Abstract B097: Tomatidine inhibits ATF4 activity and induces ferroptosis to limit pancreatic cancer progression
Authors: Mukherjee Debasmita , Chakraborty Srija , Bercz Lena , D’Alesio Liliana , Wedig Jessica , Torok Molly A. , Pfau Timothy , Lathrop Hannah , Jasani Shrina , Guenther Abigail , McGue Jake , Adu-Ampratwum Daniel , Fuchs James R. , Frankel Timothy L. , Pietrzak Maciej , Culp Stacey , Strohecker Anne M. , Skardal Aleksander , Mace Thomas A. 
Published: January 2024

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a five-year survival rate of 12%. Current therapies are often ineffective due to metastasis and an immunosuppressive tumor microenvironment (TME). Activating Transcription Factor 4 (ATF4), a master regulator of cellular stress, is exploited by cancer cells to promote their survival and protect cells from ferroptosis. Prior published work and data reported in our study provide evidence that high ATF4 expression in the PDAC TME correlates with worse overall survival in PDAC patients. Tomatidine, a natural steroidal alkaloid, has been associated with inhibition of ATF4-dependent signaling in multiple disease settings. We hypothesized that tomatidine targets ATF4-dependent signaling leading to ferroptosis mediated cell death in pancreatic cancer. We discovered in vitro tomatidine treatment of pancreatic cancer cells inhibits tumor growth by MTT cell viability assay and reduced the protein expression of p4EBP1/Total 4EBP1 (downstream of ATF4) in a dose dependent manner. In vivo tomatidine treatment (5mg/kg daily intraperitoneally) significantly reduced pancreatic tumor growth. RNA-sequencing of tomatidine treated vs. vehicle treated human and murine pancreatic cancer cells validated targeting of ATF4 related stress regulatory genes. Further, immunofluorescence revealed that ATF4 preferentially localized in the cytoplasm instead of nucleus in presence of tomatidine suggesting tomatidine inhibited nuclear translocation of ATF4, rendering ATF4 ineffective to work as a transcription factor. Further, chromatin immunoprecipitation revealed that tomatidine reduced the transcriptional binding activity of ATF4 with its downstream genes. Tomatidine significantly enhanced gemcitabine chemosensitivity in 3D ECM-hydrogels and in an orthotopic model of pancreatic cancer in vivo. RNA sequencing suggested ferroptosis mediated cell death was a top hit in tomatidine treated pancreatic cancer cells. Therefore, we validated how tomatidine affects the ferroptosis pathway in pancreatic cancer cells by looking at lipid peroxidation via C11 Bodipy staining, immunoblotting for the antioxidant protein Glutathione peroxidase 4 (GPX4) and assessing the changes in the oxygen consumption rate (OCR) using the seahorse assay. Tomatidine treatment was associated with increased lipid peroxidation, decreased GPX4 protein expression, and enhanced mitochondrial biogenesis in PDAC cells. This study highlights a possible novel therapeutic approach utilizing a plant derived metabolite, tomatidine, to target ATF4 activity and cause ferroptosis mediated cancer cell death leading to tumor burden control and chemotherapy sensitization in the PDAC tumor microenvironment.
Citation Format: Debasmita Mukherjee, Srija Chakraborty, Lena Bercz, Liliana D’Alesio, Jessica Wedig, Molly A. Torok, Timothy Pfau, Hannah Lathrop, Shrina Jasani, Abigail Guenther, Jake McGue, Daniel Adu-Ampratwum, James R. Fuchs, Timothy L. Frankel, Maciej Pietrzak, Stacey Culp, Anne M. Strohecker, Aleksander Skardal, Thomas A. Mace. Tomatidine inhibits ATF4 activity and induces ferroptosis to limit pancreatic cancer progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B097.