These observations suggest that KRAS plays an essential role in the initiation, progression and maintenance of PDA. Because of its critical importance in PDA development, oncogenic KRAS represents a good target for therapeutic intervention. as leads for subsequent development of clinically-effective treatments for pancreatic cancer. and tumor suppressors mutations represent one of the earliest genetic changes associated with the transformation of normal ductal epithelium. mutations have been detected in pancreatic duct lesions with minimal cytological and architectural atypia, and, occasionally, in the histologically normal pancreas (4;21;24;25;27). The frequency of mutations correlates with disease progression, reaching almost 100% in PDA. Targeted endogenous expression of an oncogenic allele in the mouse pancreas is sufficient to drive the development of PanINs, and subsequently, at low frequency, the progression to both locally invasive adenocarcinoma and metastatic disease with sites of spread exactly as found in human pancreatic cancer (1;12;13). These observations suggest that KRAS plays an essential role in the initiation, progression and maintenance of PDA. Because of its critical importance in PDA development, oncogenic KRAS represents a good target for therapeutic intervention. Consistent with this notion, siRNA specifically targeting oncogenic allele induces growth inhibition and apoptosis in pancreatic cancer cell lines harboring mutation. However, small molecules that inhibit RAS activation by targeting its posttranscriptional modification have so far yielded little success as these compounds also affect cellular targets/pathways other than RAS. On the other hand, novel cell-based screens that target the Achilles’ heel of PC (the dependence of PC cell’s survival on oncogenic RAS) may lead to the identification of effective inhibitors for PC. Recently, a chemical genetic screening strategy has been developed that involves searching for synthetic lethal antitumor brokers that Dye 937 selectively kill tumor cells with specific genotypes such as, the presence of a specific oncoprotein or the loss of a specific tumor suppressor (6;20). In this report, we developed a chemical genetic screen using an oncogenic KRAS-based human pancreatic ductal epithelia (HPDE) cancer model. This cell-based assay allowed us to search through existing libraries of small chemical compounds to identify mechanism-based inhibitors that show selective lethality in the presence of oncogenic alleles. Those compounds with KRASV12-selective lethality may serve as leads for subsequent development of clinically-effective drugs with a favorable therapeutic index for PC. 3. Materials and Methods 3.1. Cell lines Primary human pancreatic ductal epithelial cell (HPDE-c7) immortalized by E6/E7 genes of human papilloma virus (HPV)-16 virus was a gift from Dr. Ming S. Tsao. Stable HPDE-c7-KRASV12 cell line was generated from HPDE-c7 by the addition of oncogene using retroviral vector (18). Parental HPDE-E6E7c7 and HPDE-c7-KRASV12 cells are maintained in keratinocyte-SFM medium supplemented by bovine pituitary extract and epidermal growth factor (Gibco-BRL) at 37 C and 5% CO2. 3.2. Compound libraries Small chemical compounds from the Diversity set (1990 compounds), Mechanistic set (879 compounds), Challenge set (57 compounds), and Natural Product set (235 compounds) were obtained from the Open Chemical Repository of National Cancer Institute Developmental Therapeutics Program (DTP). Compounds were supplied in DMSO in 96-well polypropylene plates and stored at -80C. Replica daughter plates were generated by diluting original stock plates 10-fold in DMSO and used for screening. 3.3. Screening Assay plates were prepared by seeding cells in 96-well plates (4000 cells/well in 100 l) using a repetitive dispenser. Columns 2-11 were treated with compounds from Dye 937 a daughter library plate. The final compound concentration in assay plates was 10 M. Columns 1 and 12 were treated with vehicle and Dye 937 1 M Taxol as negative and positive controls, respectively. The assay plates were incubated for 24-48 hrs at 37C in a humidified incubator made up of 5% CO2 and processed for cell viability assay, as described below. 3.4. Alamar Blue cell viability assay A 100 l of the cell suspension was seeded per well in duplicates in 96-well microtiter.The robustness and reproducibility of the assay were tested using 0.1% DMSO and Doxorubicin (10 M) as vehicle and positive controls. almost 100% in PDA. Targeted endogenous expression of an oncogenic allele in the mouse pancreas is sufficient to drive the development of PanINs, and subsequently, at low frequency, the progression to both locally invasive adenocarcinoma and metastatic disease with sites of spread exactly as found in human pancreatic cancer (1;12;13). These observations suggest that KRAS plays an essential role in the initiation, progression and maintenance of PDA. Because of its critical importance in PDA development, oncogenic KRAS represents a good target for therapeutic intervention. Consistent with this notion, siRNA specifically targeting oncogenic allele induces growth inhibition and apoptosis in pancreatic cancer cell lines harboring mutation. However, small molecules that inhibit RAS activation by targeting its posttranscriptional modification have so far yielded little success as these compounds also affect cellular targets/pathways other than RAS. On the other hand, novel cell-based screens that target the Achilles’ heel of PC (the dependence of PC cell’s survival on oncogenic RAS) may lead to the identification of effective inhibitors for PC. Recently, a chemical genetic screening Cast strategy has been developed that involves searching for synthetic lethal antitumor brokers that selectively kill tumor cells with specific genotypes such as, the presence of a specific oncoprotein or the loss of a specific tumor suppressor (6;20). In this report, we developed a chemical genetic screen using an oncogenic KRAS-based human pancreatic ductal epithelia (HPDE) cancer model. This cell-based assay allowed us to search through existing libraries of small chemical compounds to identify mechanism-based inhibitors that show selective lethality in the presence of oncogenic alleles. Those compounds with KRASV12-selective lethality may serve as leads for subsequent development of clinically-effective drugs with a favorable therapeutic index for PC. 3. Materials and Methods 3.1. Cell lines Primary human pancreatic ductal epithelial cell (HPDE-c7) immortalized by E6/E7 genes of human papilloma virus (HPV)-16 virus was a gift from Dr. Ming S. Tsao. Stable HPDE-c7-KRASV12 cell line was generated from HPDE-c7 by the addition of oncogene using retroviral vector (18). Parental HPDE-E6E7c7 and HPDE-c7-KRASV12 cells are maintained in keratinocyte-SFM medium supplemented by bovine pituitary extract and epidermal growth factor (Gibco-BRL) at 37 C and 5% CO2. 3.2. Compound libraries Small chemical compounds from the Diversity set (1990 compounds), Mechanistic set (879 compounds), Challenge set (57 compounds), and Natural Product set (235 compounds) were obtained from the Open Chemical Repository of National Cancer Institute Developmental Therapeutics Program (DTP). Compounds were supplied in DMSO in 96-well polypropylene plates and stored at -80C. Replica daughter plates were generated by diluting original stock plates 10-fold in DMSO and used for screening. 3.3. Screening Assay plates were prepared by seeding cells in 96-well plates (4000 cells/well in 100 l) using a repetitive dispenser. Columns 2-11 were treated with compounds from a daughter library plate. The final compound concentration in assay plates was 10 M. Columns 1 and 12 were treated with vehicle and 1 M Taxol as negative and positive controls, respectively. The assay plates were incubated for 24-48 hrs at 37C in a humidified incubator containing 5% CO2 and processed for cell viability assay, as described below. 3.4. Alamar Blue cell viability assay A 100 l of the cell suspension was seeded per well in duplicates in 96-well microtiter plates (Corning, NY) and treated with compounds. An aliquot of 10 l of Alamar Blue was added to each well and the plates were incubated for another 4 hrs. Following the incubation, fluorescence intensity was monitored using a SpectraMax M2 microplate reader (Molecular Devices) with excitation and.