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Background A significant proportion of treatment-naive lung adenocarcinoma (LUAD) patients exhibit intrinsic resistance to neoadjuvant chemoimmunotherapy (N=816 trial cohort), underscoring an unmet need for mechanism-driven therapeutic strategies.Methods We conducted multi-omics profiling of 48 multicentric LUAD specimens from the CheckMate 816 trial cohort using single-cell RNA sequencing (scRNA-seq) and Visium spatial transcriptomics, enabling systematic identification of therapy-resistant subclones and spatially resolved immune-microenvironment dynamics. Experimental validation was performed through three complementary platforms: 1) Tet-op-hEGFR-TD/CC10rtTA bitransgenic mice for lineage tracing of EGFR-driven LUAD progression; 2) Humanized NSG mice (NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ) with reconstituted human immune systems to model immunotherapy responses; 3) Patient-derived organoids (PDO, n=18) maintaining original tumor-stroma interactions for high-throughput drug screening.Key techniques included quantitative multiplex immunohistochemistry, luminescence-based cGAS-STING activity assays, high-content drug screening platforms integrating, CRISPR-Cas9 knockout validation, and PET-CT-guided pharmacodynamic monitoring with [18F]FDG to assess tumor metabolic response in real-time.Results Multi-omics profiling of 48 LUAD specimens (scRNA-seq, N=48; spatial transcriptomics, N=12) identified Coiled-Coil-Helix-Coiled-Coil-Helix Domain Containing 3 (CHCHD3) as the top resistance driver. CHCHD3 overexpression induced immunosuppression in PDOs and EC mice (p<0.01). Mechanistically, the CHCHD3-S2 domain directly binds to the catalytic core of cGAS, reducing cytosolic cGAS by 68±5% (p<0.001) and inhibiting downstream chemokines production. CHCHD3 knockout upregulated CXCL1/CXCL5, enhancing CD8 +T cells and macrophages infiltration, which sensitized immunotherapy. Drug screening identified TN4510 as a dual-target inhibitor binding CHCHD3-S2 and cGAS-S3 domains. Combining TN4510 with anti-PD-1 and chemotherapy synergistically inhibited tumor growth in humanized NSG mice (65% reduction vs. control, p<0.001).Conclusions CHCHD3 drives immunotherapy resistance by mitochondrial sequestration of cGAS and suppression of the cGAS-STING axis. Targeting CHCHD3 with TN4510 restores antitumor immunity and enhances neoadjuvant therapy efficacy in LUAD, providing a clinical strategy for treatment-refractory patients ( figure 1).Ethics Approval The use of patients‘ specimens and information was approved by the Ethics Committee of the Tianjin Medical University Cancer Institute and Hospital (Approve number: bc2022323). All animal experiments were performed according to protocols approved by IACUC at the Tianjin Cancer Institute and Hospital, (Approve number: bc2025055).Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.Abstract 480 Figure 1Graphical abstract. The CHCHD3 inhibits the efficacy of neoadjuvant immunotherapy in lung adenocarcinoma patients by directly binding with the functional domain of cGAS