Abstract 1776: Tumor-derived endothelins regulate antitumor immune responses through macrophage endothelin B receptor
Authors: Folkert Ian Wesley , To Tsun Ki Jerrick , Devalaraja Samir , Norgard Robert J. , Haldar Malay 
Published: July 2021

Abstract
Soft tissue sarcomas (STS) comprise a heterogeneous group of solid tumors arising from mesodermal tissues. With few effective systemic therapies, advanced-stage STS is frequently fatal. Immune checkpoint blockade (ICB) is currently under investigation for the treatment of STS. However, STS harbor a highly immunosuppressive tumor microenvironment (TME) that inhibits effective antitumor T cell responses, with only a subset of patients responding to ICB. While the major focus of immunotherapy to date has been on the role of T cells, targeting innate immune cells such as tumor associated macrophages (TAMs) within the TME holds great promise.
TAMs are the most abundant leukocytes in STS, and contribute to the establishment of an immunosuppressive TME through multiple mechanisms. We identified the endothelin B receptor (EDNRB) as a potential novel therapeutic target on TAMs based on an analysis of TCGA data. EDNRB is highly expressed in “immunologically quiet” tumors with high macrophage to T cell ratios and M2-like gene expression. EDNRB encodes a G-protein-coupled receptor that binds the vasoactive peptides endothelin (EDN) 1, 2, or 3. EDNRB is upregulated during macrophage differentiation, and is highly expressed by certain macrophages subsets, including TAMs. However, the role of the EDN-EDNRB axis in regulating macrophage function and antitumor immunity is unknown.
Using a murine syngeneic transplant model with cell lines derived from methylcholanthrene-induced fibrosarcomas, we first genetically deleted or overexpressed the ligand Edn1 in sarcoma cells prior to transplant. Genetic deletion of Edn1 using CRISPR-Cas9 resulted in significantly increased major histocompatibility complex class II (MHCII) expression in TAMs, indicative of a more immunostimulatory phenotype. RNA-seq of sorted TAMs from Edn1 CRISPR tumors revealed enrichment for gene sets involved in antitumor immunity, including interferon gamma and NF-kB. Conversely, overexpression of Edn1 dramatically reduced TAM MHCII expression. Furthermore, knockout of Edn1 or pharmacologic blockade of EDNRB significantly enhanced responses to anti-PD1. To confirm these effects were mediated by macrophage EDNRB, we proceeded to generate a conditional deletion of Ednrb in TAMs by crossing Ednrbflox/flox and LysM-Cre mice. Consistent with our hypothesis, TAMs from sarcomas in LysM-Cre:Ednrbflox/flox mice also took on an immunostimulatory phenotype, with increased MHCII expression and reduced M2 markers.
Taken together, these findings support the existence of a novel regulatory axis, whereby tumor-derived endothelins promote a suppressive TAM phenotype through macrophage EDNRB. Combining endothelin receptor antagonists with immune checkpoint blockade has the potential to enhance antitumor immune responses in STS and other solid tumors by targeting both innate and adaptive immune cells within the TME.
Citation Format: Ian Wesley Folkert, Tsun Ki Jerrick To, Samir Devalaraja, Robert J. Norgard, Malay Haldar. Tumor-derived endothelins regulate antitumor immune responses through macrophage endothelin B receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1776.