Progress Report 1. Improving anti-MAPK pathway therapy in pancreatic cancer A panel of 70 pancreatic cancer cell lines was profiled for sensitivity to MEK inhibition 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 and activity area over the curve. Overall response to MEK inhibition in sensitive cell lines constitutes a cytostatic growth arrest effect rather than induction of cell death as described for MEK therapy in other solid organ cancers. To improve efficacy of future MEK treatment in pancreas cancer the following progress has been made: a. A high-throughput siRNA screen identified genes mediating resistance to MAPK pathway inhibition in pancreas cancer: 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 secondary screens mediating resistance to MEK inhibition are CNKSR1 (connector enhancer of kinase suppressor of Ras 1), WNK2, (WNK lysine deficient protein kinase 2), MAP3K8 (COT kinase of the MAPK pathway), and RPS6KA5 (ribosomal protein S6 kinase, 90kDa, polypeptide 5). Gene expression levels of CNKSR1 appear to directly correlate with resistance to MAPK inhibition making CNKSR1 both an attractive target as well as possible predictor of response to MEK inhibition in pancreas cancer. b. Next generation genomic sequencing of genes most commonly involved in cancer identified genetic variants associated with response to MAPK pathway inhibition. Interestingly, among several, specific isoforms of the RAS oncogene, which is mutated in more than 85 percent of pancreas cancers, are associated with MEK sensitivity. The mutation isoform G12R is exclusively identified in MEK sensitive cell lines and might represent a readily applicable clinical marker predicting response to MEK inhibition. c. The Erk2 inhibitor VTX-11e (NCGC00242487-01) is superior to MEK inhibition in a subset of pancreatic cancers The Erk small molecule inhibitor VTX-11e (NCGC00242487-01) induces apoptosis rather than cell cycle arrest in a third of pancreatic cancer cell lines implying a superior treatment strategy compared to MEK inhibition. Both MEK- and Erk inhibition equally effectively inhibit MAPK pathway signaling as determined by reduction of phospho Elk levels. Cell lines undergoing cell death following treatment with VTX-11e show a greater reduction of phosphorylation of the Erk target p90-RSK (ribosomal protein S6 kinase, 90kDa, polypeptide 1) than cells undergoing cell cycle arrest upon VTX-11e treatment. Gene expression profiling identified multiple novel genes involved in embryological pathways upregulated in Erk-resistant cell lines possibly maintaining p90-RSK phosphorylation which mediates survival despite Erk inhibition. 2. Targeting the PI3K-Akt pathway in pancreas cancer Nearly all of 70 pancreatic cancer lines treated with the dual PI3K/mTOR inhibitor BEZ235 displayed marked sensitivity when judged on their GI50 values in the low nanomolar range. When tested for induction of cell death upon PI3K/mTOR inhibition, about 20 percent of cell lines showed a greater than 2.5-fold induction of apoptosis and were classified as sensitive to PI3K-Akt inhibition. In vitro activity of dual PI3K/mTOR inhibition was confirmed in vivo using a heterotopic xenotransplant models established from sensitive and resistant pancreas cancer cell lines. Results of mutation testing revealed novel single nucleotide variants in intronic regions of both the PI3K andAKT genes, and others, in cell lines sensitive to PI3K/mTOR inhibition and current studies are examining the functional impact on PI3K signaling of these variants. Gene expression profiling of sensitive and resistant lines showed a number of kinases and phosphatases differently expressed between the two groups. For prospective validation of such a possible biomarker a xenobank from human pancreas cancer specimens from patients operated on at the Surgery Branch/NCI has been established. 3. Preclinical evaluation of the ITK inhibitor NCGC-00188382 in pancreas cancer In a high-throughput pharmacological screen against pancreas cancer stem-like cells, the 'ITK'inhibitor NCGC-00188382 was found to be the most active compound. Cell-based and in situ kinase screens in the pancreas cancer line Panc1 showed that the compound inhibits a number of kinases (CDK7, IRAK4, CLK1, CLK2, CaMMK2, TAOK3, aurora B, Ephrin receptor B2). These novel targets were validated in secondary screens. siRNA silencing studies probing into the polypharmacological mechanism of action of this novel compound identified 'intrinsic synergism'of some of these targets. The compound was tested in vivo and showed a strong anti-metastatic phenotype. Current studies focus on improving the selectivity and pharmacokinetic profile of this promising new molecule, and to further understand its mechanism of action. 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 as possible targets for novel treatment strategies in pancreas cancer 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 pancreas cancer in combination with knock-outs of the common tumor suppressor genes CDKN2A (p16) and Smad4 which are lost in 50% of cases. These genetically engineered mouse models resemble the human genomic landscape of pancreas cancer which is driven by alterations in one of these genes in 95% of cases. Treatment of the transgenic Kras p16 knockout pancreas cancer animal model Pdx-Cre;LSLKrasG12D;Ink4a/Arflox/lox with the TGFRbeta inhibitor LY2109761increases perfusion of pancreatic head tumors several-fold compared to control as measured by increased dextran perfusion and intratumoral gemcitabine. Additionally, the combination of the novel interleukin 10 peptide analogue 10N with gemcitabine led to a dramatic decrease in tumor volume compared to control as well as to a survival advantage in the treated animals. This anti-tumor effect is independent of increased delivery of gemcitabine to the tumor and suggests a novel, yet undioscovered synergistic effect between 10A and gemcitabine. Current studies are aiming to identify the cellular compartment targeted by anti-interleukin 10 and gemcitabine as well as gene differently expressed upon treatment with this combination. These positive findings are in the process of being extended to other transgenic animal models to probe into genotype-directed anti-microenvironment treatment strategies of combining anti-stroma with anti-cancer treatments in pancreas cancer. It is aimed to translate positive findings of gemcitabine combinations into the currently ongoing RECLAP trial.

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