Abstract 7025: Novel Insights into anti-tumor mechanisms of small-molecule ID antagonists

Abstract Inhibitors of Differentiation (ID) proteins (ID1-4), members of the helix-loop-helix (HLH) transcription regulator family, play critical roles in cancer progression and metastasis. Previously, we developed AGX51, a small-molecule ID antagonist, and its more potent derivative, AGX-A, which target ID proteins for degradation. Loss of ID proteins induces reactive oxygen species (ROS) accumulation and promotes lipid peroxidation, leading to non-ferroptotic cell death in breast cancer (BC) cells. This cell death can be rescued by the lipid scavenger alpha-tocopherol, implicating lipid ROS in the mechanism. To investigate changes in lipid composition, we performed non-targeted LC-MS lipidomic profiling of AGX-A-treated BC cells. This analysis revealed significant increases in specific lipid classes, including phosphatidylinositols (PI) (+47.06%), lysolipids (+23.53%), and phosphatidylcholine (PC) (+6.5%) correspondently, indicating a distinct lipidomic signature compared to classical ferroptotic inducers. Importantly, co-treatment with alpha-tocopherol restored lipid composition to basal levels, supporting the hypothesis that non-ferroptotic lipid peroxidation is a key mechanism underlying these changes. Furthermore, combining AGX-A with the general lipase inhibitor Orlistat significantly enhanced its cell-killing effects. Lipidomic profiling of this combination revealed notable increases in triacylglycerol species, which are likely susceptible to lipid peroxidation, amplifying AGX-A's cytotoxic activity. These findings highlight the therapeutic potential of ID antagonists in inducing a unique, non-ferroptotic cell death pathway. By altering lipid metabolism and exploiting lipid peroxidation, ID antagonists represent a promising strategy for targeting aggressive and drug-resistant tumors. Citation Format: Sergey Yaklichkin, Yohannes Ambaw, Michelle Saoi, Justin Cross, Robert Benezra. Novel Insights into anti-tumor mechanisms of small-molecule ID antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7025.