RXR agonist IRX4204 enhances BCMA CAR-T-cell efficacy by suppressing ferroptosis via CHAC1 downregulation

Abstract Background: Chimeric antigen receptor (CAR) T-cell therapy targeting B cell maturation antigen (BCMA) shows remarkable efficacy in multiple myeloma (MM), but long-term success is limited by CAR-T cell exhaustion and poor persistence, highlighting the critical need for innovative strategies to enhance CAR T-cell functionality and longevity. The Retinoid X receptors (RXRs) function as master transcriptional regulators that form heterodimers with numerous nuclear receptors to control diverse cellular processes including cell growth, differentiation, and survival. We previously reported that RXR agonists were able to upregulate T cell activation markers while suppressing exhaustion markers of T cells (Wu, J., et al, Cells, 2023, 12:1993). In the current study, we investigated the roles of RXRs in CAR-T-cell therapy and the effectiveness and molecular mechanisms of RXR agonist IRX4204 in enhancing CAR T-cell function. Methods To determine the clinical relevance of RXR expression in CAR-T cell therapy, we analyzed baseline RXR expression, T-cell populations and exhaustion markers, and clinical outcomes in 39 patients with relapsed/refractory MM who received commercial BCMA CAR-T cell therapy at our institution. Anti-BCMA CAR-T cells were co-cultured in vitro with or without IRX4204 against MM cell lines to assess tumor cell lysis, and T-cell activation/exhaustion marker expression. An MM xenograft NSG mouse model was used to further evaluate the anti-tumor effect of anti-BCMA CAR-T cells with IRX4204 in vivo. To understand the biological process through which IRX4204 enhances CAR T-cell function, we measured the effects of IRX4204 on CAR T-cell ferroptosis, a form of regulated cell death characterized by the iron dependent accumulation of lipid peroxides. Ferroptosis involvement was further examined by challenging CAR-T cells with RSL3 (Ferroptosis inducer) and Ferr-1 (Ferroptosis inhibitor) in the presence or absence of IRX4204. To explore the underlying molecular pathway, we performed gene differential analysis on the GSE182638 GEO database based on RNA sequencing of MM cell lines treated with ferroptosis inducer RSL3. Finally, to determine the role of Gutathione specific γ-glutamylcyclotransferase (CHAC1) in IRX4204 mediated ferroptosis suppression, ChIP assay,CHAC1 promoter-luciferase reporter assay, CRISPR knockout, and overexpression experiments were performed. Results Patients with higher baseline RXR expression were found to have significantly higher CD4 and CD8 T-cell counts, and improved response rates. Patients with lower baseline RXR expression were found to have higher expression of markers associated with T-cell exhaustion and higher severity of CAR-T-related toxicities. In vitro, IRX4204-treated CAR-T cells exhibited greater anti-myeloma cytotoxicity than controls, along with increased activation (elevated CD69 and cytokine production) and reduced inhibitory receptor expression (PD-1). In MM-bearing NSG mice, treatment with human anti-BCMA CAR-T plus IRX4204 exhibited superior tumor control when compared to CAR-T alone. Combination therapy also promoted CAR-T cell expansion and persistence in vivo: by day 21, mice receiving IRX4204 had higher human T-cell levels with sustained expression of markers of activation and lower expression of markers of exhaustion than those given CAR-T cells alone. Furthermore, IRX4204 attenuated CAR-T cell susceptibility to the ferroptosis inducer RSL3 and acted synergistically with Ferr-1 to protect CAR-T cells from ferroptosis-induced dysfunction. Mechanistically, IRX4204 reduced PPARα occupancy, suppressed CHAC1 promoter activity, and downregulated CHAC1 expression via RXR/PPARα-dependent transcriptional repression. Additionally, IRX4204 upregulated ferroptosis-protective factors (SLC7A11, GPX4). Ferr-1 and IRX4204 synergistically improved SLC7A11 and GPX4 expression. Similar phenotypes were also observed in CHAC1 knockout and CHAC1 overexpression models. Conclusions The clinical correlation between high RXR expression and superior CAR-T outcomes underscores the relevance of this pathway. Our findings lay the groundwork establishing RXR agonism as a promising strategy to improve CAR-T cell persistence and the durability of responses in MM. These preclinical results provide a strong rationale for clinical translation, warranting exploration of the combination strategy of IRX4204 plus CAR-T in future clinical trials.