An Unintended Effect Of The EZH2 Inhibitor GSK126:Promoting Myeloid Derived Suppressor Cell Production And Tumor Progression

Background:Epigenetic histone modifications comprise an important mechanism that controls cellular processes including tumorigenesis and immunity.Epigenetic aberrations are often associated with tumor progression and cancer development.Of particular interest,the methyltansferase enhancer of zeste homolog 2(EZH2),the catalytic component of the polycomb repressive complex 2(PRC2)that trimethylates lysine27 of histone H3 to promote transcriptional repression,is frequently overexpressed in various cancers including lung,colorectal,breast,pancreatic,and prostate cancers.Aberrant EZH2 over-expression is also associated with poor prognosis.Importantly,EZH2 is functionally critical for the growth and metastasis of cancer cells and thus pharmacological inhibition of EZH2 is proposed to represent a targeted therapy for various cancers.Currently,several EZH2 inhibitors including GSK126 and EPZ-6438 are in clinical trials to treat advanced solid tumors or B cell lymphomas.The tumor microenvironment(TME)plays critical roles in directing the outcome of tumor rejection vs.tumorigenesis.Increasing evidence suggests that to fully assess the effect of anti-cancer drugs and devise more effective therapies,it is imperative to understand the impact of the drug on the TME.Whereas GSK126 is a promising anti-cancer drug currently in multiple clinical trials,the premise has been largely based on studies using immune-deficient hosts,where it is impossible to assess the effect of the drug on the TME.Because EZH2functions in many immune cell types that might contribute to tumor immunity,it is important to address how its inhibition might affect immune cell function during tumor development,a currently unresolved question.Therefore,we suspected that EZH2 inhibition might weaken antitumor effects by affecting immune cell activity in immune-competent hosts.Thus,exploring the therapeutic effect in immune-competent hosts and the changes in immune cells within the tumor microenvironment after EZH2 inhibitor(GSK126)treatment may provide novel insights on the factors restricting the therapeutic activity of EZH2 inhibitor(GSK126).Methods:1.Explore the GSK126 antitumor effect in immune competent hosts and immune deficient hosts.1.1 LLC cells were subcutaneously injected into the flanks of C57BL/6 mice.The mice were treated with GSK126 or normal saline.1.2 LLC cells were subcutaneously injected into the flanks of nude mice.The mice were treated with GSK126 or normal saline.1.3 MC38 cells were subcutaneously injected into the flanks of C57BL/6 mice.The mice were treated with GSK126 or normal saline.1.4 MC38 cells were subcutaneously injected into the flanks of nude mice.The mice were treated with GSK126 or normal saline.2.Explore the effect of GSK126 in adaptive immune cells.2.1 Percentage of tumor-infiltrating CD4~+T cells and CD8~+T cells were determined by flowcytometry.2.2 Percentage of Ki67~+cells in CD4~+T cells and CD8~+T cells in tumor were determined by flowcytometry.2.3 Percentage of IFN-γ~+cells in CD8~+T cells in tumor were determined by flowcytometry.2.4 Percentage of Gzms-B~+cells in CD8~+T cells in tumor were determined by flowcytometry.2.5 Percentage of CD4~+T cells and CD8~+T cells in blood were determined by flowcytometry.2.6 Percentage of IFN-γ~+cells in CD8~+T cells in blood were determined by flowcytometry.2.7 Percentage of CD4~+T cells and CD8~+T cells in spleen were determined by flowcytometry.2.8 Percentage of IFN-γ~+cells in CD8~+T cells in spleen were determined by flowcytometry.3.Explore if CD8+T cells activity were suppressed by changing TME after GSK126treatment.3.1 MC38 cells expressing ovalbumin(OVA)(1?10~6)were implanted subcutaneously into female C57BL/6 mice.GSK126 was administered during the initial 4 days.Seven days later,naive CD45.1~+CD8~+T cells from OT-1 transgenic mice were purified by magnetic bead sorting and 2?10~6 activated or naive CD45.1~+CD8~+T cells were injected into CD45.2~+C57BL/6 mice.3.2 Percentage of CD45.1~+cells in CD8~+T cells in spleen were determined by flowcytometry.3.3 Percentage of IFN-γ~+cells in CD45.1~+CD8~+T cells(naive CD8~+T cells)after PMA+ionomycin or OVA stimulation in spleen were determined by flowcytometry.3.4 Percentage of IFN-γ~+cells in CD45.1~+CD8~+T cells(activated CD8~+T cells)after PMA+ionomycin or OVA stimulation in spleen were determined by flowcytometry.4.Investigate which immunosuppressive cells mediate CD8~+T cell suppression after GSK126 treatment.4.1 Percentage of Tregs in tumor were determined by flowcytometry.4.2 Percentage of TAMs in tumor were determined by flowcytometry.4.3 Percentage of DCs in tumor were determined by flowcytometry.4.4 Percentage of MDSCs in tumor were determined by flowcytometry.4.5 Percentage of G-MDSCs and M-MDSCs in tumor were determined by flowcytometry.4.6 Tumoral MDSCs were isolated from Control or GSK126 treated tumor-bearing mice,and a CD8~+T cell proliferation inhibition assay was performed.4.7 Percentage of ROS~+cells in MDSCs in tumor were determined by flowcytometry.4.8 Tumoral MDSCs were isolated from Control or GSK126 treated tumor-bearing mice,followed by examination of the mRNA expression of Arg1 and iNOS.5.Investigate if GSK126-induced MDSCs were indeed functionally critical to mask the anti-tumor effect of this compound in immune competent hosts.5.1 LLC cells(1?10~6)were implanted subcutaneously in female C57BL/6 mice.GSK126 was delivered five times per week and the Gr-1 antibody delivered twice per week until the end of the experiment.Tumor growth was measured using calipers every 2 days.5.2 Gr-1 antibody removal MDSC efficiency were detected in blood by flowcytometry.5.3 Percentage of tumor-infiltrating MDSCs were determined by flowcytometry.5.4 Percentage of tumor-infiltrating CD4~+T cells and CD8~+T cells were determined by flowcytometry.5.5 Percentage of IFN-γ~+cells in CD8~+T cells in tumor were determined by flowcytometry.5.6 Tunel assays were performed for detecting apoptotic tumor cells.6.Investigate whether combining gemcitabine or 5Fu with GSK126 could improve the efficacy of GSK126 in immune competent hosts.6.1 LLC cells(1?10~6)were implanted subcutaneously in female C57BL/6 mice.GSK126 was delivered five times per week and gemcitabine/5Fu was administered once per week until the end of the experiment.Tumor growth was measured using calipers every 2days.6.2 Percentage of tumor-infiltrating MDSCs were determined by flowcytometry.6.3 Percentage of tumor-infiltrating CD4~+T cells and CD8~+T cells were determined by flowcytometry.7.Investigated how GSK126 promotes the formation of MDSCs.7.1 BrdU incorporation was performed for examination of the proliferation of MDSCs.7.2 Percentage of MDSCs in bone marrow,blood,and spleen were determined by flowcytometry.7.3 qPCR and immunoblot were used for detecting EZH2 expression in HPCs,MDSCs,and macrophages.7.4 Immunoblot analysis of H3K27me3 expression in hematopoietic progenitor cells(HPCs)after GSK126 treatment.7.5 HPC-MDSC differentiative capacity was performed by flowcytometry after GSK126treatment.Results:1.Inhibition of EZH2 activity with GSK126 has no effect on tumor growth in immune competent mice1.1 GSK126 could effectively inhibit tumor growth in immune deficient mice in LLC model.1.2 GSK126 failed to inhibit tumor growth in immune competent mice in LLC model.1.3 GSK126 could effectively inhibit tumor growth in immune deficient mice in MC38model.1.4 GSK126 failed to inhibit tumor growth in immune competent mice in LLC model.2.GSK126 attenuates the anti-tumor immune response2.1 The percentages of tumor infiltrating CD4~+and CD8~+T cells were decreased with GSK126 treatment.2.2 Percentage of Ki67~+cells in CD8~+T cells in tumor were significantly decreased after GSK126 treatment.2.3 Percentage of IFN-γ~+cells in CD8~+T cells in tumor were significantly decreased after GSK126 treatment.2.4 There was no significant differences of Gzms-B~+cells in CD8~+T cells in tumor after GSK126 treatment.2.5 percentages of CD4~+and CD8~+T cells in blood were decreased with GSK126treatment.2.6 Percentage of IFN-γ~+cells in CD8~+T cells in blood were significantly decreased after GSK126 treatment.2.7 The percentages of CD4~+and CD8~+T cells in spleen were decreased with GSK126treatment.2.8 Percentage of IFN-γ~+cells in CD8~+T cells in spleen were significantly decreased after GSK126 treatment.3.GSK126 suppresses CD8~+T cell activity by reprograming the TME3.1 Percentage of CD45.1~+cells in CD8~+T cells were significantly decreased after GSK126 treatment.3.2 Percentage of IFN-γ~+cells in CD45.1~+CD8~+T cells(naive CD8~+T cells)were significantly decreased after GSK126 treatment.3.3 Percentage of IFN-γ~+cells in CD45.1~+CD8~+T cells(activated CD8~+T cells)were significantly decreased after GSK126 treatment.4.GSK126 results in the expansion of the MDSC population in the TME4.1 Treg cells did not appear to be affected by GSK126.4.2 TAMs did not appear to be affected by GSK126.4.3 DCs did not appear to be affected by GSK126.4.4 GSK126 led to an increase in the percentages of MDSCs in the tumor tissue.4.5 Both G-MDSC and M-MDSC sub-types were increased upon GSK126 treatment.4.6 The immunosuppressive function of MDSCs from the GSK126-treated tumor tissues appeared to be normal,because T cell proliferation was effectively and comparably inhibited by MDSCs that were isolated from GSK126-treated and untreated tumor-bearing mice.4.7 The expression of signature MDSC effector molecules including ROS,Arg1,and iNOS was similar between MDSCs isolated from GSK126-treated and untreated mice.5.MDSC depletion unmasks the anti-tumor effect of GSK126 in immune competent hosts.5.1 Tumor volume and size significantly decreased after MDSC depleted.5.2 MDSC were depleted efficiently in blood after injecting anti-Gr-1 neutralizing antibodies.5.3 Percentage of tumor infiltrating MDSCs were significantly decreased after injecting anti-Gr-1 neutralizing antibodies.5.4 Percentage of tumor infiltrating CD4~+and CD8~+T cells were significantly increased after injecting anti-Gr-1 neutralizing antibodies.5.5 Percentage of tumor infiltrating IFN-γ~+cells in CD8~+T cells were significantly increased after injecting anti-Gr-1 neutralizing antibodies.5.6 Combined treatment with GSK126 and Gr-1 neutralizing antibodies increased tumor cell apoptosis.6.Clinically-used chemotherapy drugs rescue the anti-tumor efficacy of GSK126 in immune competent hosts6.1 Gemcitabine and 5Fu greatly enhanced the efficacy of GSK126 mediated LLC tumor control.6.2 Percentage of tumor infiltrating MDSCs were significantly decreased in combined therapy group.6.3 Percentage of tumor infiltrating CD4~+and CD8~+T cells were significantly decreased in combined therapy group.7.GSK126 promotes HPC differentiation into MDSCs7.1 GSK126 unlikely directly promotes the proliferation of MDSCs as assessed by BrdU incorporation.7.2 GSK126 unlikely promotes the expression of MDSC-related chemotactic factors.7.3 Percentage of MDSCs were significantly increased in bone marrow,blood,and spleen after GSK126 treatment.7.4 The mRNA and protein expression of EZH2 were high in HPCs,the precursor of MDSCs,but was barely detectable in the committed MDSCs and macrophage.7.5 The H3k27me3 expression was decreased in HPCs after GSK126 treatment.7.6 GSK126 promotes HPC differentiation into MDSCs.Conclusions:GSK126-mediated EZH2 suppression promotes HPC differentiation to MDSCs,resulting in increased MDSCs in the tumor microenvironment,which suppress anti-tumor immunity.Importantly,MDSC depletion with an antibody or gemcitabine/5Fu can enhance the anti-tumor efficacy of GSK126.Mechanistically,our results suggest that the unintended effect of GSK126 in promoting MDSC generation,which masks its anti-tumor effect,can be suppressed by MDSC depletion;this could be exploited clinically to unleash the anti-tumor effects GSK126.