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Background In recent years, immune checkpoint blockade (ICB) therapy has demonstrated significant clinical efficacy and improved patient prognosis across various malignancies, 1 thereby fueling widespread interest and advancing research in cancer immunotherapy. However, tumor mediated immunosuppressive mechanisms continue to limit anti-tumor immune responses and are now recognized as major barriers to effective immunotherapy.2 A deeper understanding of these immune inhibitory pathways in cancer cells may reveal novel therapeutic targets and improve clinical outcomes.Methods In this study, we integrate public scRNA-seq data from breast cancer patients 3 with transcriptomic profiles of tumor-bearing mice to uncover and validate dominant mechanisms in breast cancer cells that hamper anti-tumor immunity. Our scRNA-seq results are validated using in vivo studies performed in syngeneic mouse models. We employ high-resolution single-cell spatial transcriptomics using CosMX technology to further resolve the spatial organization and cellular composition of the tumor microenvironment. Additionally, we evaluate 1300 cellular features using Cell Painting to identify compounds that mimic NEDD8 loss in human breast cancer cells, and verify the features at the subcellular level using transmission electron microscopy.Results Here, we reveal that low NEDD8 mRNA expression in cancer cells correlates with enhanced T cell expansion in pre-therapy patient samples treated with pembrolizumab. In mice bearing neddylation-deficient breast EO771 tumors, PD-1 blockade significantly elicits potent efficacy and primes strong immune memory. Analysis of scRNA-seq datasets from patients and tumor-bearing mice shows that elevated neddylation impairs antigen presentation and rewires lipid metabolism in tumor cells and macrophages, while reduced neddylation significantly enhances CD8+ T cell cytotoxicity and macrophage antigen presentation and activation following PD-1 blockade. Single-cell spatial transcriptomics analysis illustrates distinct spatial-temporal changes and the establishment of stimulatory macrophage niches in neddylation-deficient murine tumors. To test neddylation as a therapeutic target in immunotherapy, we show that the clinical-stage neddylation inhibitor, pevonedistat, significantly reduces the efficacy of PD-1 blockade at high doses, but leads to curative effects and the expansion of PD-1+TCF1+ stem-like memory T cells in vivo. Genetic deletion of neddylation in human breast cancer cells causes the loss of sensitivity to statin drugs and ferroptosis inducers. Indeed, simvastatin or RSL3 strongly synergizes with PD-1 blockade and potentiates immune memory in tumor-bearing mice.Conclusions Our work shows the novel role of protein neddylation on lipid metabolism and immunogenicity in cancer cells and macrophages, and proposes actionable strategies to reprogram tumors for improved immunotherapy responsiveness.Acknowledgements We thank the BioVis platform at Uppsala University, the Chemical Biology Consortium Sweden (CBCS), SciLifeLab, the National Genomics Infrastructure in Stockholm, and the KIGene core facility at Karolinska Institutet for technical support. We acknowledge the contributions of collaborators, facility staff, and research team members. This work was supported by grants from SciLifeLab (SLL2019/9, SciLifeLab Fellowship), the Swedish Research Council (2022-01461), the Swedish Foundation for Strategic Research (FFL-0043), and the Swedish Cancer Society (220474JIA; 232656Pj). I.P. was additionally supported by a grant from the Royal Swedish Academy of Sciences (ME2024-0042).References Shiravand Y, et al. Immune checkpoint inhibitors in cancer therapy.Curr Oncol. 2022;29(5):3044–3060.Liu D, Jenkins RW, Sullivan RJ, Mechanisms of resistance to immune checkpoint blockade.Am J Clin Dermatol. 2019;20(1):41–54.Bassez A, et al. A single-cell map of intratumoral changes during anti-PD1 treatment of patients with breast cancer. Nat Med. 2021;27(5):820-832.Ethics Approval All animal studies were approved by the Swedish Board of Agriculture at Jönköping, Sweden (Dnr: 5.8.18-06394/2020).