Loss of interferon gamma (IFNγ) signaling or antigen presentation in cancer cells can lead to primary and acquired resistance to immune checkpoint blockade (ICB) therapy. Our work aims to overcome immune resistance by targeting tumor-intrinsic vulnerabilities. Using a melanoma cell line derived from a patient that acquired resistance to pembrolizumab due to a JAK2 loss-of-function mutation1, we reveal through whole genome CRISPR/Cas9 screens that neddylation can be targeted to restore cancer cell sensitivity to PD-1 blockade in a co-culture system. Genetic deletion or pharmacological inhibition of the E1 neddylation enzyme, ubiquitin-like modifier activating enzyme 3 (UBA3), increases the immunogenicity of cancer cells in co-culture, evidenced by an increased immune cell proliferation and cytokine secretion. Using proteomics, flow cytometry, and immunoblotting, we observe that deletion of neddylation in JAK2-deficient cancer cells leads to an enriched interferon signature with an increased JAK1 expression, stabilization of chromatin-bound cGAS, and elevated HLA-I and HLA-II. In tumor-bearing mice, genetic deletion of Jak1, Jak2 and/or B2m confers resistance to PD-1 blockade, which is reverted in neddylation-deficient tumors. Single-cell RNA-Seq of the Jak2 deficient tumors reveals a heightened cancer-intrinsic interferon signaling when neddylation is impaired. Although changes in T cells are marginal, NK cells exhibit enhanced activation and cytolytic gene signatures upon PD-1 blockade in tumors lacking neddylation. Interestingly, neddylation deficiency leads to an increased infiltration and activation of migratory dendritic cells and myeloid cells, which are validated using flow cytometry. Using a single-cell RNA-Seq dataset from melanoma patients2, we observe that tumors with low neddylation have an elevated cancer-intrinsic IFNγ signature and enriched dendritic cell infiltration. Based on our in vitro and in vivo evidence, we hypothesize that the loss of neddylation unlocks interferon signaling and innate immune activation through the cGAS/STING pathway in the absence of JAK/STAT signaling. Indeed, genetic deletion of Cgas in murine cancer cells abolishes the superior response to PD-1 blockade elicited by the neddylation-deficient tumors. To conclude, tumors lacking neddylation activate cGAS/STING signaling, promoting a microenvironment with pro-inflammatory myeloid, dendritic, and NK cell infiltration, which facilitates the response to PD-1 blockade. Given the challenges of developing agonists for the cGAS/STING pathway, targeting cancer-intrinsic neddylation offers an attractive alternative to balance therapeutic efficacy and toxicity. References 1. Zaretsky, J. M. et al. Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma. New England Journal of Medicine 375, 819–829 (2016). 2. Yang, J., Wang, C., Fu, D. et al. Mature and migratory dendritic cells promote immune infiltration and response to anti-PD-1 checkpoint blockade in metastatic melanoma. Nat Commun 16, 8151 (2025). Marta Rúbies Bedós, Yonglin Lu, Eirini Voutsinou, Irineos Papakyriacou, Liam P. Alford, Rebeca Tomás Parracho, Vaishnavi Iyer, Yumeng Mao. Targeting cancer-intrinsic neddylation overcomes resistance to immune checkpoint blockade therapy in interferon-deficient tumors [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B068.

The loss of IFNγ signaling in cancer cells causes primary and acquired resistance to immunotherapy. Using a patient-derived JAK2-deficient melanoma cell line, we have developed a co-culture system to model acquired resistance to immunotherapy. Genome-wide genetic screens uncover that protein neddylation can be targeted to restore sensitivity to PD1 blockade in co-cultures, and in MHC-I and/or JAK/STAT-deficient murine tumors. Analysis of the Jak2-deficient murine tumors with scRNA-seq reveals a heightened cancer-intrinsic inflammatory signature when neddylation is absent, which causes an influx of stimulatory antigen presenting cells. In patients with melanoma, tumors with low cancer-intrinsic neddylation exhibit elevated IFN signatures and are associated with dendritic cell infiltration. Mechanistically, neddylation deletion stabilizes chromatin-bound cGAS in JAK2-deficient cancer cells, which is responsible for the superior response to PD1 blockade, and the recruitment of pro-inflammatory innate immune cells. Altogether, we demonstrate the broad therapeutic potential of protein neddylation in overcoming resistance to immunotherapy.

Supplemental Table S1: Compounds for the Cell Painting screen on control and NEDD8 KO MDA-MB-231 cells.

Supplementary Figure S5. Characterization of tumor-infiltrating T and NK cells in EO771 tumors using scRNA-seq.

Supplementary Figure S10. Assessment of combinatorial effects of simvastatin or RSL3 with PD-1 blockade in C1498 or EO771 tumor-bearing mice.

Supplementary Figure S6. Phenotypes of tumor-infiltrating immune cells are modulated by Nedd8-deficiency in EO771 cancer cells.

Supplemental Table S2: Antibodies and proteins.

Supplementary Figure S9. Simvastatin-treated HCC1937 human TNBC cells show similar phenotype in TEM as the NEDD8 deletion.

Supplementary Figure S4. Nedd8 deletion modulates gene expression in EO771 cancer cells and infiltrating macrophages.

Supplementary Figure S2. Neddylation in cancer cells regulates immune cell phenotype.

Supplementary Figure S7. Characterization of immune cell infiltration in EO771 tumors using CosMX single-cell spatial transcriptomics.

Supplemental Table S3: Other reagents.

Acute myeloid leukemia is an aggressive hematological disease, where cancer cells down-regulate antigen presentation and immune-stimulatory molecules. Here, we show that genetic deletion of protein neddylation rewires the human AML proteome towards an immunogenic phenotype, while suppressing cholesterol biosynthesis. As a result, neddylation-deficient AML cells respond more potently to TLR agonists in vitro. Using genome-wide CRISPR/Cas9 screens, we have identified mTOR as an important pathway in deficient cells. Inhibition of lipid metabolism by simvastatin in primary human monocytes amplifies response to LPS, which is governed by mTOR and JAK/STAT pathways. In mice, simvastatin fine-tunes the inflammatory response driven by LPS and polarizes myeloid cells to a stimulatory phenotype. While neither simvastatin nor LPS show efficacy against murine C1498 tumors, a combination of the agents delays tumor growth in mice. Altogether, our results uncover a previously unknown function of protein neddylation in governing the response to TLR stimulation and propose the therapeutic potential of statin drugs in immunotherapy against hematological malignancies.

Supplementary Figure S1. Pre-therapy NEDD8 mRNA levels in cancer cells are associated with T-cell expansion.

Supplemental Table S4: CRISPR RNA sequences.

Supplementary Figure S8. Simvastatin-treated MDA-MB-231 human TNBC cells show similar phenotype in TEM as the NEDD8 deletion.

Supplementary Figure S3: Characterization of tumor-infiltrating cells using scRNAseq.

Childhood neuroblastoma with MYCN amplification is classified as high risk and often relapses after intensive treatments. Immune checkpoint blockade therapy against the PD-1/L1 axis shows limited efficacy in patients with neuroblastoma, and the cancer intrinsic immune regulatory network is poorly understood. Here, we leverage genome-wide CRISPR/Cas9 screens and identify H2AFYas a resistance gene to the clinically approved PD-1 blocking antibody nivolumab. Analysis of single- cell RNA-Seq datasets reveals that H2AFYmRNA is enriched in adrenergic cancer cells and is associated with worse patient survival. Genetic deletion of H2afy in MYCN-driven neuroblastoma cells reverts in vivo resistance to PD-1 blockade by eliciting activation of the adaptive and innate immunity. Mapping of the epigenetic and translational landscape demonstrates that H2afy deletion promotes cell transition to a mesenchymal-like state. With a multiomics approach, we uncovered H2AFY- associated genes that are functionally relevant and prognostic in patients. Altogether, our study elucidates the role of H2AFY as an epigenetic gatekeeperfor cell states and immunogenicity in high-risk neuroblastoma.

Immune checkpoint blockade therapy aims to activate the immune system to eliminate cancer cells. However, clinical benefits are only recorded in a subset of patients. Here, we leverage genome-wide CRISPR/Cas9 screens in a Tumor-Immune co-Culture System focusing on triple-negative breast cancer (TNBC). We reveal that NEDD8 loss in cancer cells causes a vulnerability to nivolumab (anti-PD-1). Genetic deletion of NEDD8 only delays cell division initially but cell proliferation is unaffected after recovery. Since the NEDD8 gene is commonly essential, we validate this observation with additional CRISPR screens and uncover enhanced immunogenicity in NEDD8 deficient cells using proteomics. In female immunocompetent mice, PD-1 blockade lacks efficacy against established EO771 breast cancer tumors. In contrast, we observe tumor regression mediated by CD8+ T cells against Nedd8 deficient EO771 tumors after PD-1 blockade. In essence, we provide evidence that NEDD8 is conditionally essential in TNBC and presents as a synergistic drug target for PD-1/L1 blockade therapy. NEDD8 is a ubiquitin-like protein that governs protein neddylation, previously demonstrated to be essential for cell survival. Here the authors show that NEDD8 loss in breast cancer cells is associated with enhanced immunogenicity and increased sensitivity to PD-1 blockade in preclinical cancer models.