Targeting EZH2 enhances antigen presentation, antitumor immunity and circumvents anti-PD-1 resistance in head and neck cancers.

Purpose: Anti–programmed death-1 (PD-1) receptor–based therapeutics improve survival in patients with recurrent head and neck squamous cell carcinoma (HNSCC), but many do not benefit due to a low response rate. Herein, we identified EZH2 as a therapeutic target that enhanced tumor cell antigen presentation and subsequently sensitized resistant tumors to anti–PD-1 therapy. Experimental Design: EZH2 regulation of antigen presentation was defined using EZH2 inhibitors (GSK126 and EPZ6438) in human and mouse HNSCC cell lines. Mechanistic dissection of EZH2 in regulation of antigen presentation was investigated using flow cytometry, qRT-PCR, ELISA, and chromatin-immunoprecipitation assays. EZH2-deficient cell lines were generated using CRISPR-CAS9. GSK126 and anti–PD‐1–blocking antibody were used in testing combinatorial therapy in vivo. Results: EZH2 expression was negatively correlated with antigen-processing machinery pathway components in HNSCC data sets in The Cancer Genome Atlas. EZH2 inhibition resulted in significant upregulation of MHC class I expression in human and mouse human papillomavirus–negative HNSCC lines in vitro and in mouse models in vivo. Enhanced antigen presentation on the tumor cells by EZH2 inhibitors or CRISPR–mediated EZH2 deficiency increased antigen-specific CD8+ T-cell proliferation, IFNγ production, and tumor cell cytotoxicity. Mechanistically, EZH2 inhibition reduced the histone H3K27me3 modification on the β-2-microglobulin promoter. Finally, in an anti–PD-1–resistant model of HNSCC, tumor growth was suppressed with combination therapy. Conclusions: Our results demonstrated that targeting EZH2 enhanced antigen presentation and was able to circumvent anti–PD-1 resistance. Thus, combining EZH2 targeting with anti–PD-1 may increase therapeutic susceptibility in HNSCC.


Introduction
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide (1). Patients with advanced human papillomavirus (HPV)-negative HNSCC have poor outcomes. Programmed cell death protein-1 (PD-1) checkpoint blockade with nivolumab or pembrolizumab has shown promising clinical outcomes in patients with poor prognosis recurrent/metastatic HNSCC (2)(3)(4)(5)(6). However, the majority do not benefit with responses limited to 15% to 20% of patients. This resistance to immunotherapy is unexpected given the high mutational burden of cisplatin-resistant tumors and the presence of immune infiltrates that together should predict better response rates (2)(3)(4)(5)(6). These findings lead to the question: can combination therapies directed at nonoverlapping pathways improve outcomes of anti-PD-1 therapy in HNSCC?
Clinical responses to mAbs blocking PD-1/PD-L1 signaling are based on the reversal of the dysfunctional exhausted state of tumor-infiltrating T cells. Mechanistically, IFNg production by tumorinfiltrating T cells induces PD-L1 expression, among other downstream biologic changes, in the tumor microenvironment (7,8). Deficiency of genes in the IFNg signaling pathway or HLA class I have been shown to impair the efficacy of immunotherapy (9)(10)(11). Chan and colleagues reported that specific patient HLA class I genotypes are associated with response to immune checkpoint blockade (12). In HNSCC, dysfunctional HLA class I antigen processing and presentation has been identified as a key factor contributing to tumor progression and immunotherapy resistance (13)(14)(15). Deficiency in the expression of HLA class I components, including B2M and specific HLA alleles, and antigen-processing machinery have been correlated with poor prognosis of HNSCC (16,17). Together, these observations suggest that approaches which enhance target tumor cell antigen presentation may improve the efficacy of anti-PD-1 therapy in HNSCC.
Enhancer of zeste homolog 2 (EZH2) is the methyltransferase subunit of the polycomb repressive complex 2 (PRC2) that catalyzes histone H3 methylation on lysine 27 (H3K27). This H3K27me3 histone modification can suppress chromatin accessibility and silence downstream gene expression (18,19). The role of EZH2 in different components of antitumor immunity has been investigated by several groups. Zou and colleagues reported the contribution of EZH2 in regulating T-cell recruiting, Th1 chemokines, CXCL9 and CXCL10 in ovarian cancer cells. Consequently, combination therapy with EZH2 inhibition and adoptive T-cell transfer resulted in better tumor growth suppression than either approach alone in ovarian tumor-bearing mice (20,21). In regulatory T cells (Tregs), disruption of EZH2 can enhance antitumor immunity by diminishing the suppressive activity of Tregs and enhancing T-cell infiltration in the tumor (22,23). Combining EZH2 inhibition with anti-CTLA4 resulted in a synergistic impact in mouse bladder carcinoma and melanoma models (23).
Sommer and colleagues reported the role of EZH2 in regulating a broad range of pathways impacting immunogenicity in melanoma (24). In addition, EZH2 is also involved in natural killer (NK) cell-mediated tumor eradication in hepatocellular carcinoma by silencing the expression of NK group 2D ligands (25). Very recently, diffuse large B-cell lymphoma showed a strong correlation between EZH2 mutation enrichment and MHC class I and class II expression deficiency. EZH2 inhibition relieved suppression of NLRC5 and CIITA expression leading to increased expression but only in an EZH2mutated background (26). However, in HNSCC, the specific role of EZH2 in modulating MHC class I antigen presentation and T-cellmediated antitumor immunity has not been investigated.
In this study, we hypothesized that inhibiting EZH2 function may improve the outcome of anti-PD-1 therapy by enhancing antigen presentation in HPV-negative HNSCC. Using both pharmacologic inhibition and CRISPR-Cas9-mediated genome editing, we investigated the role of EZH2 in human HNSCC HLA class I expression and also its role in antigen presentation and T-cell-mediated killing in preclinical models. Extending these data to a novel anti-PD-1 therapyresistant preclinical model, we found that combinatorial therapy of GSK126, an EZH2 inhibitor, and anti-PD-1 showed significant tumor growth suppression compared with single-agent therapy. Together, these data identify EZH2 as a potential therapeutic target in promoting antigen presentation and antitumor immunity in HNSCC.
OT-1 T-cell isolation, expansion, and coculture killing assay CD8 þ T cells were isolated from OT-1 mouse splenocytes using CD8a þ T-cell Isolation Kit (130-104-075; Miltenyi Biotec). Isolated T cells were cultured in RPMI1640, supplemented with 10% heatinactivated FBS, 20 mmol/L HEPES (15630080; Gibco), 1 mmol/L sodium pyruvate (11360070; Gibco), 0.05 mmol/L 2-mercaptoethanol, 2 mmol/L L-glutamine (G7513; Sigma), and 100 U/mL penicillinstreptomycin. For activation, Dynabeads Mouse T-Activator CD3/ CD28 (11456D; Thermo Fisher Scientific) were added to isolated T cells at a bead-to-cell ratio of 1:2. Recombinant mouse IL2 (402-ML-020/CF; R&D Systems) was added to a final concentration of 20 ng/mL on day 1 after isolation. Medium supplemented with 20 ng/mL IL2 was changed every 2 days. T cells were split every 2 days to keep an approximate cell density of 10 6 cells/mL. Activated OT-1 T cells were used for coculture killing assay on day 6 to 8 after isolation. For coculture killing assays, mouse IFNg (20 ng/mL) prestimulated tumor cells were pulsed with 0.02 nmol/L of SIINFEKL peptide for 2 hours at 37 C. Activated OT-1 cells were counted and plated with washed tumor cells at indicated E:T ratios. After 24 hours of coculture, T cells were gently washed off using PBS. Surviving cells were harvested and stained with mouse CD45.2 antibodies to gate out residual T cells. Surviving tumor cells were counted using a MACSQuant Analyzer 10 (Miltenyi).

Immunoblotting
For the assessment of protein expression levels, RIPA buffer supplemented with protease inhibitor cocktail (11836153001; Sigma Aldrich) was used to homogenize cells. Fifty micrograms of total

Translational Relevance
Anti-PD-1 checkpoint inhibitor-based immunotherapies have shown clinical benefit in patients with recurrent/metastatic head and neck squamous cell carcinoma (HNSCC). However, new therapeutic strategies are needed to improve the low response rate. Herein, we identified EZH2 as a druggable target to enhance antigen presentation in human papillomavirus-negative HNSCC. Targeting EZH2 sensitized tumor cells to T-cell-mediated killing, increased antigen-specific CD8 þ T-cell proliferation, and IFNg production in vitro. Mechanistic studies revealed that EZH2 modulated the histone H3K27me3 on the B2M promoter region. In an anti-PD-1-resistant model of HNSCC, combination EZH2 inhibition and anti-PD-1 suppressed tumor progression. Our results highlight a combinatorial therapeutic strategy of EZH2 inhibition and anti-PD-1 to enhance immunotherapeutic approaches in patients with HNSCC.

Cell viability assay
Human or mouse cells (1,500/well) were plated in 96-well plates (3903; Corning) 1 day before starting drug treatment. Cells were treated with increasing concentrations of GSK126, EPZ6438, or vehicle (DMSO) for 72 hours. Cell viability was analyzed using CellTiter Glo-Luminescent Cell Viability Assay (G7572; Promega). Luminescent signal was measured with an Infinite M200 Pro Multimode microplate reader (Tecan Life Science).

In vivo studies
Female C57BL/6 mice (6-week-old; Taconic) were housed in a pathogen-free animal facility and all experiments performed were approved by the Institutional Animal Care and Use Committee of Dana-Farber Cancer Institute. For tumor inoculation, MOC cells were harvested and washed extensively with cold endotoxin-free PBS. Cells (10 6 MOC1-esc1 and 10 5 MOC2) were injected subcutaneously into the right flank of each mouse in a volume of 150 mL. Anti-PD-1 therapy was performed by intraperitoneal injections of anti-PD-1 (250 mg/mouse) or isotype control (250 mg/mouse) on day 3, 6, and 9 postinoculation. GSK126 was dissolved in 20% Captisol (H107; Sigma Aldrich) and administered by intraperitoneal injections 3 times a week from day 6 post-inoculation. Tumor growth was monitored using a digital caliper twice weekly. Tumor volume was calculated using the formula V ¼ (W 2 Â L)/2.

CRISPR/CAS9-mediated knockout of EZH2
LentiCas9-Blast (Addgene, #52962) was cotransfected with pCMV-VSV-G and psPAX2 into HEK293T cells for lentivirus production using TransIT-293 (MIR2700, Mirus Bio). Virus in the media was collected 48 hours after transfection and MOC1-esc1 cells were transduced for 48 hours and then subjected to Blasticidin (4 mg/mL) selection for 4 days. Pooled MOC1-esc1-cas9 cells were single-cell sorted in 96-well cell culture plate to generate single clones. Cas9 editing efficiency of each clone was tested by assessing the knockout efficiency of B2M single-guide RNA (sgRNA)-expressing lentivirustransduced cells. H2-K b class I cell surface expression levels were determined by flow cytometry as a readout of Cas9 efficiency (not shown). Clones #2 and #3 with high editing efficiency were transduced with lentivirus-expressing specific gRNAs targeting EZH2 or ROSA26, respectively. The sequences of specific sgRNAs are listed below.

T-cell proliferation assay
Na€ ve CD8 þ T cells isolated from OT-1 mouse splenocytes were stained with CFSE Cell Division Tracker Kit (423801; BioLegend) at a final concentration of 5 mmol/L in PBS supplemented with 5% heatinactivated FBS for 3 minutes in the dark at room temperature. After staining, cells were washed 3 times in PBS with 5% FBS. IFNg prestimulated tumor cells were plated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled T cells at an E:T ratio of 10:1. After 72 hours coculture, media were harvested for IFNg ELISA. CD8 þ T cells proliferation measured by CFSE-dilution was determined using flow cytometry.

TCGA dataset analysis
Data for HNSCC and lung SCC patients from The Cancer Genome Analysis (TCGA) were obtained from cBioPortal. HPV status of patients was obtained from a PanCancer Atlas study (28). Gene expression correlations between EZH2 and molecules involved in antigen presentation machinery were assessed by Spearman coefficient.

Mouse IFNg ELISA
IFNg levels in the media from T-cell proliferation coculture experiments were measured with the Mouse IFNg DuoSet ELISA (DY485; R&D Systems) according to the manufacturer's instructions.

Statistical analysis
Data are plotted as the mean AE SD or mean AE SEM as indicated in specific experiments. The statistical significance was determined by Student t test, one-way ANOVA, and two-way ANOVA using Graph-Pad Prism. Significance differences P < 0.05, 0.01, and 0.001 are symbolized as Ã , ÃÃ , and ÃÃÃ , respectively.

EZH2 expression is negatively correlated with major MHC class I antigen presentation molecules
As antigen presentation defects have been identified as a common feature in HNSCCs, we sought to identify the relationship between EZH2 expression and antigen presentation pathway components.
Analysis of 522 HNSCC HPV-negative tumors from TCGA (28) showed a significant inverse correlation between the EZH2 expression levels and major MHC class I antigen presentation molecules, including b2M, HLA-A, HLA-B, HLA-C, and HLA-E (Fig. 1), highlighting a potential regulatory function of EZH2 on antigen presentation in HNSCC. Interestingly, this negative correlation was also seen in 483 HPV-negative lung squamous cell carcinomas ( Supplementary  Fig. S1).

EZH2 inhibition promotes antigen presentation and Th1-type chemokine expression in cell line models of human HNSCC
To test the hypothesis that targeting EZH2 could promote antigen presentation in HNSCC, we used two highly selective EZH2 inhibitors, GSK126 and EPZ6438 (22,29,30). Human HNSCC cell lines (CAL27, CAL33, SCC25, and SCC9) were treated with increasing concentrations of GSK126 or EPZ6438 to test the impact of EZH2 inhibition on cell viability. On the basis of the various sensitivities of these cell lines to EZH2 inhibitors, we selected the 10 mmol/L concentration for both GSK126 and EPZ6438, at which neither inhibitor showed significant cell growth inhibition ( Fig. 2A). Although GSK126 increased IFNg-induced HLA expression in three of four lines tested, EPZ6438 increased both basal and IFNg-induced HLA cell surface expression levels compared with DMSO control in all cell lines ( Fig. 2B). Given the known function of EZH2 as a methyltransferase that silences target genes, we quantified the mRNA expression changes of MHC class I antigen presentation genes, including B2M, HLA-A, HLA-B, and HLA-C after treatment with EZH2 inhibitors. Despite different sensitivities to EZH2 inhibition, major class I antigen presentation genes were upregulated by at least one EZH2 inhibitor under either basal or exogenous IFNg-stimulated conditions or both in all tested cell lines ( Fig. 2C; Supplementary Fig. S2). Therefore, EZH2 regulated mRNA levels of antigen presentation genes. Consistent with findings in ovarian cancer (20), we also observed a significant induction of CXCL10 expression in EZH2 inhibitor-treated cells compared with DMSO control that was dramatically enhanced in combination with IFNg (Fig. 2C). Therefore, EZH2 inhibition influenced MHC class I expression and CXCL10 expression in human HNSCC lines.

EZH2 inhibition upregulated MHC class I antigen presentation in anti-PD-1-resistant mouse HNSCC cells
Aiming at reversing immunotherapy resistance in HNSCC, we tested the effect EZH2 inhibition in MOC1-esc1, a syngeneic anti-PD-1-resistant mouse model. When assayed for impact on cell viability assay, significant cytotoxicity in MOC1-esc1 was not seen with either EZH2 inhibitors at the 10 mmol/L dose (Fig. 3A). We then used GSK126 or EPZ6438 to determine the effect of EZH2 inhibition on antigen presentation in MOC1-esc1. Consistent with the data in human HNSCC, either treatment significantly upregulated MHC class I cell surface protein levels in combination with IFNg ( Fig. 3B and C).
To confirm that the role of EZH2 in antigen presentation modulation was not model specific, we performed similar experiments using MOC2, another immunotherapy-resistant mouse HNSCC model. Consistent with the results in MOC1-esc1, EZH2 inhibitor-treated MOC2 cells showed enhanced MHC class I expression (Fig. 3C). To assess whether this increased expression also resulted in increased antigen presentation, we assessed levels of MHC class I bound SIINFEKL. EZH2 inhibition also increased the presentation of SIIN-FEKL peptide by H2-K b on the MOC1-esc1 and MOC2 cell surface confirming the functional upregulation of antigen presentation (Fig. 3D). Together, these data showed that EZH2 inhibition increased antigen presentation in anti-PD-1-resistant HNSCC cells.
Pharmacologic inhibition and genetic ablation of EZH2 in tumor cells enhance T-cell-mediated killing Next, we asked whether the EZH2 inhibition based enhanced MHC class I antigen presentation sensitized tumor cells to T-cell-mediated killing. Note that, in addition to pharmacologic targeting and to further study the role of EZH2 in T-cell/tumor cell interactions, we generated CRISPR-CAS9 genetically deleted EZH2 MOC1-esc1. We used two independent clones of CAS9-expressing MOC1-esc1 to knockout EZH2 with two different sgRNA to rule out clone-specific or offtarget effects. Immunoblotting for EZH2 was performed and showed attenuated EZH2 expression relative to ROSA26 control targeted lines (Fig. 4A). We performed a 2D coculture assay using OT-1 CD8 þ T cells that were in vitro activated and expanded with SIINFEKL peptide antigen pulsed tumor cells as targets. GSK126 or EPZ6438 treatment sensitized MOC1-esc1 cells to T-cell-mediated killing (Fig. 4B). Genetic ablation of EZH2 dramatically sensitized tumor cells to T-cell-mediated killing in both clones in comparison with their parental lines and ROSA26 targeting controls ( Fig. 4B; Supplementary  Fig. S6). Consistent with the inhibitor treatment experiment results, loss of EZH2 significantly increased MHC class I cell surface expression levels, which again were enhanced in combination with IFNg without impacting PD-L1 ( Fig. 4C and D), indicating the specificity of this regulation on antigen presentation. Therefore, targeting of EZH2 sensitized tumor cells to T-cell-mediated killing. EZH2 inhibition enhances antigen presentation and Th1-type chemokine expression levels in human HNSCC lines. A, SCC9, SCC25, CAL27, and CAL33 were treated with increasing concentrations of GSK126 or EPZ6348 (1 nmol/L-100 mmol/L) for 72 hours to assess the impact on cell viability. B, Human HNSCC cells were treated with GSK126 (10 mmol/L), EPZ6438 (10 mmol/L), or DMSO as control for 72 hours. IFNg was added in the last 24 hours of drug incubation for all assays shown. HLA cell surface protein levels were measured by flow cytometry. The data are representative of two independent experiments. C, B2M and CXCL10 mRNA expression levels were quantified by qRT-PCR. Relative mRNA levels were normalized to GAPDH. Ã , P < 0.05; ÃÃ , P < 0.01; ÃÃÃ , P < 0.001. Significance was calculated by one-way ANOVA. Data are shown as mean AE SD.

EZH2 represses antigen presentation by regulating the enrichment of H3K27me3 on the B2M promoter
To start to define the mechanism of EZH2 regulation of antigen presentation, we tested H3K27me3 levels in GSK126-or EPZ6438treated cells. As expected, inhibition of EZH2 resulted in dramatic decrease of global H3K27me3 levels, without affecting the protein expression levels of EZH2 (Fig. 5A). In addition, the mRNA levels of both B2M and H2-K1 were significantly upregulated by EZH2 inhi-bition (Fig. 5B), suggesting that the regulation of EZH2 on antigen presentation is conserved between human and mouse ( Fig. 2B and C;  Supplementary Fig. S2). Interestingly, CXCL10 expression was not induced by EZH2 inhibition in this mouse model (Fig. 5B).
To test the hypothesis that EZH2 regulates H3K27me3 occupancy on the promoter region of B2M, MOC1-esc1 cells were treated with EPZ6438 or DMSO and subjected to ChIP with antibodies against EZH2, H3K27me3, or IgG control. To test the occupancy of EZH2 and H3K27me3 modification on B2M promoter, DNA samples from ChIP were quantified using two independent primers specific for B2M promoter region. The results of ChIP assay followed by qPCR showed that EPZ6438 treatment did not affect the binding of EZH2 on B2M promoter region (Fig. 5C), which is in agreement with the mode of action of EPZ6438 as an S-adenosyl-I-methionine (SAM)competitive inhibitor. Importantly, EPZ6438 treatment significantly reduced H3K27me3 enrichment on the B2M promoter region (Fig. 5D).
Combinatorial therapy of GSK126 and anti-PD-1 suppresses MOC1-esc1 tumor progression in vivo Next, we asked whether the enhanced antigen presentation in EZH2-deficient tumor cells can promote the function of antigenspecific T cells. To answer this question, we analyzed CD8 þ T-cell proliferation and IFNg production after coculture of SIINFEKL peptide pulsed tumor cells with na€ ve OT-1 T cells. These data show that both T-cell proliferation and IFNg production were significantly higher in EZH2-deficient tumor cells compared with ROSA26 control (Fig. 6A-C). To investigate whether EZH2 inhibitor can improve the outcome of anti-PD-1 therapy, we used resistant MOC1-esc1 to assess the effect of combination therapy. MOC1-esc1 tumor growth in immunocompetent wild-type mice was significantly attenuated by the combination of GSK126 (50 mg/-kg) and anti-PD-1 but not by either agent alone (Fig. 6E). To test the role of EZH2 inhibition in modulating antigen presentation of tumor cells in vivo, we analyzed the tumor samples with these treatment conditions ( Fig. 6F and G). Tumor cell MHC class I cell surface expression levels were increased by GSK126 monotherapy compared with the control group (Fig. 6F). Similarly, anti-PD-1 monotherapy also increased the MHC class I level compared with control (Fig. 6F). Interestingly, the combination of GSK126 and anti-PD-1 resulted in an additive increase of MHC class I (Fig. 6F). Total CD45 þ immune infiltration in the tumors was not affected by either monotherapy or combinatorial therapy ( Fig. 6G; Supplementary Fig. S8). In summary, enhanced antigen presentation induced by targeting of EZH2 promoted proliferation and IFNg production of antigen-specific T cells and combination therapy of GSK126 and anti-PD-1 resulted in attenuation of tumor progression in an anti-PD-1-resistant mouse model of HNSCC.
In addition, as significant numbers of patients are resistant to immunotherapy due to poor immune infiltration, we asked whether the efficacy of GSK126 and anti-PD-1 combinatorial therapy requires preexisting immune infiltration in tumors. We next assessed for combination therapy impact in the MOC2 model that displays an immune "desert" phenotype with negligible T-cell infiltration and aggressive in vivo growth (31). Consistent with previous data (31), MOC2 tumors are resistant to anti-PD-1 treatment (Fig. 6H). Neither GSK126 monotherapy nor the combination of GSK126 and anti-PD-1 suppressed tumor growth (Fig. 6H). Although the growth phenotype was not altered, we assayed for in vivo MOC2 tumor cell class I expression with different treatments. Neither the control nor the monotherapy groups displayed alteration in surface class I expression. However, consistent with our observation in MOC1esc1 tumors, the combination group induced a significant higher MHC class I on tumor cells in vivo. As for MOC1-esc1, there was minimal impact on CD45 þ T-cell infiltration. Therefore, EZH2 inhibition and anti-PD-1 combinatorial therapy upregulates tumor cell MHC Class I expression in vivo, whereas the therapeutic efficacy may require preexisting immune infiltration.

Discussion
To define approaches to improve outcomes of anti-PD-1 therapy in HNSCCs, we identified that targeting the histone methyltransferase EZH2 enhanced antigen presentation in both human and mouse HNSCC lines. Mechanistic studies revealed that EZH2 inhibition decreased the enrichment of H3K27me3 on the promoter region Relative mRNA levels were normalized to GAPDH. C and D, ChIP for EZH2, H3K27me3, and IgG, and subsequent qPCR in B2M promoter using two independent primer sets. Ã , P < 0.05; ÃÃ , P < 0.01; ÃÃÃ , P < 0.001. Significance was calculated by one-way ANOVA and Student t test. Data are shown as mean AE SD.

EZH2 Is a Negative Regulator of HNSCC Antigen Presentation
AACRJournals.org Clin Cancer Res; 26(1) January 1, 2020 of B2M. The enhanced antigen presentation induced by targeting EZH2-sensitized tumor cells to T-cell-mediated killing, resulted in higher T-cell proliferation and IFNg production in vitro. These findings translated to a synergistic impact of combination GSK126 and anti-PD-1 in attenuating the tumor progression of an anti-PD-1resistant model of HNSCC and showed that EZH2 inhibition can enhance tumor cell Class I expression in vivo including in highly resistant models. Our data define EZH2 as a therapeutic target to improve the outcome of anti-PD-1 therapy in HNSCC. EZH2 inhibition has been proposed as a therapeutic strategy in cancers with frequent gain-of-function mutation or over expression of EZH2, including melanoma, ovarian cancer, and lymphoma to induce apoptotic cell death (32)(33)(34). In HNSCC, overexpression of EZH2 has been correlated with poor prognosis, recurrence, and lymph node metastasis in HNSCC (35)(36)(37). Saunders and colleagues reported that targeting EZH2 in HNSCC attenuated tumor growth by inducing differentiation gene expression via modulating H3K27me3 occupancy on the promoter regions of genes involved in squamous differentiation (38). To test the effect of EZH2 inhibition on human HNSCC tumor progression, Cal27 was implanted in immunocompromised NOD/SCID mice, in which setting the interaction between tumor cells and host immunity was eliminated to support the human tumor growth in mice (38). These previous studies highlighted EZH2 inhibition as a potential therapeutic target in HNSCC by modulating epigenetic silencing of genes involved in tumor progression. In our study, we first demonstrated the effect of EZH2 inhibition on increasing antigen presentation is conserved in human and mouse HNSCC lines. Subsequently, a syngeneic mouse model was used to investigate the in vivo therapeutic potential of EZH2 inhibition independent from direct cytotoxicity of EZH2 on tumor cells. Thus, this study provides a new line of evidence in support of EZH2 inhibition as a new combination immunotherapy target in promoting HNSCC antitumor immunity to overcome checkpoint blockade resistance. Consistent with other studies, inhibition of EZH2 significantly induced the expression of CXCL10 in human HNSCC lines ( Fig. 2C; ref. 20). CXCL10 is a Th1-chemokine CXCL10 that is a key ligand regulating T-cell trafficking into the tumor microenvironment through CXCR3 (39,40). However, CXCL10 expression was not increased by EZH2 inhibition in the HNSCC mouse model (Fig. 5B), suggesting the regulation of CXCL10 by EZH2 is not conserved in human and mouse models used in this study. Although speculative, this suggests that in human HNSCC, the effect of EZH2 inhibition on antitumor immunity might include enhanced antigen presentation and additionally, higher immune infiltration in tumors.
One limitation of this study is the lack of well-defined endogenous antigen and antigen-specific T cells in the MOC1-esc1 HNSCC model. Therefore, we employed a commonly used model antigen SIINFEKL peptide from ovalbumin and OT-1 mouse-derived antigen-specific T cells for in vitro studies (41). Supporting the use of this approach, CRISPR-CAS9 knockout of B2M or PD-L1 in MOC1-esc1 resulted in resistance or sensitivity to T-cell-mediated killing, respectively (data not shown). Therefore, this 2D coculture system retained the relevant function of EZH2 in T-cell recognition to tumor cells. In addition, as EZH2 impacts a broad range of pathways in vivo, dissecting the specific immune contribution will require further studies. Our work supports that EZH2 has the capacity to directly regulate tumor cell Class I in vitro and that inhibition in vivo results in a similar end result of enhanced Class I expression. Further mechanistic dissection of in vivo antigen presentation is required.
Others have studied the contribution of EZH2 in augmenting tumor immunogenicity including antigen presentation regulation in melanoma and lymphoma, respectively (24,26). Our study focused on HNSCCs and defined further details of antigen presentation regulation. Differences in HNSCC include that the frequency of EZH2 overexpression in HNSCC is relatively low compared with that in cutaneous melanoma, lymphoma, and ovarian cancers. In addition, there are no EZH2 gain-of-function mutations identified in HNSCC (19). Therefore, the sensitivity of HNSCC to EZH2 inhibition induced apoptosis may be relatively low. The function of EZH2 inhibition in promoting antigen presentation thus identifies a disease-specific contextual opportunity in which to consider EZH2 inhibitor as an immunotherapy agent in HNSCC.
Interestingly, the inverse correlation of EZH2 and MHC class I antigen presentation molecule expression levels in HNSCC TGCA dataset is also found in lung squamous cell carcinoma, suggesting the function of EZH2 in regulating antigen presentation may be conserved in squamous cell carcinomas from other tissues. On the basis of our results, we propose that combination EZH2 inhibition with anti-PD-1 therapy may be beneficial for patients with HNSCC. Studies using different EZH2 inhibitors and additional HNSCC preclinical models are needed to further confirm our findings. In addition, the effect of EZH2 in regulating antigen presentation in professional antigenpresenting cells, such as macrophages and dendritic cells, also requires further investigation.