Functional characterization of candidate driver genes in pancreatic neoplasia development and maintenance using GEMM-ESC technology

• Applicant: Dr. Michael Saborowski
• Subject Area: Gastroenterology ; Hematology, Oncology
• Term: from 2015 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 271741315

Genetically engineered mouse models (GEMMs) are valuable tools to study the pathobiology of pancreatic ductal adenocarcinoma (PDAC) and explore potential treatment strategies. Unfortunately, the cost and time to generate multi-allelic transgenic mice to investigate the role of a novel candidate cancer gene or genetic dependencies in PDAC maintenance limits their practical use. To address this problem, we have established a genetically flexible embryonic stem cell (ESC) based platform (GEMM-ESCs), which facilitates the generation of multi-allelic transgenic mice directly from ESCs by blastocyst injection within a timeframe of only 2 months. These ESCs harbor well-established pancreatic cancer susceptibility genes like mutant Kras, as well as a genetic locus that can be precisely targeted with doxycycline (dox) inducible shRNAs or cDNAs in a simple electroporation step. The system enables pancreas specific and reversible expression of candidate genes in life mice. Notably, a dual fluorescent reporter system allows to identify shRNA/cDNA-expressing cells and to track these cells even in an off-dox setting. The overall objective of this research proposal is to further extend and adapt the GEMM-ESC platform by establishing additional GEMM-ESC lines harboring distinct genetic configurations that will enable us to more efficiently characterize driver genes and molecular dependencies in the development and maintenance of pancreatic neoplasia. Specifically, I will utilize GEMM-ESC technology to delineate the role of the candidate oncogene Guanine nucleotide binding protein (G protein), alpha stimulating activity polypeptide 1 (GNAS) and the candidate tumor suppressor Ubiquitin E3 ligase ring finger proteins 43 (RNF43) in the initiation and maintenance of Intrapancreatic Papillary Mucinous Neoplasia (IPMN), a precursor lesion of PDAC. Mutations in GNAS and RNF43 occur at high frequency in IPMNs (66-79% and 14-75%, respectively) (Wu et al. Sci Transl Med 2011a; Wu et al. PNAS 2011b; Sakamoto et al. Mod Pathol 2014), but their precise functional role and therapeutic potential remains to be elucidated. Therefore, I will engineer GEMM-ESCs that reversibly express the most common GNAS mutations, or an shRNA directed against RNF43, in a pancreas-specific fashion. Importantly, to mirror the genetics of the human tumors, this will be done in the presence or absence of Kras and Tp53 mutations. In addition, considering the frequent co-occurrence of GNAS and RNF43 alterations and the capability of the GEMM-ESCs to simultaneously express two distinct genetic elements, I will assess whether these lesions cooperate to accelerate the development of IPMNs. Finally, I will adopt a strictly genetic approach to assess the role of mutant GNAS and RNF43 loss in the maintenance of IPMN, which will allow me to project the potential therapeutic ramifications of therapies directed against GNAS/RNF43 signaling cascades, a topic of immediate clinical relevance.

• DFG Programme: Research Grants