{
  "abstract": "Background A major cause of death in patients with breast cancer is complications resulting from metastasis. Immune checkpoint inhibitors (ICIs) activate T cells and B cells to mount anti-tumor responses, but they have proved ineffective against metastatic breast cancer as single-agent therapies. Myeloid cell-T cell interactions contribute to this treatment inefficacy, as macrophages and myeloid-derived suppressor cells (MDSCs) are abundant in breast tumors, which suppress anti-tumoral responses by T cells. Our group has demonstrated that the histone deacetylase inhibitor, entinostat, decreases the immunosuppression by MDSCs, and when combined with anti-PD-1 and anti-CTLA-4, increases survival in HER2 + and triple-negative breast cancer (TNBC) primary tumor models.Methods NeuN mice harboring spontaneous HER2 + breast-to-lung metastases were treated with combinations of entinostat, anti-PD-1, and anti-CTLA-4 and assessed for survival. Lung metastases were analyzed using single-cell RNA-sequencing (scRNA-seq), and tumor-targeting antibodies were determined via immunostaining of cancer cells. Immune cell proportions within lung metastases from HER2+ and TNBC models were determined via flow cytometry. Myeloid immunosuppression and validation of treatment targets identified via scRNA-seq were assessed via T cell proliferation in ex-vivo co-cultures between macrophages/G-MDSCs isolated from lung metastases with CD8+ T cells. Tissues from metastatic patients treated with the triple combination from our phase 1b trial (NCI-9844) were analyzed using imaging mass cytometry (IMC).Results Triple combination of entinostat, anti-PD-1, and anti-CTLA-4 increased survival in the NT2.5-LM model of HER2 + breast cancer, which was associated with an increase in plasma cell proportions, tumor-targeting antibodies, and Th2 activation, as determined by scRNA-seq and flow cytometry using HER2+ and TNBC models. CellChat analysis revealed a decrease in ligand-receptor interactions associated with immunosuppression in myeloid cells, including Icam1_Itgam_Itgb2 and Ifng_Ifngr1_Ifngr2, in mice treated with either entinostat or triple-combination. Macrophages and G-MDSCs suppressed T cell proliferation, and blockade of ICAM1 or IFNγ receptor increased T cell proliferation, respectively. IMC results demonstrated increased proportions of CD8+ T cells, expression of pro-inflammatory marker CD137 in B cells and CD8+ T cells, and distances between CD8+ T cells and macrophages with triple combination treatment in responders. In addition, only responders had detectable germinal centers or mature tertiary lymphoid structures and increased proportions of B cells and plasma cells post-treatment.Conclusions Overall, our results suggest that B cell activation and the amelioration of myeloid immunosuppression increase the efficacy of checkpoint inhibition in metastatic breast cancer; these mechanisms should be carefully considered when using ICIs in clinical settings.Ethics Approval All mice were housed under pathogen-free conditions and handled according to protocol 21770 approved by the USC Institutional Animal Care and Use Committee, following guidelines by the American Association of Laboratory Animal Committee policies. The use of patient samples for this study was approved by the Institutional Review Boards of participating institutions (Johns Hopkins, Yale, University of Pittsburgh Medical Center and City of Hope), and participants signed a written informed consent before enrollment. The trial was conducted according to the principles of Good Clinical Practice and the Declaration of Helsinki. The NCI Central Institutional Review Board (9844) also reviewed the clinical protocol. The Sidney Kimmel Comprehensive Cancer Center Clinical Research Review Committee and Safety Monitoring Committee were responsible for reviewing accrual and safety data for this trial at least twice a year. The Protocol Chair and the study statistician reviewed the study as needed based on rate of accrual for toxicity monitoring during the dose expansion phase at least quarterly.",
  "authors": [
    {
      "affiliations": [
        "University of Southern California, Los Angeles, CA, USA"
      ],
      "name": "Edgar Gonzalez"
    },
    {
      "affiliations": [
        "University of Southern California, Los Angeles, CA, USA"
      ],
      "name": "Jesse Kreger"
    },
    {
      "affiliations": [
        "University of Southern California, Los Angeles, CA, USA"
      ],
      "name": "Yingtong Liu"
    },
    {
      "affiliations": [
        "University of Southern California, Los Angeles, CA, USA"
      ],
      "name": "Xiaojun Wu"
    },
    {
      "affiliations": [
        "University of Southern California, Los Angeles, CA, USA"
      ],
      "name": "Matthew B Jacobo"
    },
    {
      "affiliations": [
        "Keck School of Medicine, University of Southern California, Los Angeles, CA, USA"
      ],
      "name": "Julie Jang"
    },
    {
      "affiliations": [
        "University of Southern California, Los Angeles, CA, USA",
        "Keck School of Medicine, University of Southern California, Los Angeles, CA, USA",
        "Johns Hopkins University, Baltimore, MD, USA",
        "Weill Cornell Medicine, New York, NY, USA",
        "Johns Hopkins School of Medicine, Baltimore, MD, USA"
      ],
      "name": "Batul Al-zubeidy"
    },
    {
      "affiliations": [
        "University of Southern California, Los Angeles, CA, USA"
      ],
      "name": "Graduate Student"
    },
    {
      "affiliations": [
        "University of Southern California, Los Angeles, CA, USA"
      ],
      "name": "Aaron G Baugh"
    },
    {
      "affiliations": [
        "University of Southern California, Los Angeles, CA, USA"
      ],
      "name": "Arianna Barbetta"
    },
    {
      "affiliations": [
        "Johns Hopkins University, Baltimore, MD, USA"
      ],
      "name": "Sarah M Shin"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Vered Stearns"
    },
    {
      "affiliations": [
        "Johns Hopkins School of Medicine, Baltimore, MD, USA"
      ],
      "name": "Roisin Connolly"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Won Ho"
    },
    {
      "affiliations": [
        "University of Southern California, Los Angeles, CA, USA"
      ],
      "name": "Juliet Emaumaullee"
    },
    {
      "affiliations": [
        "University of Southern California, Los Angeles, CA, USA"
      ],
      "name": "Adam L MacLean"
    },
    {
      "affiliations": [
        "Keck School of Medicine, University of Southern California, Los Angeles, CA, USA"
      ],
      "name": "Evanthia T Roussos Torres"
    }
  ],
  "title": "735 T cell interactions and B cell activation mediate response to treatment with entinostat and checkpoint inhibitors in metastatic breast cancer",
  "uid": "263396fc-a82b-5a05-8888-9317d07741d2"
}