Tracy L. Gamblin

The BET bromodomain inhibitor JQ1 suppresses growth of pancreatic ductal adenocarcinoma in patient-derived xenograft models

The primary aim of this study was to evaluate the antitumor efficacy of the bromodomain inhibitor JQ1 in pancreatic ductal adenocarcinoma (PDAC) patient-derived xenograft (tumorgraft) models. A secondary aim of the study was to evaluate whether JQ1 decreases expression of the oncogene c-Myc in PDAC tumors, as has been reported for other tumor types. We used five PDAC tumorgraft models that retain specific characteristics of tumors of origin to evaluate the antitumor efficacy of JQ1. Tumor-bearing mice were treated with JQ1 (50 mg/kg daily for 21 or 28 days). Expression analyses were performed with tumors harvested from host mice after treatment with JQ1 or vehicle control. An nCounter PanCancer Pathways Panel (NanoString Technologies) of 230 cancer-related genes was used to identify gene products affected by JQ1. Quantitative RT–PCR, immunohistochemistry and immunoblots were carried out to confirm that changes in RNA expression reflected changes in protein expression. JQ1 inhibited the growth of all five tumorgraft models (P<0.05), each of which harbors a KRAS mutation; but induced no consistent change in expression of c-Myc protein. Expression profiling identified CDC25B, a regulator of cell cycle progression, as one of the three RNA species (TIMP3, LMO2 and CDC25B) downregulated by JQ1 (P<0.05). Inhibition of tumor progression was more closely related to decreased expression of nuclear CDC25B than to changes in c-Myc expression. JQ1 and other agents that inhibit the function of proteins with bromodomains merit further investigation for treating PDAC tumors. Work is ongoing in our laboratory to identify effective drug combinations that include JQ1.

Development and Histopathological Characterization of Tumorgraft Models of Pancreatic Ductal Adenocarcinoma

Pancreatic cancer is the one of the deadliest of all malignancies. The five year survival rate for patients with this disease is 3-5%. Thus, there is a compelling need for novel therapeutic strategies to improve the clinical outcome for patients with pancreatic cancer. Several groups have demonstrated for other types of solid tumors that early passage human tumor xenograft models can be used to define some genetic and molecular characteristics of specific human tumors. Published studies also suggest that murine tumorgraft models (early passage xenografts derived from direct implantation of primary tumor specimens) may be useful in identifying compounds with efficacy against specific tumor types. Because pancreatic cancer is a fatal disease and few well-characterized model systems are available for translational research, we developed and characterized a panel of pancreatic tumorgraft models for biological evaluation and therapeutic drug testing. Of the 41 primary tumor specimens implanted subcutaneously into mice, 35 produced viable tumorgraft models. We document the fidelity of histological and morphological characteristics and of KRAS mutation status among primary (F0), F1, and F2 tumors for the twenty models that have progressed to the F3 generation. Importantly, our procedures produced a take rate of 85%, higher than any reported in the literature. Primary tumor specimens that failed to produce tumorgrafts were those that either contained <10% tumor cells or that were obtained from significantly smaller primary tumors. In view of the fidelity of characteristics of primary tumor specimens through at least the F2 generation in mice, we propose that these tumorgraft models represent a useful tool for identifying critical characteristics of pancreatic tumors and for evaluating potential therapies.

JQ1 Induces DNA Damage and Apoptosis, and Inhibits Tumor Growth in a Patient-Derived Xenograft Model of Cholangiocarcinoma

Cholangiocarcinoma (CCA) is a fatal disease with a 5-year survival of <30%. For a majority of patients, chemotherapy is the only therapeutic option, and virtually all patients relapse. Gemcitabine is the first-line agent for treatment of CCA. Patients treated with gemcitabine monotherapy survive ∼8 months. Combining this agent with cisplatin increases survival by ∼3 months, but neither regimen produces durable remissions. The molecular etiology of this disease is poorly understood. To facilitate molecular characterization and development of effective therapies for CCA, we established a panel of patient-derived xenograft (PDX) models of CCA. We used two of these models to investigate the antitumor efficacy and mechanism of action of the bromodomain inhibitor JQ1, an agent that has not been evaluated for the treatment of CCA. The data show that JQ1 suppressed the growth of the CCA PDX model CCA2 and demonstrate that growth suppression was concomitant with inhibition of c-Myc protein expression. A second model (CCA1) was JQ1-insensitive, with tumor progression and c-Myc expression unaffected by exposure to this agent. Also selective to CCA2 tumors, JQ1 induced DNA damage and apoptosis and downregulated multiple c-Myc transcriptional targets that regulate cell-cycle progression and DNA repair. These findings suggest that c-Myc inhibition and several of its transcriptional targets may contribute to the mechanism of action of JQ1 in this tumor type. We conclude that BET inhibitors such as JQ1 warrant further investigation for the treatment of CCA. Mol Cancer Ther; 17(1); 107–18. ©2017 AACR.

Abstract 1075: JQ1 induces DNA damage, inhibits expression of DNA repair proteins, and synergizes with PARP inhibitors in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer. PDAC is a highly aggressive tumor with a 5-year survival of The goal of the current study was to develop effective combination therapy for PDAC by identifying agents that might be combined with the BET bromodomain inhibitor JQ1, which we have shown to inhibit the growth in vivo of PDAC patient derived xenografts (PDX). Expression profile analysis of tumors from vehicle control and JQ1 treated mice revealed that JQ1 inhibited the expression of multiple gene products involved in DNA repair. Notably, JQ1 inhibited expression of DNA double-strand break (DSB) repair proteins BRCA2 and Ku80. Immunohistochemical staining confirmed down-regulation of expression of both proteins in tumors of mice treated with JQ1. Further, immunoblot and immunofluorescence analyses demonstrated that decreased expression of BRCA2 and Ku80 was coincident with increased levels of DNA damage, as reflected by expression of the DNA DSB marker gamma-H2AX. Data generated in vivo in three independent PDX models corroborated in vitro data generated using pancreatic cancer cell lines BxPC3 and Panc1. The data suggest that JQ1 induces DNA damage by inhibiting DNA repair. Because DNA repair deficiency sensitizes cells to PARP inhibitors, we hypothesized that JQ1-induced DNA repair deficiency would sensitize PDAC cells to PARP inhibitors. To address this hypothesis, we exposed Panc1 and BxPC3 to JQ1 or to a PARP 1/2 inhibitor (veliparib or olaparib) or to the combinations, and assessed the efficacy of each. Growth inhibition data, analyzed using Compusyn software and reported as combination indices, demonstrated that the combinations of JQ1 + veliparib or olaparib exert synergistic cytotoxicity. Further, the combination of JQ1 + a PARP inhibitor increased the accumulation of DNA damage in vitro, compared to either agent alone. We conclude that JQ1 induces DNA damage due at least in part to DNA repair deficiency, and propose that this mechanism sensitizes PDAC cells to PARP inhibitors. Citation Format: Aubrey Lynn Miller, Tracy Gamblin, Leona Council, Robert van Waardenburg, Eddy Yang, James Bradner, Karina Yoon. JQ1 induces DNA damage, inhibits expression of DNA repair proteins, and synergizes with PARP inhibitors in pancreatic cancer cells [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 1075. doi:10.1158/1538-7445.AM2017-1075

Abstract 1293: JQ1 sensitivity of patient-derived xenograft models of cholangiocarcinoma

Cholangiocarcinoma (CCA) is a lethal malignancy arising from cholangiocytes in any part of the biliary tree. The incidence of CCA has been on the rise worldwide, and the prognosis and clinical outcome have remained essentially unchanged for 30 years. The majority of patients are diagnosed at late stage, and surgery continues to be the only cure. Patients receive systemic chemotherapy with the first-line combination therapy comprising gemcitabine and cisplatin. Median survival for these patients is To determine gene products whose upregulation or downregulation is responsible for the differences in sensitivity to JQ1 among our CCA models, we generated expression profiles of tumors from vehicle control and JQ1 treated mice using NanoString technology (nCounter PanCancer Pathways panel). Our data demonstrate that JQ1 inhibited the expression of c-Myc to a greater extent in the sensitive models than in the insensitive model. Expression array data showed further that gene products involved in cell cycle and DNA repair pathways were also decreased by JQ1. Of particular interest were two transcriptional targets of c-Myc, Chk1 and BRCA2, each of which is involved in DNA damage response. Immunohistochemistry staining confirmed expression profile analyses. We conclude that the inhibition of cell cycle and DNA repair genes may contribute to the mechanism of action of JQ1 in CCA tumors. Citation Format: Aubrey L. Miller, Patrick L. Garcia, Tracy L. Gamblin, Leona N. Council, Xiangqin Cui, James E. Bradner, Eddy S. Yang, Karina J. Yoon. JQ1 sensitivity of patient-derived xenograft models of cholangiocarcinoma. [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 1293.

Abstract 1779: Bromodomain inhibitor JQ1 inhibits cholangiocarcinoma tumor growth in patient-derived xenograft models

Cholangiocarcinoma (CCA) is a lethal malignancy of the biliary epithelium that can arise in any part of the biliary tree. Surgery is the only curative treatment for CCA, but only ∼30% of patients present with resectable disease. The remaining 70% of patients present with advanced or metastatic disease and, if eligible, undergo systemic chemotherapy with the first-line combination of gemcitabine and cisplatin. This combination was shown in a phase II clinical trial to significantly increase median survival from 8.1 to 11.7 months, compared to gemcitabine alone. In order to improve on current treatment, pre-clinical evaluation of novel therapeutics is essential to improving outcome. Unfortunately, the paucity of data describing characteristics common to CCA make development of targeted therapy difficult. However, as is true for other types of solid tumors, c-Myc expression likely contributes to CCA phenotype: c-Myc expression has been observed in 95% of CCA tumors, and experimental down-regulation of c-Myc decreases the invasive potential of CCA cells in vitro. Recently it has become possible to inhibit expression of c-Myc using BET inhibitors. Therefore, we evaluated the efficacy of the bromodomain (BET) inhibitor JQ1 using in vivo models of CCA. The five patient-derived xenograft (PDX) models of CCA that we developed are the first such models to be reported. These models retain the heterogeneity, architecture and specific genetic characteristics of the primary tumors from which they were derived. We used three of these models to examine whether the BET inhibitor JQ1 inhibited CCA tumor growth and generated expression profiles of vehicle- and drug-treated tumors. We administered 50 mg/kg of JQ1 i.p. daily for 20 days and monitored tumor growth. This treatment regimen was well tolerated by tumor-bearing mice, without apparent toxicity. Our data demonstrate that JQ1 suppressed tumor growth in two of the three models, compared to vehicle control treated mice. The data also showed that JQ1-treated tumors had lower levels of c-Myc RNA (↓5-fold) and protein and of RNA encoding multiple transcriptional targets downstream of this oncogenic transcription factor. We conclude that BET inhibitors such as JQ1 warrant further investigation as potentially effective drugs for the treatment of CCA. Citation Format: Patrick L. Garcia, Aubrey L. Miller, Kelly Kreitzburg, Tracy L. Gamblin, Leona N. Council, John D. Christein, Pablo Arnoletti, Marty Heslin, Sushanth Reddy, Joseph H. Richardson, Eddy S. Yang, Jun Qi, James E. Bradner, Karina J. Yoon. Bromodomain inhibitor JQ1 inhibits cholangiocarcinoma tumor growth in patient-derived xenograft models. [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 1779. doi:10.1158/1538-7445.AM2015-1779

Abstract 4612: JQ1 suppresses tumor growth in tumorgraft models of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, is one of the most deadly of malignancies. Patients often present late in the course of disease limiting treatment options, with the majority of patients receiving chemotherapy. However, currently available chemotherapy has not impacted overall patient survival, and new therapies are urgently needed. Recently, our lab has documented that tumorgrafts derived specifically from primary PDAC tumors retain characteristics of the tumors of origin. In terms of drug evaluation, tumorgraft models have been shown to be predictive of clinical utility because these models retain tumor heterogeneity, recapitulate tumor architecture, and contain human stroma. To date, most efforts to use tumorgraft models to identify effective agents for the treatment of PDAC have taken an “all comers approach”; but this approach has thus far identified no effective therapies. We proposed that JQ1, a compound that targets the oncogene c-Myc, would be efficacious for the treatment of PDAC, based on molecular characteristics of these tumors. The oncogene c-Myc is amplified or overexpressed in a 30-45% of primary or metastatic PDAC tumors. Also, overexpression of this oncogene is sufficient to induce tumor formation in a genetically engineered mouse model of PDAC, suggesting a critical role in the tumorigenesis of this tumor type. JQ1 is a novel compound in that it is a relatively specific bromodomain inhibitor. Bromodomain containing proteins recognize acetylated lysine residues on histones and direct the assembly of macromolecular molecules to the chromatin for transcription. A specific subfamily of these proteins, known as BETs (bromodomain and extra-terminal), recruit c-Myc to specific sites for transcription. Exposure of PDAC cells to JQ1 decreases the expression and therefore the activity of c-Myc. To test the effectiveness of JQ1 in vivo, we administered 50 mg/kg of JQ1 i.p. once a day for 28 days and monitored tumor growth in a panel of 5 PDAC tumorgraft models. This dose and schedule of JQ1 administration was well tolerated in mice with no significant toxicity. Our data indicate that JQ1 suppressed tumor growth in all 5 models, compared to vehicle control treated mice. The data also showed a modest, JQ1-induced down regulation of c-Myc and NFkB. We conclude that JQ1 and other bromodomain inhibitors warrant further investigation as potentially effective agents for the treatment of PDAC. This work was supported by UAB/UMN SPORE in pancreatic cancer (P50 CA101955). Citation Format: Patrick L. Garcia, Tracy Gamblin, Leona N. Council, John D. Christein, J. Pablo Arnoletti, Martin J. Heslin, Joseph H. Richardson, Jun Qi, Jay E. Bradner, Karina J. Yoon. JQ1 suppresses tumor growth in tumorgraft models of pancreatic ductal adenocarcinoma. [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 4612. doi:10.1158/1538-7445.AM2014-4612

Abstract 2725: Establishment of the primary human tumor xenograft (tumorgraft) mouse model of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer continues to be plagued by a low overall 5 year survival rate, which has remained at a stagnant 5% over the past 30 years. In addition, pancreatic cancer is the 10 th leading diagnosed cancer, yet is the 4 th leading cause of cancer related deaths in the US, in both men and women. Recent research efforts have been focused on increasing our understanding of the molecular and genetic aspects of pancreatic cancer, in order to improve outcome for these patients. A major tool of pancreatic cancer research has been the use of established cancer cell lines, although the inherent ability of these cell lines to recapitulate human pancreatic disease is not ideal, due to growth in artificial environment and homogenous nature. Implantation of primary human tumor samples into immune compromised mice have been shown to be more genetically heterogeneous, and allow for more relevant genetic profiling data when compared to established cell lines. To better understand the molecular and genetic events in pancreatic cancer, we established a patient derived primary human tumor xenograft (tumorgraft) model system. Briefly, non-diagnostic tumor tissue was procured from previously consented patients at the University of Alabama at Birmingham Hospital, under IRB approved protocols. The tumors were then examined grossly by a pathologist, and immediately implanted into the flank of immune compromised mice. Here we report, a set of 10 PDAC tumors, with a mean age of patients at diagnosis of 68.3 (± 11.6) years. Primarily, these tumors were stage II (six IIA and four IIB) and with a histological grade reported as five moderately differentiated and five poorly differentiated. Lymph node involvement at the time of surgical resection was also assessed and six patients were positive, while four patients were negative. The tumorgrafts retained the morphological and histological characteristics of the primary tumor, even after serial passage in mice, as determined by a pathologist blinded to tumor identity. Currently, we are using next generation sequencing to generate detailed gene expression, epigenetic, and genotyping profiles of these established PDAC tumors. This work is supported by UAB/UMN SPORE in Pancreatic Cancer-Career Development Award and the Department of Pharmacology/Toxicology, UAB. Citation Format: Patrick L. Garcia, Tracy Gamblin, Leona N. Council, John D. Christein, Martin J. Heslin, Joseph Richardson, Karina J. Yoon. Establishment of the primary human tumor xenograft (tumorgraft) mouse model of pancreatic ductal adenocarcinoma. [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 2725. doi:10.1158/1538-7445.AM2013-2725