Progress Report1.Improving anti-MAPK pathway therapy in pancreatic cancerA 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. Overall response to MEK inhibition in sensitive cell lines constitutes a cytostatic growth arrest effect rather than mediation of induction of 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 identified genes overcoming 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). b.Multiplexed gene reporter assays identified NF-kB signaling as the main escape pathway switched on upon MEK inhibition. Current experiments aim to link the identified genes mediating MEK resistance in the siRNA screen to NF-kB activation and pursue in vivo validation of compensatory NF-kB activation using a xenobank of human pancreatic cancer xenotransplants established from patients operated on for pancreatic cancer at the Surgery Branch/NCI.c.The Erk2 inhibitor VTX-11e (NCGC00242487-01) is superior to MEK inhibition in a subset of pancreatic cancerThe 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. 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 cancerNearly 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 sensitivity to BEZ235 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 pancreas cancer cell lines. Results of mutation testing revealed novel single nucleotide variants in intronic regions of both the PI3K and AKT genes 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 patientsoperated on at the Surgery Branch/NCI has been established. 3.Preclinical evaluation of the ITK inhibitor NCGC00188382 in pancreas cancerThe ITK inhibitor NCGC00188382 was shown to have ?pan-activity? against a large panel of pancreas cancer lines with IC50 values in the 20 ? 200 nanomolar range. A cell-based kinase screen in the pancreas cancer line Panc1 showed that the compound inhibited 5 kinases (CDK7, IRAK4, CLK1, CLK2, CaMMK2) greater than 90% and another 12 kinases greater than 80%. Cell cycle analysis showed a unique G2-M arrest and combination siRNA silencing studies are currently under way to elicit the polypharmacological mechanism of action of this compound aiming to identify the main targets of NCGC00188382. 4.The impact of the tumor environment on the efficacy of anticancer therapy in ductal adenocarcinoma of the pancreasThe 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 (p16) 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.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 5-fold compared to control as measured by increased dextran perfusion. Currently, LY2109761 is combined with gemcitabine to see if the anti-microenvironment agent LY2109761 can increase delivery of chemotherapeutic agents to pancreatic tumors. These findings are in the process of being extended to other transgenic animal models to probe into genotype-directed anti-microenvironment treatment strategies. It is aimed to translate positive findings of gemcitabine combinations into the currently ongoing RECLAP trial.

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