Lymphoma mutations in HVEM (TNFRSF14) disrupt bi-directional activation of BTLA and CD160 singaling within the tumor microenvironment

Abstract

Abstract Herpesvirus entry mediator (HVEM; TNFRSF14) is frequently mutated in human cancer with a high degree of mutations in follicular and diffuse large B cell lymphoma. In lymphocytes, HVEM receptors are activated by its ligands LIGHT (TNFSF14) and Lymphotoxin-α, and engage in bi-directional signaling with the receptors B and T lymphocyte attenuator (BTLA) and CD160. We hypothesized that mutations in HVEM disrupted binding to either of its ligands resulting in altered cell-intrinsic HVEM signaling in lymphoma, or in altered activation of BTLA or CD160 in tumor infiltrating lymphocytes. Here, we show that point mutations identified in human lymphoma were localized to the extracellular domain and specifically target ligand binding, resulting in preferential loss of HVEM interactions with CD160 and BTLA compared to LIGHT (TNFSF14). Disrupted binding correlated with loss of receptor functionality resulting in abrogated cell-intrinsic HVEM activation of NF-κB, as well as reduced CD160 activation of ERK and AKT phosphorylation by HVEM ligation. We next identified HVEM-activated CD160 and BTLA signaling pathways through AP/MS and phospho-proteomic analysis in NK and T cells. Activation of CD160 versus BTLA resulted in contrasting phosphorylation status in a number of phospho-proteins, illustrating opposing functions of these receptors. In a mouse model of lymphoma we tested the impact of BTLA versus CD160 in regulating antigen-specific anti-tumor responses, demonstrating regulatory effects of these receptors in vivo. Together these data illustrate how selective pressures within the tumor microenvironment may drive cancer mutations that alter HVEM network signaling in vivo, and indicate potential targets for onco-immunotherapy.