ABSTRACT
PURPOSE: Immunotherapy has recently been shown to improve outcomes for advanced PD-L1-positive triple-negative breast cancer (TNBC) in the Impassion130 trial, leading to FDA approval of the first immune checkpoint inhibitor in combination with taxane chemotherapy. To further develop predictive biomarkers and improve therapeutic efficacy of the combination, interrogation of the tumor immune microenvironment before therapy as well as during each component of treatment is crucial. Here we use single-cell RNA sequencing (scRNA-seq) on tumor biopsies to assess immune cell changes from two patients with advanced TNBC treated in a prospective trial at predefined serial time points, before treatment, on taxane chemotherapy and on chemo-immunotherapy. METHODS: Both patients (one responder and one progressor) received the trial therapy, in cycle 1 nab-paclitaxel given as single agent, in cycle 2 nab-paclitaxel in combination with pembrolizumab. Tumor core biopsies were obtained at baseline, 3 weeks (after cycle 1, chemotherapy alone) and 6 weeks (after cycle 2, chemo-immunotherapy). Single-cell RNA sequencing (scRNA-seq) of both cancer cells and infiltrating immune cells isolated were performed from fresh tumor core biopsy specimens by 10 × chromium sequencing. RESULTS: ScRNA-seq analysis showed significant baseline heterogeneity of tumor-infiltrating immune cell populations between the two patients as well as modulation of the tumor microenvironment by chemotherapy and immunotherapy. In the responding patient there was a population of PD-1high-expressing T cells which significantly decreased after nab-paclitaxel plus pembrolizumab treatment as well as a presence of tissue-resident memory T cells (TRM). In contrast, tumors from the patient with rapid disease progression showed a prevalent and persistent myeloid compartment. CONCLUSIONS: Our study provides a deep cellular analysis of on-treatment changes during chemo-immunotherapy for advanced TNBC, demonstrating not only feasibility of single-cell analyses on serial tumor biopsies but also the heterogeneity of TNBC and differences in on-treatment changes in responder versus progressor.
Subject(s)
Triple Negative Breast Neoplasms , Albumins , Antibodies, Monoclonal, Humanized , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Humans , Paclitaxel , Prospective Studies , Single-Cell Analysis , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/genetics , Tumor MicroenvironmentABSTRACT
Despite substantial progress in lung cancer immunotherapy, the overall response rate in patients with KRAS-mutant lung adenocarcinoma (LUAD) remains low. Combining standard immunotherapy with adjuvant approaches that enhance adaptive immune responses-such as epigenetic modulation of antitumor immunity-is therefore an attractive strategy. To identify epigenetic regulators of tumor immunity, we constructed an epigenetic-focused single guide RNA library and performed an in vivo CRISPR screen in a Kras G12D/Trp53 -/- LUAD model. Our data showed that loss of the histone chaperone Asf1a in tumor cells sensitizes tumors to anti-PD-1 treatment. Mechanistic studies revealed that tumor cell-intrinsic Asf1a deficiency induced immunogenic macrophage differentiation in the tumor microenvironment by upregulating GM-CSF expression and potentiated T-cell activation in combination with anti-PD-1. Our results provide a rationale for a novel combination therapy consisting of ASF1A inhibition and anti-PD-1 immunotherapy. SIGNIFICANCE: Using an in vivo epigenetic CRISPR screen, we identified Asf1a as a critical regulator of LUAD sensitivity to anti-PD-1 therapy. Asf1a deficiency synergized with anti-PD-1 immunotherapy by promoting M1-like macrophage polarization and T-cell activation. Thus, we provide a new immunotherapeutic strategy for this subtype of patients with LUAD.See related commentary by Menzel and Black, p. 179.This article is highlighted in the In This Issue feature, p. 161.
Subject(s)
Adenocarcinoma of Lung/drug therapy , Cell Cycle Proteins/metabolism , Drug Resistance, Neoplasm/genetics , Immune Checkpoint Inhibitors/pharmacology , Lung Neoplasms/drug therapy , Molecular Chaperones/metabolism , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/immunology , Adenocarcinoma of Lung/pathology , Animals , CRISPR-Cas Systems/genetics , Cell Cycle Proteins/genetics , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Line, Tumor , Disease Models, Animal , Epigenesis, Genetic/immunology , Gene Expression Regulation, Neoplastic/immunology , Gene Knockout Techniques , HEK293 Cells , Humans , Immune Checkpoint Inhibitors/therapeutic use , Lung/pathology , Lung Neoplasms/genetics , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Male , Mice , Molecular Chaperones/genetics , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , Proto-Oncogene Proteins p21(ras)/genetics , RNA, Guide, Kinetoplastida/genetics , RNA, Small Interfering/metabolism , RNA-Seq , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunology , Tumor Suppressor Protein p53/geneticsABSTRACT
Pancreatic ductal adenocarcinoma (PDA) is characterized by immune tolerance and immunotherapeutic resistance. We discovered upregulation of receptor-interacting serine/threonine protein kinase 1 (RIP1) in tumor-associated macrophages (TAMs) in PDA. To study its role in oncogenic progression, we developed a selective small-molecule RIP1 inhibitor with high in vivo exposure. Targeting RIP1 reprogrammed TAMs toward an MHCIIhiTNFα+IFNγ+ immunogenic phenotype in a STAT1-dependent manner. RIP1 inhibition in TAMs resulted in cytotoxic T cell activation and T helper cell differentiation toward a mixed Th1/Th17 phenotype, leading to tumor immunity in mice and in organotypic models of human PDA. Targeting RIP1 synergized with PD1-and inducible co-stimulator-based immunotherapies. Tumor-promoting effects of RIP1 were independent of its co-association with RIP3. Collectively, our work describes RIP1 as a checkpoint kinase governing tumor immunity.
