Search

Supplementary Figure from Inhibiting Type I Arginine Methyltransferase Activity Promotes T Cell–Mediated Antitumor Immune Responses

Supplementary Table from Inhibiting Type I Arginine Methyltransferase Activity Promotes T Cell–Mediated Antitumor Immune Responses

Protein arginine methyltransferases (PRMT) are a widely expressed class of enzymes responsible for catalyzing arginine methylation on numerous protein substrates. Among them, type I PRMTs are responsible for generating asymmetric dimethylarginine. By controlling multiple basic cellular processes, such as DNA damage responses, transcriptional regulation, and mRNA splicing, type I PRMTs contribute to cancer initiation and progression. A type I PRMT inhibitor, GSK3368715, has been developed and has entered clinical trials for solid and hematologic malignancies. Although type I PRMTs have been reported to play roles in modulating immune cell function, the immunologic role of tumor-intrinsic pathways controlled by type I PRMTs remains uncharacterized. Here, our The Cancer Genome Atlas dataset analysis revealed that expression of type I PRMTs associated with poor clinical response and decreased immune infiltration in patients with melanoma. In cancer cell lines, inhibition of type I PRMTs induced an IFN gene signature, amplified responses to IFN and innate immune signaling, and decreased expression of the immunosuppressive cytokine VEGF. In immunocompetent mouse tumor models, including a model of T-cell exclusion that represents a common mechanism of anti–programmed cell death protein 1 (PD-1) resistance in humans, type I PRMT inhibition increased T-cell infiltration, produced durable responses dependent on CD8+ T cells, and enhanced efficacy of anti–PD-1 therapy. These data indicate that type I PRMT inhibition exhibits immunomodulatory properties and synergizes with immune checkpoint blockade (ICB) to induce durable antitumor responses in a T cell–dependent manner, suggesting that type I PRMT inhibition can potentiate an antitumor immunity in refractory settings.

PTEN loss in melanoma promotes resistance to T cell-mediated anti-tumor activity in the context of concurrent a selective inhibitor for mutant BRAF.

Supplementary Methods

Supplementary tables

Immune-checkpoint inhibitors and adoptive tumor-infiltrating lymphocyte (TIL) therapies have profoundly improved the survival of patients with melanoma. However, a majority of patients do not respond to these agents, and many responders experience disease relapse. Although numerous innovative treatments are being explored to offset the limitations of these agents, novel therapeutic combinations with immunotherapies have the potential to improve patient responses. In this study, we evaluated the antimelanoma activity of immunotherapy combinations with Telaglenastat (CB-839), a potent glutaminase inhibitor (GLSi) that has favorable systemic tolerance. In in vitro TIL:tumor coculture studies, CB-839 treatment improved the cytotoxic activity of autologous TILs on patient-derived melanoma cells. CB-839 treatment decreased the conversion of glutamine to alpha-ketoglutarate (αKGA) more potently in tumor cells versus TILs in these cocultures. These results suggest that CB-839 may improve immune function in a tumor microenvironment by differentially altering tumor and immune cell metabolism. In vivo CB-839 treatment activated melanoma antigen–specific T cells and improved their tumor killing activity in an immune-competent mouse model of adoptive T-cell therapy. Additionally, the combination of CB-839 with anti-PD1 or anti-CTLA4 antibodies increased tumor infiltration by effector T cells and improved the antitumor activity of these checkpoint inhibitors in a high mutation burden mouse melanoma model. Responsiveness to these treatments was also accompanied by an increase of interferon gamma (IFNγ)–associated gene expression in the tumors. Together, these results provide a strong rationale for combining CB-839 with immune therapies to improve efficacy of these treatments against melanoma.

Supplementary figures

Supplementary Table S1. Patient characteristics of the single-agent anti-PD-1 cohort. Supplementary Table S2. List of primers used for gene expression analysis by real-time PCR. Supplementary Table S3. List of autophagy-related genes for overexpressing experiment.

The cytokine changes in mice receiving PI3K inhibition in combination with anti-OX40 treatment.

Supplementary Figure legends

A list of all 384 ORFs in the library

Purpose:OX40 agonist–based combinations are emerging as a novel avenue to improve the effectiveness of cancer immunotherapy. To better guide its clinical development, we characterized the role of the OX40 pathway in tumor-reactive immune cells. We also evaluated combining OX40 agonists with targeted therapy to combat resistance to cancer immunotherapy.Experimental Design: We utilized patient-derived tumor-infiltrating lymphocytes (TILs) and multiple preclinical models to determine the direct effect of anti-OX40 agonistic antibodies on tumor-reactive CD8+ T cells. We also evaluated the antitumor activity of an anti-OX40 antibody plus PI3Kβ inhibition in a transgenic murine melanoma model (Braf mutant, PTEN null), which spontaneously develops immunotherapy-resistant melanomas.Results:We observed elevated expression of OX40 in tumor-reactive CD8+ TILs upon encountering tumors; activation of OX40 signaling enhanced their cytotoxic function. OX40 agonist antibody improved the antitumor activity of CD8+ T cells and the generation of tumor-specific T-cell memory in vivo. Furthermore, combining anti-OX40 with GSK2636771, a PI3Kβ-selective inhibitor, delayed tumor growth and extended the survival of mice with PTEN-null melanomas. This combination treatment did not increase the number of TILs, but it instead significantly enhanced proliferation of CD8+ TILs and elevated the serum levels of CCL4, CXCL10, and IFNγ, which are mainly produced by memory and/or effector T cells.Conclusions:These results highlight a critical role of OX40 activation in potentiating the effector function of tumor-reactive CD8+ T cells and suggest further evaluation of OX40 agonist–based combinations in patients with immune-resistant tumors.

Purpose: Cancer immunotherapy has shown promising clinical outcomes in many patients. However, some patients still fail to respond, and new strategies are needed to overcome resistance. The purpose of this study was to identify novel genes and understand the mechanisms that confer resistance to cancer immunotherapy.Experimental Design: To identify genes mediating resistance to T-cell killing, we performed an open reading frame (ORF) screen of a kinome library to study whether overexpression of a gene in patient-derived melanoma cells could inhibit their susceptibility to killing by autologous tumor-infiltrating lymphocytes (TIL).Results: The RNA-binding protein MEX3B was identified as a top candidate that decreased the susceptibility of melanoma cells to killing by TILs. Further analyses of anti–PD-1–treated melanoma patient tumor samples suggested that higher MEX3B expression is associated with resistance to PD-1 blockade. In addition, significantly decreased levels of IFNγ were secreted from TILs incubated with MEX3B-overexpressing tumor cells. Interestingly, this phenotype was rescued upon overexpression of exogenous HLA-A2. Consistent with this, we observed decreased HLA-A expression in MEX3B-overexpressing tumor cells. Finally, luciferase reporter assays and RNA-binding protein immunoprecipitation assays suggest that this is due to MEX3B binding to the 3′ untranslated region (UTR) of HLA-A to destabilize the mRNA.Conclusions: MEX3B mediates resistance to cancer immunotherapy by binding to the 3′ UTR of HLA-A to destabilize the HLA-A mRNA and thus downregulate HLA-A expression on the surface of tumor cells, thereby making the tumor cells unable to be recognized and killed by T cells. Clin Cancer Res; 24(14); 3366–76. ©2018 AACR.See related commentary by Kalbasi and Ribas, p. 3239

Supplementary figure and table legends

Protein arginine methyltransferases (PRMT) are a widely expressed class of enzymes responsible for catalyzing arginine methylation on numerous protein substrates. Among them, type I PRMTs are responsible for generating asymmetric dimethylarginine. By controlling multiple basic cellular processes, such as DNA damage responses, transcriptional regulation, and mRNA splicing, type I PRMTs contribute to cancer initiation and progression. A type I PRMT inhibitor, GSK3368715, has been developed and has entered clinical trials for solid and hematologic malignancies. Although type I PRMTs have been reported to play roles in modulating immune cell function, the immunologic role of tumor-intrinsic pathways controlled by type I PRMTs remains uncharacterized. Here, our The Cancer Genome Atlas dataset analysis revealed that expression of type I PRMTs associated with poor clinical response and decreased immune infiltration in patients with melanoma. In cancer cell lines, inhibition of type I PRMTs induced an IFN gene signature, amplified responses to IFN and innate immune signaling, and decreased expression of the immunosuppressive cytokine VEGF. In immunocompetent mouse tumor models, including a model of T-cell exclusion that represents a common mechanism of anti–programmed cell death protein 1 (PD-1) resistance in humans, type I PRMT inhibition increased T-cell infiltration, produced durable responses dependent on CD8+ T cells, and enhanced efficacy of anti–PD-1 therapy. These data indicate that type I PRMT inhibition exhibits immunomodulatory properties and synergizes with immune checkpoint blockade (ICB) to induce durable antitumor responses in a T cell–dependent manner, suggesting that type I PRMT inhibition can potentiate an antitumor immunity in refractory settings.