Our results provide further rationale for future preclinical and clinical trials in pancreatic cancer using nab-paclitaxel as a backbone therapy in combination with novel experimental and targeted agents. Acknowledgements: The study was supported by funding from Celgene Corporation and AACR-Stand Up To Cancer Dream Team Translational Cancer Research Grant (SU2C-AACR-DT0509). Citation Format: N.V Rajeshkumar, Shinichi Yabuuchi, Shweta G. Pai, Scott Bateman, Ellen Filvaroff, Daniel W. Pierce, Carla Heise, Daniel D. Von Hoff, Anirban Maitra, Manuel Hidalgo. Highlights of innovative preclinical studies which guided the rapid bench to bedside development of nab-paclitaxel plus gemcitabine combination for the treatment of pancreatic cancer. [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 LB-94. doi:10.1158/1538-7445.AM2014-LB-94

<div>Abstract<p><b>Purpose:</b> To identify effective metabolic inhibitors to suppress the aggressive growth of pancreatic ductal adenocarcinoma (PDAC), we explored the <i>in vivo</i> antitumor efficacy of metabolic inhibitors, as single agents, in a panel of patient-derived PDAC xenograft models (PDX) and investigated whether genomic alterations of tumors correlate with the sensitivity to metabolic inhibitors.</p><p><b>Experimental Design:</b> Mice with established PDAC tumors from 6 to 13 individual PDXs were randomized and treated, once daily for 4 weeks, with either sterile PBS (vehicle) or the glutaminase inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), transaminase inhibitor aminooxyacetate (AOA), pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA), autophagy inhibitor chloroquine (CQ), and mitochondrial complex I inhibitor phenformin/metformin.</p><p><b>Results:</b> Among the agents tested, phenformin showed significant tumor growth inhibition (>30% compared with vehicle) in 5 of 12 individual PDXs. Metformin, at a fivefold higher dose, displayed significant tumor growth inhibition in 3 of 12 PDXs similar to BPTES (2/8 PDXs) and DCA (2/6 PDXs). AOA and CQ had the lowest response rates. Gene set enrichment analysis conducted using the baseline gene expression profile of pancreatic tumors identified a gene expression signature that inversely correlated with phenformin sensitivity, which is in agreement with the phenformin gene expression signature of NIH Library of Integrated Network-based Cellular Signatures (LINCS). The PDXs that were more sensitive to phenformin showed a baseline reduction in amino acids and elevation in oxidized glutathione. There was no correlation between phenformin response and genetic alterations in <i>KRAS, TP53, SMAD4</i>, or <i>PTEN</i>.</p><p><b>Conclusions:</b> Phenformin treatment showed relatively higher antitumor efficacy against established PDAC tumors, compared with the efficacy of other metabolic inhibitors and metformin. Phenformin treatment significantly diminished PDAC tumor progression and prolonged tumor doubling time. Overall, our results serve as a foundation for further evaluation of phenformin as a therapeutic agent in pancreatic cancer. <i>Clin Cancer Res; 23(18); 5639–47. ©2017 AACR</i>.</p></div>