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Mutations of the oncogene KRAS are important drivers of pancreatic cancer progression. Activation of epidermal growth factor receptor (EGFR) and human EGFR2 (HER2) is observed frequent in pancreatic adenocarcinomas. Because of co-activation of these two signaling pathways, we assessed the efficacy of inhibition of EGFR/HER2 receptors and the downstream KRAS effector, mitogen-activated protein kinase/extracellular-signal regulated kinase (ERK) kinase 1 and 2 (MEK1/2), on pancreatic cancer proliferation in vitro and in a murine orthotopic xenograft model. Treatment of established and patient-derived pancreatic cancer cell lines with the MEK1/2 inhibitor trametinib (GSK1120212) inhibited proliferation, and addition of the EGFR/HER2 inhibitor lapatinib enhanced the inhibition elicited by trametinib in three of eight cell lines. Importantly, in the orthotopic xenograft model, treatment with lapatinib and trametinib resulted in significantly enhanced inhibition of tumor growth relative to trametinib treatment alone in four of five patient-derived tumors tested and was, in all cases, significantly more effective in reducing the size of established tumors than treatment with lapatinib or trametinib alone. Acute treatment of established tumors with trametinib resulted in an increase in AKT2 phosphorylation that was blunted in mice treated with both trametinib and lapatinib. These data indicate that inhibition of the EGFR family receptor signaling may contribute to the effectiveness of MEK1/2 inhibition of tumor growth possibly through the inhibition of feedback activation of receptor tyrosine kinases in response to inhibition of the RAS-RAF-MEK-ERK pathway. These studies provide a rationale for assessing the co-inhibition of these pathways in the treatment of pancreatic cancer patients.

208 Background: Mutations of the oncogene KRAS and activation of cell-surface receptor tyrosine kinases are important and preserved mechanisms of tumorgenicity in pancreatic cancer. Dual inhibition of the downstream KRAS effector MEK 1/2 and tyrosine kinases EGFR and Her2 results in effective inhibition of patient-derived tumor growth in a murine orthotopic transplantation model. Because combinatorial therapies are moving rapidly into clinical trials, we sought to develop a model of acquired tumor resistance to this combination therapy. Methods: Patient-derived pancreatic tumor xenografts MAD 09-366 (KRAS mut), MAD 08-608 (KRAS mut) and MAD 08-738 (KRAS wt) were implanted orthotopically in nude mice and treated with combination lapatinib (EGFR/Her2 inhibitor) and GSK1120212 (MEK1/2 inhibitor) and tumor volume was measured by MRI. Following 4-6wks of treatment, tumors were reimplanted in second and third generation mice and retreated. Tumors were evaluated by phospho-RTK and phospho-MAPKinase array. Results: Acquired resistance developed in all three tumor xenografts. Treated tumors demonstrated a relative volume decrease in the original (F0) generation. All second re-implantation (F2) tumors, demonstrated relative tumor volume increases despite treatment. A comparison of pre-treatment mean tumor volumes showed a significant decrease in tumor size from the F0 to F2 generations suggesting selection for slower growing tumors. Array data demonstrated increased activation of FGFR1, VEGFR1/3, GSK-3β, p38, and Akt in resistant tumors as compared to their pre-treatment controls. These may represent mechanisms of tumor resistance and warrant further investigation. Conclusions: Repeated tumor exposure in vivo to combination treatment with GSK1120212 and lapatinib was used to develop a preclinical, orthotopic murine model of acquired drug resistance in patient-derived pancreatic cancers. This model provides the opportunity to define the mechanism of resistance in an appropriate tumor microenvironment and to develop alternative strategies for treating tumors resistant to this and other emerging therapies.

Pancreatic ductal adenocarcinoma (PDAC) is associated with the shortest survival duration of any solid cancer. To improve treatment for this disease, better understanding of the proliferative signals that drive pancreatic cancer progression is imperative. To this end, we have established a transplantation model in which surgically resected human pancreatic cancers are propagated orthotopically in the pancreas of immunocompromised mice, permitting detailed molecular and cellular analysis. Approximately 70-85% of engrafted tumors grow and can be serially propagated in multiple generations of mice. Tumor architecture, desmoplastic character, and the metastatic properties of tumors are preserved between the original patient tumor and orthopically propagated tumors. The initial cohort of tumors reported herein was comprised of 4 early stage (Ib/IIb) and 3 late stage (IV) tumors. Five of 7 tumors had KRAS mutations in codon 12. Interrogation of tumor lysates with phospho-receptor tyrosine kinase arrays (R & D Systems) revealed activation of EGFR in all tumors, ERB2/HER2 receptors in 2 of 7 tumors, FGFR1/3 in 6 of 7 tumors, and Tie-2 receptor in 4 of 7 tumors. Based on the ubiquitous expression of EGFR family receptors and the presence of activated KRAS we assessed the efficacy of EGFR and MEK inhibitors on pancreatic cancer cell growth in culture and in the orthotopic xenograft model. A subset of patient-derived tumors adapted for growth in cell culture as well as 4 well established PDAC cell lines (MPanc96, L3.6pl, BxPC-3, PANC-1) were treated with erlotinib (EGFR inhibitor) or lapatinib (EGFR/HER2 inhibitor) and cell proliferation was assessed in a 5 day growth assay. None of the pancreatic cancer cell lines exhibited significant growth inhibition to either drug at concentrations known to be inhibitory in a sensitive cell line, SKBr3. In contrast, treatment of the pancreatic cancer cells with lapatinib in addition to a MEK inhibitor, GSK1120212, significantly inhibited cell growth in all but one of the cell lines tested. Three patient-derived tumors, including both KRAS wild type and mutant tumors, were tested in the orthotopic xenograft model for sensitivity to lapatinib, GSK1120212, or the combination of the two inhibitors. Treatment with lapatinib induced modest inhibition of tumor growth (approximately 30-50%), GSK1120212 treatment led to an approximate 60-80% reduction in tumor volume, whereas treatment with both lapatinib and GSK1120212 led to tumor regression in two tumors and greater than 80% inhibition in another. These data indicate that simultaneous inhibition of the EGF receptor and MAP kinase pathways may prove efficacious in inhibiting tumor proliferation in some primary PDACs. The effectiveness of this approach in inhibiting growth of other patient-derived tumors is under study. Supported by funding from NCI, ACS and UVa Cancer Center. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1592. doi:10.1158/1538-7445.AM2011-1592