Inhibition of EZH2 methyltransferase decreases immunoediting of mesothelioma cells by autologous macrophages through a PD-1–dependent mechanism
Malik Hamaidia, Hélène Gazon, Clotilde Hoyos, Gabriela Brunsting Hoffmann, Renaud Louis, Bernard Duysinx, Luc Willems
Malik Hamaidia, Hélène Gazon, Clotilde Hoyos, Gabriela Brunsting Hoffmann, Renaud Louis, Bernard Duysinx, Luc Willems
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Research Article Cell biology Immunology

Inhibition of EZH2 methyltransferase decreases immunoediting of mesothelioma cells by autologous macrophages through a PD-1–dependent mechanism

Abstract

The roles of macrophages in orchestrating innate immunity through phagocytosis and T lymphocyte activation have been extensively investigated. Much less understood is the unexpected role of macrophages in direct tumor regression. Tumoricidal macrophages can indeed manifest cancer immunoediting activity in the absence of adaptive immunity. We investigated direct macrophage cytotoxicity in malignant pleural mesothelioma, a lethal cancer that develops from mesothelial cells of the pleural cavity after occupational asbestos exposure. In particular, we analyzed the cytotoxic activity of mouse RAW264.7 macrophages upon cell-cell contact with autologous AB1/AB12 mesothelioma cells. We show that macrophages killed mesothelioma cells by oxeiptosis via a mechanism involving enhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27–specific (H3K27-specific) methyltransferase of the polycomb repressive complex 2 (PRC2). A selective inhibitor of EZH2 indeed impaired RAW264.7-directed cytotoxicity and concomitantly stimulated the PD-1 immune checkpoint. In the immunocompetent BALB/c model, RAW264.7 macrophages pretreated with the EZH2 inhibitor failed to control tumor growth of AB1 and AB12 mesothelioma cells. Blockade of PD-1 engagement restored macrophage-dependent antitumor activity. We conclude that macrophages can be directly cytotoxic for mesothelioma cells independent of phagocytosis. Inhibition of the PRC2 EZH2 methyltransferase reduces this activity because of PD-1 overexpression. Combination of PD-1 blockade and EZH2 inhibition restores macrophage cytotoxicity.

Authors

Malik Hamaidia, Hélène Gazon, Clotilde Hoyos, Gabriela Brunsting Hoffmann, Renaud Louis, Bernard Duysinx, Luc Willems

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Figure 1

Cytotoxicity of RAW264.7-conditioned SN toward AB1 mesothelioma cells.

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Cytotoxicity of RAW264.7-conditioned SN toward AB1 mesothelioma cells. (...
(A) Experimental design of RAW264.7 macrophages treated with mock agent, NG-monomethyl-l-arginine (L-NMMA), or apocynin for 24 hours in a 24-well plate. After stimulation with LPS for 24 hours, the cell culture SN was collected and clarified by centrifugation for 6 minutes at 500 g. AB1 cells were cultivated for 48 hours in 25% macrophage-conditioned SN mixed with 75% complete RPMI 1640 containing 10% FCS and 1% penicillin and streptavidin. The peroxynitrite scavenger [5,10,15,20-tetrakis(4-sulfonatophenyl)-prophyrinato iron (III) chloride; FeTTPS] was directly added to the culture medium. (B) Apoptosis was evaluated after labeling with annexin V-FITC (Becton Dickinson) and propidium iodide (PI, MilliporeSigma). Data were collected with an FACSAria cytometer and analyzed by the FACSDiva software. Treatment with a tyrosine kinase inhibitor (10 μM of lapatinib) for 24 hours was used as a positive control. Both PI–annexin V+ and PI+annexin V+ cells were considered to have undergone apoptosis. (C) Apoptotic rates of AB1 cells were quantified by flow cytometry after labeling with annexin V-FITC. Mean values and standard deviations were deduced from 8 independent experiments. (D) Nitrites (μM) were quantified in the cell SN using the Griess reaction assay. (E) Intracellular ROS were measured by flow cytometry using the cell-permeant 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCFDA) probe. Each bar represents the mean ± SEM from 8 independent experiments performed in triplicate. Statistical significance was evaluated using 1-way ANOVA followed by Tukey’s multiple-comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001. MFI, mean fluorescence intensity.