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Enhancer of Zeste Homologue 2 in Cancer Treatments

The oncogene EZH2 drives tumorigenesis and metastasis through multiple mechanisms

  1. Introduction

Chromatin regulators control gene expression by controlling the availability of DNA to the basal transcriptional machinery. One class of chromatin regulators, the polycomb group proteins, accomplish this by methylating  histone tails to promote transcriptional repression through chromatin condensation (Fan et al., 2011). Enhancer of Zeste Homologue 2 (EZH2), a histone methyltransferase which preferentially methylates histone 3 at lysine 27 (H3K27), is the main subunit of Polycomb Repressive Complex 2 (PRC2), which is essential for inactivation of the X-chromosome in females and epigenetic silencing of differentiation genes. In 2002, EZH2 was first associated with cancer when it was found to be over-expressed in metastatic prostate tumor samples (Varambally et al., 2002). In the following years, several mutations and copy number alterations affecting EZH2 would be described across a diverse range of cancers. The tumor promoting activities of  EZH2 have become the object of much scrutiny as its over-activity is correlated with cancer aggression and metastasis (Völkel et al., 2015). While originally thought to drive cancer by epigenetically silencing of tumor suppressor genes (TSGs) to generate de-differentiated cancer stem cells (CSCs), additional driver roles for the protein have been revealed. Surprisingly, EZH2 can methylate non-histone proteins involved in critical cell processes and even and drive tumorigenesis through other, non-enzymatic functions. This paper presents the ways EZH2 becomes activated in cancer, its numerous tumorigenic activities, and several current and possible future anti-EZH2 treatments.

  1. Cancer-associated EZH2 mutations and CNVs

Several cancer-associated alterations of EZH2 gene expression have been found across a broad spectrum of cancers which activate its oncogenic properties. The EZH2 locus is located at 7q36.1, spans approximately 77 kb, and consists of 20 exons. Notably, 7q36 hosts other oncogenes (e.g. BRAF) and is frequently amplified in solid tumors, ultimately leading to  increased EZH2 mRNA transcript and H3K27me3 levels (Tiffen et al., 2016). While EZH2 copy-number variations (CNVs) are common across a broad spectrum of cancers, they are all but absent in hematological tumors, in which several gain-of function mutations have been described. Recurrent nonsynonymous mutations resulting in single amino acid substitutions at Y641, A677, or A687 hotspots of the 746 residue EZH2 protein are most frequently observed in liquid tumors. These mutations are located in the conserved C-terminal SET domain responsible for EZH2 histone methyltransferase activity and alter the function of the enzyme by reducing its affinity for unmethylated or mono-methylated H3K27 while simultaneously increasing its affinity for di-methylated H3K27 (Yap et al., 2011). In vitro experiments revealed that EZH2Y641N gain-of-function mutants methylated H3K27me2 more effectively than wild-type EZH2 yet could not methylate unmethylated or monomethylated H3K27 (Yap et al., 2011).Thus, in hematological cancer cells, mutant EZH2 methylates the lysine residues already di-methylated by wild-type EZH2 present, shifting the methylation “steady state” of H3K27 to that of a repressive, tri-methylated state to epigenetically silence Polycomb target genes (Yap et al., 2011).

  1. EZH2 is transcriptionally overexpressed in cancer cells

Gene amplification events and gain-of-function mutations are not the only events that drive EZH2 overexpression – aberrant activation of several signal transduction pathways could result in the transcriptional over-activation of EZH2. In 2013, Coe et al. investigated the factors behind the hyper-activation of EZH2 in squamous cell lung carcinoma (SCLC) which typically does not feature somatic copy number gain of EZH2. (Coe et al., 2013). The authors hypothesized the observed aberrant EZH2 activity in these cancers was due to dysregulation of the E2F family of transcription factors, since copy number loss of RB1 is a hallmark of SCLC (Coe et al., 2013). RT-PCR revealed that RB1 expression was much lower in SCLC tumor samples compared to NSCLC samples, in which EZH2 is not hyper-activated. RB1 binds to the activating region of E2Fs, repressing their ability to transcriptionally activate their downstream targets. To determine whether EZH2 is a downstream target of E2Fs, several E2F genes were either knocked down in SCLC cells or overexpressed in the HBEC cell line.  E2F knockdown decreased EZH2 protein levels in SCLC cells (and reduced their viability) while overexpression of E2Fs had the opposite effect in HBEC cells, showing that EZH2 lies downstream of E2F and is vital to cancer cell viability. RB1 is not the only cancer-associated regulator of E2F expression, as this family of transcription factors can be transcriptionally activated by the oncoprotein c-MYC (Koh et al., 2011). Moreover, supporting evidence from investigations of castrate resistant prostate cancer revealed that c-MYC-driven activation of E2F ultimately results in increased EZH2 levels (Leone et al., 1997). Together, these results demonstrate that transcriptional activation of E2F members via c-MYC activation or RB1 loss of function can result in transcriptional activation of EZH2 in tumors.

Downstream activation of E2F is not the only way by which c-MYC can activate EZH2. c-MYC can activate EZH2 by directly binding to its promoter region to activate its transcription. Chromatin immunoprecipitation assays revealed that c-MYC bound immediately upstream of the EZH2 transcriptional start site in prostate cancer cell lines while luciferase reporter assays confirmed that EZH2 promoter activity is decreased following c-MYC depletion (Koh et al., 2011). Interestingly, c-MYC can also inhibit expression of miR-26a/b, two microRNAs which specifically deplete EZH2. Inhibition of c-MYC in Burkitt Lymphoma cells increased the availability of these miRNA species and diminished EZH2 protein levels while transfection of the miRNAs into prostate cancer cell lines similarly reduced EZH2 expression (Sander et al., 2008) . C-MYC was also found to be enriched at the promoters of the genes in which miR-26a/b is embedded, where it presumably acts to shut down their expression (Sander et al., 2008). Thus, c-MYC is a notable factor driving EZH2 expression as it can directly activate its transcription and indirectly act to maintain its cellular levels.

In addition to c-MYC, other oncogenes can activate other intracellular signaling pathways ultimately resulting in aberrant EZH2 activity. Oncogenic KRAS mutants activate the MAPK/ERK and PI3K/AKT signaling pathways upstream of EZH2 as knockdown of KRAS mutants in lung adenocarcinoma cell lines reduced the signaling activity of these pathways as well as EZH2 protein levels (Riquelme et al., 2016). Additionally, AKT1 or MEK1 inhibitor treatment of KRASG12 -harboring colon and prostate cancer cell lines dramatically reduced the appropriate signaling pathway and EZH2 protein levels, definitively showing that EZH2 is a downstream target of these two signaling pathways and not a direct target of oncogenic KRAS itself (Riquelme et al., 2016).  Several studies into the downstream effector genes of the MAPK/ERK and PI3K/AKT pathways reveal that these two pathways activate E2F and ETS family transcription factors, which both transcriptionally activate EZH2 (Chaussepied and Ginsberg, 2005); (Kar and Gutierrez-Hartmann, 2017); (Kunderfranco et al., 2010).

Aside from aberrantly activated signaling axes, the tumor microenvironment itself can work to transcriptionally upregulate EZH2. Hypoxia-inducible factor-1α (HIF-1α) can bind to a consensus HIF-1α response element sequence (HRE) in the EZH2 promoter under hypoxic conditions, transcriptionally activating EZH2.  CHiP assays in primary human breast tumor cells confirmed that HIF-1α bound specifically to the HRE in the EZH2 promoter, and this binding corresponded to an increase in EZH2 mRNA transcript abundance (Chang et al., 2011). To conclude this section, dysregulation of intracellular signaling pathways can aberrantly activate regulators/factors upstream of EZH2 such as those presented above (as well as others not discussed here), which in turn lead to EZH2 hyper-activity in cancer cells.

  1. Mutations in critical antagonists/regulators functionally upregulate EZH2 “activity”

EZH2 does not have to be “overexpressed” or mutated for cancer cells to acquire increased EZH2 activity. Mutations in chromatin regulators that antagonize EZH2 enzymatic function, such as UTX and SWI/SNF have been found in several cancer types, including hematological cancers. Inactivating mutations of UTX, a histone demethylase with high specificity for H3K27me3, changes the protein near its JmjC domain which is necessary for its histone demethylase activity, thus inhibiting its enzymatic function (Jankowska et al., 2011). Several subunits of the SWI/SNF chromatin remodeling complex, which hydrolyzes ATP to promote the formation of euchromatin state, are also frequently mutated (e.g. ARID1A and SMARCA4). Loss of these EZH2 antagonists cause “unchecked” EZH2/PRC2 activity and are functionally analogous to EZH2 overexpression/gain-of-function.

Cells can also gain EZH2 function through its increased recruitment to target sites. Co-immunoprecipitation assays revealed that BRCA1 binds to EZH2 at the same region it binds the long non-coding RNA (lncRNA) “HOTAIR” (Wang et al., 2013). This lncRNA targets EZH2 to Polycomb target loci, a role usually performed by the PRC2 subunit SED12. Loss of BRCA1 in breast cancer cells allows EZH2 to bind HOTAIR, suggesting that in cancer types driven by BRCA1 loss of function, EZH2 can be recruited to target sites without associating with the rest of the PRC2 complex. As cancer types driven by BRCA1 loss of function are associated with increased EZH2 activity, this “sequestering” role of BRCA1 may be crucial in EZH2 regulation (Wang et al., 2013). Since chromatin remodelers normally lack innate target specificity and need to be recruited to genomic regions, HOTAIR may be a promising drug target to preventing EZH2 from acting on target genes even when it is aberrantly activated.

  1. Tumorigenic Mechanisms of EZH2

EZH2 provides cancer cells with a selective advantage by promoting their de-differentiation and generating a population of CSCs. EZH2 activity can promote an epithelial-to-mesenchymal transition (EMT) by both directly and indirectly repressing E-cadherin transcription. ChIP assays reveal that EZH2 is enriched at the promoter region of E-cadherin in many cancers and directs its silencing by catalyzing the deposition of H3K27me3 at the locus (Cao et al., 2008). Additionally, EZH2 was found to be enriched at the PTEN promoter where it transcriptionally silences this tumor suppressor gene in an identical manner. Silencing of PTEN activates AKT signaling and downregulates E-cadherin expression (Gan et al., 2018). Absence or downregulation of the E-cadherin epithelial adhesion protein results in loss of cell-cell adhesion and allows cancer cells to break free from the primary tumor to migrate to and invade distant tissues (Petrova et al., 2016).

EZH2 also promotes the formation CSCs by providing cancer cells an increased proliferative capacity and an escape from senescence and cell death. ChIP assays confirmed that EZH2 associates with the promoters of cell cycle regulator genes including GSK3.  Epigenetic silencing of GSK3 activates the Wnt/β-Catenin pathway by preventing the proteasomal degradation of β-catenin. Consequently, stabilized β-catenin can translocate into the nucleus to activate transcription of target genes such as c-MYC and Cyclin D1 which deregulate the cell cycle and increase cell proliferation (Krishnamurthy and Kurzrock, 2018). Stabilization of β-catenin also promotes cancer migration and invasion by transcriptionally upregulating expression of cell migration molecular markers such as the adhesion molecule ICAM-1 and the cytokines CXCR4 and CCL18 (Yang et al., 2017).  Inhibition of the Wnt/β-Catenin pathway with XAV939, which stimulates β-catenin degradation by stabilizing the Axin protein, significantly reduced the viability and proliferation of EZH2-overexpressing HEK293T cells (Chen et al., 2016), showing that deregulation of Wnt signaling to be crucial to the tumorigenic activity of EZH2.

EZH2 can also de-regulate the cell cycle by inhibiting p53CDKN1A, and CDKN2A TSGs. While EZH2 transcriptionally represses p53 and CDKN2A by directly methylating their promoter regions, inhibition of CDKN1A is achieved through a different mechanism. ChIP analysis revealed that after knockdown of EZH2, HDAC1 levels, but not H3K27me3 levels, were reduced at the CDKN1A promoter suggesting that EZH2 promotes the silencing of CDKN1A by recruiting and/or maintaining HDAC1 at the locus (Fan et al., 2011). HDAC1 in turn removes activating histone acetylation marks, promoting heterochromatin formation to deny the transcriptional machinery access to the promoter region. Inhibition of these cell cycle checkpoints is presumably essential to cancer cell viability, as EZH2 also epigenetically represses DNA damage repair genes such as RAD51, RUNX3, CDKN1C, and FOXC1. Without a compromised genome surveillance system, these cancer cells would have little chance to proliferate with their damaged genomes.

  1. Non-canonical tumorigenic activities of EZH2

One big surprise is that EZH2 has tumorigenic activities outside of its canonical enzymatic activity. AKT kinase can phosphorylate EZH2 at serine 21 (S21), causing the protein to localize to the cytoplasm (instead of the nucleus) where it methylates non-histone proteins such as STAT3, androgen receptor (AR), estrogen receptor (ER), GATA4, ROR-, and talin (Kim et al., 2013a). Methylation of of ROR- promotes its degradation via the ubiquitin-proteasome pathway, resulting in the de-repression of Wnt/β-catenin signaling (Lee et al., 2012). Meanwhile, methylation of the cell adhesion protein talin promotes its post-translational degradation to promote EMT (Gunawan et al., 2015), and methylation of STAT3 and AR transcription factors activates their trans-activator activities (Gan et al., 2018). Additionally, EZH2 can form complexes with AR, ER, or NFκB in its non-phosphorylated form, helping to activate their downstream target genes(Gan et al., 2018). Intriguingly, EZH2 can bind to the promoter of NOTCH1 where it acts as a transcriptional activator rather than a transcriptional repressor. Much is still unknown about this co-activator role of EZH2, however it is crucial as inhibiting the NOTCH pathway in EZH2-overexpressing triple-negative breast cancer (TNBC) cells reduced their proliferation (Gonzalez et al., 2014). Finally, the hypoxic tumor microenvironment can activate non-canonical EZH2 roles by inducing its release from PRC2 and promoting its binding to the transcription factor FOXM1 (Mahara et al., 2016). This transactivation complex binds promoters of matrix metalloprotease genes, transcriptionally activating them resulting in the disruption of the extracellular matrix which promotes an EMT and cancer metastasis.

  1. Future

The observation that EZH2 has oncogenic properties outside of its normal histone methyltransferase activity has inspired several pressing questions. Among the most salient involve the identity of the “molecular switches” that allow EZH2 to carry out its non-canonical tumorigenic activities outside of the context of PRC2. As described earlier, one of these switches is the AKT-dependent phosphorylation of EZH2 at serine 21, which confers upon the enzyme the ability to methylate non-histone proteins. However, this event alone cannot explain the ability of EZH2 to form complexes with other transcriptional activators such as STAT3 and AR. Since these activities are PRC2 independent, it may be that mutations in one or more of the PRC2 accessory proteins can inhibit the assembly of the protein complex, thereby promoting the independence of EZH2 and allowing its association with these transcription factors. In the case of androgen receptor, it may be that EZH2 only interacts with certain AR isoforms present in androgen-sensitive cancers (e.g. prostate cancer). One last way to explain the ability of EZH2 to form these trans-activating complexes is that its dissociation from EZH2 exposes crucial domains normally “hidden” away, thus allowing these interactions to occur.

Perhaps the most confusing non-canonical EZH2 function is its transcriptional activation of the NOTCH1 gene. As this phenomenon was discovered in TNBCs (which notably lack expression of ER, PR, and HER2), when EZH2 is overexpressed and independent of PRC2, it may adopt a novel trans-activator function when its molecular partners (PRC2, AR, ER) are unavailable (The Cancer Genome Atlas, 2012). Finally, it would be interesting to determine if lncRNAs aid in targeting of EZH2 to the NOTCH1 promoter or if previously undiscovered factors are responsible. The following pages will detail hypothetical experiments that could (at least partly) provide answers to these questions.

EZH2 interaction with AR

To determine the essential domains through which EZH2 can form a complex with AR, lentiviral constructs containing full length FLAG-tagged EZH2 and tagged EZH2 truncation mutants could be transfected into prostate cancer cell lines, such as LNCaP, which are typically androgen sensitive and contain active androgen receptor (Abeshouse et al., 2015). Whole cell and nuclear lysates could be prepared from transfected cells followed by the immunoprecipitation of full-length or truncated EZH2 mutants using an anti-FLAG antibody. Resulting immunoprecipitates could be probed via western blotting to determine the ability of the different forms of EZH2 to pull down AR. As an essential control, cell and nuclear lysates from each transfection condition could be analyzed by western blot using antibodies for AR to prove its presence in the cells. Additionally, immunoprecipitates could be analyzed by mass spectrometry to identify additional, novel EZH2 interacting proteins and identify possible adaptor proteins allowing for an indirect interaction between EZH2 and AR. If AR is able to be pulled down by full-length EZH2 but not certain truncation mutants, it suggests that these mutants lack the essential domain(s) for the interaction to take place. After pinpointing the crucial domain(s), structural analysis tools (such as Cn3D) could be used to visualize whether or not these EZH2 domains are embedded within the rest of the PRC2 complex. If this is the case, the PRC2 complex could physically shield these domains from the rest of the nucleoplasm, preventing EZH2 from associating with AR. This would also suggest that mutations in PRC2 accessory subunits that favor the dissociation of the complex could be present in cancer cells, de-regulating EZH2 activity and allowing its interaction with nuclear factors such as AR and NF-kB.

PRC2 as a molecular switch

To determine if cancer-associated mutations are present in PRC2 accessory subunits preventing the proper assembly and function of the complex, whole exome sequencing data (WES) from a broad spectrum of cancers present in The Cancer Genome Atlas (TCGA) could be analyzed to identify whether mutations in these accessory subunits are common across all or select cancers. Special consideration should be given to EED and SUZ12, as these subunits are required for the complete stability and proper targeting of the complex, respectively. If mutations in these proteins are found in cancer samples, several steps would need to be taken to show that they are specifically cancer-associated and affect the activity of the proteins. Mutations could be checked against one or more genome databases to ensure that they are not natural polymorphisms present in the population. Additionally, mutations could be analyzed using protein-prediction software such as PolyPhen, which work to assess whether a particular mutation is likely to have deleterious effects on proper protein function.

After this preliminary screening process, tagged EZH2, EED, and SUZ12 could be cloned into lentiviral vectors which could then be co-transfected into HEK293T cells (or a more appropriate cell line). Three experimental conditions could be used: co-transfection with (1) mutant SUZ12, WT EZH2 and WT EED constructs; (2) mutant EED, WT EZH2 and WT SUZ12 constructs; and mutant SUZ12, mutant EED, and WT EZH2 and EED constructs. To determine whether mutant SUZ12 or EED inhibited the assembly of PRC2, the ability of EZH2 to pull down PRC2 accessory subunits could be tested through co-immunoprecipitation assays in each of the three experimental conditions. Finally, to determine whether mutant SUZ12 or EED inhibited the histone methyltransferase activity of EZH2, the enrichment of H3K27me3 at Polycomb target genes could be assessed through chromatin immunoprecipitation (using antibodies specific for H3K27me3) in tandem with WES or qPCR. If the results of these experiments suggest that the candidate mutations prevent PRC2 assembly, this may be indicative of a role for these mutants as a molecular switch that promotes EZH2 PRC2-independent activity irrespective of their ability to change histone methylation patterns. However, if the results suggest these mutants alter genome-wide H3K27me3 methylation levels and not PRC2, that would suggest that these mutations impair the proper targeting of PRC2 rather than function as a molecular switch.

Does EZH2 preferentially interact with AR splice variants?

EZH2 is aberrantly activated in prostate cancers, which are typically androgen sensitive and express multiple AR splice variants, including the splice variant AR-V7. To determine if EZH2 could specifically interact with AR-V7 in vitro, co-immunoprecipitation assays could be performed using the VCaP cell line made to express a FLAG-tagged EZH2. The VCaP cell line is the only prostate cancer cell line that exclusively expresses the AR-V7 splice variant (Sprenger and Plymate, 2014). Cell and nuclear lysates could be immunoprecipitated using an anti-FLAG antibody and immunoprecipitates could be subsequently analyzed via western blot to determine if EZH2 pulled down AR-V7.

Additionally, ELISA assays could be performed to determine if EZH2 directly binds to AR-V7 and if it does so with a greater affinity than it does wild-type AR. For this assay, HEK293T cells could be made that separately express tagged versions of EZH2, AR, and AR-V7. The tagged nature of these proteins is to aid in their subsequent isolation and purification as the assay requires the use of purified proteins. Briefly, purified EZH2 could be immobilized in the wells of a 96 well plate, after which increasing concentrations of AR or AR-V7 are added to successive wells. After washing away inbound substrate (AR/AR-V7), the levels of substrate bound to EZH2 can be detected using primary antibodies to AR and an HRP-conjugated secondary antibody.  This ELISA technique is powerful because it can conclusively show if EZH2 can directly bind AR/AR-V7 (without cofactors) and provides a quantitative assessment of the binding affinity. If it is discovered that EZH2 can bind to AR-V7 with a higher affinity than it can bind AR, it would suggest that its ability to form a trans-activating complex with AR would rely on two molecular switches being flipped in tandem: (1) EZH2 dissociation from PRC2 and (2) the availability of high affinity AR splice variants.

What factors drive EZH2 trans-activation of NOTCH1?

One hypothesis that potentially explains the ability of EZH2 to transcriptionally activate NOTCH1 expression in TNBCs is that this phenomenon is the result of both EZH2 hyperactivity and “absence” of its interacting proteins (PRC2, AR, ER, etc.). One ambitious way to test this is by using an in vitro cell-free system containing in vitro translated versions of EZH2, AR, ER, and PRC2 subunits along with a luciferase reporter plasmid containing the NOTCH1 promoter sequence. A control condition could be set up in which the NOTCH1 luciferase reporter plasmid is added to an artificial cell (containing essential transcription and translation machinery) together with levels of EZH2 sufficient to activate its expression (detected by luciferase production). Once an optimal artificial cell is designed in which EZH2 can activate NOTCH1 expression, several test conditions could be designed in which varying levels of AR, ER, and PRC2 subunits are added to “compete” for EZH2 and prevent its trans-activation role.

The Role of lncRNAs in EZH2 activity

Finally, to determine if there are lncRNAs involved in targeting EZH2 or PRC2 that are upregulated in cancer, RNA-seq data across a broad spectrum of EZH2 associated cancers (most likely present in TCGA data files) could be used to determine if any lncRNAs are more abundant in EZH2-overexpressing cancers to identify candidate “recruiter” lncRNAs. Identification of these “cancer-specific” lncRNAs could provide an effective therapeutic target as they can be targeted for destruction by specific miRNA’s, preventing EZH2 and PRC2 recruitment to target loci. This approach is also intriguing as miRNA-based therapies have generally been shown to be effective in cancer treatment.

  1. Discussion

EZH2 is an oncogene that promotes cancer through diverse mechanisms and is associated with cancer progression and metastasis. EZH2 can drive cancer by epigenetically silencing of TSGs enzymatic, co-activating cancer associated genes, and methylating cancer-associated proteins. While not discussed here, recent reports have demonstrated a tumor suppressor activity for the protein, further complicating its role in oncogenesis. Research is still being done to clarify the molecular mechanisms of EZH2 and identify patients in whom pharmacologically targeting EZH2 would be most effective. Targeting EZH2 in smokers may be effective as carcinogens in cigarette smoke increase EZH2 transcriptional silencing of Dickorpf-1, increasing Wnt signaling (Hussain et al., 2009).

There are currently several anti-EZH2 treatments available on the market. The majority of these drugs are competitive inhibitors of the methyl group donor S-adenosyl-L-methionine (SAM) (Wen et al., 2017). These drugs prevent the binding of the methyl donor to EZH2, preventing it from performing its enzymatic activity. While the concept of blocking the donor-enzyme interaction has been around for some time, several groups are currently developing SAM inhibitors with improved specific affinity for EZH2 (as compared with other methyltransferase enzymes) that can also be easily administered. One such drug currently being tested is EPZ-6438 (Tazemetostat), which is highly potent and can be administered orally. Tazemetostat has progressed through Phase I/II trials involving patients with B-cell lymphomas with little cytotoxic effects, and several patients achieved partial or complete responses (Italiano et al., 2018); NCT01897571). Other classes of drugs being tested are those that disrupt the interaction between EZH2 and the PRC2 complex. The peptide named stabilized alpha-helix of EZH2 (SAH-EZH2) derived from the EED interacting domain of EZH2 has been effective in leukemia and lymphoma cell lines (Kim et al., 2013b).  EZH2 activity can also be controlled through diet as sulforaphane, derived from broccoli, transcriptionally and post-translationally inhibits of EZH2 expression. This compound is a promising future therapy as it has yet to demonstrate significant side effects (Fisher et al., 2016).

One area of cancer treatment that has exhibited constant growth and has provided a beacon of hope for many patients is immunotherapy. Research has shown that immune checkpoint inhibitors work well in tumors that have a higher nonsynonymous burden (Rizvi et al., 2015). Since EZH2 negatively impacts several DNA repair genes and pathways, it would be reasonable to suggest that tumors bearing EZH2 mutations would shoulder a larger nonsynonymous mutational burden and respond more favorably to available immunotherapies such as CTLA-4 or PD-1 blockade. However, several studies report that EZH2/PRC2 activity acts to inhibit natural antitumor responses. Investigations in B-16 melanoma cells revealed that the downstream effector genes of EZH2 include immunoproteasome subunits and MHC class I molecules, reducing tumor cell immunogenicity by limiting the diversity and presentation of the antigenic peptidome (Zingg et al., 2017). This suggests that combination therapies involving immune checkpoint therapies given in tandem with the EZH2 inhibitors described above could provide patients whose tumors are driven in part by EZH2 hyper-activity with a long term clinical benefit. In conclusion, Better understanding of EZH2 and its cancer driver mechanisms can help researchers develop better anti-cancer drugs and treat patients with later stage cancers in a new and promising way.


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