Abstract 6187: The combination of Trifluridine/Tipiracil and a WEE1 inhibitor is an effective and tolerable candidate strategy against ESCC
Authors: Vu Hoang Trang Nguyen , Kikuchi Osamu , Saito Tomoki , Nakai Yukie , Ida Tomomi , Kondo Yuki , Kataoka Shigeki , Mitani Yosuke , Ohashi Shinya , Muto Manabu 
Published: April 2023

Background: Recently we reported that trifluridine (FTD)/Tipiracil (TPI) is tolerable for unresectable Esophageal squamous cell carcinoma (ESCC) patients although the anti-tumor effect was modest (Mori Y, et al. Esophagus 2022). Therefore, we aimed at developing a combination therapy with FTD/TPI with another small molecule to achieve better efficacy against ESCC. CHK1 inhibitor was considered to be the candidate, because combination treatment with FTD/TPI and prexasertib showed potent antitumor effects in p53-mutant ESCC cells through the synthetic lethality (Ohashi S, et al. Mol Cancer Ther 2020); however, CHK1 inhibitors are not clinically available for further clinical development. Here we explored the concept of synthetic lethality with CHK1 perturbation to the whole ATR-CHK1-WEE1 pathway, especially to the downward WEE1 which directly targets a cell cycle regulator CDK1. The aim of this study is to elucidate the efficacy of the combination of a WEE1 inhibitor (WEE1i) MK1775 with FTD/TPI in ESCC.

Methods: ESCC cells (TE-8 and TE-11) are used for in vitro assay, with compounds including FTD, MK1775 (WEE1i). Mitosis assay (flowcytometry with phospho-Histone H3 [p-hH3] antibody), cell viability assay (WST-1 and clonogenic assays), cytotoxicity assay (CytoTox-Glo assay), and western blotting (double-strand DNA break [γ-H2AX], DDR activity [phospho-CHK1], and CDK1 activity [phospho-Tyr15-CDK1]) were performed. Antitumor effects and tolerability were observed in vivo with TE-8 xenograft model with nu/nu nude mice.

Results: FTD induced activation of CHK1 and inhibition of CDK1 sequentially in ESCC cells. FTD also decreased the proportion of p-hH3 positive cells in mitosis assay. Combination treatment with WEE1i and FTD had activated CDK1, increased p-hH3 positive cells, and induced γ-H2AX. The WST-1 cell viability assay showed significant sensitizing effect of WEE1i to FTD in ESCC cells. The cytotoxicity assay also revealed the significant increase of dead cells by the combination treatment (p = 0.003). Furthermore, we confirmed the combination treatment significantly suppressed xenografted tumor growth (-86%) without major adverse events in vivo (two-way ANOVA and Tukey post-hoc analyses: FTD/TPI vs. control, P &lt; 0.05; MK1775 vs. control, P &lt; 0.05, without significant interaction between the FTD/TPI treatment and MK1775 treatment).

Conclusion: FTD/TPI and WEE1i combination showed potent cytotoxicity, and is considered as a candidate treatment strategy against ESCC.

Citation Format: Hoang Trang Nguyen Vu, Osamu Kikuchi, Tomoki Saito, Yukie Nakai, Tomomi Ida, Yuki Kondo, Shigeki Kataoka, Yosuke Mitani, Shinya Ohashi, Manabu Muto. The combination of Trifluridine/Tipiracil and a WEE1 inhibitor is an effective and tolerable candidate strategy against ESCC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6187.