Targeting Key Vulnerabilities in Pancreatic Cancer

Abstracts

Keynote Presentation
Steven D. Leach, MD, Memorial Sloan Kettering Cancer Center

Pancreatic intra-epithelial neoplasia (PanIN) represents a common precursor lesion for invasive pancreatic cancer. Beyond the ability of oncogenic Kras to initiate PanIN formation, little is known regarding fundamental mechanisms of PanIN origin and progression. Our group has utilized different tamoxifen-inducible, pancreatic cell type-specific Cre driver lines to simultaneously activate oncogenic Kras and an mTmG lineage label in different adult pancreatic cell types.  These studies have led to new insights regarding the ability of different cell types to serve as PanIN “cells-of-origin”, and also allowed identification of PanIN cell subpopulations with unique functional capacities.  In addition, this approach has allowed us to identify critical interactions between epithelial and non-epithelial cell types during the earliest stages of PanIN initiation.  Using whole genome transcriptional profiling of FACS-sorted PanIN epithelial cells, we observed significant upregulation in IL-17RA, a receptor for the pro-inflammatory cytokine IL-17.  At the same time, FACS analysis of immune cells infiltrating early PanIN lesions demonstrated expanded populations of IL-17 producing TH17 and gdT-cells. Forced IL-17 overexpression dramatically accelerates PanIN initiation and progression, while inhibition of IL-17 signaling using either genetic or pharmacologic techniques effectively prevents PanIN formation. These studies suggest that a hematopoietic-to-epithelial IL-17 signaling axis is a potent and requisite driver of PanIN formation, and that therapeutic targeting of IL-17 signaling may represent a potentially effective strategy for the chemoprevention and/or treatment of early pancreatic cancer.
 

Hypoxia & Precision Medicine in Pancreatic Adenocarcinoma (PDAC): Defining the Target before Targeting the Therapy
Neesha C. Dhani¹

The relevance of hypoxia in promoting aggressive tumour biology is well accepted but variable across cancer types. Although PDAC has been historically considered hypoxic, direct evidence is limited. Up-regulation of indirect markers of hypoxia suggests an importance of hypoxia-related pathways, however may also reflect oxygen independent, oncogenic signaling.
 
We have developed a translational program to explore the clinical relevance of hypoxia across the spectrum of PDAC. Histology-based analyses and functional imaging using nitroimidazole markers identify tissue at p02 < 10mmHg, allowing for evaluation of severe hypoxia and its biological consequences. Patient-derived xenograft (PDX) models are used to explore the efficacy of hypoxia-directed therapeutic strategies.
 
Resected tumours from patients receiving pre-operative pimonidazole demonstrated a range of hypoxia (hypoxic percentage (HP): 0 to 26%) in both stromal and epithelial compartments. There was significant heterogeneity within and across tumours and a small number (3 of 10 tumours) demonstrated minimal to no measureable pimonidazole. Similar trends were noted with [18F] FAZA-PET imaging of patients with advanced disease, with 5 of 20 patients demonstrating no significant hypoxia. These clinical observations are consistent with those made in PDX models where 6 of 16 demonstrated minimal hypoxia by EF5. The more hypoxic PDX models also had faster growth rates with a predilection for distant metastases. Further, these models demonstrated greater sensitivity to hypoxia targeting therapy with TH-302. This data suggests that nitroimidazole hypoxia markers are able to differentiate amongst PDAC with different levels of hypoxia. This has implications for the further development of hypoxia-targeting therapies in PDAC.
 
Coauthors: Cristiane Metran-Nascente¹, Stefano Serra², Ines Lohes¹, Melania Pintilie³, Ivan Yeung⁴, Ines Lohes¹, Trevor McKee⁴, Michael Milosevic⁴, Steven Gallinger⁵, Ming-Sound Tsao², Richard Hill⁴, David Hedley¹
1. Division of Medical Oncology & Hematology
2. Department of Laboratory Medicine & Pathobiology
3. Department of Biostatistics
4. Radiation Medicine Program
5. Division of Hepato-biliary Pancreatic Surgical Oncology, University Health Network and Mount Sinai Hospital
University Health Network, Princess Margaret Cancer Centre/Ontario Cancer Institute, 610 University Ave, Toronto ON M5G 2M9
 

Macrophages — Master Regulators of the Immune Reaction to Pancreatic Cancer
Gregory L. Beatty, MD PhD, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA

Macrophages accumulate at the earliest stages of neoplasia in pancreatic ductal adenocarcinoma (PDAC). Because of their potential to promote tumor growth, invasion and metastasis, strategies that block macrophage pro-tumor activity or re-direct macrophages with anti-tumor properties may hold promise as a therapeutic approach in cancer. We have previously demonstrated the capacity of a CD40 agonist to induce major tumor regressions in both mice and humans with PDAC. This anti-tumor activity is dependent on macrophages which reside outside of the tumor microenvironment and never infiltrate tumor lesions. We hypothesized that these extratumoral macrophages may coordinate anti-tumor activity mediated by CD40 agonists by regulating the trafficking of leukocytes into tumor lesions. Consistent with this hypothesis, we have found that extratumoral macrophages direct the infiltration of anti-tumor inflammatory monocytes into tumor tissue while inhibiting the infiltration of T cells with anti-tumor properties. Our findings demonstrate a key role for extratumoral macrophages in orchestrating immune biology within the tumor microenvironment of PDAC and suggest that tumor immunogenicity can be regulated, at least in part, by macrophages residing outside of the tumor microenvironment.
 

Imaging Pancreatic Cancer and its Microenvironment in Living Mice
Mikala Egeblad, PhD, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

A characteristic feature of pancreatic cancer is its stroma, consisting of fibroblasts, immune cells and extracellular matrix (ECM), including fibrillar type I collagen. To determine how type I collagen influences cancer cell behavior, we developed approaches to perform confocal microscopy in live mice. We used mice that expressed GFPtopaz fused to the α2 chain of type I collagen, expressed under control of the COL1A1 promoter [α2(I)-col-GFP mice]. Live imaging of pancreatic tumors (formed after orthotopic injection of cells from the KPC mouse model) in α2(I)-col-GFP mice showed that cancer cells invaded on linear type I collagen.
 
Cross-linking and linearization of type I collagen by lysyl oxidases (LOXs) is inversely correlated with metastasis-free survival in breast cancer. We examined whether inhibiting the LOXs could limit pancreatic cancer progression. LOX and LOX-like 2 (LOXL2) were expressed at higher levels in tumors than in normal pancreatic tissue. However, inhibition of LOX activity, using a small molecule inhibitor (β-aminoproprionitirile [BAPN]) or RNA interference against either LOX or LOXL2, significantly increased tumor size and metastasis. Using live imaging, we found that LOX inhibition increased the thickness of the collagen fibers in the tumors. LOX inhibition also altered the activities of signaling molecules downstream of integrins, with more cancer cells staining positive for active focal adhesion kinase.
 
In conclusion, our data strongly suggest that targeting collagen cross-linking enzymes would be a poor strategy for the treatment of pancreatic cancer.
 
Coauthors: Mario Shields¹, Pascal Maguin¹, Priyanka Kumar¹, Sarah L. Dallas²
1. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
2. University of Missouri, Kansas City, Missouri
 

Control of Stress Granules by Oncogenic KRas Drives Tumor Drug Resistance in PDAC
Elda Grabocka, PhD¹

Oncogenic mutations in KRAS are prevalent in PDAC and critical for tumor initiation and maintenance. It is widely accepted that oncogenic KRas leads to the acquisition of stress-resistant properties. Consequently, stress-coping mechanisms represent a vulnerability of KRAS-driven tumors and may function as therapeutically beneficial targets. A major hurdle for the successful implementation of such therapies is the substantial sub-clonal heterogeneity and heterotypic interactions within tumors. Tumorigenic fitness and resistance to endogenous and exogenous stresses require cooperation between cancer cell sub-clones and cancer cells and the surrounding stromal cells. We have found that oncogenic KRas drives the response to a variety of stresses by upregulating stress granules (SGs), which are intracellular granules composed of protein and RNA that have emerged as critical components of the cellular stress response. Using a variety of preclinical mouse models, we have established that KRas-mutant PDAC cells display robust SG formation in vivo. Furthermore, we have found that KRas-induced SGs confer cytoprotection against different stressors and chemotherapeutic agents. Importantly, we have discovered that oncogenic KRas promotes SG formation in a paracrine manner, thus conferring stress resistance to multiple cell types within the tumor. The paracrine regulation of SGs occurs through Ras-dependent secretion of PGJ2, a critical parameter for SG assembly. Consistent with this finding, disruption of the Ras-mediated PGJ2 secretory loop impairs SG formation and confers sensitivity to chemotherapeutic agents. These results suggest that KRas regulates SG formation as a means of hijacking the stress response within PDAC tumors and point to the possibility of exploiting this dependence for therapeutic benefit.
 
Coauthors: Yuliya Pylayeva-Gupta, PhD¹, Cosimo Commisso, PhD¹, Dafna Bar-Sagi, PhD¹
1. Department of Biochemistry and Molecular Pharmacology, NYU Langone School of Medicine, New York, NY
 

Exploiting Glutamine Addiction in Pancreatic Cancer
Costas A Lyssiotis, PhD, Cancer Center, Weill Cornell Medical College, New York, New York

Cancer cells exhibit metabolic dependencies that distinguish them from their normal counterparts. Among these addictions is an increased utilization of glutamine (Gln). We identified a non-canonical pathway of Gln utilization in human pancreatic cancer that is required for tumor growth. While most cells utilize glutamate dehydrogenase (GLUD1) to fuel the TCA cycle, pancreatic cancer relies on a distinct pathway such that Gln-derived aspartate is transported into the cytoplasm where it can be converted into oxaloacetate (OAA) by aspartate transaminase (GOT1). Subsequently, this OAA is converted into malate and then pyruvate, increasing the NADPH/NADP+ ratio and thereby maintaining the cellular redox state. Pancreatic cancer cells are dependent on this series of reactions, as Gln deprivation or genetic inhibition of any enzyme in this pathway leads to a redox imbalance and a pronounced suppression of growth. Furthermore, the reprogramming of Gln metabolism is mediated by mutant Kras, the signature genetic alteration in pancreatic cancer, via the transcriptional upregulation and repression of key metabolic enzymes in this pathway. The essentiality of this pathway in pancreatic cancer and the fact that it is dispensable in normal cells may provide novel therapeutic approaches to treat these refractory tumors.
 

Modulation of the Pancreatic Tumor Microenvironment by SBRT and Cell Death
George Miller, MD, New York University School of Medicine, New York, New York, United States

Pancreatic ductal carcinoma is the 4th most common cause of cancer death in the United States and is lethal in more than 95% of caes. Unlike most adenocarcinomas, pancreatic cancer is composed mainly of stromal and desmoplastic elements interspersed with islands of neoplastic epithelium. Recent evidence suggests that far from being a passive observer, pancreatic tumor stroma has impact both on cancer progression and clinical outcome. In particular, by releasing nutrient growth factors into the tumor microenvironment, such as insulin-like growth factor and platelet-derived growth factor (PDGF), the stromal component of pancreatic cancer has been closely linked to both tumor growth and invasiveness. In addition, chemotherapy resistance has been correlated with the extent of tumor desmoplasia as the stroma is thought to be a physical barrier to cytotoxic agents reaching the neoplastic epithelial cells. Hence the delineation of the regulatory circuitry responsible for the activation of pancreatic cancer stroma has the potential to greatly expand our capabilities for therapeutic intervention.  Various elements of the tumor microenvironment induce immune suppression enabling tumor progression. For example, myeloid derived suppressor cells veto cytotoxic CD8 T cells tumoricidal activity. Conversely, Th2-deviated T cells can generate tumor-promoting inflammation. Similarly, selected subsets of tumor associated macrophages promote tumor growth by facilitating immune suppression. In the current work we investigate the collateral effects of tumor-directed radiation on changes to the inflammatory microenvironment in pancreatic cancer. We also focus on the effect of radiation-induced cell death on the surround inflammatory milieu and tumor progression.
 
Coauthors: Lena Tomkötter, Susanna Nguy, Elliot Levie, Rocky Barilla, Andrew Eisenthal, Matthew Pergamo, Nina Avanzi, Atsuo Ochi, Donnele Daley, Alejandro Torres-Hernandez, Mauricio Rendon, Sara Alothman, Dalia Qunaibit, Daniel Tippens, Mridul Pansari, Atif Salyana, Kevin Du
New York University School of Medicine, New York, New York, United States
 

Targeting the Inflammatory Aspect of Pancreatic Cancer through PI3Kδ Inhibition
Michael O. Ports¹

Inflammation in pancreatic ductal adenocarcinoma (PDA) is apparent at the earliest stages of neoplastic development and contributes to a microenvironment that is conducive for disease progression.  PDA-mediated inflammation extends its reach systemically, contributing to the emergence of paraneoplastic syndromes like cachexia that increase morbidity, while decreasing opportunity for aggressive therapeutic intervention.  Zydelig® (idelalisib), a potent and selective PI3Kδ inhibitor, was recently approved for treatment of relapsed chronic lymphocytic leukemia, follicular lymphoma, and small lymphocytic leukemia.  The PI3Kδ isoform, generally expressed by leukocytes, is an attractive target for hematologic malignancies or inflammatory disorders with pathologic activation of the PI3K pathway; however, little is known regarding the utility of targeting PI3Kδ for the treatment of solid tumors.  To this end, we evaluated a potent and selective PI3Kδ inhibitor (GS-9820), with similar properties to Zydelig, on primary PDA patient immune cells and KrasLSL-G12D/+; Trp53LSL‑R172H/+; Pdx-Cre (KPC) mice with advanced disease.  Peripheral myeloid cells from patients and KPC mice displayed enhanced PI3Kδ signaling relative to myeloid cells isolated from healthy donors or control mice.  PI3K signaling in myeloid cells was inhibited by GS-9820, resulting in decreased inflammatory cytokine expression.  GS-9820 treatment of KPC mice with detectable tumor, improved median survival to 30.5 days relative to gemcitabine (17 days) and vehicle controls (13.5 days) (p<0.001).  PI3Kδ inhibitor decreased tumor burden by more than 50% (p<0.05), and decreased fibrillar collagen organization. In depth histologic evaluation of KPC tumors revealed no effect on CD11b, CD8 and T-regulatory numbers in the primary tumor.  Systemically, PI3Kδ inhibition altered inflammatory gene expression in PDA-associated myeloid cells.  Additionally, a systemic response to treatment was observed through increased KPC mice quadriceps weight and a normalization of increased glycolysis and oxidative damage metabolites in skeletal muscle predicted to be associated with cachexia.  Our results provide proof-of-concept for PI3Kδ inhibition as a target for pancreatic cancer, normalizing deregulated systemic inflammatory and metabolic pathologies associated with the etiology of PDA.
 
Coauthors: Maryam Jangani^2a, Essam Ghazaly^2b, Juliana Candido^2a, Jun Wang^2c, Ai Nagano^2c, Andrew Campbell³, Terry Gentzler¹, Adam Kashishian¹, Owen Sansom³, Nicholas R Lemoine^2c, Christophe Quéva¹, Thorsten Hagemann^2a
1. Gilead Sciences, Seattle, WA 98102, USA
2. Centre for Cancer and Inflammationa, Centre for Hemato-Oncologyb, Centre for Molecular Oncologyc, Barts Cancer Institute - a CR-UK Centre of Excellence, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London, EC1M 6BQ, United Kingdom
3. Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, Scotland
 

Enzymatic Remodeling of the Pancreatic Ductal Adenocarcinoma Tumor Microenvironment to Improve Chemotherapeutic Efficacy
Curtis B. Thompson, PhD¹

Pancreatic ductal adenocarcinoma (PDA) is characterized by a tumor microenvironment (TME) with high stromal desmoplasia, a reduction in vessel density, and poor perfusion, all instrumental in PDAs observed resistance to chemotherapeutic intervention. Hyaluronan (HA), an extracellular matrix glycosaminoglycan, has been shown to accumulate to high levels in approximately 90% of PDA tumors, and is thought to be a key component of this desmoplastic response. In preclinical mouse models, including tumor xenografts, patient-derived tumor xenografts, and genetically engineered mouse models of pancreatic cancer (KPC), depletion of HA from the TME with a pegylated recombinant human hyaluronidase PH20 (PEGPH20) is associated with remodeling of the tumor stroma, reduction of tumor interstitial fluid pressure, expansion of tumor blood vessels and facilitated delivery of chemotherapy. Tumor remodeling following HA depletion appears to be sustained. Studies in the KPC model have shown that increased tumor perfusion following PEGPH20 treatment in combination with gemcitabine persists for weeks after therapy cessation. In other mouse models, PEGPH20-mediated HA removal also induces a partial reversal of the classical epithelial-mesenchymal transition process associated with the progression of malignancy. In view of these findings, a Phase 1b/2 clinical trial to evaluate PEGPH20 in combination with gemcitabine in patients with stage IV metastatic pancreatic cancer was conducted. PEGPH20 plus gemcitabine was well tolerated and showed promising efficacy in patients with high tumor HA, leading to the current clinical phase 2 study of PEGPH20 in combination with nab-paclitaxel and gemcitabine for the treatment of stage IV PDA.
 
Coauthors: H. Michael Shepard, PhD¹ and Daniel C. Maneval, PhD¹
1. Halozyme Therapeutics, Inc., San Diego, CA