Abstract 1214: The polymeric fluoropyrimidine F10 is effective towards cell and organoid models of 5-FU-resistant colorectal cancer

Abstract

Abstract Background: Fluoropyrimidine drugs (FPs, e.g. 5-fluorouracil (5-FU)) constitute the backbone of most chemotherapy regimens for treating locally-advanced and metastatic colon cancer (e.g. FOLFOX, FOLFIRI, FOLFOXIRI). While current FPs improve survival and are the preferred drugs for most colorectal (CRC) cases, drug resistance is a serious problem. One of the most prevalent causes of 5-FU resistance is mutation in TP53 which occur in more than one-third of stage III CRC tumors. 5-FU requires wtp53 for initiation of apoptosis and CRC cells with mtp53 are markedly less sensitive to 5-FU. Methods: To test if F10 induced p53-independent apoptosis in CRC cells, we obtained p53-/- and p53+/+ HCT-116 cells from Professor Vogelstein (Johns Hopkins) and evaluated drug-response in cell-based, organoid, and xenograft models. Cell viability and apoptosis were assessed using Cell-Titer Glo and Caspase Glo assays (Promega). Cell-cycle distribution was evaluated by flow cytometry analysis of propidium iodide stained cells. Thymidylate synthase (TS) activity was assessed using a 3H-release assay and Topoisomerase 1 cleavage complex (Top1cc) formation was assessed using an in vivo complex of enzyme (ICE) bioassay. Cell division was quantified by time-lapse video microscopy. Organoids were developed by bioprinting cells on a biogel matrix to produce a center comprised of tumor cells encompassed by stromal cells. Drug effects in each cell type were evaluated using a “Live-Dead” assay. Drug effects in vivo were evaluated using xenograft models. Results: p53-/- HCT-116 cells were resistant to 5-FU, but sensitive to F10 and underwent p53-independent apoptosis. While F10 is more efficient than 5-FU at inhibiting TS, it uniquely supported continued DNA synthesis, mitosis, and cell division in p53-null cells while 5-FU-treated cells underwent S-phase arrest under thymineless conditions. Following cell division, F10 induced high levels of Top1cc in daughter cells causing massive DNA damage. Using a DT40 knockout system we determined that Tdp1 and PARP-1 were important for F10-induced DNA damage. Organoid models demonstrated selective killing of malignant cells relative to stroma by F10, but not 5-FU. Xenograft models are in progress. Conclusions: F10 is much more potent than 5-FU and induces p53-independent apoptosis. F10 differs from 5-FU in supporting continued DNA synthesis under thymineless conditions leading to Top1-mediated DNA damage, Top1 depletion, genomic instability and activation of the extrinsic apoptotic pathway. Citation Format: William H. Gmeiner, Anthony Dominijanni, Olcay Boyacioglu, Steven Forsyth, Aleks Skardal, Boris Pasche. The polymeric fluoropyrimidine F10 is effective towards cell and organoid models of 5-FU-resistant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1214. doi:10.1158/1538-7445.AM2017-1214