A Novel Oral Arginase 1/2 Inhibitor Enhances the Antitumor Effect of PD-1 Inhibition in Murine Experimental Gliomas by Altering the Immunosuppressive Environment.

Paulina Pilanc,Paulina S Stańczak,Roman Błaszczyk,Marcin M Grzybowski,Paweł Dobrzański,Natalia Ochocka,Bozena Kaminska,Salwador Cyranowski,Bartłomiej Gielniewski,Aleksandra Ellert-Miklaszewska,Adria-Jaume Roura,Kamil Wojnicki

doi:10.3389/fonc.2021.703465

Abstract

Glioblastomas (GBM) are the common and aggressive primary brain tumors that are incurable by conventional therapies. Immunotherapy with immune checkpoint inhibitors is not effective in GBM patients due to the highly immunosuppressive tumor microenvironment (TME) restraining the infiltration and activation of cytotoxic T cells. Clinical and experimental studies showed the upregulation of expression of the arginase 1 and 2 (ARG1 and ARG2, respectively) in murine and human GBMs. The elevated arginase activity leads to the depletion of L-arginine, an amino-acid required for the proliferation of T lymphocytes and natural killer cells. Inhibition of ARG1/2 in the TME may unblock T cell proliferation and activate effective antitumor responses. To explore the antitumor potential of ARG1/2 inhibition, we analyzed bulk and single-cell RNA sequencing (scRNA-seq) data from human and murine gliomas. We found the upregulation of ARG1/2 expression in GBMs, both in tumor cells and in tumor infiltrating microglia and monocytes/macrophages. We employed selective arginase inhibitors to evaluate if ARG1/2 inhibition in vitro and in vivo exerts the antitumor effects. A novel, selective ARG1/2 inhibitor - OAT-1746 blocked microglia-dependent invasion of U87-MG and LN18 glioma cells in a Matrigel invasion assay better than reference compounds, without affecting the cell viability. OAT-1746 effectively crossed the blood brain barrier in mice and increased arginine levels in the brains of GL261 glioma bearing mice. We evaluated its antitumor efficacy against GL261 intracranial gliomas as a monotherapy and in combination with the PD-1 inhibition. The oral treatment with OAT-1746 did not affect the immune composition of TME, it induced profound transcriptomic changes in CD11b+ cells immunosorted from tumor-bearing brains as demonstrated by RNA sequencing analyses. Treatment with OAT-1746 modified the TME resulting in reduced glioma growth and increased antitumor effects of the anti-PD-1 antibody. Our findings provide the evidence that inhibition of ARG1/2 activity in tumor cells and myeloid cells in the TME unblocks antitumor responses in myeloid cells and NK cells, and improves the efficacy of the PD-1 inhibition.

Highlights

Glioblastoma (GBM, WHO grade IV glioma) is the most common and aggressive primary brain tumor in adults
We took advantage of having in-house sc-RNA-seq data of CD11b+ immunosorted from murine GL261 gliomas, which provided resolution to distinguish resident microglia from CNS-border associated macrophages (BAMs) or monocytes/macrophages (Mo/MF) [34]
There was a low number of microglial cells expressing either Arg1 (< 1%) or Arg2 (< 0.1%) mRNA and both genes were more abundantly expressed in the Mo/MF population (11% and 6%, respectively) (Figure 1C and Supplementary Figure 1C)

Summary

Introduction

Glioblastoma (GBM, WHO grade IV glioma) is the most common and aggressive primary brain tumor in adults. While the available treatments may slow down the progression of GBM and reduce neurological symptoms, the disease remains incurable. The standard treatment for GBM patients is surgical resection followed by radiation and oral chemotherapy with temozolomide (TMZ). Surgical techniques, radiotherapy and chemotherapy, GBM inevitably recurs and the prognosis of patients with GBM remains poor, with a median overall survival of 15 months [1, 2]. The ability of tumors to modify the surrounding microenvironment and evade the immune system is increasingly recognized as an important determinant of cancer progression and patient prognosis [3]. GBMs are infiltrated with various myeloid cells which do not activate their proper functions but instead they are tumor supportive and create the immunosuppressive tumor microenvironment (TME), poorly infiltrated with cytotoxic T lymphocytes and natural killer (NK) cells being frequently deficient in their antitumor activity [4]

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Results

Conclusion