EZH2 inhibitors restore epigenetically silenced CD58 expression in B-cell lymphomas

Highlights

• Loss of CD58 is a common mechanism for immune evasion in lymphoid malignancies.

• EZH2 inhibitors restore epigenetically suppressed CD58 expression of lymphoma cells.

• EZH2 inhibitors enhance IFN-γ production of T and NK cells against lymphoma cells.

Abstract

Loss of CD58 is a common mechanism for tumor immune evasion in lymphoid malignancies. CD58 loss is known to occur due to both genetic and non-genetic causes; therefore, we hypothesized that restoring CD58 expression in lymphoma cells may be an effective treatment approach. To explore the potential for restoring CD58 expression, we first screened 11 B-cell lymphoma lines and found that 3 had decreased CD58 expression. Among these, CD58 was genetically damaged in two lines but not in the third line. Using the cell line with downregulated CD58 without a genetic abnormality, we performed epigenetic library screening and found that two EZH2 inhibitors, EPZ6438 and GSK126, specifically enhanced CD58 expression. By examining the effect of three EZH2 inhibitors with different selectivity profiles in different B-cell lines, EZH2 inhibition was shown to have a common activity in upregulating CD58 expression. Restoring the expression of CD58 in lymphoma cells using an EZH2 inhibitor was shown to enhance interferon-γ production of T and NK cells against lymphoma cells. H3K27 was shown to be highly trimethylated in the CD58 promoter region, and EZH2 inhibition induced its demethylation and activated transcription of the CD58 gene. These results indicated that EZH2 is involved in the epigenetic silencing of CD58 in lymphoma cells as a mechanism for tumor immune escape, and EZH2 inhibitors are able to restore epigenetically suppressed CD58 expression. Our findings provide a molecular basis for the combination of an EZH2 inhibitor and immunotherapy for lymphoma treatment.

Introduction

The tumor microenvironment plays a critical role in the development and progression of lymphoid malignancies. Lymphoma cell survival and proliferation in vivo are often dependent on interactions with the non-tumor cell components of the microenvironment to some degree. On the other hand, lymphoma cells also modulate these cellular interactions to escape from immune surveillance.

Understanding of these interactions has contributed to the development of novel immunotherapies. For instance, immune checkpoint inhibitors have been highly effective for the treatment of Hodgkin lymphoma (Ansell et al., 2015); Chen et al., 2017; Armand et al., 2018. Hodgkin lymphoma is characterized by malignant Hodgkin/Reed-Sternberg (H/RS) cells dispersed within the immune cell infiltrates, and a PD-1-blocking antibody inhibited the interaction between PD-1 and its ligands expressed on tumor-infiltrating T cells and H/RS cells, respectively (Ansell et al., 2015; Yamamoto et al., 2008; Carey et al., 2017). Although PD-1 signaling pathway is also suggested to be involved in other lymphoma subtypes, PD-1 blockade therapy is not as effective in Hodgkin lymphoma, at least when it is used as a single agent (Ansell et al., 2019; Lesokhin et al., 2016). One possible reason for the limited efficacy of immune checkpoint inhibitors in non-Hodgkin lymphomas is the frequent involvement of additional immune escape mechanisms. In the comprehensive genomic analysis of diffuse large B-cell lymphoma (DLBCL), molecules involved in antigen presentation and costimulatory signaling, such as HLA class I/II, CD58, B2M, CD70, and 4-1BBL, were shown to be recurrently mutated (Challa-Malladi et al., 2011); Dubois et al., 2016; Pasqualucci et al., 2011; Karube et al., 2018; Schmitz et al., 2018; Reddy et al., 2017; Chapuy et al., 2018. These findings suggested that defective immune synapse formation between lymphoma cells and immune effector cells is an important underlying mechanism of immune evasion in DLBCL.

The CD58 gene is one of the recurrent targets of genetic abnormalities in DLBCL and in other lymphoid malignancies such as peripheral T-cell lymphoma (Palomero et al., 2014) and adult T-cell leukemia/lymphoma (Kataoka et al., 2015; Yoshida et al., 2014). CD58 gene abnormality is also suggested to appear in the process of transformation of follicular lymphoma (FL) into DLBCL (Pasqualucci et al., 2014). CD58 is a member of the immunoglobulin superfamily, which has a function to bind to CD2 expressed on T and NK cells (Wang et al., 1999); Grakoui et al., 1999; Moingeon et al., 1989, and the CD2-CD58 interaction is especially important for tumor recognition by T and NK cells. Loss of CD58 expression is indicated to be associated with worse overall and event-free survivals in patients with DLBCL (Cao et al., 2016) and acute lymphoblastic leukemia (Li et al., 2016). CD58 expression is reported to be lost through both genetic and non-genetic mechanisms (Challa-Malladi et al., 2011); therefore, we hypothesized that restored expression of CD58 may facilitate T and NK cell-immune recognition of lymphoma cells and increase the efficacy of immune-targeted therapy.

To explore the non-genetic mechanism of defective CD58 expression in lymphoma cells, we performed epigenetic compound library screening using a B-cell lymphoma line with decreased CD58 expression without any CD58 gene abnormality. We found that EZH2 inhibitors specifically restored CD58 expression in these cells, and upregulation of CD58 by an EZH2 inhibitor enabled lymphoma cells to strongly stimulate T and NK cells. H3K27 was shown to be highly trimethylated in the CD58 promoter region, and EZH2 inhibition induced its demethylation and increased CD58 gene transcription. These results indicated that EZH2 is involved in the epigenetic silencing of CD58 in lymphoma cells as a mechanism for tumor immune escape, and an EZH2 inhibitor was effective for the restoration of epigenetically downregulated CD58 expression. Our findings provide a molecular basis for the effectiveness of EZH2 inhibitors and a rationale for their combination with immune-targeted therapies for the treatment of lymphomas.