Abstract PO-013: Combined epigenetic therapy to induce latency II/III antigen expression in latency I EBV+ lymphoma
Authors: Kong Isabella Y , Alonso Vicenta Trujillo , Clark Sarah , Sun Suhong , Alonso Alicia , Whitehouse Rebecca , Trezise Stephanie , Zappasodi Roberta , Cesarman Ethel , Roth Lisa Giulino 
Published: June 2024

Abstract
In EBV+ lymphomas the virus exists in one of three latent states. Cellular therapy with EBV cytotoxic T cells (CTLs) is effective in latency II/III tumors that express the viral proteins LMP1/2 and/or EBNA2-6 but not in latency I tumors. Previously, we reported that EBV latency restriction is regulated through DNA methyltransferase 1 (DNMT1, Guo at al, Nat Micrbiol 2020) and that treatment with decitabine (DCB) induces latency II/III antigen expression in latency I Burkitt lymphoma (BL), rendering BL sensitive to EBV-specific CTLs (Dalton et al, Blood 2020). We have also found that a DNMT1 specific inhibitor (DNMT1i) converts BL to latency II/III, highlighting the key role of DNMT1 in latency regulation. One limitation to 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 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 viral-directed immunotherapy. To determine if epigenetic mechanisms are responsible for resistance to latency conversion, we performed ATACseq and ChIP-qPCR on sensitive vs resistant KemI cells. ATACseq revealed open chromatin at Cp, which controls expression of EBNA2-6, in DCB-sensitive cells, but closed-chromatin in DCB-resistant cells, suggesting chromatin compaction may play a role in suppression of EBNA2-6. We observed no difference in chromatin accessibility at the LMP1 promoter, suggesting an independent mechanism may regulate LMP1. We performed ChIP-qPCR to evaluate activating (H3K27ac, H3K4me3 and H3K36me2) and repressive (H2K27me3 and H3K9me3) marks at Cp and LMP1p. We observed an increase in H3K27me3 and H3K9me3 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 vs. sensitive cells. H3K9me3 was increased a 2.4-fold (adj-p&lt;0.0001) and 2.3-fold (adj-p&lt;0.0001) on Cp in DCB and DNMT1i resistant cell respectively. H3K9me3 was increased 3.46-fold (adj-p=0.0013) on LMP1p in DCB resistant cells. Given our observation of increased H3K27me3 resistant cells, we next evaluated the effect of the EZH2 inhibitor tazemetostat, which targets H3K27me3, alone and in combination with DCB or DNMT1i in KemI. Tazemetostat treatment alone did not induce expression of latency II/III. However, when combined with DCB or DNMT1i, tazemetostat increases the percentage of LMP1+ EBNA2+ cells compared to DCB or DNMT1i alone (DCB: 32.3% to 44.5% adj-p=0.0006; DNMT1i: 22.2% to 35.6% adj-p=0.0002). Our data suggests that repressive histone marks contribute to resistance to DNMT1 inhibition-induced latency switch and that the addition of tazemetostat to DCB or DNMT1i can improve latency switch, potentially 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.
Citation Format: Isabella Y Kong, Vicenta Trujillo Alonso, Sarah Clark, Suhong Sun, Alicia Alonso, Rebecca Whitehouse, Stephanie Trezise, Roberta Zappasodi, Ethel Cesarman, Lisa Giulino Roth. Combined epigenetic therapy to induce latency II/III antigen expression in latency I EBV+ lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-013.