Activation of the receptor tyrosine kinase Axl is associated with poor outcomes in pancreatic cancer (PDAC), where it coordinately mediates immune evasion and drug resistance. Here, we demonstrate that the selective Axl kinase inhibitor BGB324 targets the tumor-immune interface to blunt the aggressive traits of PDAC cells in vitro and enhance gemcitibine efficacy in vivo. Axl signaling stimulates the TBK1-NFkB pathway and innate immune suppression in the tumor microenvironment. In tumor cells, BGB324 treatment drove epithelial differentiation, expression of nucleoside transporters affecting gemcitabine response, and an immune stimulatory microenvironment. Our results establish a preclinical mechanistic rationale for the clinical development of Axl inhibitors to improve the treatment of PDAC patients.Significance: These results establish a preclinical mechanistic rationale for the clinical development of AXL inhibitors to improve the treatment of PDAC patients. Cancer Res; 78(1); 246-55. Ó2017 AACR.

Pancreatic ductal adenocarcinoma (PDA) is a highly metastatic disease that resists most current therapies. A defining characteristic of PDA is an intense fibrotic response that promotes tumor cell invasion and chemoresistance. Efforts to understand the complex relationship between the tumor and its extracellular network and to therapeutically perturb tumor-stroma interactions are ongoing. Fibronectin (FN), a provisional matrix protein abundant in PDA stroma but not normal tissues, supports metastatic spread and chemoresistance of this deadly disease. FN also supports angiogenesis, which is required for even hypovascular tumors such as PDA to develop and progress. Targeting components of the tumor stroma, such as FN, can effectively reduce tumor growth and spread while also enhancing delivery of chemotherapy. Here, we review the molecular mechanisms by which FN drives angiogenesis, metastasis and chemoresistance in PDA. In light of these new findings, we also discuss therapeutic strategies to inhibit FN signaling.

Elevated levels of TGF-β are a negative prognostic indicator for patients diagnosed with pancreatic cancer; as a result the TGF-β pathway is an attractive target for therapy. However, clinical application of pharmacologic inhibition of TGF-β remains challenging because TGF-β has tumor suppressor functions in many epithelial malignancies including pancreatic cancer. In fact, direct neutralization of TGF-β promotes tumor progression of genetic murine models of pancreatic cancer. Here we report that neutralizing the activity of murine TGF-β receptor 2 using a monoclonal antibody (2G8) has potent anti-metastatic activity in orthotopic human tumor xenografts, syngenic tumors and a genetic model of pancreatic cancer. 2G8 reduced activated fibroblasts, collagen deposition, microvessel density and vascular function. These stromal specific changes resulted in tumor cell epithelial differentiation and a potent reduction in metastases. We conclude that TGF-β signaling within stromal cells participates directly in tumor cell phenotype and pancreatic cancer progression. Thus, strategies that inhibit TGF-β dependent effector functions of stromal cells could be efficacious for the therapy of pancreatic tumors.

Elevated oxidative stress is an aberration seen in many solid tumors, and exploiting this biochemical difference has the potential to enhance the efficacy of anti-cancer agents. Homeostasis of reactive oxygen species (ROS) is important for normal cell function, but excessive production of ROS can result in cellular toxicity and therefore ROS levels must be balanced finely. Here, we highlight the relationship between the extracellular matrix and ROS production by reporting a novel function of the matricellular protein Fibulin-5 (Fbln5). We employed genetically engineered mouse models of pancreatic ductal adenocarcinoma (PDA) and found that mutation of the integrin-binding domain of Fbln5 led to decreased tumor growth, increased survival, and enhanced chemoresponse to standard PDA therapies. Through mechanistic investigations, we found that improved survival was due to increased levels of oxidative stress in Fbln5 mutant tumors. Furthermore, loss of the Fbln5-integrin interaction augmented fibronectin signaling, driving integrin-induced ROS production in a 5-lipooxygenase-dependent manner. These data indicate that Fbln5 promotes PDA progression by functioning as a molecular rheostat that modulates cell-ECM interactions to reduce ROS production and thus tip the balance in favor of tumor cell survival and treatment-refractory disease.

The deposition of extracellular matrix (ECM) is a defining feature of pancreatic ductal adenocarcinoma (PDA), where ECM signaling can promote cancer cell survival and epithelial plasticity programs. However, ECM signaling can also limit PDA tumor growth by producing cytotoxic levels of reactive oxygen species. For example, excess fibronectin stimulation of ␣5␤1 integrin on stromal cells in PDA results in reduced angiogenesis and increased tumor cell apoptosis because of oxidative stress. Fibulin-5 (Fbln5) is a matricellular protein that blocks fibronectin-integrin interaction and thus directly limits ECMdriven reactive oxygen species production and supports PDA progression. Compared with normal pancreatic tissue, Fbln5 is expressed abundantly in the stroma of PDA; however, the mechanisms underlying the stimulation of Fbln5 expression in PDA are undefined. Using in vitro and in vivo approaches, we report that hypoxia triggers Fbln5 expression in a TGF-␤-and PI3K-dependent manner. Pharmacologic inhibition of TGF-␤ receptor, PI3K, or protein kinase B (AKT) was found to block hypoxia-induced Fbln5 expression in mouse embryonic fibroblasts and 3T3 fibroblasts. Moreover, tumor-associated fibroblasts from mouse PDA were also responsive to TGF-␤ receptor and PI3K/AKT inhibition with regard to suppression of Fbln5. In genetically engineered mouse models of PDA, therapy-induced hypoxia elevated Fbln5 expression, whereas pharmacologic inhibition of TGF-␤ signaling reduced Fbln5 expression. These findings offer insight into the signaling axis that induces Fbln5 expression in PDA and a potential strategy to block its production.The maintenance of solid tumors relies heavily on cues received from the surrounding environment. The ECM 2 is composed of structural proteins such as FN and collagen, which promote signaling through integrins and receptor tyrosine kinases (1, 2). ECM signaling is modulated by matricellular proteins, which regulate ECM-cell interactions without serving a direct structural function (3-5). The composition of the ECM is dynamic and varies between tumors and tumor types, contributing to intra-and intertumor heterogeneity, which presents challenges for effective therapeutic strategies. Furthermore, mouse studies have revealed anti-and pro-tumorigenic functions for ECM (5-7). In addition to ECM, stromal cells, including endothelial cells, immune cells, and fibroblasts, are present in the tumor microenvironment. These cells contribute to tumor growth, invasion, and chemoresponse (8 -10).During tumor progression, cancer cells release factors that maintain a microenvironment conducive for growth. For example, TGF-␤ is a cytokine expressed in many cancers that enhances the expression of multiple ECM molecules, including but not limited to FN, collagen, elastin, and fibulins (11-13). Fbln5 is a 448-amino acid secretory glycoprotein of the fibulin family of matricellular proteins. Fbln5 is unique among its members as it contains an RGD-integrin binding domain and can ligate a number of integrins (14). Fbln5 is ex...

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<p>Supplemental Figures S1-S12. 2G8 does not bind to human TGFβR2 (S1); 2G8 treated stromal cells do not induce tumor cell proliferation (S2); 2G8 inhibits smad2 dependent signaling in a dose dependent fashion (S3); Tgfβr2 inhibition induces apoptosis and inhibits proliferation in a murine transgenic model of PDAC (S4); Tgfβr2 inhibition reduces perfusion and permeability in KIC tumors (S5); Collagen deposition and fibroblast infiltration is reduced after Tgfβr2 inhibition in PDAC (S6); 2G8 preserves M1 macrophage phenotype in the presence of exogenous TGF-β (S7); 2G8 promotes a proinflammatory immune cell phenotype in an immunocompetent model of PDAC (S8); 2G8 reduces metastatic burden in KIC mice (S9); 2G8 inhibition of tumor cell Tgfβr2 alone is insufficient to alter tumor cell epithelial/mesenchymal phenotype in vitro (S10); 2G8 inhibits EMT in multiple in vivo human xenograft models (S11); Cytokine changes in stromal cells in vitro after inhibition of Tgfβr2 (S12).</p>

<p>Supplementary Figures 1-7 and Supplementary Table 1. Supplementary Figure 1 H&E stain and trichrome stain of representative 7-week-old KIC tumor tissue. Supplementary Figure 2 Axl gene expression in pancreatic cancer patient-derived xenografts. Supplementary Figure 3 Immunofluorescence of cleaved-caspase 3, p-histon-3, endomucin and EMT markers in Pan02 tumor tissue. Supplementary Figure 4 Western blot of TBK1/NF-κB signaling in tumor lysates from KIC mice treated with/without BGB324. Supplementary Figure 5 F4/80 stain in tumor tissue from KIC mice treated with BGB324 +/- gemcitabine. Supplementary Figure 6 Arginase 1 mRNA expression level in bone marrow-derived macrophages treated with IL-4 +/- BGB324. Supplementary Figure 7 Flow cytometry of KPC-M09 subcutaneous tumors treated with/without BGB324. Supplementary Table 1 Cytokine/Chemokine Concentration in KIC tumor lysates from different treatment groups.</p>