Combined Epigenetic Therapy to Induce Latency II/III Antigen Expression in Latency I EBV+ Lymphoma
Authors: Kong Isabella Y , Clark Sarah E , Sun Suhong , Trujillo-Alonso Vicenta , Alonso Alicia , Zappasodi Roberta , Cesarman Ethel , Roth Lisa Giulino 
Published: December 2024

Epstein-Barr virus (EBV) infection contributes to the development of a significant subset of human lymphomas. In EBV+ lymphomas, the virus exists in one of three latent states: latency I, in which only EBNA1 viral protein is expressed. Conversely, latency II and III tumors express immunogenic viral proteins such as EBV latent membrane protein 1 or 2 (LMP1/2) and EBV nuclear antigen 2 or 3 (EBNA2/3) that can be recognized by cytotoxic T cells (CTLs). As such, cellular therapy with EBV-CTLs is effective in latency II/III tumors but not in latency I tumors, such as EBV+ Burkitt lymphoma (BL).

Previously, we reported that EBV latency restriction is regulated through DNA methyltransferase 1 (DNMT1) (Guo et al, Nat Microbiol 2020), and treatment with decitabine (DCB) induces latency II/III antigen expression in latency I BL, rendering BL tumors sensitive to EBV-specific CTLs (Dalton et al, Blood 2020). We also found that a selective DNMT1 inhibitor (DNMT1i) converts BL to latency II/III, highlighting the key role of DNMT1 in latency regulation. One limitation of DNMT1 inhibition as a therapeutic approach in latency I EBV+ lymphoma is that the induction of latency II/III is only observed in a subset of cells. Here, we investigated the mechanisms of resistance to latency conversion to develop rational combinations to maximize the conversion of latency I tumors to latency II/III thereby sensitizing them to EBV-directed immunotherapy.

To determine whether epigenetic mechanisms are responsible for resistance to latency conversion, we performed ATAC-seq and ChIP-qPCR on BL cell line cells sensitive or resistant to DCB-induced latency conversion. We observed minor reduction in chromatin accessibility at the promoter region of EBNA3A (logFC=-0.06, p=4.98E-21) and LMP2A (logFC=-0.13, p=0.002), but no significant differences in other EBV promoter regions, suggesting that chromatin compaction plays a minor role in regulating resistance to DCB-induced latency conversion. Next we performed ChIP-qPCR to evaluate activating (H3K27ac, H3K4me3, H3K4me and H3K36me2) and repressive (H2K27me3 and H3K9me3) marks at Cp (encodes for EBNA2) and LMP1p. We observed an increase in H3K27me3 on Cp and LMP1p in resistant cells. H3K27me3 was increased 2-fold on Cp (adj-p=0.0009) and 2.45-fold on LMP1p (adj-p=0.0086) in DNMT1i resistant versus sensitive cells.

Given our observation of increased H3K27me3 resistant cells, we next evaluated the effect of the EZH2 inhibitor tazemetostat (TAZ), which targets H3K27me3, alone and in combination with DCB or DNMT1i. TAZ treatment alone did not induce expression of latency II/III. However, when combined with DCB or DNMT1i, TAZ increases the percentage of EBNA2+ LMP1+ cells compared to DCB or DNMT1i alone (DCB vs DCB+TAZ: 32.3% vs 44.5% adj-p=0.0006; DNMT1i vs DNMT1i+TAZ: 22.2% vs 35.6% adj-p=0.0002). To examine this in vivo, we treated mice with DCB and/or TAZ and found that the combination of DCB and TAZ significantly increases the proportion of LMP1+ cells (DCB vs DCB+TAZ: 4.58% vs 6.62% adj-p≤0.0001).

Our data suggests that repressive histone mark H3K27me3 contributes to resistance to DNMT1 inhibition-induced latency switch and that the addition of TAZ to DCB or DNMT1i can overcome resistance to latency switch, resulting in a more immunogenic tumor. These findings will guide the development of therapies to improve the treatment of otherwise immune refractory EBV+ latency I lymphoma.