A Novel Fluoropyrimidine Drug to Treat Recalcitrant Colorectal Cancer

Abstract ID 22462 Poster Board 441 Purpose: Colorectal cancers (CRCs) are the third leading cause of cancer-related death. Most of these deaths are exclusively due to recalcitrant disease. The sensitive form of CRCs progresses to a more metastatic and resistant form mainly due to a lack of more potent and safer chemotherapeutics as chemotherapeutic agents are the mainstay treatment for CRCs. The fluoropyrimidine (FP) drug 5-fluorouracil (5-FU) is the backbone of conventional chemotherapy regimens (e.g., FOLFOX and FOLFIRI). The several serious side effects and ineffectiveness of 5-FU along with the urgency to combat these recalcitrant CRCs necessitates the need to develop safer and more potent FP. We aimed to develop a new second-generation nanoscale FP polymer (CF10) which is safer and more potent in several preclinical models. Additionally, we also aimed to identify if CF10 blocks thymidylate synthase (TS)-mediated pro-metastatic activities and overcomes 5-FU resistance in CRCs. Methods: We performed immunoblot to identify the markers for stalled replication forks (FANCD2, pChk1-S317) and DNA double-strand breaks (pChk2-T68, Rad51, pH2AX). We used an alkaline comet assay and immunofluorescence imaging to visualize the extent of DNA damage, followed by different treatments. The organoid culture of primary colon cancer cells was used to demonstrate the attenuation of primary colon cancer organoids. We used the Tet-on system to regulate TS expression in HCT-116 cells to further study the significance of enhanced TS as a factor in CRC metastatic progression and 5-FU resistance. Finally, we performed an invasion and migration assay to demonstrate that TS promotes pro-metastatic activities. Results: In CRCs cells, CF10 effectively inhibited thymidylate synthase (TS), increased Top1 cleavage complex formation, and induced replication stress, whereas identical dosages of 5-FU were ineffective. The CF10 induces more replication stress in rat, mouse, human-established cell lines, and primary CRC cells compared to 5-FU. In vivo, we observed that CF10 possesses significantly higher anti-tumor activity when compared with the control. Additionally, CF10 treatment improved survival (84.5 days vs 32 days; P < 0.0001) relative to 5-FU in an orthotopic HCT-116-luc colorectal cancer mice model that spontaneously metastasized to the liver. Interestingly, we saw a reduction in metastatic tumors in the CF10 treatment group. This led us to hypothesize further that CF10 could b