Lalita A. Shevde

Despite substantial advances in early detection and therapeutic strategies, breast cancer remains a major cause of cancer-related morbidity and mortality worldwide. Changes in cytoskeletal proteins that typically regulate cellular architecture underpin tumor progression. Merlin, encoded by the NF2 gene and a member of the ezrin–radixin–moesin protein family, functions as a critical cytoskeletal linker protein and a tumor suppressor that governs multiple oncogenic pathways and restricts epithelial–mesenchymal plasticity. While Merlin deficiency alters cellular architecture, the precise impact of a Merlin-deficient breast tumor on remodeling its immune microenvironment remains largely undefined. In this study, we demonstrate that in breast cancer, Merlin deficiency displays attributes consistent with enhanced epithelial–mesenchymal plasticity and enrichment of Hedgehog signaling. In vivo, Merlin-deficient tumors displayed accelerated growth and an immunosuppressive milieu characterized by increased abundance of regulatory T cells and M2-like macrophages. Treatment with Vismodegib, a pharmacological Hedgehog pathway inhibitor, reduced tumor growth of Merlin-deficient basal-like tumors, accompanied by a decrease in tumor-infiltrating regulatory T cells and immunosuppressive M2-like macrophages, and increased recruitment of pro-inflammatory M1-like macrophages. The data present Hedgehog inhibition as a targetable vulnerability of Merlin-deficient basal-like, triple-negative breast cancer.

The imbalance of pro-inflammatory and immunosuppressive constituents in the tumor microenvironment (TME) significantly dictates cancer progression, immune invasion, and treatment response. Immunosuppressive regulatory T cells (Tregs) are associated with decreased disease-free survival due to their ability to suppress anti-tumor immunity. Notably, in response to environmental cues, Tregs display functional and phenotypic plasticity with inflammatory helper T cell subsets. Given their predominant role in sustaining immune suppression, it is remarkable that multiple developmental pathways converge to regulate Treg development and function. In this study, we designed, validated, and employed a novel genetically modified mouse model to conditionally ablate the Hedgehog (Hh) gene, Gli2, specifically in Tregs. Ablation of Gli2 activity in Tregs significantly reduced tumor burden, impaired Treg suppressive function, and shifted the transcriptional balance of Foxo3 and Rorγt, transcription factors essential for Tregs and Th17 cells. Spatial mapping highlighted that Gli2 ablation in Tregs enhances the immunogenicity of the tumor and promotes a pro-inflammatory milieu of the TME. This was underscored by a higher tumor immune signature score and enhanced infiltration of cytotoxic CD8 ⁺ T cells into the tumor. These findings highlight Hh/Gli2 signaling in Tregs as a mechanistic regulator of immunogenicity in the TME and a potential therapeutic target to prime tumors for enhanced responsiveness for adjuvant treatments.

CIBERSORTx Pre and Post IR Total Cell Abundance.

Supplemental Figure 8 M2 macrophages are increased in both tumor and pre-neoplastic lesions.

Supplemental Figure 3 shows BMH-21 treatment has no impact on cell cycle or ribosomal proteins.

Supplemental Methods detailing the scripts used for data analysis

Supplemental Figure 4 shows BMH21 induces expression of CD80 and CD86 in M2 macrophages

Supplemental Figure 5 shows BMH-21treatment promotes M1 like phenotypes in M2 macrophages

Supplemental Figure 7 shows radiation in combination with BMH-21 treatment blunts tumor growth and enhances inflammatory cell infiltrate.