Progress Report 1. Improving anti-MAPK pathway therapy in pancreatic cancer A panel of 70 pancreatic cancer cell lines was profiled for MEK sensitivity using the allosteric small molecule inhibitor AZD6244 which is currently in phase II clinical trials. About 40 percent of profiled pancreatic cancer lines exhibit marked MEK sensitivity according to their half growth inhibitory concentration (GI50) of less than 1 micromolar. Sensitivity was determined by luminescent measurement of intracellular ATP concentration after 72 hours treatment with AZD6244. Induction of apoptosis was measured by determining lethal dose values of AZD6244 using the same assay. Overall, response to MEK inhibition in sensitive cell lines is a cytostatic growth arrest effect rather than mediated by induction cell death as described for MEK therapy in other solid organ cancers. To improve efficacy of MEK treatment the following progress has been made: a. A high-throughput siRNA screen has been carried out in the MEK-resistant cell line YAPC: To identify intracellular signaling pathways and targets which are used or switched on by pancreas cancer cells to escape MAPK pathway blockade and MEK inhibition a synthetic lethality drug sensitization screen in the cell line YAPC inhibited with AZD6244 has been carried out by the RNAi Screening Center, NIH Chemical Genomics Center, NIH Center for Translational Therapeutics, NHGRI/NIH. Targets validated in two independent screens mediating resistance to MEK inhibition which are promising opportunities for future effective combinational targeted therapy strategies are e.g. MAP3K8 (COT kinase of the MAPK pathway), PKMYT1 (regulating kinase of the G2-M cell cycle checkpoint), or GSK3B (regulator of the intracellular Wnt signaling pathway) among others. b. Gene expression arrays from sensitive and resistant cell lines have been carried. Using a variety of computational methods including gene set enrichment analysis (GSE), prioritization of networks of intercorrelated expression, and linkage of dynamic gene expression a gene classifier predicting sensitivity and resistance to MEK inhibition is developed. Validation is carried out on a xenobank of human pancreatic cancer xenotransplants established from patients operated on for pancreatic cancer at the Surgery Branch/NCI. c. Addition of the novel Erk2 inhibitor NCGC00242487-01 to improve anti-MAPK therapy To further improve shutdown of the MAPK pathway the novel Erk2 inhibitor NCGC00242487-01 was tested in a panel of MEK-resistant pancreas cancer lines. Addition of NCGC00242487-01 can induce apoptosis rather than cell cycle arrest in some cell lines implying a superior treatment strategy compared to MEK inhibition in a subset of pancreatic cancers. The cause for this different therapeutically potentially valuable mechanism of action is currently investigated. 2. Targeting the PI3K-Akt pathway in pancreas cancer Nearly all pancreatic cancer lines treated with BEZ235 displayed marked sensitivity to PI3K/mTOR inhibition when judged on their GI50 values in the low nanomolar range. It was therefore decided to use induction of apoptosis after treatment with BEZ235 rather than the absolute GI50 value to determine response to dual PI3K/mTOR inhibition. Using a combination of Annexin V staining and cell cycle analysis in cell lines exposed for 72 hours to BEZ235, about 20 percent of cell lines showed a greater than 2.5-fold induction of apoptosis which was a match to prior LD50 determination in the lethal dose assay. In vitro activity of dual PI3K/mTOR inhibition was confirmed in vivo using a heterotopic xenotransplant models established from sensitive and resistant cell lines. Tumor volume was markedly decreased in mice growing tumors from sensitive cell lines after treatment for 21 days. Results of gene expression profiling are currently combined with the expression levels of the negative PI3K-Akt pathway regulator PTEN as well as the mutation status of common oncogenes in pancreas cancer to develop a clinically readily applicable biomarker for predicting response to dual PI3K/mTOR inhibition. For prospective validation of such a biomarker a xenobank from human xenotransplants from patients with pancreatic cancer operated on at the Surgery Branch/NCI is currently expanded. 3. Preclinical evaluation of the novel Myrk/Dyrk1B inhibitor NCGC00185981-02 in pancreas cancer Pancreatic cancer lines Panc1 and SU86.86 have been previously shown to be dependent on the function of Myrk/Dyrk1B kinase when resting in the G0 phase. Using a proliferation assay under serum-starved conditions to enhance the fraction of cells in G0, cell growth was significantly reduced in the presence of NCGC00185981-02. Testing in additional cancer biology assays is currently ongoing and will be followed by - testing for synergy when combined with gemcitabine or choroquine to augment ROS stress response and Myrk/Dyrk1B dependency - in vivo models 4. The impact of the tumor environment on the efficacy of anticancer therapy in ductal adenocarcinoma of the pancreas The role of the microenvironment cannot be studied in an in vitro cell system. To evaluate the complex interactions of the various cellular components, the tumor micro-vasculature, and the extracellular matrix of the tumor microenvironment as possible targets for novel treatment strategies in PDAC requires an in vivo model: Transgenic/knockout mice who develop pancreatic cancer are well-established models for studying possible modulators of carcinogenesis. These models contain conditional knock-in mutations of the Kras oncogene which is present in >85% of PDAC in combination with knock-outs of the common tumor suppressor genes CDKN2A and Smad4 which are lost in >50% of PDAC. These genetically engineered mouse models resemble the human genomic landscape of PDAC which is driven by alterations in one of these genes in >95% of cases: Pdx-cre;LSL-KrasG12D;LSL-p53R172H Pdx-cre;LSLKrasG12D;Ink4a/Arflox/lox Pdx-cre;LSL-KrasG12D;Ink4a/Arflox/lox;Smad4lox/lox The efficacy of the following three anti-pancreas cancer treatments/strategies: 1. Control (normal saline) 2. Gemcitabine 3. MEK inhibition (AZD6244) is tested in combination with treatment of the microenviroment using the following compounds / small molecules: 4. TGFRbeta inhibitors LY2109761 and LY2157299 5. BMPR inhibitor LDN-193189 6. discoidin domain receptor tyrosine kinase 2 (DDR2) inhibitor 409100 Study endpoint: A novel ultrasound technique has been developed to accurately assess tumor size in mice. 3D-ultrasound is the main cross-sectional technique to sequentially monitor tumor growth and tumor growth inhibition. Ultrasound will be complemented with CT and functional imaging after mice have been bred into ROSA 26 PdxCre mice which allows following and quantification of tumor progression via luciferase activity by Xenogen imaging. Ultimate endpoint for analysis of the impact of the different anti-microenvironment treatments on efficacy of anti-cancer therapy is/are: - increase in intratumoral gemcitabine concentration - tumor size - overall survival of the tumor-bearing mice

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