Exploring the role of EZH2 modulation in shaping the tumor microenvironment

1. Introduction

Epigenetic modifications include all the changes in gene expression caused by events like DNA methylation, chromatin remodeling and histone modifications (e.g., phosphorylation, acetylation, methylation and ubiquitination). It is largely demonstrated that alterations and aberrations of epigenetic processes are involved in tumorigenesis and tumor progression. Indeed, a plethora of molecular mechanisms involving epigenetic alterations can induce and actively contribute to several hallmarks of cancer [1], making the study of epigenetics an essential tool to identify new targets for cancer therapy.

EZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2) which is responsible for the trimethylation of histone 3 on the lysine 27 (H3K27me3), thus mediating transcriptional repression. The proteins SUZ12, EED, and EZH1 constitute the other components of the complex. The PRC2 complex is involved in several physiological processes mainly related to cell cycle progression and cell differentiation [2]. Moreover, different studies have shown that the PRC2 complex is overexpressed in several malignant tumors, including breast, melanoma, ovarian, prostate, lung, hepatocellular and colorectal cancer and its activity has been correlated with poor prognosis [3]. EZH2 is involved in cancer initiation, growth, metastasis, epithelial to mesenchymal transition, apoptosis and aggressive disease stage [4–6]. Additionally, it can also mediate DNA damage repair, drug resistance, and immunity regulation, as well as inhibition of cellular senescence [7,8].

For these reasons, there is a high interest in studying PRC2, and in particular EZH2, as a tumor promoter, as well as a druggable therapeutic target. Indeed, several clinical trials are investigating EZH2 inhibitors, such as tazemetostat, lirametostat, valemetostat, CPI-0209, SHR2554 and MAK683, for efficacy and safety in targeting solid tumors [9].

Besides the above described functions in tumor cells, it has been deeply confirmed that EZH2 activity impacts also the tumor immune microenvironment (TME), with different functions on lymphoid or myeloid cells. Indeed, EZH2 inhibition has proven the ability to either boost or suppress the immune response against tumors according to their type and depending on the targeted immune cell subset, as detailed below.

2. Anti tumorigenic effects of EZH2 inhibition in the lymphoid compartment

Regarding the T cell compartment, it has been shown in murine ovarian cancer models that EZH2 inhibition enhances T cell recruitment within the TME, by increasing the production of Th1-type chemokines such as CXCL9 and CXCL10. Also, the combination with an EZH2 inhibitor improved the therapeutic efficacy of PD-L1 checkpoint blockade. In the same work, the authors observed a negative correlation between EZH2 expression and both CD8⁺ T cells infiltration and survival in ovarian cancer patients [10]. Interestingly, Tumes and colleagues used Ezh2 knockout mice to demonstrate that EZH2 is involved in the regulation of the differentiation of CD4⁺ T cells in Th1/Th2 phenotypes. In particular, EZH2 binds TBX21 and GATA3 transcription factors mediating Th1 and Th2 fate, respectively, and therefore controlling the plasticity of these subsets [11].

Moreover, EZH2 activity is needed for inducing the expression of FOXP3, the main transcription factor regulating differentiation and activity of regulatory T cells (Tregs) [12]. Indeed, the inhibition of EZH2 in mice leads to an upregulation of IL-4 and INFγ, and to a consequent downregulation of FOXP3. Together with the shrinkage of the Treg compartment, EZH2 inhibition induces an expansion of memory T cells [12]. Concordantly, EZH2 is essential to maintain the functionality of Tregs. DuPage and colleagues demonstrated that mature Tregs depleted for EZH2 in vivo showed altered transcription and stability despite appearing phenotypically normal. They hypothesize that EZH2 collaborates with FOXP3 to regulate the expression of genes responsible for Tregs activity [13]. The role of EZH2 in the functionality of Tregs was also confirmed by Wang and colleagues [14], who showed that Tregs from a mouse model (MC38 colorectal carcinoma model) and human patients (colorectal, lung and breast cancer) express high levels of EZH2. In this context, they demonstrated that the inhibition of EZH2 reduces Treg-mediated immunosuppression and tumor proliferation. In our recent work, we also demonstrated that an inhibitor of EZH2, GSK-126, resensitizes castration-resistant prostate cancer cells to enzalutamide, an androgen-receptor pathway inhibitor, also reducing the frequency of tumors with aggressive neuroendocrine differentiation. Moreover, we unveiled that this combination therapy awaked the cytotoxic activity of CD8⁺ T cells against the tumor, apparently also enhancing the accumulation of Th17 cells [15].

EZH2 is also involved in the function of B cells, although it has not yet been studied in the context of the TME. In mice, the selective depletion of Ezh2 in the B cell compartment significantly impaired B cell maturation. This is because EZH2 is involved in the rearrangement of the immunoglobulin heavy chain gene (Igh), which is critical for early B cell development [16]. Moreover, it has been recently discovered that EZH2 activity is needed for correct proliferation and functionality of antibody-secreting B cells, in which Ezh2 deficiency causes a defective expression of genes involved in the cell cycle [17]. The potential restriction of B cell maturation due to EZH2 inhibition warrants investigation in the TME.

EZH2 can also restrain differentiation and function of natural-killer (NK) cells by negatively regulating the expression of CD122 (IL-15R), which regulates the activity of several transcriptional factors essential for the generation of NK cell precursors, as well as for the development of immature and mature NK cells. Finally, EZH2 can negatively regulate the expression of the NKG2D activating receptor [18].

3. Anti tumorigenic effects of EZH2 inhibition in the myeloid compartment

M1-like tumor-associated macrophages (TAMs) are proinflammatory cells that can boost immune response and contribute to restrain cancer development. In contrast, M2-like TAMs play a pro-tumorigenic role, facilitating tumor cell proliferation, migration and metastasis by enhancing immunosuppression, angiogenesis and extracellular matrix remodeling [19]. An in vitro study showed that EZH2 inhibition in glioblastoma cell lines induces the polarization of co-cultured microglia toward M1 phenotype, increasing also their phagocytic activity [20]. Similarly, in a colorectal cancer model, EZH2 inhibition stimulated M1 macrophages that contributed to the suppression of tumor growth [21]. Furthermore, in a murine hepatocellular carcinoma model, it has been shown that EZH2 silences the miR-144/miR-451 cluster, thus regulating TAMs polarization in a paracrine way. These miRNAs downregulate hepatocyte growth factor, which stimulates M2 polarization, and macrophage migration inhibitory factor, which promotes TAM immunosuppressive functions. Yet, high EZH2 activity resulted in lower levels of miR-144/miR-451 cluster, in higher levels of both hepatocyte growth factor and migration inhibitory factor, and consequently in M2 polarization [22]. M2 polarization can also be affected by other cytokines, such as CCL5 and CCL2, whose production is also regulated by EZH2 activity in tumor cells [23].

4. Protumorigenic effects of EZH2 inhibition in the TME

All the above reported studies demonstrate that the pharmacologic inhibition of EZH2 has the potential to positively activate the immune system toward tumor suppression. However, in some instances, EZH2 inhibition may have detrimental effects in the TME.

It has been shown in murine subcutaneous models of lung and colon cancers that EZH2 can restrain the maturation of myeloid-derived suppressor cells (MDSCs), a heterogeneous population of immune cells which contributes to sustain a tumor immunosuppressive microenvironment through inhibition of T cell activity and direct production of tumor promoting factors. Consequently, the inhibition of EZH2 with GSK-126 was able to promote differentiation of hematopoietic progenitor cells into MDSCs, thus fostering immunosuppression [24]. Thus, EZH2 inhibition could be deleterious in tumors in which the activity of MDSC is relevant, for example prostate and breast cancer.

Moreover, the effect exerted by EZH2 inhibition on TAMs can be either positive, as described in the former chapter, or negative. Indeed, it has been reported that the crosstalk with breast cancer cell lines treated in vitro with an EZH2 inhibitor switched macrophages toward an M2-like phenotype. This polarization was mediated via CCL2 released by breast cancer cells and induced the production of IL-10 by TAMs, eventually leading to tumor cells migration [25]. These controversial results on TAMs could be due to a tissue-dependent activity of EZH2. Moreover, it should be considered that these studies are mainly performed in vitro and this could be a significant limitation for the broad understanding of the effects of altering EZH2 related functions in the TME.

5. Conclusion & perspective

All these data support the essential role of EZH2 in the modulation of the TME and reinforce the idea of targeting this factor to boost the immune response against the tumor. Nevertheless, EZH2 inhibition could also negatively regulate the antitumor immunity. Both these positive and side effects need further preclinical and clinical investigation to improve the therapeutic efficacy of anti EZH2 drugs in cancer patients.

Particularly, a deep analysis is needed to clarify the effects of EZH2 inhibition in different immune cell populations and in different tumor contexts. This could help defining more specific schedules of treatments and combination therapies. For example, a successful strategy could be obtained by combining EZH2 inhibition with immunotherapy, as for example immune checkpoint blockade, as shown in several preclinical studies [26,27]. Indeed, it has been shown that the administration of the anti CTLA-4 antibody ipilimumab can increase the expression of EZH2 in peripheral T cells across various tumor models and in cancer patients. Consequently, the inhibition of EZH2 potentiates the effectiveness of anti–CTLA-4 therapy in a murine model of bladder cancer [26]. Similarly, in hepatocellular carcinoma, the pharmacological depletion of the cell cycle related kinase CCRK reduces EZH2 activation leading to an increased PD-L1 expression, thus likely rendering the tumor more susceptible to PD-L1 blockade [27].

In conclusion, despite the fact that the therapeutic potential of different inhibitors of EZH2 is already under investigation in different Phase I/II clinical trials of solid tumors and lymphomas (ClinicalTrials.gov), however, further studies to dissect the specific mechanisms of action on either the tumor or the TME, as well as possible combinations with immunotherapy, are needed to enhance the therapeutic efficacy and the benefit for patients.

Author contributions

I Fischetti: writing, MD Luca: writing, E Jachetti: writing, revision and supervision.

Competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.