319 - Cholesterol Flux Rewiring through LDLR Palmitoylation Establishes a Mitochondrial Shield and Immune Desert in Radioresistant Tumors
Presenter(s)
W. Zhong Sr1, Y. Hu1, J. Wang1, C. Wan1, K. Yang1, and L. Kong2; 1Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, 2Huazhong University of Science and Technology, Wuhan, China
Purpose/Objective(s): Radiotherapy remodels tumor metabolism in NSCLC, inducing adaptive changes that may drive therapeutic resistance—with cholesterol metabolic rewiring emerging as a key yet mechanistically undefined axis. This study identifies LDLR post-translational modification as a central regulator coordinating mitochondrial stress adaptation and immune evasion, establishing its dual checkpoint role for targeted radiosensitization.
Materials/Methods: Radiation-induced metabolic changes were profiled via UHPLC-QTOF-MS (TIF) and multi-omics (RNA-seq/proteomics) in radioresistant NSCLC models (H1299/Lewis). Cholesterol flux was assessed by HPLC/DIL-LDL tracking. Radiosensitivity was tested via clonogenic/CCK-8/?-H2AX assays. LDLR interactome (CISD2/CPT1a/ZDHHC4) was mapped by Co-IP-MS and validated via siRNA/OE and GST-pull down. Site-specific palmitoylation of LDLR was analyzed by acyl-biotin exchange (ABE) and structural domain mutagenesis. Mitochondrial cholesterol/mitophagy were evaluated via WB, live imaging, and ROS quantification (MitoSOX and ROS). In vivo, CRISPR-LDLR-KO tumors were analyzed by FACS (CD8+/Tregs/MDSCs) and multispectral imaging.
Results: Multi-omics profiling of irradiated tumor microenvironments identified LDLR as the pivotal regulator, with enhanced membrane localization driven by site-specific palmitoylation. Comparative analysis across multiple cell lines revealed a critical palmitoylation site within LDLR’s transmembrane-proximal domain, where radiation promotes palmitoylation via CPT1a-mediated recruitment of the ZDHHC4 enzyme. This modification stabilizes LDLR within lipid raft microdomains, amplifying cholesterol uptake—a conserved mechanism validated through super-resolution imaging and membrane trafficking assays. Radiation upregulates CISD2 to tether LDLR (specific domain interaction) to mitochondria, elevating cholesterol levels that trigger selective mitophagy. This attenuates radiotherapy-induced oxidative damage (ROS reduction confirmed by MitoSOX flow cytometry and linked to diminished DNA strand breaks) while reducing cytosolic mtDNA leakage and cGAS-STING activation. Universal immune remodeling emerges through LDLR ablation, characterized by cytotoxic T cell infiltration—effects conserved across adenocarcinoma and squamous subtypes.
Conclusion: This study reveals LDLR palmitoylation as a radiotherapy-triggered switch that reprograms cholesterol metabolism and mitochondrial mitophagy to fuel resistance. Radiation stabilizes LDLR via CPT1a-ZDHHC4-mediated palmitoylation, amplifying cholesterol uptake and mitochondrial shuttling to activate protective autophagy while suppressing ROS-driven DNA damage. LDLR ablation disrupts metabolic adaptation, restores radiosensitivity, and reactivates antitumor immunity in NSCLC—through cholesterol depletion impairing immunosuppressive cell function while potentiating CD8+ T cell activation.