Sara J. Adair

Inhibition of Focal Adhesion Kinase by PF-562,271 Inhibits the Growth and Metastasis of Pancreatic Cancer Concomitant with Altering the Tumor Microenvironment

Current therapies for pancreatic ductal adenocarcinoma (PDA) target individual tumor cells. Focal adhesion kinase (FAK) is activated in PDA, and levels are inversely associated with survival. We investigated the effects of PF-562,271 (a small-molecule inhibitor of FAK/PYK2) on (i) in vitro migration, invasion, and proliferation; (ii) tumor proliferation, invasion, and metastasis in a murine model; and (iii) stromal cell composition in the PDA microenvironment. Migration assays were conducted to assess tumor and stromal cell migration in response to cellular factors, collagen, and the effects of PF-562,271. An orthotopic murine model was used to assess the effects of PF-562,271 on tumor growth, invasion, and metastasis. Proliferation assays measured PF-562,271 effects on in vitro growth. Immunohistochemistry was used to examine the effects of FAK inhibition on the cellular composition of the tumor microenvironment. FAK and PYK2 were activated and expressed in patient-derived PDA tumors, stromal components, and human PDA cell lines. PF-562,271 blocked phosphorylation of FAK (phospho-FAK or Y397) in a dose-dependent manner. PF-562,271 inhibited migration of tumor cells, cancer-associated fibroblasts, and macrophages. Treatment of mice with PF-562,271 resulted in reduced tumor growth, invasion, and metastases. PF-562,271 had no effect on tumor necrosis, angiogenesis, or apoptosis, but it did decrease tumor cell proliferation and resulted in fewer tumor-associated macrophages and fibroblasts than control or gemcitabine. These data support a role for FAK in PDA and suggest that inhibitors of FAK may contribute to efficacious treatment of patients with PDA. Mol Cancer Ther; 10(11); 2135–45. ©2011 AACR.

Clinical, Molecular and Genetic Validation of a Murine Orthotopic Xenograft Model of Pancreatic Adenocarcinoma Using Fresh Human Specimens

Background Relevant preclinical models that recapitulate the key features of human pancreatic ductal adenocarcinoma (PDAC) are needed in order to provide biologically tractable models to probe disease progression and therapeutic responses and ultimately improve patient outcomes for this disease. Here, we describe the establishment and clinical, pathological, molecular and genetic validation of a murine, orthotopic xenograft model of PDAC. Methods Human PDACs were resected and orthotopically implanted and propagated in immunocompromised mice. Patient survival was correlated with xenograft growth and metastatic rate in mice. Human and mouse tumor pathology were compared. Tumors were analyzed for genetic mutations, gene expression, receptor tyrosine kinase activation, and cytokine expression. Results Fifteen human PDACs were propagated orthotopically in mice. Xenograft-bearing mice developed peritoneal and liver metastases. Time to tumor growth and metastatic efficiency in mice each correlated with patient survival. Tumor architecture, nuclear grade and stromal content were similar in patient and xenografted tumors. Propagated tumors closely exhibited the genetic and molecular features known to characterize pancreatic cancer (e.g. high rate of KRAS, P53, SMAD4 mutation and EGFR activation). The correlation coefficient of gene expression between patient tumors and xenografts propagated through multiple generations was 93 to 99%. Analysis of gene expression demonstrated distinct differences between xenografts from fresh patient tumors versus commercially available PDAC cell lines. Conclusions The orthotopic xenograft model derived from fresh human PDACs closely recapitulates the clinical, pathologic, genetic and molecular aspects of human disease. This model has resulted in the identification of rational therapeutic strategies to be tested in clinical trials and will permit additional therapeutic approaches and identification of biomarkers of response to therapy.

A thirteen-gene expression signature predicts survival of patients with pancreatic cancer and identifies new genes of interest.

Background Currently, prognostication for pancreatic ductal adenocarcinoma (PDAC) is based upon a coarse clinical staging system. Thus, more accurate prognostic tests are needed for PDAC patients to aid treatment decisions. Methods and Findings Affymetrix gene expression profiling was carried out on 15 human PDAC tumors and from the data we identified a 13-gene expression signature (risk score) that correlated with patient survival. The gene expression risk score was then independently validated using published gene expression data and survival data for an additional 101 patients with pancreatic cancer. Patients with high-risk scores had significantly higher risk of death compared to patients with low-risk scores (HR 2.27, p = 0.002). When the 13-gene score was combined with lymph node status the risk-score further discriminated the length of patient survival time (p<0.001). Patients with a high-risk score had poor survival independent of nodal status; however, nodal status increased predictability for survival in patients with a low-risk gene signature score (low-risk N1 vs. low-risk N0: HR = 2.0, p = 0.002). While AJCC stage correlated with patient survival (p = 0.03), the 13-gene score was superior at predicting survival. Of the 13 genes comprising the predictive model, four have been shown to be important in PDAC, six are unreported in PDAC but important in other cancers, and three are unreported in any cancer. Conclusions We identified a 13-gene expression signature that predicts survival of PDAC patients and could prove useful for making treatment decisions. This risk score should be evaluated prospectively in clinical trials for prognostication and for predicting response to chemotherapy. Investigation of new genes identified in our model may lead to novel therapeutic targets.

The TAG Family of Cancer/Testis Antigens is Widely Expressed in a Variety of Malignancies and Gives Rise to HLA-A2-Restricted Epitopes

The TAG-1, TAG-2a, TAG-2b, and TAG-2c cancer/testis genes, known to be expressed in an unusually high percentage of melanoma cell lines, are shown here to be expressed in a variety of tumor lines of diverse histologic type, including cancers of the brain, breast, colon, lung, ovary, pharynx, and tongue. The genes are also expressed in fresh, uncultured melanoma, and ovarian cancer cells. Epitope prediction algorithms were used to identify potential HLA-A1, HLA-A2, HLA-A3, HLA-B7, and HLA-B8 epitopes, and these potential epitopes were tested for their ability to stimulate a peptide-specific cytotoxic T lymphocyte response using lymphocytes from healthy donors. Two HLA-A2–restricted epitopes (SLGWLFLLL and LLLRLECNV) were identified using this approach. Cytotoxic T lymphocytes specific for each of these peptides were capable of recognizing tumor cells expressing both the corresponding class I major histocompatibility complex encoded molecule and the TAG genes. These results indicate that TAG-derived peptides may be good components of a therapeutic vaccine designed to target melanoma and a variety of epithelial cell-derived malignancies.

Evaluation of SAS1B as a target for antibody-drug conjugate therapy in the treatment of pancreatic cancer

Successful therapeutic options remain elusive for pancreatic cancer. The exquisite sensitivity and specificity of humoral and cellular immunity may provide therapeutic approaches if antigens specific for pancreatic cancer cells can be identified. Here we characterize SAS1B (ovastacin, ASTL, astacin-like), a cancer-oocyte antigen, as an attractive immunotoxin target expressed at the surface of human pancreatic cancer cells, with limited expression among normal tissues. Immunohistochemistry shows that most pancreatic cancers are SAS1Bpos (68%), while normal pancreatic ductal epithelium is SAS1Bneg. Pancreatic cancer cell lines developed from patient-derived xenograft models display SAS1B cell surface localization, in addition to cytoplasmic expression, suggesting utility for SAS1B in multiple immunotherapeutic approaches. When pancreatic cancer cells were treated with an anti-SAS1B antibody-drug conjugate, significant cell death was observed at 0.01-0.1 μg/mL, while SAS1Bneg human keratinocytes were resistant. Cytotoxicity was correlated with SAS1B cell surface expression; substantial killing was observed for tumors with low steady state SAS1B expression, suggesting a substantial proportion of SAS1Bpos tumors can be targeted in this manner. These results demonstrate SAS1B is a surface target in pancreatic cancer cells capable of binding monoclonal antibodies, internalization, and delivering cytotoxic drug payloads, supporting further development of SAS1B as a novel target for pancreatic cancer.

CRISPR knockout screening identifies combinatorial drug targets in pancreatic cancer and models cellular drug response.

Predicting the response and identifying additional targets that will improve the efficacy of chemotherapy is a major goal in cancer research. Through large-scale in vivo and in vitro CRISPR knockout screens in pancreatic ductal adenocarcinoma cells, we identified genes whose genetic deletion or pharmacologic inhibition synergistically increase the cytotoxicity of MEK signaling inhibitors. Furthermore, we show that CRISPR viability scores combined with basal gene expression levels could model global cellular responses to the drug treatment. We develop drug response evaluation by in vivo CRISPR screening (DREBIC) method and validated its efficacy using large-scale experimental data from independent experiments. Comparative analyses demonstrate that DREBIC predicts drug response in cancer cells from a wide range of tissues with high accuracy and identifies therapeutic vulnerabilities of cancer-causing mutations to MEK inhibitors in various cancer types.Predicting the response to chemotherapy is a major goal of cancer research. Here the authors use CRISPR knockout screens in pancreatic ductal adenocarcinoma cells to identify deletions synergistic with MEK inhibitors.

Abstract C19: The role of resident liver macrophages in suppressing the progression of hepatic micrometastases from pancreatic ductal adenocarcinoma

Background: The majority of patients with localized pancreatic ductal adenocarcinoma (PDAC) die from metastatic disease, typically of the liver, despite a margin-negative (R0) resection. Therefore, these patients likely harbor occult metastatic disease in the liver at the time of surgery. In this study, we evaluated the role of resident liver macrophages in suppressing the progression of hepatic micrometastases using a murine model of micrometastatic PDAC with patient-derived xenografts (PDXs). Methods and Results: Low-passage, patient-derived KRAS-mutant tumor cells expressing firefly luciferase were injected into the spleens of athymic nude mice resulting in liver metastases, followed by splenectomy to remove the primary tumor. Hepatic tumor burden and metastatic growth kinetics were evaluated by bioluminescent imaging on post-injection days 1, 2, 3, 7, and weekly thereafter. Each of the PDX tumors exhibited a decline in tumor burden over the initial days after injection followed by a period of quiescence with a reproducible, PDX-specific time to proliferative outgrowth ranging from 15 days to greater than 200 days. To assess the role of apoptosis in initial tumor cell clearance and suppression of outgrowth, mouse liver preparations were analyzed for expression of cleaved caspase-3 and cleaved PARP in tumor cells using flow cytometry. There was low expression of both apoptosis markers, suggesting that apoptosis is not a major cell-clearance mechanism. Athymic nude mice lack cell-mediated immunity but have functioning innate immunity. Because there is a large population of resident macrophages in the liver, we hypothesized that macrophages play an important role in the clearance and suppression of hepatic PDAC metastases. To test this, hepatic metastasis outgrowth was assessed following macrophage ablation with liposomal clodronate treatment of mice 48 hours prior to tumor cell injection. Following macrophage ablation, there was a trend toward less robust initial tumor cell clearance and a significantly decreased time to proliferative outgrowth compared with control (13 days vs 26 days, p =0.039). HE PDX 366: 40.4% vs 76.3% clearance at seven days, p =0.024). Average relative hepatic bioluminescence at 21 days was significantly increased for PDX 608 in the NSG mice (14.7 vs 0.355, p =0.014) and there was a trend toward increase for PDX 366 (3.07 vs 0.025, p =0.065). Conclusions: In a preclinical model of hepatic micrometastatic PDAC, resident liver macrophages are implicated in the initial clearance and the suppression of proliferation of tumor cells. Further investigation of the interaction of resident hepatic macrophages and micrometastatic PDAC cells may lead to novel strategies for therapy. Citation Format: Alex D. Michaels, Timothy E. Newhook, James M. Lindberg, Sara J. Adair, Sarbajeet Nagdas, Matthew G. Mullen, Edward B. Stelow, J. Thomas Parsons, Todd W. Bauer. The role of resident liver macrophages in suppressing the progression of hepatic micrometastases from pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C19.

Abstract 808: Combination therapy with a MEK inhibitor plus T-type calcium channel inhibitor is highly effective in patient-derived pancreatic ductal adenocarcinomas

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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 4040: The MEK inhibitor trametinib delays tumor outgrowth and prolongs survival in a patient-derived mouse model of occult hepatic metastatic pancreatic cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Survival for patients with pancreatic ductal adenocarcinoma (PDAC) remains dismal and the majority of patients succumb to metastatic disease. Even for those with localized PDAC, most will die from metastatic disease despite margin-negative resection and adjuvant therapy. Therefore, these patients must harbor occult metastatic PDAC at presentation. We have developed a PDAC model of occult liver metastases using patient-derived xenografts (PDXs) to study the growth of PDAC within the metastatic microenvironment of the liver and evaluated the role of KRAS-MEK-ERK signaling on tumor progression. Methods and Results: Extensively characterized low passage, patient-derived KRAS-mutant (Tumors 608, 366, and 654) and wild-type (Tumors 738 and 215) PDAC cells expressing luciferase were injected into the spleens of athymic, nude mice and allowed to circulate for 10 minutes, after which a splenectomy was performed. To evaluate metastatic cell growth kinetics in the liver, tumor burden was monitored by sequential bioluminescent imaging. Each of the PDX tumors exhibited a characteristic and reproducible time to proliferative outgrowth ranging from 20 days (Tumor 608) to greater than 100 days (Tumor 654). To evaluate the role of KRAS signaling in maintaining dormant cell survival and proliferative outgrowth, tumor 608 cells were injected and mice were treated with the MEK inhibitor trametinib (0.3 mg/kg, daily) or control beginning 48 hours post-injection. Trametinib significantly reduced metastatic tumor burden, delayed time to proliferative outgrowth, and greatly prolonged survival as compared to control (med. survival: 114 vs. 43 days, p<0.001). In contrast, in an orthotopic model with 250-500 mm3 tumors trametinib led to limited inhibition in tumor growth for Tumor 608. To characterize these PDAC cells, we isolated Tumor 608 cells from the liver 48 and 72 hours, 10 and 28 days after splenic injection using magnetic column separation with human EpCAM (CD326)-targeted magnetically labeled microbeads. Flow cytometric analyses of retrieved cells revealed that decreased cellular markers of proliferation and increased PARP cleavage correlated with decreased tumor burden observed at these timepoints in mice treated with trametinib. Conclusions: Using a model of occult liver metastatic PDAC, patient-derived tumors exhibited characteristic, albeit different growth kinetics in the liver microenvironment. Further, inhibition of KRAS-MEK-ERK signaling with the MEK inhibitor trametinib decreased metastatic cellular proliferation, increased apoptosis, prolonged metastatic tumor outgrowth, and significantly increased survival. Further investigation into factors promoting PDAC cell survival within the hepatic microenvironment will lead to development of rational therapeutic strategies for patients with occult metastatic PDAC. Citation Format: Timothy Eric Newhook, James M. Lindberg, Sara J. Adair, J. Thomas Parsons, Todd W. Bauer. The MEK inhibitor trametinib delays tumor outgrowth and prolongs survival in a patient-derived mouse model of occult hepatic metastatic 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 4040. doi:10.1158/1538-7445.AM2014-4040

Targeting occult metastatic disease: A hematogenously derived xenograft model of human pancreatic tumor growth in the murine liver.

198 Background: Most pancreatic cancer patients will die following surgery due to recurrent metastatic disease. Thus, better systemic therapies are needed to treat occult metastases to improve survival. We have developed a model of occult liver metastasis from pancreatic cancer in order to evaluate novel treatment strategies. Methods: Pancreatic cancer cells (MAD 09-366, 08-608, MPanc96) transduced with green fluorescent protein (GFP) and luciferase were injected into the spleens of athymic, nude mice to generate hepatic metastases. Ninety-six hours after injection, tumor-bearing mice were treated with MEK1/2 inhibitor (trametinib, 0.3mg/kg, daily), gemcitabine (100mg/kg, twice weekly), or vehicle control. Sequential bioluminescence imaging, flow cytometry, and histologic evaluation were used to assess hepatic tumor growth and behavior. Results: All injected cell lines generated hepatic metastases. Different cell lines exhibited different growth kinetics. MPanc96 injected mice demonstrated a 64% decrease ...