Figure S5: Evaluation of SETDB1 knockdown in human melanoma cells, related to Figure 4:

Figure S2: CRISPR screen guide distributions, related to Figure 1

List of primers used to perform RT-qPCR on YUDOSO cells related to Figure S6.

Figure S6: Characterization of possible SKO tumor antigens, related to Figure 4.

Figure S3: Validation and characterization of Setdb1-/- cell lines, tumors, and microenvironments, related to Figure 2.

Ranked CRISPR screen dropout hits, related to Figure 1. Analysis was performed using MaGeCK.

Differential expression of SKO vs YRG cells, by RNAseq.

Abstract Glioblastoma (GBM), the most frequent and aggressive primary tumor of the brain, escapes all standard-of-care treatments that are provided after resection, including chemotherapy, radiotherapy or immunotherapy. The GBM tumor microenvironment is regulated by immunosuppressive mechanisms of the innate immune system, thereby contributing to the lack of anti-tumor adaptive immunity. We found that tumor associated macrophages (TAMs) can be repolarized into a M1 anti-tumor phenotype via agonist stimulation of the retinoic acid-inducible gene I (RIG-I), a cytosolic double-stranded RNA pattern recognition receptor (PRR). Innate immunity is governed by PRRs, which act as sensors of foreign and danger-associated molecular patterns and initiate the innate immune response. In silico analysis of adult GBM datasets available in the public domain revealed that RIGI-I expression by a subset of activated M1 TAMs positively correlated with patient survival. Studies in syngenic mouse models of GBM showed that intratumoral delivery of stem-loop RNA 14 (SLR14), a RIG-I agonist, improved the efficacy of all standard anti-GBM treatments, beyond the effects of other nuclei acid sensor agonists. We found that the combined activation of M1 TAMs and priming of functional cytotoxic CD8+ T lymphocytes and NK cells were accounting for the anti-GBM effect of SLR14, opening a significant new avenue for adult GBM treatment. Citation Format: Han Xu, Sungwoon Lee, Felipe Saceanu Leser, Olga Federova, Peiwen Lu, Eric Song, Anne Eichmann, Akiko Iwasaki, Anna Anna Marie Pyle, Jean-Leon Thomas. Agonist activation of RIG-I+tumor associated macrophages enhances anti-tumor immunity and therapeutic response in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2918.

Figure S4: Evaluation of differences in SKO tumor gene expression and microenvironment profiles, related to Figure 3:

Differential expression of SKO vs YRG cells ± IFNAR blockade by RNAseq.

Summary of exonic mutations in YRG cells compared with YMG and WT C57BL6/J cells.

Glioblastoma (GBM), the most frequent and aggressive primary brain tumor, remains refractory to all current therapies including surgical resection, chemotherapy, radiotherapy and immunotherapy. Immunosuppressive mechanisms in the GBM tumor microenvironment contribute to the lack of anti-tumor adaptive immunity. We found that a subset of tumor associated macrophages (TAMs) can be repolarized into an anti-tumor phenotype via agonist stimulation of the retinoic acid-inducible gene I (RIGI), a cytosolic double-stranded RNA pattern recognition receptor (PRR). In silico analysis of adult GBM datasets available in the public domain revealed that RIGI expression by a subset of activated TAMs positively correlated with patient survival. Studies in syngeneic mouse models of GBM showed that intratumoral delivery of stem-loop RNA 14 (SLR14), a RIG-I agonist, improved the efficacy of chemotherapy, radiotherapy and immunotherapy treatments, beyond the effects of other nuclei acid sensor agonists. We found that RIGI + macrophages are the main drivers of SLR14 effect, combining activation of TAMs and priming of functional cytotoxic CD8+ T lymphocytes and NK cells. The anti-GBM effect of SLR14 is opening a significant new avenue for adult GBM treatment.

Figure S1: Experimental validation of SETDB1 knockout and flow gating schemes, related to Figure 4

Supplementary Table S3: Predicted pathogenic/deleterious variants by exome sequencing.

Supplementary Table S2: HLA genotype calls.

Supplementary Figure S1: Additional characterization of the clinical trial cohort.Supplementary Figure S2: Additional characterization of exome profiles and defining MLH1 methylation status.Supplementary Figure S3: JAK1 alterations are most frequent in endometrial cancer.Supplementary Figure S4: Final cell counts in each scRNA-seq sample.Supplementary Figure S5: Longitudinal transcriptional profiling of circulating immune cells before and after PD-1 blockade.Supplementary Figure S6: Differential abundance of cell neighborhoods after PD-1 immunotherapy.Supplementary Figure S7: Differential abundance of cell neighborhoods in JAK1-mutant tumors.Supplementary Figure S8: Transcriptional profiles of T and NK cells in responders vs non-responders.Supplementary Figure S9: Gene expression changes in T and NK cells following PD-1 immunotherapy.Supplementary Figure S10: Additional transcriptional characterization of CD16+ NK cells in epiR patients.

Supplementary Table S1: Genomic and clinical characteristics of the study cohort.

Supplementary Table S4: Metadata annotations for the scRNA-seq dataset.

Despite recent advances in the treatment of melanoma, many patients with metastatic disease still succumb to their disease. To identify tumor-intrinsic modulators of immunity to melanoma, we performed a whole-genome CRISPR screen in melanoma and identified Setdb1 as well as all components of the human silencing hub complex. We found that loss of Setdb1 leads to increased immunogenicity and complete tumor clearance in a CD8(+) T cell-dependent manner. Mechanistically, loss of Setdb1 causes de-repression of endogenous retroviruses (ERV) in melanoma cells and triggers tumor cell-intrinsic type I IFN signaling, upregulation of MHC-I expression, and increased CD8(+) T-cell infiltration. Importantly, spontaneous immune clearance observed in Setdb1(-/-) tumors results in subsequent protection from other ERV-expressing tumor lines, supporting the functional antitumor role of ERV-specific CD8(+) T cells found in the Setdb1(-/-) microenvironment. Blocking the type I IFN receptor in mice grafted with Setdb1(-/-) tumors decreases immunogenicity by decreasing MHC-I expression, leading to decreased T-cell infiltration and increased melanoma growth, comparable with Setdb1(wt) tumors. Together, these results provide key in vivo evidence of a critical role for Setdb1 and type I IFNs in generating an inflamed tumor microenvironment and potentiating tumor cell-intrinsic immunogenicity in melanoma. This study further emphasizes regulators of ERV expression and type I IFN expression as potential therapeutic targets for augmenting anticancer immune responses.

Purpose:Alteration of visual acuity in wet age-related macular degeneration (AMD) is mostly driven by vascular endothelial growth factor A (VEGF-A)-induced edema from leaky newly forming blood vessels below the retina layers. To date, all therapies aimed at alleviation of this process have relied on inhibition of VEGF-A activity. Although effective in preventing vascular leak and edema, this approach also leads to the loss of normal vasculature and multiple related side effects. Methods:We have developed an alternative strategy that uses anti-syndecan-2 polyclonal antibody (anti-Sdc2 pAb) to block VEGF-A-induced permeability without interfering with other VEGF-A activities. The effect of anti-Sdc2 pAb therapy was assessed in vitro using a transendothelial electrical resistance (TEER) assay, as well as staining of the endothelial cell junction, and in vivo in the laser-induced choroidal neovascularization (CNV) model. Results:Anti-Sdc2 pAb blocked VEGF-A-induced permeability in vitro, and both local intravitreal injections and systemic intravenous treatments with anti-Sdc2 pAb were as effective as intravitreal anti-VEGF therapy in reducing edema, size of retinal lesions, and local inflammation in this model. Post-injury neovascularization was not affected by treatment with anti-Sdc2 pAb. Conclusions:These findings indicate that anti-Sdc2 pAb therapy can be an effective alternative to anti-VEGF-A approaches for suppression of edema and to prevent retinal lesions in wet neovascular AMD (nAMD). Translational Relevance:Intravitreal anti-Sdc2 treatment may avoid side effects observed with the long-term anti-VEGF therapy, and systemic treatment with an anti-Sdc2 pAb antibody can address the issues associated with repeated intravitreal injections.