Abstract Background: Medulloblastoma is the most common pediatric brain tumor, and Group 3 medulloblastoma (G3 MB) represents the most aggressive subtype with poor prognosis and limited therapeutic options. We found that the multi-kinase inhibitor regorafenib combined with MEK inhibitor demonstrates preclinical efficacy; however, its mechanism of action and specific impact on the tumor immune microenvironment remain poorly defined, particularly in an immunocompetent context. Methods: We utilized CTD, a genetically engineered syngeneic mouse model of G3 MB that recapitulates human disease histologically and molecularly, to evaluate the efficacy of regorafenib plus mirdametinib (MEK inhibitor) in vivo. Tumor burden was monitored by in vivo imaging, and survival was assessed. To characterize tumor cell response and immune remodeling, we performed single-cell RNA sequencing (scRNA-seq) on endpoint tumors enriched for immune cells (CD45+). Additionally, we tested the synergistic effect of adding the immune checkpoint inhibitor anti-PD1 to the regorafenib and mirdametinib combination in vivo. Results: Regorafenib-mirdametinib combination therapy significantly reduced tumor burden and extended survival compared to monotherapy or control. scRNA-seq revealed that tumor cells under combination therapy upregulated MHC class I/II genes and downregulated Lgals3, enhancing antigen presentation. Immune profiling showed increased CD8+ T effector cells, reduced Tregs, and enrichment of M1 macrophage signatures in the combination treatment group. CD8+ T cells from the combination group exhibited activation markers (Ifng, Prf1) and chemokines (Ccl3, Ccl4), but also expressed exhaustion markers (Pd1, Lag3, Tim3). These findings were validated by observing additional benefit from adding anti-PD1 antibody to the drug combination. Conclusions: Regorafenib plus MEK inhibition not only improves survival in G3 MB but also reprograms the suppressive TME, promoting anti-tumor immunity. This effect is further enhanced by adding the immune checkpoint inhibitor anti-PD1. These findings support combining targeted therapy with immunotherapy to achieve durable responses in G3 MB. Citation Format: JINGJING LIU, Mingrui Zhu, Xu Yang, Honglei Zhou, Amar Gajjar, Jiyang Yu. Regorafenib–MEK inhibitor combination reshapes tumor cell states and reprograms the tumor microenvironment to enable immunotherapy in group 3 medulloblastoma 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 A032.
Liu et al. (Wed,) studied this question.