Immune Circuit Rewiring by HDAC Inhibition Enables Response to Dual Checkpoint Blockade in Metastatic Breast Cancer

High levels of immune suppression are a common intrinsic mechanism of resistance in metastatic breast cancer that calls for developing immunotherapeutic combinations to broaden treatment responses. Histone deacetylase (HDAC) inhibitors can sensitize tumors to dual checkpoint inhibition in patients. Here, we investigated the tumor microenvironment (TME) of breast metastases by combining experimental and clinical data with theory to elucidate the mechanism of response to treatment with the HDAC inhibitor entinostat combined with anti-PD-1 and anti-CTLA-4. Knowledge-guided subclustering of single-cell RNA-sequencing (scRNA-seq) data and cell circuit analyses from murine breast-to-lung metastases identified 39 cell states and salient interactions, of which myeloid, T cell, and B cell subpopulations were most affected. Analyses of patient biopsies and blood via spatial proteomics and flow cytometry corroborated the preclinical findings, showing increased T cell and B cell activation, mature tertiary lymphoid structures, and increased CD8+ T cell-macrophage distances in responders to entinostat + nivolumab + ipilimumab. Combination treatment increased immunoglobulin production in patients and mice, and murine studies demonstrated increased tumor-targeting IgG and implicated B cells as necessary for treatment response. Inhibition of the ICAM1 and IFNγ pathways in myeloid cells partially recapitulated treatment effects on CD8+ T cells observed via scRNA-seq. Mathematical modeling of tumor-immune dynamics implicated simultaneous modulation of multiple TME interactions as required for response to the combination treatment. Overall, this study identifies lymphoid and myeloid cell contributions to response to treatment with HDAC inhibitors and immune checkpoint blockade, providing a framework for discovering interactions driving responses in complex TMEs.