New pathway could restore immunotherapy effectiveness in resistant cancers

Abstract 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). Citation Format: 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.