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.

Keywords: Cancer immunotherapy; Cell Biology; Epigenetics; Immunology; Macrophages.

Figure 1. Cytotoxicity of RAW264.7-conditioned SN toward AB1 mesothelioma cells.

(A) Experimental design of RAW264.7 macrophages treated with mock agent, N^G-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.

Figure 2. Direct cytotoxicity of RAW264.7 macrophages upon cell-to-cell contact with syngeneic mesothelioma AB1 cells.

(A) Experimental design. RAW264.7 macrophages were treated with L-NMMA or apocynin for 24 hours and then further cultivated in the presence or absence of LPS for 24 hours. After 3 washes in PBS, RAW264.7 macrophages were cocultivated with AB1 cells at a 10:1 ratio for 48 hours. (B) AB1 cells and CFSE-labeled RAW264.7 macrophages were monitored by time-lapse microscopy using an LSM 510 (Zeiss) equipped with an environmental chamber maintained at 37°C in a humidified 5% CO₂ atmosphere. (C) Cells were fixed, permeabilized, and stained for F4/80 (shown in green) and nitrosylated tyrosine (N-Tyr; shown in blue). Images were acquired using a Zeiss LSM 510 confocal microscope equipped with a ×63-1.4 oil immersion objective. (D) Apoptotic rates of AB1 cells were determined by flow cytometry after staining with the annexin V-FITC kit (Becton Dickinson). Each bar represents the mean ± SD from 8 independent experiments performed in triplicate. (E) AB1 cells were transduced by lentivectors encoding PGAM5 shRNAs (#2 and #5) or a scramble control. The levels of PGAM5 transcripts were measured by reverse transcription quantitative PCR. (F) RAW264.7-induced apoptosis of shRNA-transduced AB1 cells was measured as described in D. Each bar represents the mean ± SD from 6 independent experiments. Statistical significance was evaluated using 1-way ANOVA followed by Tukey’s multiple-comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. Effect of EZH2 inhibition by EPZ on RAW264.7-mediated killing of AB1 cells.

RAW264.7 macrophages were incubated with the EZH2 inhibitor (10 μM EPZ5687) for 48 hours and treated or not with LPS for 24 hours. (A) RAW264.7 macrophages were labeled with an anti-H3K27me3 antibody, stained with Draq5, and analyzed by confocal microscopy. Magnification 40×. (B) The rMFI corresponds to the ratio of fluorescence intensities associated with H3K27me3 and Draq5. (C) RAW264.7 macrophages were fluorescently labeled with antibodies against pan-Histone 3 or H3K27me3 and analyzed by flow cytometry using a FACSaria flow cytometer (Becton Dickinson). The rMFI represents the ratio of fluorescence intensities associated with H3K27me3 and pan-H3. (D) Intracellular ROS were measured by flow cytometry using the cell-permeant H₂DCFDA probe. MFI, mean fluorescence intensity. (E) Nitrites (in μM) in the cell SNs were quantified by the Griess reaction assay. (F) RAW264.7 macrophage–conditioned SNs were added to AB1 cell cultures as described in Figure 1A. Apoptosis was determined by flow cytometry after labeling with annexin V-FITC. (G) CFSE-labeled RAW264.7 macrophages were cocultivated for 48 hours with AB1 cells at a 10:1 ratio. Apoptotic rates of CFSE^– AB1 cells were determined by flow cytometry after staining with the annexin V-FITC kit (Becton Dickinson). Each bar represents the mean ± SD. Statistical significance was evaluated using the paired Student’s t test (B and C) and 1-way ANOVA followed by Tukey’s multiple-comparisons test (D–G). *P < 0.05; **P < 0.01; ***P < 0.001. (H) CFSE-labeled RAW264.7 macrophages were cocultivated for 24 hours with AB1 cells at a 1:1 ratio in the presence of annexin V-APC (red fluorescence). The cells were monitored by the IncuCyte S3 Live-Cell imaging system (Essen Bioscience) placed in an incubator maintained at 37°C in a humidified 5% CO₂ atmosphere. (I) RAW264.7 macrophages pretreated with EPZ and/or LPS were cocultured with CFSE-labeled AB1 cells. The number of CFSE⁺ (AB1) annexin V⁺ events was determined every 10 minutes for 24 hours using the IncuCyte S3 Live-Cell imaging system. Each curve is the average of 5 independent experiments performed in triplicate.

Figure 4. Effect of EPZ on RAW264.7-mediated inhibition of AB1 tumor growth.

(A) RAW264.7 macrophages were cultured with the EZH2 inhibitor (10 μM EPZ5687) for 24 hours and stimulated with LPS for an additional day. After 3 washes, RAW264.7 cells were coimplanted subcutaneously (SC) in BALB/c mice with 2 × 10⁶ AB1 or AB12 cells at a 1:3 ratio. In B, D, F, and H, tumor volumes (in mm³) were calculated weekly using the following formula: 4/3 × π × (diameter/2)³. C, E, G, and I represent the corresponding survival curves. Groups of at least 6 mice were tested in each experimental condition. All data are plotted as mean ± SEM (n = 6). Statistical significance was evaluated using 2-way ANOVA with Bonferroni’s post test (B, D, F, and H) and log-rank χ² test (survival curves). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5. Immunohistochemistry of AB1 mesothelioma tumors.

(A) Histochemical analyses of tumors from BALB/c mice inoculated with AB1 tumor cells and RAW264.7 macrophages differentiated in the presence or absence of EPZ and/or LPS. Sections from tumors with similar volumes were stained with hematoxylin and eosin (original magnification, ×20). (B) Representative immunohistochemistry micrographs of apoptotic cells and F4/80⁺ macrophages. Apoptotic cells were labeled with an antibody directed against cleaved caspase-3 (active) and an Alexa Fluor 546 conjugate (in red). Infiltrating macrophages were identified by F4/80 and anti-IgG Alexa Fluor 488 antibodies (in green). Cell nuclei were stained with DAPI (in blue). The higher magnification (original magnification, ×40) reveals macrophage pseudopodia interacting with an apoptotic tumor cell (white arrows). (C) The cleaved caspase-3 fluorescence intensity (FI) was quantified from 25 images (3 × 3 segmentation at original magnification ×40) by using ImageJ and FSX100. Each bar represents the mean ± SD. *P < 0.05, evaluated using the 2-tailed Mann-Whitney U test.

Figure 6. Effect of EPZ on PD-1 expression and activity of anti–PD-1 blockade in AB1/RAW264.7 cocultures.

(A) RAW264.7 macrophages were cultured in the presence of LPS and/or EPZ as described in Figure 3. After labeling with an anti–mouse PD-1 antibody, fluorescence emission was analyzed by flow cytometry using a BD FACSAria. Representative dot plots of FSC-H (x axis) and percentage of PD-1 cells (y axis) are shown. (B) Percentages of RAW264.7 macrophages expressing PD-1 were deduced from 4 independent experiments. Each bar represents the mean ± SD. **P < 0.01, and ***P < 0.001, calculated using 1-way ANOVA followed by Tukey’s multiple-comparisons test. (C) Time-lapse analysis of CFSE-labeled AB1 cells cocultured with RAW264.7 macrophages for 24 hours at a 1:1 ratio in the presence of PE/Cy7–conjugated anti–mouse PD-1 antibody (red). (D) RAW264.7 macrophages were incubated with anti–PD-1 antibody (10 μg/mL; InVivoMAb, BioXcell) or rat IgG2a isotype control for 6 hours and then cocultivated with CFSE-labeled AB1 cells in the presence of annexin V-APC. The cells were monitored by the IncuCyte S3 Live-Cell imaging system (Essen Bioscience) placed in an incubator maintained at 37°C in a humidified 5% CO₂ atmosphere. The number of annexin V⁺ AB1 cells (percentage) was quantified every 10 minutes for 24 hours. Statistical significance was evaluated using 2-way ANOVA with Bonferroni’s post test. ***P < 0.001. (E) RAW264.7 macrophages were cultured with the EZH2 inhibitor (10 μM EPZ5687) for 24 hours and with anti–PD-1 antibody (10 μg/mL; InVivoMAb, BioXcell) or Rat IgG2a isotype control (10 μg/mL, BD) for 6 hours before inoculation. After 3 washes, RAW264.7 macrophages were coimplanted SC into BALB/c mice with 2 × 10⁶ AB1 or AB12 cells at a 1:3 ratio. Tumor volumes (in mm³) were calculated weekly using the following formula: 4/3 × π × (diameter/2)³. (F) The corresponding survival curve for E. Groups of at least 6 mice were tested in each experimental condition. All data are plotted as mean ± SEM (n = 6). Statistical significance was evaluated using 2-way ANOVA with Bonferroni’s post test (E) and log-rank χ² test (F). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7. A model for EZH2-dependent immunoediting of mesothelioma cells by autologous macrophages.

Schematic representation of direct and indirect cytotoxicities mediated by ROS and peroxynitrites produced by macrophages. Oxidative stress induces tyrosine nitration in tumor cells, leading to apoptosis and phagocytosis by macrophages. Inhibition of EZH2 reduces direct cytotoxicity exerted by macrophages without affecting ROS production but increases PD-1 expression. The engagement of PD-1 at the synapse between macrophages and mesothelioma cells impairs killing activity of macrophages. The combination of an EZH2 inhibitor (such as tazemetostat) and immune checkpoint inhibitor that targets PD-1 (e.g., pembrolizumab, nivolumab) restores immunoediting activity of macrophages.