Inhibition of microglial EZH2 leads to anti-tumoral effects in pediatric diffuse midline gliomas

Abstract

Background: Diffuse intrinsic pontine gliomas (DIPG), within diffuse midline gliomas are aggressive pediatric brain tumors characterized by histone H3-K27M mutation. Small-molecule inhibitors for the EZH2-H3K27 histone methyltransferase have shown promise in preclinical animal models of DIPG, despite having little effect on DIPG cells in vitro. Therefore, we hypothesized that the effect of EZH2 inhibition could be mediated through targeting of this histone modifying enzyme in tumor-associated microglia.

Methods: Primary DIPG tissues, and cocultures between microglia and patient-derived DIPG or -pediatric high-grade glioma (pHGG) cell lines, were used to establish the H3-K27M status of each cell type. Antisense RNA strategies were used to target EZH2 gene expression in both microglia and glioma cells. Microglia anti-tumoral properties were assessed by gene expression profile, tumor cell invasion capacity, microglial phagocytic activity, and associated tumor cell death.

Results: In primary DIPG tissues, microglia do not carry the H3-K27M mutation, otherwise characteristic of the cancer cells. Activation of a microglial tumor-supportive phenotype by pHGG, independently of their H3-K27M status, is associated with a transient H3K27me3 downregulation. Repression of EZH2 in DIPG cells has no impact on tumor cell survival or their ability to activate microglia. However, repression of EZH2 in microglia induces an anti-tumor phenotype resulting in decreased cancer cell invasion capability, increased microglial phagocytosis, and tumor-related cell death.

Conclusions: These results indicate that microglia, beyond the tumor cells, contribute to the observed response of DIPG to EZH2 inhibition. Results highlight the potential importance of microglia as a new therapeutic avenue in DIPG.

Keywords: DIPG; EZH2; H3K27me3; anti-tumoral; microglia.

Figure 1.

Microglia do not carry the H3K-27M mutation in the DIPG microenvironment. (A) Confocal microscopy of SF188 and SF8628 glioma cells immunostained with H3K-27M mutation and DAPI, scale bar, 50 µm. (B) Immunoblot analysis of H3K-27M mutation and β-actin (ACTB) (right margin, molecular size, in kilodaltons [kDa]). Quantification of immunoblots are shown relative to β-actin expression. (C) and (D) Confocal microscopy of human DIPG tumors obtained from three individual patients with immunostaining for H3K-27M mutation, IBA1 and DAPI, scale bar, 30 µm. P value: ***<.001.

Figure 2.

DIPG-induced microglia activation is associated with the transient downregulation of H3K27me3. (A) Quantification of the invasion capacity of SF188 and SF8628 glioma cells into matrigel coated transwells with BV2 microglia or 10% complete DMEM as control in the lower compartment; results are presented relative to 10% media only control, set as 1. (B) qPCR analysis of Nos2 expression in BV2 microglia grown as monocultures or with SF188 or SF8628 glioma cells as segregated co-cultures for 4 h, results are shown relative to BV2 monocultures, set as 1. (C) Immunoblot analysis of BV2 microglia cells for H3K27me3 and β-actin following segregated co-culture with SF188 and SF8628 glioma cells. Results shown relative to monoculture set as 1. P value: *<.05, and ***<.001.

Figure 3.

Repression of EZH2 gene expression in H3K-27M DIPG cancer cells does not impact on their survival or capability to activate microglia. (A) qPCR analysis of EZH2 in SF188 and SF8628 glioma cells transfected with either EZH2-specific siRNA (siEZH2) or scramble siRNA (siCtrl). Fold change is shown relative to siCtrl with siCtrl set as 1. Immunoblot analysis of siEZH2 and siCtrl transfected SF188 and SF8628 glioma cells for (B) EZH2, H3K27me3, H3K27ac and β-actin, (C) H3K-27M mutation and β-actin and (D) and (E) PCNA and cleaved PARP. (F) qPCR analysis of Nos2 expression in BV2 microglia grown as monocultures or with siEZH2 or siCtrl SF188 or siEZH2 or siCtrl SF8628 glioma cells as segregated co-cultures for 4 h, results are shown relative to BV2 monocultures, set as 1. P value: ***<.001.

Figure 4.

Repression of Ezh2 gene expression in microglia promotes the expression of anti-tumoral factors. (A) qPCR analysis of Ezh2 in BV2 microglia cells transfected with either Ezh2-specific siRNA (siEZH2) or scramble siRNA (siCtrl). Fold change is shown relative to siCtrl with siCtrl set as 1. (B) Immunoblot analysis of siEzh2 and siCtrl transfected microglia cells for EZH2, H3K27me3, H3K27ac and β-actin. qPCR analysis of (C) Nos2 expression in siEzh2 or siEzh2 BV2 microglia (D) Chromatin Immunoprecipitation (ChIP) analysis of H3K27me3 and H3K27ac on the Nos2 promoter in siCtrl or siEzh2 BV2 cells (E) Il1b expression of BV2 microglia grown as monocultures or with SF188, SF8628 or SU-DIPG-XIII glioma cells as segregated co-cultures for 4 h, results are shown relative to BV2 monocultures, set as 1. qPCR analysis of Il1b for (F) siEzh2 or siCtrl microglia grown as monocultures or with SF188 as segregated co-cultures for 4 h, results are shown relative to siCtrl BV2 monocultures, set as 1 and (G) LPS-treated siEzh2 or siCtrl BV2 4 h (H) Immunoblot analysis of siEzh2 and siCtrl transfected microglia cells following 4 h co-culture with SF188 or SF8628 glioma cells for phospho-S6, Total S6 and β-actin. P value: *<.05, and ***<.001.

Figure 5.

Repression of Ezh2 gene expression in microglia promotes a cell phenotype holding anti-tumoral properties. (A) Quantification of the invasion capacity of SF188 and SF8628 glioma cells into matrigel coated transwells with siCtrl or siEzh2 BV2 microglia in the lower compartment; results are presented relative to siCtrl BV2 control, set as 1. (B) Quantification of the migration capacity of SU-DIPG-XIII glioma cells into transwells with siCtrl or siEzh2 BV2 microglia in the lower compartment; results are presented relative to siCtrl BV2 control, set as 1. (C) Quantification of the migration capacity of SF188 and SF8628 glioma cells into transwells with siCtrl or siEZH2 HMC3 human microglia in the lower compartment; results are presented relative to siCtrl HMC3 control, set as 1. (D, E) Capacity of siCtrl or siEzh2 BV2 to phagocytosis CFSE-labelled (D) SF188 and SF8628 glioma cells and (E) CFSE-labelled SU-DIPG-XIII when co-cultured for 4 h at a ratio of 2:1, results are presented relative to siCtrl BV2 set as 1. (F) Microglia-induced glioma cell death measured by counting the number of cell-tracker labelled SF188 or SF8628 glioma cells after co-culture with siCtrl or siEzh2 BV2 microglia for 4 h. results are presented relative to siCtrl BV2 set as 1. P value: *<.05, **<.01 and ***<.001.

Figure 6.

Schematic illustration showing proposed mechanism for how EZH2 inhibition can lead to apoptotic tumor cell death in the context of DIPG upon activation of a microglial anti-tumoral phenotype.