Abstract Copper overload induced by specific ionophores can trigger cuproptosis, a newly defined form of regulated cell death that offers a novel strategy to target cancer. While this mechanism has been well characterized in cultured cells, its relevance in vivo and potential for therapeutic use remain unclear. The best-characterized ionophore, elesclomol (ES), had been previously tested in clinical trials with limited success; however, these studies were conducted without knowledge or consideration of its copper-binding mechanism or its ability to induce cuproptosis. Here, we define the biological and pharmacologic determinants of ES-induced cuproptosis in vivo, guiding the design of a more effective and tolerable ES analog. We first established that pre-bound ES (ES-Cu) is more potent and pharmacologically active than ES alone, maintaining on-target cuproptosis activity in culture and showing superior efficacy in a xenograft model. Although ES-Cu treatment produced marked tumor responses, complete regression was not achieved. To uncover mechanisms limiting response, we performed a genome-scale ORF overexpression screen and identified CYP11A1 and CYP27A1, mitochondrial partners of FDX1, as resistance factors. These findings indicate that FDX1 expression alone is not sufficient for sensitivity and that effective cuproptosis requires FDX1 activity uncoupled from CYP metabolism, a relationship that may also protect FDX1-high organs such as the adrenal gland and kidney from toxicity. To further overcome ES’s poor solubility and pharmacokinetic limitations, we synthesized a new copper ES analog, which exhibits improved solubility, enhanced antitumor efficacy, and reduced systemic toxicity. Multiplexed imaging confirmed intratumoral aggregation of lipoylated proteins consistent with on-target cuproptosis induction. Together, these findings uncover a previously unrecognized role of FDX1-CYP coupling in regulating cuproptosis sensitivity and define a path toward biomarker-guided development of copper ionophore-based cancer therapies. Citation Format: Piyush Mishra, Mainak Banerjee, Jian-Ren Lin, Shannon Coy, Jeffrey Hsiao, Jonah Lee, John Clohessey, Alexandre Detappe, Todd R. Golub, Loic Charbonniere, Sandro Santagata, Peter Tsvetkov. Selective induction of cuproptosis in vivo by a soluble Elesclomol analog 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 553.
Mishra et al. (Fri,) studied this question.