Abstract A29: A selective HDAC3 inhibitor synergizes with 5-azacytidine in diffuse large B-cell lymphoma
Authors: Dominguez Pilar M , Cluse Leonie A , Tawfic Noura , Fraser Peter J , Rutaquio Sophia , Lim Rachel , Inghirami Giorgio , Gregory Gareth , Dickinson Michael , Melnick Ari M , Johnstone Ricky W 
Published: September 2022

Abstract
Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoma, with 40% of patients relapsing or refractory to the conventional chemotherapy treatment, usually with fatal consequences. Therefore, more targeted therapies are needed. DLBCL are characterized by profound alterations in the epigenome (histone modifications and DNA methylation) that are correlated with poor survival. While epigenetic drugs are used as anti-cancer treatments, their full potential has not been achieved. One limitation for the implementation of epigenetic-based therapies in the clinic has been its use as single agents; therefore, we explored new combinatorial therapies with the goal to increase efficacy. Our previous studies showed that lymphoma epigenetic programming involves repression of BCL6 target genes. This BCL6-mediated gene silencing in DLBCL is driven by 1) reduced H3K27 acetylation through recruitment of histone deacetylase 3 (HDAC3) and 2) reduced 5-hydroxymethylcytosine (5hmC). We thus tested a therapy consisting of the hypomethylating agent 5-azacytidine (5-Aza) and a specific HDAC3 inhibitor (HDAC3i). DLBCL cell line MD901 was treated with 200nM 5-Aza or vehicle daily for 5 days, with 10uM HDAC3i or vehicle added at days 3 and 5. Analysis at day 8 showed that whereas single agent did not induce cell death based on propidium iodide (PI) staining (vehicle=13.5%, 5-Aza=18%, HDAC3i=14.8% PI+ cells), the combination induced significantly higher cell death (5-Aza+HDAC3i=43.6% PI+ cells). We observed similar increased efficacy of 5-Aza+HDAC3i in OCI-Ly7 and SUDHL-4 cells. We also demonstrated that 5-Aza+HDAC3i acted synergistically in MD901, OCI-Ly7 and SUDHL-4 (combination index (CI)&lt;1), with a dose-reduction index (DRI)=3.1 for 5-Aza and DRI=2.6 for HDAC3i to achieve 50% cell death in MD901; similar results were obtained in the other cell lines tested. We established xenografts -transplanting lymphoma cells in NSG mice through intraperitoneal injection- to test the efficacy of the combinatorial therapy in vivo. Mice engrafted with OCI-Ly7 cells received daily treatment with 1mg/kg 5-Aza or vehicle for 1 week, and were also treated with 25mg/kg HDAC3i or vehicle for other 3 weeks. 80% mice treated with 5-Aza+HDAC3i survived after 150 days, compared to 50% mice treated with HDAC3i alone; 5-Aza did not induce survival advantage compared to vehicle-treated mice. At the molecular level, we found that 5-Aza as single agent reduced global 5hmC levels compared to vehicle-treated cells; interestingly, this was also observed when cells were treated with HDAC3i alone. Intriguingly, the level of 5hmC when combining 5-Aza+HDAC3i was comparable to vehicle-treated cells. In summary, we demonstrate superior efficacy of 5-Aza in combination with HDAC3i in DLBCL. We are currently performing genome-wide assays to dissect the molecular mechanisms underpinning this synergistic anti-tumor activity. At the same time, we are generating xenograft models with primary DLBCL samples to confirm the potential of this new therapeutic approach to improve the outcomes of DLBCL patients.
Citation Format: Pilar M Dominguez, Leonie A Cluse, Noura Tawfic, Peter J Fraser, Sophia Rutaquio, Rachel Lim, Giorgio Inghirami, Gareth Gregory, Michael Dickinson, Ari M Melnick, Ricky W Johnstone. A selective HDAC3 inhibitor synergizes with 5-azacytidine in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A29.