RXR agonist IRX4204 induces ferroptosis in multiple myeloma via HMOX1/GPX4 axis and enhances lenalidomide efficacy

Abstract Background: Multiple myeloma (MM) remains incurable despite therapeutic advances, necessitating novel treatment strategies. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, represents an emerging therapeutic vulnerability in hematologic malignancies. Our previous studies demonstrated that retinoid X receptor (RXR) agonists enhance lenalidomide efficacy in MM (Wu, J., et al, Cells, 2023, 12:1993). Importantly, RXR agonists promote T cell activation and can lower glucose levels, providing additional benefits during MM treatment. IRX4204 (Io Therapeutics, Houston, TX) is a GMP pharmaceutical-grade, highly selective RXR agonist with demonstrated safety and anti-cancer activities in preclinical and phase I/II clinical studies. To ultimately combine IRX4204 with lenalidomide for clinical application, our current study investigated the effects and underlying mechanism of IRX4204 in enhancing lenalidomide anti-myeloma activity. Methods Human MM cell lines (MM1.R and U266) were treated with IRX4204 (1µM), the ferroptosis inducer/GPX4 inhibitor RSL3 (2 µM), lenalidomide, or combinations. Ferroptosis was assessed by measuring lipid reactive oxygen species (ROS) using BODIPY C11 and intracellular ferrous iron (Fe²⁺) using BioTracker Far-red dye. Protein expression of ferroptosis regulators (GPX4, SLC7A11) was examined by western blot and immunofluorescence. Differentially expressed genes (DEGs) were analyzed comparing RSL3-treated MM cell lines to controls using GEO182638 and GSE6477. CRISPR/Cas9-mediated HMOX1 knockout validated functional dependency. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays evaluated PPARα/RXR binding and transcriptional activity at the HMOX1 promoter. A subcutaneous MM1.R xenograft model assessed therapeutic efficacy of IRX4204 plus lenalidomide. Clinical relevance was evaluated using MM patient datasets (GSE9782, GSE6477) to correlate HMOX1 expression with survival outcomes. Results IRX4204 alone induced modest anti-myeloma effects but significantly enhanced ferroptotic cell death when combined with RSL3, with combination index values indicating synergy. This combination markedly increased lipid ROS accumulation and ferrous iron levels. Heme oxygenase 1 (HMOX1) was identified as a crucial ferroptosis-related DEG through bioinformatic analysis. Mechanistically, IRX4204 treatment downregulated the ferroptosis suppressors GPX4 and SLC7A11 while robustly upregulating HMOX1 expression. ChIP assays confirmed enhanced PPARα/RXR occupancy at the HMOX1 promoter following IRX4204 exposure, and HMOX1 promoter-luciferase assays validated transcriptional activation of HMOX1. Critically, CRISPR-mediated HMOX1 knockout completely abolished IRX4204-induced ferroptosis, confirming HMOX1 as essential for this pathway. HMOX1-deficient cells showed no increase in lipid peroxidation or iron accumulation upon IRX4204 treatment, demonstrating mechanistic dependence. In vivo, combination treatment with IRX4204 and lenalidomide significantly reduced tumor growth compared to lenalidomide alone and prolonged median survival without increased systemic toxicity. Tumor analysis confirmed increased HMOX1 and decreased GPX4 expression in combination-treated mice. Bioinformatic analysis of MM patient datasets revealed that high HMOX1 expression correlated with significantly improved overall survival (HR=0.51, p<0.001), while advanced-stage MM showed progressively lower HMOX1 levels compared to normal plasma cells. Conclusion IRX4204 promotes ferroptosis in MM cells by activating the PPARα/RXRα-HMOX1 axis and suppressing GPX4/SLC7A11-mediated antioxidant defense. This effect enhances the therapeutic efficacy of lenalidomide, both in vitro and in vivo. The correlation between high HMOX1 expression and improved patient survival suggests clinical relevance of this pathway. These findings establish ferroptosis induction as a novel mechanism of RXR agonist anti-myeloma activity and provide strong rationale for clinical evaluation of IRX4204 as a ferroptosis-sensitizing agent in combination with standard MM therapies. Clinical Implications This work identifies a druggable ferroptosis pathway in MM and provides mechanistic rationale for combining RXR agonists with established therapies. The correlation between HMOX1 expression and survival suggests potential for biomarker-guided therapy selection.