EZH2 inhibition strengthens existing immunotherapy

EZH2 inhibition strengthens existing immunotherapy.   The complexity and diversity of the TME modification potential of EZH2 inhibition emphasizes the necessity of reasonable combined immunotherapy.

Preface

The latest developments in tumor immunotherapy emphasize the ability of the immune system to control tumors, but only a small percentage of patients respond to current immunotherapy. In order to overcome the primary and acquired resistance of immunotherapy, such as immune checkpoint blockade (ICB), more methods are needed to mobilize anti-tumor immunity. New evidence shows that targeting epigenetic factors that promote tumor progression and suppress immune cell activity can enhance anti-tumor immunity by reshaping the tumor microenvironment (TME). The latest research has discovered the pleiotropic function of the zeste homolog 2 (EZH2) enhancer in tumors and immune cells. EZH2 inhibition is a promising method that can strengthen the existing immunotherapy to improve the health of some cancer patients. Prognosis.

Epigenetics and tumor immunity

Epigenetic regulation of gene expression can make immune cells change their phenotype according to the environment, including the environmental factors provided by TME. The latest findings indicate the contribution of epigenetic mechanisms in tumor immunity, such as DNA methylation and histone modification, to regulate anti-tumor immunity and self-tolerance. On the other hand, tumors can also use epigenetic modifications to disrupt key interactions that promote effective anti-tumor responses to suppress immunity. Epigenetic therapies against these immune evasion strategies can stimulate an immune response to make up for the shortcomings of current checkpoints or other immunotherapies.

EZH2 is the catalytic subunit of histone methyltransferase and polycomb inhibitory complex 2 (PRC2). EZH2 catalyzes the monomethylation, dimethylation and trimethylation of lysine 27 of histone H3 (H3K27me3), and histone markers are related to tight chromatin and transcriptional inhibition. EZH2 plays a role in the normal biology of many cell types including immune cells. Dysfunctional EZH2 is related to the occurrence and development of multiple cancer types in mice and humans, and can promote immune evasion by inhibiting tumor antigen presentation, immune cell migration, and enhancing the inhibitory activity of CD4+ T regulatory cells (Treg). These features make EZH2 an attractive therapeutic target that can complement existing immunotherapy methods. Several EZH2 inhibitors (EZH2i) have been developed, including tazemetostat, which has been approved by the FDA for the treatment of epithelioid sarcoma and follicular lymphoma.

The role of EZH2 in tumor immunity

The role of EZH2 in the differentiation and function of CD4+ T cells: CD4+ Th cells usually coordinate the activation of immune responses by differentiating into different lineages, including Th1, Th2, Th17 and T follicular helper cells (Tfh) Group; each type of cell plays a different role in anti-tumor immunity. Ezh2 has an inhibitory effect on the differentiation of Th1 and Th2. EZH2-dependent gene expression regulation may also help inhibit Th17 differentiation. Inhibition of EZH2 can cause CD4+ T cells to increase the expression of effector cytokines.

Tfh cells are a special subset of CD4+ T cells, which play a central role in inducing protective antibody responses to pathogens and have become a key component of TME. The differentiation of Tfh in mice and humans depends on the expression of the main transcription factor of BCL6. Ezh2 acts as a co-activator of Bcl6 transcription to promote the differentiation of Tfh.

EZH2’s regulation of Tregs: Tregs maintain immune self-tolerance and homeostasis by inhibiting inflammatory response, and play an important role in suppressing anti-tumor immunity. Recent studies have shown that Ezh2 is the chromatin modifier with the highest induction rate in mouse Treg cells after the participation of CD28 costimulatory receptor, and its expression helps stabilize the functional phenotype of activated Tregs. Studies on mouse MC38 tumor models and human colorectal cancer, non-small cell lung cancer and breast cancer have shown that EZH2 expression and related H3K27me3 markers are present in tumor-infiltrating Tregs. This in turn indicates that targeting the expression of EZH2 in Tregs in tumors may be a potentially effective way to enhance anti-tumor immunity.

The role of EZH2 in the differentiation of CD8+ T cells: The development of effective and durable anti-tumor immunity partly depends on the highly controlled CD8+ T cell activation, proliferation, terminal differentiation and memory process. In the process of T cell differentiation, the epigenetics of original CD8+ effector T cells and CD8+ memory T cells (Tmem) includes characteristic changes from activating H3K4me3 to inhibitory H3K27me3 labeling patterns. This indicates that EZH2 has a complicated role in the differentiation of CD8+Teff and Tmem. The effect of EZH2 on CD8+ T cells activated by TAAs requires more in-depth analysis of clinically relevant tumor models and more accurate identification of CD8+ T cells at different stages of differentiation within the tumor.

The role of EZH2 in the differentiation and function of NK and NKT cells: NK cells are innate lymphocytes with strong cytolytic activity and can prevent microbial infection and tumor growth. EZH2-dependent epigenetic control of gene expression plays a crucial role in the differentiation and function of NK cell lines. Gene deletion of Ezh2 activity in hematopoietic stem progenitor cells (HSPC) (Vav1-Cre, EZH2fl/fl mice) or drug inhibition (UNC1999 and EPZ005687) increase the survival and differentiation of NK cell lines by promoting the survival and differentiation of mouse NK precursor cells development. EZH2 may also negatively regulate the pro-inflammatory differentiation of invariant natural killer T cells (iNKT); NKT cells are a group of innate T cells that can recognize lipid antigens and play an important role in anti-tumor immunity .

Inhibition and remodeling of TME by EZH2

The contribution of EZH2 to the differentiation and function of lymphocyte subsets indicates that inhibition of EZH2 may improve the anti-tumor immunity of certain cancers. However, regarding the potential impact of EZH2 inhibition on anti-tumor immunity, it is necessary to consider the multiple effects of EZH2 and EZH2 inhibition on tumor cells and immune cells that form TME.

EZH2 inhibition promotes the function of CD8+Teff cells and transfers to TME: The entry of CD8+T cells into TME and killing tumor cells are the key steps of tumor immunity. Excluding T cells from TME is a key strategy for immune evasion. Tumor-infiltrating dendritic cells (DC) secrete chemokines (including CXCL9 and CXCL10) to promote T cell recruitment. EZH2’s inhibition of these chemokines impairs the transport of T cells to TME, and the inhibition of EZH2 can reverse this effect.

In some cancers, increased CD8+ T cell infiltration is associated with a poor prognosis, suggesting immunosuppressive TME. It is worth noting that studies have shown that infiltrating T cells can lead to increased expression of EZH2 and epigenetic-mediated tumor immunosuppression. EZH2 can balance T cell activation and inhibit CD8+Teff-mediated anti-tumor immunity in TME.

Since the lack of effective TIL is a key obstacle to current immunotherapy, the TME modification effect of EZH2i may help improve the ICB efficacy of patients who have failed immunotherapy. As mentioned above, the expression of EZH2 in tumors and immune cells limits the metastasis of CD8+Teff to tumors, and the increase in EZH2 expression after T cell activation may also lead to innate and acquired resistance to ICB. In summary, the clinical observations and experimental studies so far have highlighted the potential of EZH2i to overcome this obstacle and expanded the scope of ICB clinical applications.

EZH2 inhibition can reduce the effect of Tregs on TME: In TME, Treg with increased activity may hinder the effectiveness of ICB, and EZH2i is thought to help overcome this obstacle. Targeting the deletion of Ezh2 in Tregs (Foxp3creEzh2fl/fl mice) or the use of EZH2i can enhance the anti-tumor immune response by reducing the stability of Tregs in TME. Studies have also shown that EZH2i can selectively target Tregs in tumors without causing systemic changes in Treg function, which indicates that its clinical application may only produce minor adverse autoimmune reactions, although this has yet to be verified.

In addition, compared with the baseline expression before treatment, the use of ipilimumab in patients with metastatic melanoma or metastatic prostate cancer resulted in increased expression of EZH2 in peripheral blood Tregs. EZH2i improved the response of anti-CTLA-4 antibody treatment in MB49 and B16-F10 mouse models by reducing Treg inhibitory activity and increasing CD4+ and CD8+ Teff activities. The above data shows that EZH2 can be induced in activated T cells, and under certain circumstances, helps maintain Tregs and reduce the pro-inflammatory response to CD8+ T cells. Inhibition of EZH2 is an attractive candidate treatment strategy that may be combined with ICB to treat certain types of tumors.

The effect of inhibiting EZH2 on TME innate immunity: NK cell-mediated tumor cell killing is the first immune activation pathway that induces the pro-inflammatory cascade in TME. There is evidence that inhibiting EZH2 can change tumor cells and NK cells. Promote this. In addition to the role of EZH2 in limiting the maturation and activation of NK cells, EZH2 inhibition can also regulate the expression of NK cell activation ligands. Inhibition of EZH2 in a xenograft mouse model leads to tumor cell death, which is mediated to a certain extent by activating NK cells. RNA-Seq and quantitative PCR studies on HT1376 tumors treated with EZH2i showed that compared with control mice, NK cell activation markers including IFNγ increased. In addition, immunohistochemistry confirmed that compared with the control group, EZH2 inhibition resulted in increased expression of NK cell markers CD56 and NCR1.

Tumor-associated macrophages (TAMs) can improve the survival and proliferation of tumor cells, and promote the immunosuppressive microenvironment that supports tumor progression. Studies have shown that EZH2 is involved in maintaining the survival and polarization of pro-inflammatory macrophages by inhibiting miRNA let7-c and PAK1, a key determinant of pro-inflammatory macrophage phenotype. EZH2i treatment overcomes ICB resistance in a mouse model of HiMYC PCa transgenic prostate cancer tissue transplantation by increasing pro-inflammatory and reducing anti-inflammatory TAMs.

Although there is evidence that EZH2 may promote DC function in autoimmune models, there are limited data on the potential effect of EZH2 inhibition on DC function in TME and its effect on anti-tumor immunity. In view of the importance of dendritic cells in anti-tumor immunity, it is necessary to further understand the influence of EZH2 inhibition on tumor-infiltrating DC and myeloid cell recruitment and function.

Inhibition of EZH2 can increase the expression, processing and presentation of antigens in TME: reduced tumor antigen presentation of MHC class I and class II molecules will impair the recognition and targeting of activated Teff cells, which is a key immune evasion mechanism. Some studies have shown that EZH2 mediates the inhibition of MHC I and MHC Ⅱ, and the inhibition of EZH2 can restore the immunogenicity of certain tumors and enhance the response to ICB. In another preliminary study, EZH2 inhibition can overcome the ICB resistance mechanism in the anti-PD-1 mouse HNSCC model MOC1-esc1. Compared with the two intervention methods alone, the combined treatment of EZH2i and anti-PD-1 antibody can Significantly inhibit tumor growth.

Research progress of EZH2 inhibitor drugs

EZH2 inhibitors have become an active area of ​​research. The first EZH2 inhibitor with anti-tumor activity is 3-deazacyanin A (DZNeP), a drug originally developed against Ebola virus. In terms of mechanism, DZNeP inhibits S-adenosine-L-homocysteinase, thereby reducing systemic H3K27me3 levels. Preliminary studies have shown that DZNeP induces significant apoptosis in cancer cells, but not in normal cells. However, due to its hydrophilicity and rapid renal metabolism, DZNeP has little clinical value. Therefore, more and more effective and cell-permeable compounds targeting EZH2 have been developed and evaluated in clinical trials.

Tazemetostat: To date, Tazemetostat (Tazverik™) is the most widely studied EZH2 inhibitor in clinical studies. It is worth noting that it has been approved as a first-line treatment for epithelioid sarcoma in the United States in January 2020.

A total of 22 clinical trials have been initiated, including two Phase III trials, and patients are currently being recruited. In the first phase of the study, Tazemetostat showed good safety (NCT01897571). Adverse events are generally grade 1 and grade 2, mainly including fatigue (33%), anemia (14%), anorexia (6%), muscle cramps (14%), nausea (20%) and vomiting (9%), none In the death record, only one case of grade 4 thrombocytopenia occurred. In this study, the anti-cancer activity of Tazemetostat was also evaluated, and the results showed that 38% of B-cell non-Hodgkin’s lymphoma patients had a durable objective response, including some complete responses. However, only 5% of patients with solid tumors show a durable objective response.

In the phase II trial, Tazemetostat induced a response in 15% of patients, of which 67% had a response lasting more than 6 months. A multi-center, double-blind, placebo-controlled, randomized phase III study (NCT04204941), prompting the accelerated approval of the use of Tazemetostat in epithelioid sarcoma, making Tazemetostat the first molecule to treat this aggressive tumor Targeted therapy.

GSK2816126: GSK2816126 is another EZH2 inhibitor that has entered clinical trials. It was originally developed to treat lymphomas with activating mutations in EZH2. In preclinical studies, GSK2816126 showed high selectivity for EZH2 (more than 100 times that of EZH1). In addition, GSK2816126 induced in vitro cancer cell death at nanomolar concentrations and showed significant results in a xenograft model of EZH2 mutant DLBCL The anti-tumor activity.

Currently, GSK2816126 has conducted an open-label dose escalation study to evaluate the pharmacodynamics and efficacy of GSK2816126 in 41 cases of advanced non-Hodgkin’s lymphoma (20 cases) or refractory solid tumors (21 cases) And clinical activity. This is a two-part study to determine the recommended phase II dose (RP2D) of GSK2816126. The most serious limiting factor is liver transaminase, which limits the maximum tolerated dose to 2400 mg. All patients have at least one adverse reaction, the most common of these are nausea and vomiting in about half of the patients. In addition, 32% of patients had severe grade III or lower complications. In terms of anticancer activity, only one lymphoma patient had a partial response, 34% of patients had stable disease, and 51% of patients had progressed.

CPI-1205: CPI-1205 is an oral, indole-based small molecule EZH2 inhibitor that selectively binds to the EZH2 catalytic pocket. The first clinical trial of CPI-1205 (NCT02395601) started in 2015 and has now ended, and the results are yet to be announced. This study is a phase I sequential dose escalation and expansion study of CPI-1205 in patients with progressive B-cell lymphoma. Two other clinical trials involving CPI-1205 are ongoing, and there are no results yet. One is ProStar, which is a phase Ib/II trial that studies oral CPI-1205 combined with enzalutamide or abirenone/prednisone in the treatment of metastatic CRPC (NCT03480646). The second is a phase I/II trial to evaluate the efficacy of CPI-1205+ipilimumab in the treatment of advanced solid tumors (NCT03525795).

Other EZH2 inhibitors: In addition to the above compounds, three other compounds are undergoing clinical trials, and patients are currently being recruited. CPI-0209 is a second-generation EZH2 inhibitor that has shown preclinical evidence of anti-cancer activity in a bladder cancer model. In the sequential dose escalation and expansion study of patients with advanced solid tumors (NCT04104776), it will be evaluated once a day orally alone or in combination with irinotecan. Secondly, PF-06821497 is an effective oral EZH2 antagonist and is currently undergoing phase I testing for advanced malignant tumors (NCT03460977). Another oral EZH2 inhibitor, SHR2554, is also undergoing a phase I clinical trial (NCT03603951) and is currently recruiting patients with recurrent tumors.

There are also some research groups that are actively continuing to develop new EZH2 inhibitors. It is worth noting that efforts are currently being made to optimize existing compounds through reasonable structure activity and drug docking methods to improve pharmacokinetics and pharmacodynamic properties. In addition, another emerging treatment strategy is to design small molecules to disrupt the interaction between EZH2 and EED, resulting in inactivation of PRC2. Therefore, the rapid development and success of EZH2 targeted therapy is likely to continue in the future and will become the standard for other epigenetic drug targets.

issues that need resolving

How is the role of EZH2 in B cell biology related to TME and anti-tumor immunity? EZH2 plays an important role in the process of B cells forming germinal centers and transforming into plasma cells. Since the formation of tertiary lymphatic structure may be a positive prognostic indicator for certain tumors, further studies need to determine whether the inhibition of EZH2 in the context of tumor development can regulate the response of B cells in lymphoid organs in TME to tumors.

How does EZH2 inhibition affect the non-immune areas that form TME, such as tumor-associated fibroblasts (CAFs)? CAFs are composed of heterogeneous cell populations involved in tissue remodeling to support cancer invasion and metastasis. CAFs support immune tolerance in TME by promoting suppressor cells such as bone marrow-derived suppressor cells (MDSCs), TAMs and Tregs, and by eradicating Teff cells and suppressing NK cells. Therefore, CAFs are the main participants in the formation of TME and may be beneficial to the progression of cancer. There are limited and conflicting data regarding the role of EZH2 and CAFs in TME. In order to maximize the TME regulatory potential of EZH2 inhibition, it is important to understand the role of EZH2 in various indications in CAFs.

In different TME environments, how does EZH2 inhibition affect myeloid cells? The method of releasing anti-tumor myeloid cells in TME may broaden the scope of immunotherapy. In view of the conflicting reports on the role of EZH2 in these cells and the conflicting reports of EZH2 in the field, it is important to clarify the effect of EZH2 inhibition on TME inner myeloid cells; this may help to understand EZH2i and ICB and/or bone marrow targeting Potential combination therapy of immunotherapy methods.

What is the best combination of EZH2i+ICB? More preclinical trials are needed to determine the most effective EZH2i treatment plan that is guaranteed for ICB. For example, initial treatment with EZH2i may make TME perform ICB more effectively, while continuous EZH2i treatment may cause negative feedback loops in certain cell types in TME. It is important to increase our understanding of the effect of EZH2i on other cell subpopulations within TME. These preclinical data and the current comprehensive evaluation of TIL in the EZH2i trial can provide valuable information for optimizing the combination regimen.

In addition to its combined application with ICB, what is the potential of EZH2 inhibition? In view of the role of EZH2 in multiple immune cell types, it is important to determine which current immuno-oncology therapies will benefit from EZH2i’s TME modification effect, and whether the increase in tumor antigen presentation and T cell transport and activation caused by EZH2 inhibition will enhance CAR -What is the effect of T cell therapy or therapeutic cancer vaccines? A deeper understanding of how other immune components of TME are affected by EZH2 inhibition will help rational design of clinical trials.

Outlook

The complexity and diversity of the TME modification potential of EZH2 inhibition emphasizes the necessity of reasonable combined immunotherapy. It is crucial to determine how the various TME modification effects of EZH2 inhibition enhance immunotherapy in the context of different tumor types. Some small molecule EZH2i clinical trials are underway. Since EZH2 inhibition affects both tumor cells and TME, an in-depth understanding of the multiple effects of EZH2i in these patients may help predict clinical response.

In addition, understanding the indication-specific and patient-specific TME modification effects of EZH2i may better guide a reasonable combination of immunotherapy. As with current immunotherapy methods, the effect of EZH2i on TME may not only target different cancers, but also for individuals of the same cancer type. It is important to consider this when designing joint trials. The EZH2i currently being studied in clinical trials are generally well tolerated, making them attractive as a combination partner. Joint trials of EZH2i and ICB are emerging, which can provide information for future combined designs.

Tumor types with poor immunogenicity and low mutation rates, including prostate cancer, ovarian cancer, and breast cancer, may be associated with the combined use of EZH2 inhibition and ICB. In addition, the potential of inhibiting the combination of EZH2 with other immunotherapies, such as CAR-T cell therapy and therapeutic cancer vaccines, remains to be explored. We also need to understand how EZH2 inhibition affects less studied TME cell types, including B cells and cancer-associated fibroblasts, to clarify the full potential of this therapeutic strategy to expand the scope of ICB.

Inhibition of EZH2 clearly has the potential to overcome key ICB resistance mechanisms, such as low immune cell infiltration of TME, immunosuppression and reduced antigen expression. The multiple TME modification effects of EZH2 inhibition may help release the potential of current immunotherapy, and further research is needed to determine the most effective strategy to combine EZH2 inhibition with current immunotherapy methods.

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