Abstract 4681: Unlocking the anticancer potential of calix[4]arene-based Cu(I) ionophores in vitro and in vivo
Authors: Lelievre Pierre , Nogier Cyril , Renier Nathan , Coll Jean-Luc , Jabin Ivan , Sancey Lucie , Valkenier Hennie , Deniaud Aurelien , Busser Benoit 
Published: March 2024

Abstract
Introduction: Cuproptosis is a novel programmed cell death pathway triggered by increased intracellular Copper (Cu) levels. This process offers promising avenues for cancer therapy and underscores the pivotal role of Cu-targeting molecules, such as chelators and ionophores. Despite the progress made, there is still a critical need for novel molecules capable of disrupting copper homeostasis within cancer cells.
Methods: We developed and patented a novel class of Cu(I) ionophores characterized by calix[4]arene structures. The anticancer effects of these compounds were evaluated through proliferation assays (IC50), cell cycle assays, and the mechanisms of cell death were explored, including apoptosis, senescence, autophagy, and cuproptosis. To elucidate the mechanisms of action, we conducted proteomic and phosphoproteomic analyses in three different lung adenocarcinoma cell lines (A549, H322, and PC9). Intracellular concentrations of Zn, Fe, and Cu were determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Animal experiments were conducted on nude mice bearing subcutaneous A549 lung cancer xenografts.
Results: Our lead compound, Cuphoralix, demonstrated potent cytostatic effect across a panel of 60 cancer cell lines. This effect was attributed to an early and irreversible blockade of the cell cycle initiation at 24 hours post-treatment. Cuphoralix treatment did not induce senescence or apoptosis. Proteomic analyses confirmed the differential regulation—both upregulation and downregulation—of over 500 proteins and phosphopeptides following treatment with Cuphoralix. Notably, Cuphoralix treatment resulted in decreased expression levels of proteins implicated in cell cycle progression and DNA replication. We discovered that Cuphoralix induced cuproptosis and autophagy, as evidenced by the overexpression of LC3B-II. Intracellular Cu level was significantly increased after treatment with Cuphoralix, suggesting the initiation of cuproptosis. In vivo, the toxicity of liposomal Cuphoralix was assessed following three intravenous injections/week for 4 weeks. Animal weights remained stable, with no observed toxicity or signs of neurological or functional impairment. The anticancer efficacy of Cuphoralix, both alone and in combination with other treatments, was corroborated in vivo by measuring the volumes of subcutaneous xenografts.
Conclusions: Our novel Cu(I) ionophore, Cuphoralix, has demonstrated significant potential in inducing cuproptosis and disrupting copper homeostasis, presenting a novel approach to cancer therapy. The compound's capacity to initiate an early and irreversible cell cycle blockade and to modulate the expression of key proteins underscores its therapeutic promise. The profound in vitro and in vivo anticancer effects observed encourage further development and investigation of cuproptosis as a targeted cancer treatment strategy.
Citation Format: Pierre Lelievre, Cyril Nogier, Nathan Renier, Jean-Luc Coll, Ivan Jabin, Lucie Sancey, Hennie Valkenier, Aurelien Deniaud, Benoit Busser. Unlocking the anticancer potential of calix[4]arene-based Cu(I) ionophores in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4681.