Selective HDAC3 Inhibition Induces Apoptosis in B Cell Lymphoma through Protein Acetylation
Authors: Matsui Hiroyuki , Shirakawa Kotaro , Chang Hsin-Yi , Sarca Anamaria Daniela , Kazuma Yasuhiro , Fukuda Hirofumi , Yamazaki Hiroyuki , Matsumoto Tadahiko , Ishihama Yasushi , Takaori-Kondo Akifumi 
Published: October 2019

Abstract
Acetylation is a reversible process under the control of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Acetyl proteome analysis revealed that acetylation regulates various cellular processes through both histone and non-histone proteins (Choudhary et al., Science, 2008). Subsets of diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) have inactivating mutations of HATs, CBP and p300 (Pasqualucci et al., Nature, 2011). In addition, p300 mutation is a poor prognostic factor in FL patients (Pastore et al., Lancet Oncol, 2015). In a mouse model, CBP deficiency promotes B cell lymphomagenesis through H3K27 deacetylation of enhancer mediated by the HDAC3, which forms repressor complex with Bcl-6, SMRT and NCoR (Jiang et al., Cancer Discov, 2016).
Human HDACs consist of 18 isoenzymes that are classified into 4 classes (I-IV). Among the 4 classes, class I includes 4 HDACs (HDAC1, 2, 3 and 8) that are ubiquitous. Four HDAC inhibitors are FDA approved for the treatment of cutaneous T cell lymphoma (romidepsin, vorinostat), peripheral T cell lymphoma (belinostat) and multiple myeloma (panobinostat). These inhibitors mainly target class I and class II HDACs, each with a different specificity. This non-specific nature of current HDAC inhibitors limits their efficacy and causes adverse effects, therefore, several selective HDAC inhibitors has been developed. Selective HDAC3 inhibition restricts myeloma cell growth in vitro and in vivo more efficiently than selective HDAC1 or HDAC2 inhibition, allowing for DNMT1 acetylation, accelerating its degradation by the proteasome (Harada et al., Leukemia, 2017). However, the efficacy of selective HDAC3 inhibition against B cell lymphoma remains unclear.
We first knocked down HDAC3 by shRNA in a B-cell lymphoma cell line (KIS1). We found that selective HDAC3 knock down suppressed cell growth and induced apoptosis. We next tested selective HDAC3 inhibitors (RGFP966 and AA-1) using five B cell lymphoma cell lines (Raji, Ramos, KIS1, SUDHL-6 and Granta519). We confirmed that these inhibitors reduced cell viability in all treated cell lines, significantly increased the number of apoptotic cells in Ramos, KIS1 and SUDHL-6 and induced cell cycle arrest in Raji and Granta519, rather than apoptosis. Lentiviral shRNA against HDAC3 or the inhibitors also led to the cleavage and subsequent activation of caspase9 and caspase3. These results show that HDAC3 inhibition induces apoptosis by activating the intrinsic apoptotic pathway.
To clarify how HDAC3 inhibitors affect global protein acetylation and how they induce apoptosis in B cell lymphoma, we conducted acetyl proteome analysis using LC-MS/MS. Briefly, we immunoprecipitated acetyl lysine peptides from whole cell lysates of SUDHL-6 with or without RGFP966 treatment, and analyzed them by mass spectrometry. We identified 673 and 1,328 acetylation sites in RGFP966 treated and untreated samples, respectively, and 1,425 acetylation sites in total. Among these 1,425 sites, 153, including histones and HATs, were more than two fold upregulated in the RGFP966 treated sample. To identify which cellular processes are affected by RGFP966 treatment, we performed Gene Ontology (GO) enrichment analysis of the proteins with upregulated acetylation. GO enrichment analysis revealed that bromodomain, glycolysis and unfolded protein binding were significantly enriched terms upon RGFP966 treatment.
Our data show that selective HDAC3 inhibition is also effective against B cell lymphoma and that protein folding and metabolic processes as well as epigenetic mechanisms might be involved in activation of the intrinsic apoptosis pathway upon HDAC3 inhibition. We discuss how acetylation of these proteins leads to cell growth inhibition and apoptosis in B cell lymphoma.

Disclosures
Takaori-Kondo: Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria; Janssen Pharmaceuticals: Honoraria.