TIMELESS Promotes LUAD Growth via Suppressing Transferrin-Mediated Ferroptosis and Reprograms the Tumor Microenvironment against Anti-PD-1 Immunotherapy

<b>Background:</b> Lung cancer remains a major global health burden. RNA-binding proteins (RBPs) play crucial roles in post-transcriptional gene regulation, and their dysregulation is frequently implicated in tumorigenesis. The present study aimed to elucidate the molecular network governed by the highly expressed RBP TIMELESS in lung adenocarcinoma (LUAD) and determine its mechanistic role in LUAD progression. <b>Methods:</b> The Cancer Genome Atlas-LUAD, Gene Expression Omnibus, and single-cell RNA sequencing datasets were analyzed to identify aberrantly expressed RBP genes. The RBP gene <i>TIMELESS</i> exhibited the most significant effect on LUAD cell death and was selected for further study. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation sequencing and RNA sequencing were employed to identify ferroptosis-related targets directly bound by TIMELESS. Molecular mechanisms underlying the TIMELESS-mediated regulation of ferroptosis in LUAD were investigated via immunoprecipitation-mass spectrometry, glutathione <i>S</i>-transferase pull-down, immunofluorescence-fluorescence in situ hybridization, RNA immunoprecipitation, poly(A)-tail, and RNA stability assays. In an orthotopic lung cancer mouse model treated with erastin (a ferroptosis inducer) and programmed cell death protein 1 (PD-1) blockade, the role of TIMELESS in therapeutic response was assessed via flow cytometry and multiplex immunofluorescence (mIF). Infiltrating immune cells in LUAD were analyzed by tissue microarrays (TMAs) via mIF. <b>Results:</b> TIMELESS significantly affected LUAD cell proliferation and death, and <i>TIMELESS</i> knockdown significantly enriched RNA-binding and ferroptosis pathways. Transferrin (TF) was identified as a direct TIMELESS target governing ferroptosis. TIMELESS was revealed to bind Ccr4-Not transcription complex subunit 3 (CNOT3) to promote TF mRNA degradation. TIMELESS depletion combined with erastin and PD-1 blockade enhances efficacy, prolongs survival, increases T cell and M1 macrophage infiltration, and reduces M2 macrophage infiltration. Further, high TIMELESS expression was inversely correlated with ferroptosis marker 4-hydroxynonenal but positively correlated with programmed cell death ligand 1 (PD-L1), reduced T cell and M1 macrophage infiltration, and increased M2 macrophage infiltration. <b>Conclusions:</b> TIMELESS recruits CNOT3 to accelerate TF mRNA degradation, thereby suppressing ferroptosis and promoting LUAD growth. These findings suggest that the TIMELESS/TF regulatory axis may be a promising therapeutic target for LUAD.