Abstract B034: Characterization of MHC-I-mediated immune evasion in triple-negative breast cancer using human transcriptomic datasets and immunocompetent mouse models
Authors: O'Connell Constandina E , Felsheim Brooke M , Fernandez-Martinez Aranzazu , Mott Kevin R , Perou Charles M 
Published: February 2024

Abstract
In Triple-Negative Breast Cancer (TNBC) patients, adaptive immune system activity in the tumor microenvironment is associated with improved prognosis. However, tumors can evolve to evade immune detection, likely leading to worse patient outcomes. Multi-omic data of paired primary and metastatic breast tumors demonstrate that TNBC metastases frequently lose expression of genes involved in major histocompatibility complex class I (MHC-I) antigen presentation, a key pathway for adaptive immune activation (PMID: 36585450). Therefore, understanding and addressing MHC-I-mediated immune evasion may have profound clinical benefits for TNBC patients. We hypothesize that breast tumor MHC-I loss results in a unique immune-evasive phenotype, which we are characterizing using human clinical datasets and immunocompetent mouse models of breast cancer. First, using large transcriptomic datasets (i.e., SCAN-B and TCGA), we are characterizing molecular and clinical features of MHC-I-low human breast tumors using expression of the MHC-I gene human leukocyte antigen A (HLA-A) as a continuous variable. We found HLA-A expression varies significantly between PAM50 molecular and clinical subtypes of breast cancer and has differing impacts on patient survival based on clinical subtype. In TNBCs, low HLA-A expression is associated with worse outcomes, while in HR+/HER2- tumors, low HLA-A predicts improved outcomes. We also identified genes highly correlated with HLA-A regardless of molecular subtype. This list was functionally rich, related to antigen processing machinery, adaptive immune-mediated cytotoxicity, and T cell exhaustion features. To complement human genomic data, we are empirically testing the role of MHC-I in syngeneic mouse mammary tumor models by individually knocking out murine MHC-I genes–H2-K, H2-D, and B2m–through CRISPR/Cas9-mediated genome editing. We have generated and validated knockouts (KOs) of H2-K and B2m, as well as empty vector control clones from the “immune-hot” and immune checkpoint inhibitor (ICI) responsive mammary tumor cell lines KPB25L-UV and T11-APOBEC. RNA sequencing of KPB25L-UV H2-K and B2m KO versus wildtype cell lines in vitro identified lists of differentially expressed genes, many of which are related to proteolytic and peptide antigen processing. Next, these CRISPR-modified cell lines were injected into the mammary fat pad of syngeneic immunocompetent mice, and the resulting tumors were harvested for RNA sequencing. Differentially expressed genes in KPB25L-UV H2-K KO versus wildtype tumors were related to increased MHC Class II antigen presentation and dendritic cell activity. Preliminary data suggests that mice bearing KPB25L-UV H2-K KO tumors have shortened survival compared to H2-K wildtype tumors. Experiments are ongoing to further characterize the growth dynamics, metastatic potential, single-cell transcriptomic profiles, and ICI sensitivities of these KO tumors in vivo. Our long-term goal is to identify therapeutic means to overcome this common immune-evasive phenotype to improve TNBC patient outcomes.
Citation Format: Constandina E O'Connell, Brooke M Felsheim, Aranzazu Fernandez-Martinez, Kevin R Mott, Charles M Perou. Characterization of MHC-I-mediated immune evasion in triple-negative breast cancer using human transcriptomic datasets and immunocompetent mouse models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B034.