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Defining drivers of tumour initiation can provide opportunities to control cancer progression. Here we report that lysophosphatidic acid receptor 4 (LPAR4) becomes transiently upregulated on pancreatic cancer cells exposed to environmental stress or chemotherapy where it promotes stress tolerance, drug resistance, self-renewal and tumour initiation. Pancreatic cancer cells gain LPAR4 expression in response to stress by downregulating a tumour suppressor, miR-139-5p. Even in the absence of exogenous lysophosphatidic acid, LPAR4-expressing tumour cells display an enrichment of extracellular matrix genes that are established drivers of cancer stemness. Mechanistically, upregulation of fibronectin via an LPAR4/AKT/CREB axis is indispensable for LPAR4-induced tumour initiation and stress tolerance. Moreover, ligation of this fibronectin-containing matrix via integrins α5β1 or αVβ3 can transfer stress tolerance to LPAR4-negative cells. Therefore, stress- or drug-induced LPAR4 enhances cell-autonomous production of a fibronectin-rich extracellular matrix, allowing cells to survive 'isolation stress' and compensate for the absence of stromal-derived factors by creating their own tumour-initiating niche.

The MST1R (RON) kinase is overexpressed in >80% of human pancreatic cancers, but its role in pancreatic carcinogenesis is unknown. In this study, we examined the relevance of Mst1r kinase to Kras driven pancreatic carcinogenesis using genetically engineered mouse models. In the setting of mutant Kras, Mst1r overexpression increased acinar-ductal metaplasia (ADM), accelerated the progression of pancreatic intraepithelial neoplasia (PanIN), and resulted in the accumulation of (mannose receptor C type 1) MRC1+, (arginase 1) Arg+ macrophages in the tumor microenvironment. Conversely, absence of a functional Mst1r kinase slowed PanIN initiation, resulted in smaller tumors, prolonged survival and a reduced tumor-associated macrophage content. Mst1r expression was associated with increased production of its ligand Mst1, and in orthotopic models, suppression of Mst1 expression resulted in reduced tumor size, changes in macrophage polarization and enhanced T cell infiltration. This study demonstrates the functional significance of Mst1r during pancreatic cancer initiation and progression. Further, it provides proof of concept that targeting Mst1r can modulate pancreatic cancer growth and the microenvironment. This study provides further rationale for targeting Mst1r as a therapeutic strategy.

// Jeffery Chakedis 1 , Randall French 1 , Michele Babicky 1 , Dawn Jaquish 1 , Evangeline Mose 1 , Peter Cheng 1 , Patrick Holman 1 , Haleigh Howard 1 , Jaclyn Miyamoto 1 , Paula Porras 1 , Zakk Walterscheid 1 , Carsten Schultz-Fademrecht 2 , Christina Esdar 3 , Oliver Schadt 3 , Jan Eickhoff 2 , Andrew M. Lowy 1 1 Department of Surgery, Division of Surgical Oncology, Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA 2 Lead Discovery Center GmbH, Dortmund, Germany 3 Merck KGaA, Darmstadt, Germany Correspondence to: Andrew M. Lowy, email: alowy@ucsd.edu Keywords: RON, pancreatic cancer, isoform, alternative splicing, tyrosine kinase inhibitor Received: March 03, 2016 Accepted: May 23, 2016 Published: June 14, 2016 ABSTRACT The RON tyrosine kinase receptor is under investigation as a novel target in pancreatic cancer. While RON mutations are uncommon, RON isoforms are produced in cancer cells via a variety of mechanisms. In this study we sought to: 1) characterize RON isoform expression in pancreatic cancer, 2) investigate mechanisms that regulate isoform expression, and 3) determine how various isoforms effect gene expression, oncogenic phenotypes and responses to RON directed therapies. We quantified RON transcripts in human pancreatic cancer and found expression levels 2500 fold that of normal pancreas with RON isoform expression comprising nearly 50% of total transcript. RNA seq studies revealed that the short form (sfRON) and P5P6 isoforms which have ligand independent activity, induce markedly different patterns of gene expression than wild type RON. We found that transcription of RON isoforms is regulated by promoter hypermethylation as the DNA demethylating agent 5-aza-2’-deoxycytidine decreased all RON transcripts in a subset of pancreatic cancer cell lines. The viability of sfRON-expressing HPDE cells was reduced by a RON specific small molecule inhibitor, while a therapeutic monoclonal antibody had no demonstrable effects. In summary, RON isoforms may comprise half of total RON transcript in human pancreatic cancer and their expression is regulated at least in part by promoter hypermethylation. RON isoforms activate distinct patterns of gene expression, have transforming activity and differential responses to RON directed therapies. These findings further our understanding of RON biology in pancreatic cancer and have implications for therapeutic strategies to target RON activity.

The MST1R gene is overexpressed in pancreatic cancer producing elevated levels of the RON tyrosine kinase receptor protein. While mutations in MST1R are rare, alternative splice variants have been previously reported in epithelial cancers. We report the discovery of a novel RON isoform discovered in human pancreatic cancer. Partial splicing of exons 5 and 6 (P5P6) produces a RON isoform that lacks the first extracellular immunoglobulin-plexin-transcription (IPT) domain. The splice variant is detected in 73% of pancreatic adenocarcinoma patient derived xenografts and 71% of pancreatic cancer cell lines. Peptides specific to RON P5P6 detected in human pancreatic cancer specimens by mass spectrometry confirms translation of the protein isoform. The P5P6 isoform is found to be constitutively phosphorylated, present in the cytoplasm, and it traffics to the plasma membrane. Expression of P5P6 in immortalized human pancreatic duct epithelial (HPDE) cells activates downstream AKT, and in human pancreatic epithelial nestin-expressing (HPNE) cells activates both the AKT and MAPK pathways. Inhibiting RON P5P6 in HPDE cells using a small molecule inhibitor BMS-777607 blocked constitutive activation and decreased AKT signaling. P5P6 transforms NIH3T3 cells and induces tumorigenicity in HPDE cells. Resultant HPDE-P5P6 tumors develop a dense stromal compartment similar to that seen in pancreatic cancer. In summary, we have identified a novel and constitutively active isoform of the RON tyrosine kinase receptor that has transforming activity and is expressed in human pancreatic cancer. These findings provide additional insight into the biology of the RON receptor in pancreatic cancer and are clinically relevant to the study of RON as a potential therapeutic target.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a low 5-year survival rate, yet new immunotherapeutic modalities may offer hope for this and other intractable cancers. Here, we report that inhibitory targeting of PI3Kγ, a key macrophage lipid kinase, stimulates antitumor immune responses, leading to improved survival and responsiveness to standard-of-care chemotherapy in animal models of PDAC. PI3Kγ selectively drives immunosuppressive transcriptional programming in macrophages that inhibits adaptive immune responses and promotes tumor cell invasion and desmoplasia in PDAC. Blockade of PI3Kγ in PDAC-bearing mice reprograms tumor-associated macrophages to stimulate CD8+ T-cell–mediated tumor suppression and to inhibit tumor cell invasion, metastasis, and desmoplasia. These data indicate the central role that macrophage PI3Kγ plays in PDAC progression and demonstrate that pharmacologic inhibition of PI3Kγ represents a new therapeutic modality for this devastating tumor type. Significance: We report here that PI3Kγ regulates macrophage transcriptional programming, leading to T-cell suppression, desmoplasia, and metastasis in pancreas adenocarcinoma. Genetic or pharmacologic inhibition of PI3Kγ restores antitumor immune responses and improves responsiveness to standard-of-care chemotherapy. PI3Kγ represents a new therapeutic immune target for pancreas cancer. Cancer Discov; 6(8); 870–85. ©2016 AACR. This article is highlighted in the In This Issue feature, p. 803

Background Mucinous neoplasms of the appendix (MNA) are rare tumors which may progress from benign to malignant disease with an aggressive biological behavior. MNA is often diagnosed after metastasis to the peritoneal surfaces resulting in mucinous carcinomatosis peritonei (MCP). Genetic alterations in MNA are poorly characterized due to its low incidence, the hypo-cellularity of MCPs, and a lack of relevant pre-clinical models. As such, application of targeted therapies to this disease is limited to those developed for colorectal cancer and not based on molecular rationale. Methods We sequenced the whole exomes of 10 MCPs of appendiceal origin to identify genome-wide somatic mutations and copy number aberrations and validated significant findings in 19 additional cases. Results Our study demonstrates that MNA has a different molecular makeup than colorectal cancer. Most tumors have co-existing oncogenic mutations in KRAS (26/29) and GNAS (20/29) and are characterized by downstream PKA activation. High-grade tumors are GNAS wild-type (5/6), suggesting they do not progress from low-grade tumors. MNAs do share some genetic alterations with colorectal cancer including gain of 1q (5/10), Wnt, and TGFβ pathway alterations. In contrast, mutations in TP53 (1/10) and APC (0/10), common in colorectal cancer, are rare in MNA. Concurrent activation of the KRAS and GNAS mediated signaling pathways appears to be shared with pancreatic intraductal papillary mucinous neoplasm. Conclusions MNA genome-wide mutational analysis reveals genetic alterations distinct from colorectal cancer, in support of its unique pathophysiology and suggests new targeted therapeutic opportunities.

Objectives To develop a mouse model for multispectral fluorescence imaging of the pancreas and pancreatic microenvironment. Methods Cre/loxP technology was used to develop this model. We crossed mT/mG indicator mice, engineered to constitutively express a conditional tdTomato transgene that converts to green fluorescent protein (GFP) expression after exposure to Cre recombinase, with Pdx1-Cre transgenic mice. To characterize this model for studies of pancreas biology, we performed bright light and fluorescence imaging of body cavities and intact organs and confocal microscopy of pancreata from offspring of Pdx1-Cre and mT/mG crosses. Results Pdx1-Cre-mT/mG mice demonstrated bright GFP expression within the pancreas and duodenum and intense tdTomato expression in all other organs. Green fluorescent protein expression was mosaic in Pdx1-Cre-mT/mG pancreata, with most showing extensive conversion from tdTomato to GFP expression within the epithelial-derived elements of the pancreatic parenchyma. Because both GFP and tdTomato are membrane targeted, individual cell borders were clearly outlined in confocal images of mT/mG pancreata. Conclusions This mouse model enables multispectral fluorescence imaging of individual cells and cell processes at the microscopic level of the pancreatic microenvironment; it should prove valuable for a variety of fluorescence imaging studies, ranging from pancreatic development to pancreatic cancer biology.

Introduction: The RON (receptor d'origine nantais) tyrosine kinase is overexpressed in >80% of human pancreatic cancers, but its effect on pancreatic carcinogenesis is largely unknown. Methods: We developed a transgenic mouse that overexpresses RON specifically in the pancreas (Pdx-1-RON). Additionally, we utilized an existing transgenic mouse with a functionally inactive RON tyrosine kinase domain (Pdx-1-RTK-/-). These strains were crossed with the established CK murine model of pancreatic cancer yielding compound strains, RCK and CK/TK-/-. We performed immunofluorescent staining, flow cytometry, and western blot analysis on the tissues from these animals to study the effects of RON overexpression and the loss of RON signaling in the setting of oncogenic Kras. Results: RON overexpression results in increased acinar-ductal metaplasia, accelerates PanIN progression to invasive cancer, and results in an accumulation of alternatively-activated (MRC1+) macrophages in the tumor microenvironment. We found that RON overexpression results in upregulation of its own ligand, macrophage-stimulating protein (MSP), activating a positive autocrine/paracrine signaling loop that effects tumor growth and the differentiation of tumor-associated macrophages. Knockdown of RON expression and/or MSP expression results in decreased tumor growth in vivo. Additionally, loss of RON signaling in CK/TK-/- mice leads to a profound decrease in alternatively-activated (MRC1+) macrophages in the tumor microenvironment. In contrast, these animals demonstrate an accumulation of classically activated (CD11c+) macrophages. RON signaling is also associated with downregulation of the chemokine, monocyte chemoattractant protein-1 (MCP-1), a known driver of CD11c+ macrophage activation. The correlation of RON and MSP and the inverse correlation with MCP-1 expression were validated in a database analysis of human pancreatic tumor specimens. Conclusion: Our studies demonstrate that murine RON overexpression accelerates the development of invasive pancreatic cancer in the setting of oncogenic Kras. Active RON signaling results in upregulation of its own ligand, MSP, initiating a positive autocrine/paracrine feedback loop that promotes tumor growth and alters the differentiation of macrophages in the tumor microenvironment. Citation Format: Michele L. Babicky, Megan M. Harper, Evangeline S. Mose, Dawn J. Jaquish, Randall P. French, Susan E. Waltz. The RON tyrosine kinase accelerates pancreatic duct neoplasia and activates a positive autocrine/paracrine signaling loop that promotes alternative macrophage activation. [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 A26.

The RON receptor is frequently over-expressed in pancreatic cancer. We developed a mouse model of spontaneous pancreatic carcinogenesis driven by RON overexpression and oncogenic KRAS (RCK). RCK mice demonstrate accelerated PanIN progression to invasive cancer marked by desmoplasia that is highly characteristic of human disease. RON signaling in macrophages induces differentiation towards the alternatively activated (M2) phenotype, which promotes tumorigenesis. We hypothesized that pancreatic RON overexpression in RCK mice may promote changes in the tumor microenvironment, specifically by driving the differentiation of infiltrating macrophages towards the M2 phenotype. Methods: Pancreatic tissues from RCK and CK controls (3-12 months) were analyzed via immunofluorescent (IMF) staining to quantify myeloid infiltration. Single cell suspensions were generated from pancreatic and splenic tissues. CD11b+F4/80+ myeloid cell populations, as well as CD11c+ M1 and MRC1+ M2 subpopulations, were analyzed via FACS. Results: RCK pancreata demonstrated increased CD11b+F4/80+ myeloid infiltration at 6-12 months. In RCK, pancreatic myeloid cells were skewed toward the M2 phenotype compared with CK controls (MRC1+ 39% vs. 10% p

Early biomarkers and effective therapeutic strategies are desperately needed to treat pancreatic ductal adenocarcinoma (PDAC), which has a dismal 5-year patient survival rate. Here, we report that the novel tyrosine kinase PEAK1 is upregulated in human malignancies, including human PDACs and pancreatic intraepithelial neoplasia (PanIN). Oncogenic KRas induced a PEAK1-dependent kinase amplification loop between Src, PEAK1, and ErbB2 to drive PDAC tumor growth and metastasis in vivo. Surprisingly, blockade of ErbB2 expression increased Src-dependent PEAK1 expression, PEAK1-dependent Src activation, and tumor growth in vivo, suggesting a mechanism for the observed resistance of patients with PDACs to therapeutic intervention. Importantly, PEAK1 inactivation sensitized PDAC cells to trastuzumab and gemcitabine therapy. Our findings, therefore, suggest that PEAK1 is a novel biomarker, critical signaling hub, and new therapeutic target in PDACs. Cancer Res; 72(10); 2554–64. ©2012 AACR.

Introduction:The RON tyrosine kinase receptor is increasingly overexpressed during progression to Pancreatic Intraepithelial Neoplasia (PanIN) and pancreatic duct adenocarcinoma (PDAC). RON expression in these neoplastic cells is associated with a highly invasive phenotype and resistance to gemcitabine. Mutations in the gene for RON, MST1R, are rare but alternative splicing produces a wide variety of protein isoforms. Exon skipping is the most common and a skipping of exon 11 produces RONΔ165. Alternative transcription start in intron 10 produces short-form RON (sfRON) which promotes metastasis in breast cancer. Eight RON isoforms have previously been reported in different epithelial cancer types (Colon, Breast, Stomach) and we initially sought to characterize their expression in PDAC. Using low passage patient derived xenografts (PDXs), we found that the known RON isoforms Δ165 and sfRON were expressed in the majority of PDX’s. Results: This study revealed a novel RON isoform resulting from partial splicing of exons 5 and 6 (P5P6) to produce a protein that lacks the first extracellular immunoglobulin-plexin-transcription (IPT) domain. Peptides specific to RON P5P6 were detected in pancreatic cancer specimens by mass spectrometry which confirms translation of the protein. Using end point PCR we determined that RON is overexpressed in 90% of PDX and P5P6 expressed in 73%. In PDXs absolute quantification PCR determined RON isoforms P5P6, sfRON, and RONΔ165 make up 15 – 50% of total RON transcripts. This puts total isoform expression at up to 500 times more than the full length protein in normal pancreas. To determine function of these isoforms we overexpressed sfRON, RON P5P6, and RONΔ165 in a Human Pancreatic Ductal Epithelial (HPDE) cell line using a lentiviral vector. We found that all isoforms are constitutively phosphorylated, present in the cytoplasm in a reticular pattern, and RON P5P6 traffics to the plasma membrane. RON P5P6 is spliced into α and β subunits and can bind its ligand Macrophage Stimulating Protein similar to full length RON. Expression of P5P6 in HPDE cells activates downstream AKT and MAPK signaling pathways and increases cell motility, VEGF secretion, and markers of EMT. HPDE cells expressing P5P6 and short form RON were injected orthotopically into NOD SCID Gamma mouse pancreata and are tumorigenic while the parental cell line is not. P5P6 and short form RON tumors induce a denser stroma not characteristically observed in cell line derived xenografts. In HPDE sfRON tumors, host mouse acini adjacent to the tumor undergo acinar to ductal metaplasia. As the tumor invades into the mouse pancreas these newly formed ducts become increasingly dysplastic and begin to express mesenchymal markers. Conclusion: We have identified protein isoforms of the RON tyrosine kinase receptor which are expressed in human pancreatic cancer. A novel isoform RON P5P6 and short form RON transform human pancreatic ductal epithelial cells to become tumorigenic. Expression of short form RON in these tumors causes host mouse pancreas to undergo acinar to ductal metaplasia. Ultimately these findings demonstrate that RON isoform expression by pancreatic cancer cells has significant biologic consequences and promotes interaction with the adjacent normal pancreas. Further work to characterize the soluble factors responsible for RON paracrine signaling is currently underway. Citation Format: Jeffery M. Chakedis, Randall French, Michele Babicky, Dawn Jaquish, Haleigh Howard, Evangeline Mose, Jaclyn Miyamoto, Zakk Walterscheid, Patrick Holman, Andrew M. Lowy. Protein isoforms of the RON tyrosine kinase receptor transform human pancreatic ductal epithelial cells and induce acinar to ductal metaplasia. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A72.

Introduction - The RON tyrosine kinase receptor is overexpressed in 80% of Pancreatic Ductal Adenocarcinomas (PDAC) and mediates invasiveness, resistance to apoptosis, and VEGF secretion. Though point mutations are rare a wide variety of RON protein isoforms have been identified, some of which are constitutively active and oncogenic. Previous studies of the DNA methylation patterns of pancreatic cancer specimens identified the gene for RON, MST1R, to be controlled by epigenetic modification. Methylation is one mechanism to regulate expression of RON, and we seek to investigate the effects of hypomethylating therapy on isoform expression and alteration of PDAC biology. Methods - Established pancreatic cancer cell lines (MiaPaca2, BxPC3, AsPC1, HPDE, Panc1) and 2 novel cell lines derived from patient tumor specimens (34E, 79E) were treated with 5-Aza-2′-deoxycytidine (Decitabine, DAC) for 96 hours. Genomic DNA was analyzed using the Methylflash (Epigentek) assay to determine genomic methylation percentage. Reverse Transcriptase PCR and western blot analysis was used to determine treatment effects on RON transcription and expression. Results - Genomic methylation analysis of pancreatic cancer cell lines revealed that compared to the control HPDE line (1.2% methylation) some lines were hypomethylated (ASPC1-1.13%, 34E-1.17%, Panc1-1.17%) while others were hypermethylated (BxPC3-1.6%, MiaPaca2-1.98%, 79E-2.06%). Hypomethylating therapy was effective in decreasing genomic methylation to below normal levels (0.4%-0.9%) in all cell lines and depleted DNA MethylTransferase 1 (DNMT1) protein levels. The effects of demethylating therapy on RON expression were differential. The amount of RON transcripts and protein expression in the RON expressing lines (79E and 34E) was decreased but increased in MiaPaca2 and Panc1. MiaPaca2 does not normally express RON, but demethylation induced RON expression. Transcripts specific for the Δ55 isoform of RON decreased in 34E, 79E, and HPDE cell lines but did not change in the others. Transcription and expression of other RON isoforms which are constitutively active decreased in 34E, 79E, ASPC1, and BxPC3 lines, with less exon 11 skipping and partial splicing of exon 5 and 6. Though RON protein expression was decreased overall in the expressing lines, phosphorylation was still detectable following DAC treatment. Conclusions - Genomic methylation of pancreatic cancer cell lines is variable and RON expression is modulated by the methylation status of its promoter. Demethylating therapy has variable effects on RON expression and is context specific. RON isoform expression was decreased in response to treatment, linking DNA methylation to alternative splicing events. These findings suggest that the consequences of demethylating therapy on PDAC biology are complex and additional studies are required to further elucidate definitive mechanisms. Citation Format: Jeffery Chakedis, Randall French, Dawn Jaquish, Evangeline Mose, Andrew Lowy. Demethylating therapy modulates expression of the RON tyrosine kinase receptor in 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 2315. doi:10.1158/1538-7445.AM2014-2315

Despite recent advances in our understanding of pancreatic ductal adenocarcinoma (PDA), it remains a poorly understood, devastating disease that is largely resistant to all standard treatment modalities. Much effort has been made to identify novel tumor suppressor genes, biomarkers of metastatic behavior, and targets for molecular therapeutics that can improve prognosis and quality of life for PDA patients. Despite the finding that the pro-inflammatory C-X-C family chemokines CXCL1 and CXCL2 are expressed in a significant fraction of human PDA tissues and are known to promote metastasis in bladder and breast cancer, very little is known of its regulation and its role in the progression of PDA. Our laboratory has identified a mechanism by which CXCL1 expression is regulated in human PDA. CXCL1/2 expression is repressed by the Signal Transducer and Activator of Transcription (STAT)-3 and conversely, the Suppressor of Cytokine Signaling (SOCS)-3, a STAT3 inhibitor, promotes CXCL1/2 gene activation and pro-inflammatory signaling in murine embryonic fibroblasts (MEFs) and human PDA cell lines. Ectopic expression of SOCS3 in the ASPC1 PDA cell line induces CXCL1 and CXCL2 expression and drives resistance to radiation and gemcitibine treatment. Further, using an orthotopic xenograft model of human PDA in immunodeficient mice, we demonstrated the in vivo relevance of this pathway. We found that mice injected with human SOCS3-positive human PDA cells have a survival time compared and develop more aggressive disease compared to mice injected with control cells. We hypothesize that CXCL1and CXCL2 promote human PDA pathogenesis by two mechanisms: (i) autocrine activation of the CXCR2 receptor expressed on human pancreatic cancer cells and (ii) pro-inflammatory recruitment of neutrophils to the tumor microenvironment. Citation Format: Sisi Jiang, Evangeline Mose, Giovanni Coppola, Andrew Lowy, Christina Jamieson, Nicholas A. Cacalano. Suppressor of Cytokine Signaling (SOCS)-3 and the C-X-C chemokines CXCL1 and CXCL2 promote tumor aggessiveness and radiation resistance in 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 4208. doi:10.1158/1538-7445.AM2014-4208

tumor type, and pancreatic cancer is associated with the highest risk. Virchow’s triad describes three factors contributing to Vte: hypercoagulability, stasis, and endothelial injury. Rotational thromboelastography (ROTEM) is a new point of care test that allows rapid quantification of coagulation status, for which there is limited information in cancer. We used ROTEM to compare preoperative clot kinetics/strength in patients undergoing surgery for malignant disease.

Early biomarkers and effective therapeutic strategies are desperately needed to treat pancreatic ductal adenocarcinoma (PDAC), which has a dismal 5-year patient survival rate. We recently identified PEAK1 (pseudopodium-enriched atypical kinase one, SGK269) as a catalytically active tyrosine kinase that associates with the cytoskeleton and regulates cell migration and proliferation. Tyrosine kinases are highly sought after candidates as biomarkers and therapeutic targets because they induce specific phoshorylation signatures and because their catalytic activity and downstream signals can be inhibited by small molecules. The goal of this current study was to assess the role of PEAK1 expression as a diagnostic/prognostic biomarker and potential therapeutic target in human malignancies. Here, we report that the novel tyrosine kinase PEAK1 is upregulated in human malignancies, including human PDAC and pancreatic intraepithelial neoplasia (PanIN). Oncogenic KRas induced a PEAK1-dependent kinase amplification loop between Src, PEAK1 and ErbB2 to drive PDAC tumor growth and metastasis in vivo. Suprisingly, blockade of ErbB2 increased Src-dependent PEAK1 expression, PEAK1-dependent Src activation and tumor growth in vivo, suggesting a mechanism for the resistance of PDAC to therapeutic intervention. Indeed, PEAK1 mediates multiple therapy resistance phenotypes that present major hurdles in the clinical treatment of this disease. Thus, PEAK1 is a novel biomarker, critical signaling hub, and new therapeutic target in PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4167. doi:1538-7445.AM2012-4167

Introduction The RON receptor is increasingly over-expressed during pancreatic cancer progression and has been implicated as a mediator of KRAS oncogene addiction. It remains unclear if RON promotes disease progression or simply represents an epiphenomenon. Methods To address this, we generated a transgenic mouse which over-expressed wt-RON in a pancreas-specific manner using Pdx-1, the identical promoter used to drive cre expression in the Kras-LSLGD12/Pdx-1-cre (KC) strain that develops pancreatic duct neoplasia. RON over-expression was verified via Western and IHC. Pdx-1/RON mice were crossed with LSL-KRASG12D mice to yield mice (RK), then bred to Pdx-1-Cre mice to combine RON over-expression in the presence of oncogenic KRAS (RCK). RCK mice were aged and sacrificed at various time points and histology compared to KC controls. Results Pdx-1-RON mice developed no pancreatic phenotype prior to 12 months. At 18 months, one of 4 animals developed primary pancreatic adenocarcinoma with lung metastasis. RON overexpression in KRAS mutant mice (RCK) led to accelerated PanIN progression compared to KC (p 1yr). Metastatic disease was visible in RCK mice at 9 months. Conclusions RON overexpression alone results in pancreatic cancer at long latency. In the presence of KRAS mutation, RON overexpression markedly accelerates PanIN progression to primary and metastatic pancreatic duct cancer. This data supports a role for RON in pancreatic carcinogenesis and this model may prove useful for investigations regarding the role of the RON receptor in pancreatic cancer biology.