Abstract B05: Thymidylate synthase cooperates with oncogenic KRAS to markedly accelerate pancreatic cancer progression in a novel KrasG12D/+ mouse model
Authors: Francois Rony A. , Nawab Akbar , Chen Min , Reinhard Mary K. , Kaye Frederic J. , Zajac-Kaye Maria 
Published: May 2015

Abstract
Oncogenic KRAS is a key initiator in the development of pancreatic cancer; however, other aberrations are necessary for the progression into advanced metastatic disease. Our lab was the first to show that overexpression of Thymidylate Synthase (TS), an essential DNA synthesis and repair enzyme that is elevated in many common adult cancers, plays a direct role in promoting tumorigenesis. These findings suggest that TS is not a passive marker of proliferation, but rather an important oncogenic driver in cancer. To determine whether TS enhances the neoplastic effects of oncogenic KRAS, we established a novel animal model for pancreatic ductal adenocarcinoma (PDAC) by generating conditional KrasG12D mutant mice that express high levels of human TS (hTS) in the pancreas. We discovered that overexpression of hTS in the pancreas of Pdx1-cre-KrasG12D/+ mice significantly reduced survival of the hTS/KrasG12D/+ mice. Median survival of hTS/KrasG12D/+ mice was reduced by 50% as compared to KrasG12D/+ mice (112.5 vs 222 days, p=0.0005). To test whether TS accelerates the development of preneoplastic pancreatic ductal lesions and tumors, hTS/KrasG12D/+ and KrasG12D/+ mice were euthanized at 1 month intervals from 1.0 – 6.0 months of age and the percentage of ducts displaying normal, murine pancreatic intraepithelial neoplasia (mPanIN), or invasive PDAC was scored by a masked veterinary pathologist using world health organization (WHO) criteria. We found that hTS/KrasG12D/+ mice displayed a 10 fold increase in mPanIN2 and mPanIN3 lesions at 2 months of age as compared to KrasG12D/+ mice alone. At 3 months of age the hTS/KrasG12D/+ mice showed a 10 fold increase in PDAC formation as compared to KrasG12D/+ mice alone. To determine whether TS enhances the metastatic potential of oncogenic KRAS, lung and liver tissues were harvested from these mice and analyzed for the presence of metastasis. We found that hTS/KrasG12D/+ mice developed lung metastasis as early as 2 months of age, while KrasG12D/+ mice did not develop lung metastasis until 4 months of age. In addition, 60% of hTS/KrasG12D/+ mice in the 4 month age group displayed liver metastasis compared to 20% of KrasG12D/+ mice. Taken together, our data demonstrate that hTS overexpression in the pancreas 1) enhances the initiation of preneoplastic pancreatic ductal lesions, 2) accelerates pancreatic ductal adenocarcinoma development, 3) reduces survival, and 4) increases metastasis in hTS/KrasG12D/+ mice. These data demonstrate that hTS cooperates with oncogenic KRAS to accelerate the progression of pancreatic cancer. Therefore, we predict that combined inhibition of TS and KRAS signaling will maximize tumor regression. Our lab has identified 4 new TS allosteric inhibitors (provisional patent filed in 2013), which inhibit the catalytic activity of TS through a different binding site than 5-fluorouracil (5-FU). These lead allosteric inhibitors display mid-nanomolar to low micromolar activity in KRAS-mutant pancreatic cancer cell lines. Our goal is to combine these allosteric TS inhibitors with inhibitors of KRAS effectors, such as PI3K/AKT/mTOR and RAF/MEK/ERK pathways. Given the frequency of aberrant KRAS activation in pancreatic cancer and the striking effect of TS to enhance KRAS driven tumors in our hTS/KrasG12D/+ model, this combination strategy will be an important step in identifying novel treatments for pancreatic cancer.
Citation Format: Rony A. Francois, Akbar Nawab, Min Chen, Mary K. Reinhard, Frederic J. Kaye, Maria Zajac-Kaye. Thymidylate synthase cooperates with oncogenic KRAS to markedly accelerate pancreatic cancer progression in a novel KrasG12D/+ mouse model. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B05. doi: 10.1158/1557-3125.RASONC14-B05