Abstract 6484: Divergent roles of SPOP and CHD1 in ACSL4 regulation reveal context-dependent vulnerabilities for targeting ferroptosis in prostate cancer

Abstract Background: Genetic heterogeneity fundamentally shapes therapeutic responses in prostate cancer (PCa). Among the most recurrent alterations, SPOP mutations and CHD1 loss define molecularly distinct PCa subtypes that respond differently to androgen signaling-directed therapies. Ferroptosis, an iron-dependent form of cell death driven by unchecked lipid peroxidation, has recently emerged as a promising therapeutic vulnerability; however, the genetic determinants that govern ferroptosis responsiveness in PCa remain poorly defined. Methods: We integrated engineered human cell lines, 3D organoid systems, genetically engineered mouse models (GEMMs) and isogenic xenografts to dissect how SPOP mutation and CHD1 deletion modulate ferroptotic sensitivity. Ferroptosis induction and GPX4 dependence were assessed using pharmacologic GPX4 inhibitors (RSL3, ML162, and JKE1674), lipid peroxidation assays, and ferroptosis rescue controls. Transcriptomic, lipidomic, and biochemical analyses, together with ChIP-seq, ChIP-qPCR, and ACSL4 promoter-luciferase reporter assays, were used to delineate how SPOP mutations and CHD1 deletion modulate the MYC-ACSL4 transcriptional axis. Results: We uncover opposing roles for SPOP and CHD1 in regulating ferroptosis. SPOP mutations markedly sensitize PCa cells and tumors to GPX4 inhibition, whereas CHD1 loss abrogates this vulnerability and confers ferroptosis resistance. Mechanistically, SPOP mutation enhances, while CHD1 deletion suppresses, activation of the MYC-ACSL4 axis. SPOP mutation promotes, and CHD1 loss diminishes, MYC occupancy and transcriptional activation of ACSL4, a key enzyme governing polyunsaturated fatty acid metabolism and ferroptotic lipid peroxidation. Given our prior discovery that CHD1 loss upregulates cholesterol biosynthesis, we asked whether inhibiting this pathway could reverse ferroptosis resistance. Notably, pharmacologic blockade of cholesterol biosynthesis reinstates ACSL4 expression and restores ferroptosis sensitivity in SPOP-mutant/CHD1-deficient tumors in vivo, identifying a targetable metabolic checkpoint underlying ferroptosis resistance. Conclusions: These findings define SPOP mutation and CHD1 loss as antagonistic genetic determinants of ferroptosis and uncover a MYC-ACSL4-dependent mechanism that governs GPX4 reliance. Pharmacologic inhibition of cholesterol biosynthesis overcomes CHD1-driven ferroptosis resistance, enabling a biomarker-guided combination strategy that couples ferroptosis induction with cholesterol-pathway blockade. This work establishes a precision strategy for exploiting ferroptosis-based combination therapies in genetically stratified PCa. Citation Format: Feiyu Chen, Qidong Li, Qianlin Gu, Javier Leo, Xin Liang, Naayaa Mehta, Yin Wang, Francisco R. Saenz, Maya M. Phillips, Wei Shi, Chenling Meng, Jie Zhang, Boyi Gan, Di Zhao. Divergent roles of SPOP and CHD1 in ACSL4 regulation reveal context-dependent vulnerabilities for targeting ferroptosis in prostate cancer 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 6484.