Abstract 2974: The anticancer effect of TROP2-targeted antibody-drug conjugates (ADC) is potentially attenuated by dysregulated cholesterol metabolism in cancer cells.

Abstract Background: Trop-2 is a widely expressed glycoprotein on a variety of different tumors. Datopotamab deruxtecan (Dato-DXd) is a Trop-2-targeted ADC that has demonstrated anticancer efficacy in patients with EGFR-mutated non-small cell lung cancer (NSCLC). However, their efficacy is inconsistent. In cancer cells, cholesterol synthesis is promoted, and cholesterol localizes to the cell membrane, playing a crucial role in the endocytosis of various receptors on the cell membrane. The current study aimed to explore the mechanisms underlying the limited efficacy of Dato-DXd using EGFR-mutated NSCLC cells. Materials and methods: We established a Dato-DXd-resistant PC9DR cell line by long-term exposure of EGFR-mutated NSCLC PC9 cells to Dato-DXd. A colony formation assay was performed to evaluate anticancer effects. Trop-2 expression on the cell membrane was measured immunologically. Intracellular uptake of Dato-DXd was measured using a pH-Rhod method. Microarray data were analyzed using Transcriptome Analysis Console Software to examine differentially expressed genes and related-pathways. Result: According to the colony formation assay, 1µg/mL Dato-DXd inhibited 89% colony formation in PC9 cells, but inhibited 5% colony formation in PC9DR cells. In PC9DR cells, cell surface Trop-2 expression was greater than PC9 cells (434,338 vs 400,722 molecules per cell, p = 0.0012). However, intracellular uptake of Dato-DXd was significantly reduced in PC9DR cells compared to PC9 cells (two-way ANOVA, p 0.0001). These results suggested that reduced internalization of Trop-2 protein on the cell membrane is hypothetically associated with resistance to Dato-DXd. Second, pathway analysis revealed that the cholesterol metabolism pathway showed the most significant change in PC9DR cells compared to PC9 cells. Specifically, compared to PC9 cells, PC9DR cells showed a marked decrease in the expression of cholesterol synthesis-related enzymes, including HMGCR, MVD, and SQLE, while conversely exhibiting increased expression of the cholesterol transporter ABCA1 (ANOVA p0.001, greater than two-fold change). Therefore, we hypothesized that impaired cholesterol metabolism cause resistance to Dato-DXd. Finally, we exposed the Dato-DXd-sensitive cells including PC9 and HCC827 to the HMGCoA reductase inhibitor, 1µM mevastatin, and evaluated anticancer effect of 1µg/mL Dato-DXd using a colony formation assay. In PC9 cells, Dato-DXd potently inhibited colony formation (relative colony formation area, Control; 100%, Dato-DXd; 3.6%). However, PC9 cells treated with mevastatin, Dato-DXd minimally inhibited colony formation (Control; 100%, Dato-DXd; 77.4%). Similar trend was observed in HCC827 cells. Conclusion: These results suggest that impaired cholesterol metabolism may cause resistance to Dato-DXd in EGFR-mutated NSCLC. Citation Format: Kimio Yonesaka, Yusuke Kawanaka, Takashi Kurosaki, Satomi Watanabe, Junko Tanizaki, Kazuko Sakai, Takeshi Teramura, Kazuto Nishio, Hidetoshi Hayashi. The anticancer effect of TROP2-targeted antibody-drug conjugates (ADC) is potentially attenuated by dysregulated cholesterol metabolism in cancer cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2974.