HDAC inhibitor chidamide synergizes with venetoclax to inhibit the growth of diffuse large B-cell lymphoma via down-regulation of MYC, BCL2, and TP53 expression

HDAC inhibitor chidamide synergizes with venetoclax to inhibit the growth of diffuse large B-cell lymphoma via down-regulation of MYC, BCL2, and TP53 expression 组蛋白去乙酰化酶（HDAC）抑制剂西达本胺联合维奈托克通过下调 MYC 、 BCL2 和 TP53 的表达协同抑制弥漫性大B细胞淋巴瘤的生长 1 2 first-author 1 2 3 1 2 corresp Li YU, ndefy02021@ncu.edu.cn Li YU, ndefy02021@ncu.edu.cn ndefy02021@ncu.edu.cn https://orcid.org/0000-0001-5307-8782 Department of Hematology, the Second Affiliated Hospital of Nanchang University, Nanchang 330006, China Department of Hematology, the Second Affiliated Hospital of Nanchang University, Nanchang 330006, China 南昌大学第二附属医院血液科，中国南昌，330006 南昌大学第二附属医院血液科，中国南昌，330006 Jiangxi Province Key Laboratory of Hematology, Nanchang 330006, China Jiangxi Province Key Laboratory of Hematology, Nanchang 330006, China 江西省血液重点实验室，中国南昌，330006 江西省血液重点实验室，中国南昌，330006 Department of Hematopathology, Duke University School of Medicine, Duke University Medical Center, Durham, NC 27710, USA Department of Hematopathology, Duke University School of Medicine, Duke University Medical Center, Durham, NC 27710, USA 美国杜克大学医学院，美国达勒姆，NC 27710 美国杜克大学医学院，美国达勒姆，NC 27710 Diffuse large B-cell lymphoma (DLBCL) is an aggressive type of non-Hodgkin’s lymphoma. A total of 10%‒15% of DLBCL cases are associated with myelocytomatosis viral oncogene homolog ( MYC ) and/or B-cell lymphoma-2 ( BCL2 ) translocation or amplification. BCL2 inhibitors have potent anti-tumor effects in DLBCL; however, resistance can be acquired through up-regulation of alternative anti-apoptotic proteins. The histone deacetylase (HDAC) inhibitor chidamide can induce BIM expression, leading to apoptosis of lymphoma cells with good efficacy in refractory recurrent DLBCL. In this study, the synergistic mechanism of chidamide and venetoclax in DLBCL was determined through in vitro and in vivo models. We found that combination therapy significantly reduced the protein levels of MYC, TP53, and BCL2 in activated apoptotic-related pathways in DLBCL cells by increasing BIM levels and inducing cell apoptosis. Moreover, combination therapy regulated expression of multiple transcriptomes in DLBCL cells, involving apoptosis, cell cycle, phosphorylation, and other biological processes, and significantly inhibited tumor growth in DLBCL-bearing xenograft mice. Taken together, these findings verify the in vivo therapeutic potential of chidamide and venetoclax combination therapy in DLBCL, warranting pre-clinical trials for patients with DLBCL. 目的： 2 探讨表观遗传学组蛋白去乙酰化酶（HDAC）抑制剂西达本胺与BCL2抑制剂维奈托克的联合对弥漫性大B细胞淋巴瘤（DLBCL）生长的影响及相关机制。 创新点： 2 本研究首次探索了将西达本胺和维奈托克联合作用于 MYC + / BCL2 + DLBCL，使用二代测序（NGS）开创性地从表观遗传学和基因蛋白层面探讨这种联合用药的效果及作用机制。 方法： 2 利用生物信息学技术分析表观遗传学HDAC基因与BCL2基因之间的相关性；体外应用DHL细胞株DB（ MYC / BCL2 重排）和DEL细胞株SUDHL-4（ MYC 、 BCL2 表达）分别进行单药和联合用药处理，通过CCK-8法检测细胞活力，流式细胞术检测细胞凋亡和周期，RNA测序和蛋白质印迹（western blot）检测 MYC 、 BCL2 、 TP53 等相关基因的mRNA水平及蛋白的表达；体内建立DLBCL异种移植小鼠模型进行单药和联合用药治疗，分析并评价药物治疗后皮下瘤的大小和病理切片。 结论： 2 西达本胺与维奈托克协同抑制DLBCL的生长，通过调控表观遗传的改变，沉默 MYC 、 TP53 的表达，降低抗凋亡蛋白BCL2表达以及增加促凋亡蛋白BIM表达，诱导肿瘤细胞的凋亡和细胞周期阻滞。 Diffuse large B-cell lymphoma (DLBCL) Histone deacetylase (HDAC) inhibitor Venetoclax MYC BCL2 TP53 弥漫性大B细胞淋巴瘤（DLBCL） MYC BCL2 TP53 维奈托克 西达本胺 1 Introduction 1 Diffuse large B-cell lymphoma (DLBCL) is a subtype of heterogeneous and aggressive non-Hodgkin's lymphoma ( Li et al., 2018 Li et al., 2018 ). Myelocytomatosis viral oncogene homolog (MYC) and B-cell lymphoma-2(BCL2) proteins are up-regulated in 20%‍‒‍30% of DLBCL cases, irrespective of the MYC and BCL2 chromosomal rearrangement. This is called double-expressed lymphoma (DEL) and has a poor prognosis ( Swerdlowet al., 2016 Swerdlow et al., 2016 ; Riedell and Smith, 2018 Riedell and Smith, 2018 ). In addition, based on genetic and proteomics studies, 7%‍‒‍10% of DLBCLs harbor MYC , BCL2 , and/or BCL6 rearrangements, and are known as double-hit lymphoma (DHL) or triple-hit lymphoma ( Sarkozy et al., 2015 Sarkozy et al., 2015 ; Burotto et al., 2016 Burotto et al., 2016 ; Rosenthal and Younes, 2017 Rosenthal and Younes, 2017 ). The characteristics of DEL and DHL are rapid clinical progression that is refractory to aggressive treatment and has poor outcome following standard R-CHOP (rituximab plus cyclophosphamide, doxorubicin vincristine, and prednisone) therapy ( Nowakowski et al., 2016 Nowakowski et al., 2016 ; Friedberg, 2017 Friedberg, 2017 ). Therefore, current therapeutic outcomes are not ideal, and development of new targeted therapies is imperative. In recent years, there have been several novel studies on the treatment of DLBCL, using approaches such as bispecific chimeric antigen receptor (CAR)-T cell therapy, or monoclonal antibodies like tafasitamab and loncastuximab tesirine to target specific cell-surface antigens for direct cytotoxic activity, or altering immune-mediated mechanisms ( Huang et al., 2020 Huang et al., 2020 ; Patriarca and Gaidano, 2021 Patriarca and Gaidano, 2021 ). It is also worth noting that some new small-molecule inhibitors have shown promise as treatments for DLBCL. For example, a recent two-part phase-I/II trial of a Bruton's tyrosine kinase (BTK) inhibitor in patients with relapsed/refractory (R/R) B cell malignancies, including DLBCL, showed an overall response rate (ORR) of 50% and a disease control rate of 78%; and two patients achieved a complete response (CR). Tumorigenesis factors of hematological tumor diseases include genetic aberrations. For instance, B- or T-cell lymphoma occurrence is reportedly driven by epigenetic disorders ( Rodríguez-Paredes and Esteller, 2011 Rodríguez-Paredes and Esteller, 2011 ; Sermeret al., 2019 Sermer et al., 2019 ). DLBCL pathogenesis is also strongly related to epigenetic perturbations, and high epigenomic heterogeneity correlates with a higher relapse rate and poorer outcome ( Pan et al., 2015 Pan et al., 2015 ; Morin et al., 2016 Morin et al., 2016 ). Nevertheless, histone deacetylase (HDAC) has been indicated as a key enzyme in epigenetic regulation. Previous studies have shown that HDAC inhibitors are effective in treating refractory relapsed DLBCL by reducing expression of c-Myc and increasing phosphorylation of TP53 ( Heideman et al., 2013 Heideman et al., 2013 ; Santoro et al., 2013 Santoro et al., 2013 ; Adams et al., 2016 Adams et al., 2016 ). Specifically, chidamide, an oral HDAC inhibitor, selectively inhibited HDAC1‒3 and HDAC10 ( Ning et al., 2012 Ning et al., 2012 ; Xu et al., 2017 Xu et al., 2017 ), and has been approved for treatment of R/R peripheral T-cell lymphoma ( Chan et al., 2017 Chan et al., 2017 ; Shi et al., 2017 Shi et al., 2017 ). Furthermore, both in vitro and in vivo studies have shown that chidamide has good antitumor effects on lymphoma, leukemia, and multiple myeloma ( Liet al., 2017 Li et al., 2017 ; Yuan et al., 2019 Yuan et al., 2019 ). Targeting anti-apoptotic pathways involving BCL2 family proteins represents a treatment strategy for hematologic malignancies ( Hata et al., 2015 Hata et al., 2015 ). BCL2 is an anti-apoptotic protein and an important determinant of cell survival in various hematological malignancies ( Hata et al., 2015 Hata et al., 2015 ; Croce and Reed, 2016 Croce and Reed, 2016 ; Perini et al., 2018 Perini et al., 2018 ). The oral treatment venetoclax (ABT-199), a highly selective small-molecule BH3 mimetic with an even greater affinity for BCL2 but a lower affinity for (BCL-XL, has shown clinical potential in hematologic malignancies ( Souers et al., 2013 Souers et al., 2013 ; Vandenberg and Cory, 2013 Vandenberg and Cory, 2013 ). However, monotherapy has rarely been sufficient to provide sustained therapeutic responses. As BCL2 overexpression is synergistic with MYC and other oncogenes in promoting both progression of lymphoma and resistance to chemotherapy, targeting these genes along with epigenetic regulation might have therapeutic potential ( Berendsen et al., 2020 Berendsen et al., 2020 ). In this study, we sought to evaluate potential anti-tumor synergistic effects of a regimen combining chidamide with ABT-199 in DLBCL. 2 Materials and methods 1 2.1 Data acquisition and processing 2 Data of 47 lymph node samples from patients with DLBCL were obtained from The Cancer Genome Atlas (TCGA) database ( https://portal.gdc.cancer.gov https://portal.gdc.cancer.gov ), and the whole-blood-cell sample data of 337 healthy subjects were obtained from the Genotype-Tissue Expression (GTEx) database ( https://commonfund. https://commonfund. nih.gov/GTEx). The datasets were matched using the "SVA" package in R software (Version 3.61) and batch effects were removed. The "limma" package was used to analyze the differences between the two datasets, and |log 2 (fold-change)|≥1 with P < 0.05 was used to determine the significant expression changes in BCL2 and HDAC genes. We used the "clusterProfiler" package for gene function enrichment analysis and pathway enrichment analysis of these genes, and conducted correlation analysis of BCL2 and HDAC family genes. B-cell receptor (BCR) pathway genes were obtained from the gene set enrichment analysis (GSEA) website ( http://www. http://www. ‍gsea-msigdb.org/gsea/index.jsp). DLBCL-related data were selected in cBioPortal ( http://cbioportal.org http://cbioportal.org ), and the gene mutation option was selected from the sub item column to view the mutation of the target gene. 2.2 Cell lines and culture 2 The DLBCL cell line SUDHL-4 ( MYC / BCL2 positive in DLBCL cells with wild type (WT)- TP53 ) (Figs. S1a–S1c) was kindly provided by the Stem Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The DLBCL cell line DB ( MYC/BCL2 -rearranged DHL cells with mutant (MUT)- TP53 ) (Figs. S1d–S1f) was purchased from Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China). Both cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 complete medium at 37 ℃ in a 5% CO 2 humidified incubator, authenticated by short tandem repeat (STR) profiling, and tested for mycoplasma contamination. 2.3 Cell viability assay 2 Cells were inoculated into 96-well plates and treated with the designated drug, and cell counting kit-8 (CCK-8; Dojindo, Kumamoto, Japan) was added after 24, 48, or 72 h of action. The absorbance was read at 450 nm with a microplate analyzer (Thermo Fisher Scientific, USA). 2.4 Flow cytometry 2 Cells were inoculated into six-well plates and cultured as mentioned above. Cells were then collected and stained for either Annexin V or prodium iodide (PI) (eBioscience, San Diego, CA, USA) according to the manufacturer's instructions. Then, the cells were placed on a flow cytometer (BD FACSCanto, Franklin Lakes, NJ, USA) for detection of apoptosis. 2.5 Western blotting 2 Cells were inoculated into a culture flask and treated with the designated drug for 72 h. Cells were collected, total proteins were extracted using cell lysates (Sigma-Aldrich, St. Louis, MO, USA), and samples were quantified using a bicinchoninic acid (BCA) kit (ServiceBio, Wuhan, China). The same amount of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The PVDF membrane was sealed with 5% (0.05 g/mL) skim milk and incubated overnight with a primary antibody at 4 ℃. Following the wash steps, the PVDF membrane was incubated with a secondary antibody for 70 min and finally detected with a chemiluminescence substrate. 2.6 RNA sequencing analysis 2 The SUDHL-4 and DB cell lines were treated with drugs for 72 h, and then divided into the following groups: control, chidamide single-drug, venetoclax single-drug, and combination. We collected three biological replicates of RNA samples, and used total RNA for RNA sequencing (RNA-seq) analysis. Construction and sequencing of the complementary DNA (cDNA) library were conducted by the Wuhan Medical Laboratory of BGI using the DNBseq platform. The Bowtie 2 tool was used to compare the high-quality reads with the human reference genome (GRCh38). Using the expected maximization RNA-seq, the expression of each gene was normalized to the fragments per kilobase of transcript per million mapped reads (FPKM) exon model. The BGI bioinformatics service ( https://biosys. https://biosys. ‍bgi.‍com) used the combination of log 2 (ratio absolute value)≥1 and P ≤0.05 to determine the significance of the differentially expressed genes (DEGs). 2.7 Xenograft mouse model of human DLBCL 2 This study used six-week-old female BALB/c nude mice (purchased from Hunan SJA Laboratory Animal Co., Ltd., China). Ethical approval for this study was obtained from the Medical Research Ethics Committee of the Second Affiliated Hospital of Nanchang University. Mice were subcutaneously inoculated with 1×10 7 SUDHL-4 cells on the right dorsal side and monitored daily. After two weeks, tumor-bearing mice (tumor size > 100 mm 3 ) were randomly divided into four groups (five mice per group) receiving daily oral treatments: (1) control group (dimethylsulfoxide (DMSO), distilled water); (2) chidamide group (15 mg/kg); (3) venetoclax group (50 mg/kg); (4) combination group (15 mg/kg chidamide and 50 mg/kg venetoclax). Tumor size was measured daily to monitor treatment effectiveness. The mice were sacrificed on Day 21 of treatment and the tumors collected for further analysis. 2.8 Immunohistochemistry 2 The biopsy specimens of DLBCL xenograft mice were fixed with formalin, embedded with paraffin, and resected (thickness=4 μm). The slices were dewaxed in xylene and rehydrated into water by gradient ethanol. All sections were treated with 5 mmol/L citrate buffer (pH=6.0) for anti-genic repair and treated with 3% (volume fraction) H 2 O 2 to inactivate endogenous peroxidase. After sealing for 30 min, sections and primary antibodies were incubated overnight at 4 ℃. After washing, sections were stained with secondary antibodies at 25 ℃ for 30 min. Diaminobenzidine and hematoxylin were used as either chromogenic substrates or for nuclear complex staining. 2.9 Statistical analysis 2 Data are presented as mean±standard deviation (SD) or median (quartile intervals). GraphPad Prism (Version 8.0) was used for statistical analysis, processing of image data, and calculating half maximal inhibitory concentration (IC 50 ) data. Student's t -test was used for pair-wise comparison between groups, and one-way analysis of variance (ANOVA) was used for multivariate mean comparison. CompuSyn (Version 3.0.1) was used to calculate the dose–effect curves and generate the combination index (CI). P < 0.05 was considered statistically significant. 3 Results 1 3.1 Correlation between BCL2 and HDAC in DLBCL 2 By comparing 47 lymph-node sample data from patients with DLBCL with 337 normal whole-blood B-lymphocyte sample data, we obtained 2408 down-regulated and 2601 up-regulated genes ( Fig. 1 Fig. 1 a). Expression of HDAC4 , HDAC5 , and HDAC10 decreased in lymphoma, while expression of HDAC1 , HDAC7 , HDAC9 , and BCL2 increased in lymphoma tissue (Figs. 1 1 a and 1 1 b). The Gene Ontology (GO) enrichment analysis of the seven genes above had shown that these genes were closely related to the process of histone deacetylation ( Fig. 1 Fig. 1 c). By using cBioPortal, we were able to find high mutation rates (including somatic mutations, DNA copy number alterations, and gene abundance changes) for BCL2 , HDAC4 , HDAC7 , HDAC9 , and HDAC10 in the DLBCL samples ( Fig. 1 Fig. 1 d). Finally, the correlation analysis of the seven genes showed that HDAC1 , HDAC7 , HDAC9 , and HDAC10 were strongly correlated with BCL2 ( Fig. 1 Fig. 1 e). 3.2 Effects of chidamide and venetoclax on proliferation of DLBCL cells 2 We examined the sensitivity of chidamide and venetoclax in DLBCL cell lines. First, we assessed the effects of different doses of chidamide or venetoclax monotherapy on SUDHL-4 and DB cells. The single-dose agents showed dose- and time-dependent proliferative inhibitory effects on both cell lines ( Fig. 2 Fig. 2 a). Chidamide had the maximum inhibitory effect on both cell lines at 48 and 72 h, while venetoclax's maximum inhibitory effect occurred at 72 h. The IC 50 values were calculated at 24 h ( Fig. 2 Fig. 2 b). Next, we assessed the effect of drug combination on SUDHL-4 and DB cells over 24 h. Based on the Chou-Talalay method for drug combination ( Chou, 2010 Chou, 2010 ), we set constant-ratio drug combinations using two-fold serial dilutions with several concentration points above or below the 24 h IC 50 value. The results indicated that the inhibitory effect of the combination of chidamide with venetoclax was higher than those of the single agents ( Fig. 2 Fig. 2 c). We graphically analyzed the CI value according to the Chou-Talalay method, as illustrated in Fig. 2 Fig. 2 c (right). At 50% cell proliferation (fraction affected ( F a )=0.5), the CI values were 0.7 and 0.3 in the SUDHL-4 and DB cell lines, respectively, suggesting strong synergism (CI < 1) of the two drugs. 3.3 Effects of combination of chidamide and venetoclax on apoptosis in DLBCL cells and cell cycle at the G0/G1 phase 2 To further explore the synergistic effects of drug combinations, we first conducted apoptosis detection after combined drug treatments. As shown in Figs. 3 3 a and 4 4 a, SUDHL-4 cells were treated with chidamide (2 μmol/L) and venetoclax (0.5 μmol/L) for 24, 48, and 72 h; DB cells were treated with chidamide (5 μmol/L) and venetoclax (0.05 μmol/L) for 24, 48, and 72 h; and DMSO was used as the negative control. Either drug on its own could induce apoptosis of SUDHL-4 and DB cells, but the rate of apoptosis induced by either chidamide or venetoclax was significantly lower than that induced by a combination of the two drugs ( P < 0.05; Figs. 3 3 b and 4 4 b). Next, we exposed the corresponding cell lines to the same drug concentration as in the apoptosis assay for 72 h, followed by flow cytometry detection for cell-cycle distribution. The results showed that the combination of the two drugs could block progression of the DLBCL cell cycle in the G0/G1 phase (Figs. 3 3 c, 3 3 d, 4 4 c, and 4d). 3.4 Effects of chidamide on transcriptomes in multiple genes and signaling pathways in DLBCL cells, including MYC , BCL2 , and HDAC mRNA 2 The acetylated state of histones can affect gene silencing and expression; therefore, we measured transcriptome levels. As shown in Fig. 5 Fig. 5 a, for DB cells, chidamide regulated 6809 genes, venetoclax only 15 genes, and the combination group, a total of 6646 genes. For SUDHL-4 cells, chidamide regulated 4272 genes, venetoclax regulated 1041 genes, and the combination group, a total of 5200 genes. Therefore, it was evident that regulation of genes in the two DLBCL cell lines was primarily mediated by chidamide. We analyzed the transcription levels of genes in the related signaling pathways, and the results showed that the mRNA levels of MYC , TP53 , HDAC1 , HDAC2 , HDAC6 , HDAC7 , and HDAC10 in DB cells were down-regulated in the combination group, while the mRNA levels of HDAC3 , HDAC4 , HDAC5 , HDAC9 , and membrane spanning 4-domains A1 ( MS4A1 , CD20 ) were up-regulated ( Fig. 5 Fig. 5 b). For SUDHL-4 cells, the mRNA levels of TP53 , HDAC3 , and HDAC6 were down-regulated, while the mRNA levels of HDAC2 , HDAC4 , HDAC9 , HDAC11 , and MS4A1 were up-regulated in the combination group. The drug combination thus had different effects on HDAC subtypes and showed inconsistency in its regulation of MYC and TP53 between the two cell types. GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the DEGs between the combined and control groups showed that most DEGs were concentrated in phosphorylation modification, cell cycle, immune regulation, apoptosis, DNA damage repair, and chromatin remodeling (Figs. 5 5 c and 5 5 d). These results suggested that chidamide regulates a variety of biological processes in DLBCL cells together with venetoclax, and thus produces a series of anti-tumor effects. We obtained 75 BCR pathway genes from the GSEA website and constructed a heatmap to describe the mRNA levels of these genes after different treatments in DB and SUDHL-4 cells (Fig. S2). The results showed that expression of key genes ( BTK , phosphatidylinositol-3-kinase ( PI3K ), spleen tyrosine kinase ( SYK ), and protein kinase B ( AKT )) of the BCR pathway was down-regulated in the combination group. This indicated that the combined use of chidamide and venetoclax may play a therapeutic role in DLBCL by inhibiting the BCR pathway. 3.5 Effects of chidamide on c-Myc and TP53 proteins in DLBCL 2 To determine the changes in MYC, BCL2, and TP53 protein levels in response to chidamide, SUDHL-4 and DB cell lines were treated with chidamide and venetoclax for 72 h. We observed a consistent decrease in c-Myc protein levels across the two cell lines evaluated, suggesting that chidamide suppressed MYC regardless of MYC rearrangement status ( Fig. 6 Fig. 6 a). Simultaneously, the combination of chidamide and venetoclax significantly reduced the level of TP53 protein and significantly increased levels of acetyl-histone-H3 and -H4 proteins ( Fig. 6 Fig. 6 b). The BCL2 protein level in the combination group also decreased, while that of the pro-apoptotic protein BIM increased. Furthermore, the increased ratio of BCL2/BIM protein levels is one of the mechanisms of drug resistance when using BCL2 inhibitors ( Niuet al., 2016 Niu et al . , 2016 ). Therefore, we speculated that the combination of chidamide and venetoclax could reduce the occurrence of BCL2 inhibitor resistance by increasing levels of pro-apoptotic proteins. 3.6 Effects of chidamide and venetoclax on tumor growth in the DLBCL xenograft mouse model 2 To determine whether chidamide enhances venetoclax-mediated DLBCL tumor growth inhibition in vivo, we inoculated six-week-old female BALB/c nude mice with human DLBCL SUDHL-4 cells. Two weeks later, tumor-bearing xenograft mice were treated daily with 15 mg/kg chidamide and/or 50 mg/kg venetoclax for another three weeks. The tumors were collected, and the tumor load was measured ( Fig. 7 Fig. 7 a). Treatment with chidamide or venetoclax alone did not significantly reduce DLBCL tumor size, but the combination of the two drugs significantly inhibited tumor growth ( Fig. 7 Fig. 7 b). Immunohistochemical analysis confirmed that co-treatment with venetoclax and chidamide significantly increased levels of acetyl-histone-H3 in mouse DLBCL tissues, decreased BCL2 protein levels, and increased CD20 levels ( Fig. 7 Fig. 7 c). Taken together, these results indicated that chidamide enhanced venetoclax activity in the DLBCL xenograft mouse model in vivo. 4 Discussion 1 In this study, chidamide, a novel HDAC inhibitor, increased the level of BIM, decreased expression of c-Myc and TP53 , and significantly synergized venetoclax-induced tumor-growth inhibition. Evidently, we identified a synergistic effect of chidamide and venetoclax in the treatment of DLBCL in vivo and in vitro, which effectively controls the growth of DLBCL. Our biological information analysis showed that expression of the epigenetic HDAC genes was increased in lymphoma tissues, and was strongly correlated with BCL2 expression. Meanwhile, the mutation rates of BCL2 , HDAC7 , and HDAC10 in DLBCL tissues reached 21%, 19%, and 10%, respectively. HDACs as epigenetic regulators can regulate the histone tail, chromatin conformation, transcription, and protein–DNA interaction. It is known that a direct effect on chromatin involves pro-apoptotic BCL2 family activation ( Matthewset al., 2012 Matthews et al., 2012 ; Staziet al., 2019 Stazi et al., 2019 ). Therefore, these data suggest that dual inhibition of BCL2 and HDAC might provide an effective strategy for cancer treatment. Rearrangement of MYC and BCL2 is a hallmark of DHL and leads to an increase in c-Myc and BCL2 protein levels. c-Myc is a transcription factor involved in regulation of the cell cycle, DNA damage repair, metabolism, protein synthesis, and other processes ( Baluapuriet al., 2020 Baluapuri et al., 2020 ; Duffy et al., 2021 Duffy et al., 2021 ). In this study, SUDHL-4 was used as a model of DEL DLBCL cells with WT-TP53 , without MYC / BCL2 chromosome rearrangement, whereas DB was used as a model that represented MYC / BCL2 -rearranged DHL cells with MUT-TP53 . Both cell lines were of the germinal center B-cell-like lymphoma (GCB) subtype. Li et al. (2019) Li et al. (2019) showed that targeting epigenetic regions is effective in reducing MYC expression, i.e., bromodomain and extra-terminal domain (BET) inhibitors can mitigate the effects of MYC overexpression by blocking cell–signal transduction. Furthermore, HDAC inhibitors can reduce c-Myc expression by affecting the chromatin structure of MYC ( Nebbioso A et al., 2017 Nebbioso A et al., 2017 ; Eckeret al., 2021 Ecker et al . , 2021 ). MYC rearrangement status may lead to stronger cell tolerance and drug sensitivity. Our experiments also confirmed that DB cells with MYC / BCL2 rearrangement were less sensitive to chidamide than SUDHL-4 cells. However, when SUDHL-4 and DB cells were treated with a combination of chidamide and venetoclax, the inhibition rate of cell proliferation was higher than those in the single-drug groups ( P < 0.05). Although different MYC states can lead to different drug sensitivities in cells, our synergistic indices (all CI < 1) indicated that the combination of chidamide and venetoclax had synergistic effects on proliferation inhibition of both DLBCL cell types. HDAC inhibitors have been employed to reverse post-translational deacetylation of cancer cells. Studies have also shown that HDAC inhibitors reduce expression of c-Myc and TP53 , and subsequently arrest the cell cycle in the G0/G1 phase by influencing the cyclin pathway, while promoting levels of the BH3-only proteins BIM and BCL2-binding component 3 (PUMA) ( Muthalaguet al., 2014 Muthalagu et al., 2014 ; Mrakovcic et al., 2019 Mrakovcic et al., 2019 ). In this study, both the transcription and translation processes of the oncoproteins c-Myc and TP53 were reduced by the combined chidamide and venetoclax treatment. For BCL2, it appears that the inhibition occurred on the post-translational level. Studies have shown that anti-apoptotic protein overexpression represents a key compensatory factor for adaptive resistance to ABT-199; for example, Mcl-1 can act to re-sequester BIM released from BCL2 ( Kapooret al., 2020 Kapoor et al., 2020 ; Hafezi and Rahmani, 2021 Hafezi and Rahmani, 2021 ). HDAC inhibitors may reverse the ratio of anti-apoptotic to pro-apoptotic proteins by increasing the level of BIM and down-regulating BCL2 or Mcl-1 ( Gong et al., 2019 Gong et al., 2019 ; Chen et al., 2020 Chen et al., 2020 ; Laszig et al., 2020 Laszig et al., 2020 ). This information inspired us to speculate that chidamide could affect post-transcriptional modifications to affect the expression of BCL2 protein. Venetoclax has a high affinity for BCL2, and can displace BCL2-bound pro-apoptotic proteins such as BIM, BCL2-associated X (BAX), and BCL2 antagonist/killer (BAK), leading to permeabilization of the mitochondrial outer membrane, pro-apoptotic cytochrome c release, and caspase activation, and eventually resulting in apoptosis ( Bhola and Letai, 2016 Bhola and Letai, 2016 ). Mechanistically, the synergy between HDAC inhibitors and venetoclax induced cellular apoptosis through down-regulation of gene expression ( MYC / TP53 ) and the anti-apoptotic signaling pathway, as well as up-regulation of the pro-apoptotic protein BIM in DLBCL cells ( Fig. 8 Fig. 8 ). HDAC mainly induces cell differentiation, growth arrest, apoptosis, senescence, suppression of the immune system, and angiogenesis ( Wang P et al., 2020 Wang P et al., 2020 ; Wang XG et al., 2020 Wang XG et al., 2020 ). We found down-regulated mRNA levels of TP53 , HDAC6 , and HDAC7 in the combination group and the chidamide group . The biological processes were analyzed by GO enrichment, and the results showed that the combination of chidamide and venetoclax affected cell phosphorylation, apoptosis, cell cycle, immune system, and DNA damage repair. In addition, compared with the untreated controls, the combination group showed better anti-tumor effects in the tumor-bearing nude mice. The IHC staining results showed that levels of BCL2 were decreased while those of acetylated histones H3 and CD20 were increased in the combination group. Guan et al. (2020) Guan et al. (2020) previously demonstrated that HDAC can mediate gene silencing to decrease CD20 expression, and use of the HDAC inhibitor chidamide can up-regulate CD20 gene expression. Moreover, several in vivo and in vitro trials of DLBCL treated with combinations of HDAC inhibitors and rituximab (targeting CD20) have shown good anti-tumor effects ( Shimizuet al., 2010 Shimizu et al., 2010 ; Bobrowicz et al., 2017 Bobrowicz et al., 2017 ). Notably, studies have revealed that the combination of HDACs and small-molecule inhibitors, such as PI3K inhibitors, can achieve a better ORR and median response duration in patients with DLBCL than single small-molecule inhibitors ( Patriarca and Gaidano, 2021 Patriarca and Gaidano, 2021 ). Therefore, combination therapy with small-molecule inhibitors, especially epigenetic inhibitors, could be a more effective way to treat DLBCL in the future. 5 Conclusions 1 In summary, our study determined the synergistic anti-tumor effect of a combination regimen of the epigenetic modulator chidamide and the BCL2 inhibitor venetoclax in DLBCL. This synergistic effect was also observed in DHL cells with MYC / BCL2 rearrangement. The combination of chidamide and venetoclax simultaneously regulates epigenetic changes and affects the expression of genes such as MYC , TP53 , and BCL2 to achieve highly effective and precise therapeutic effects. Our data strongly supports the potential therapeutic effect of epigenetic modulators with secondary agents in DLBCL, and possibly in patients with DHL. Therefore, a pre-clinical trial using a combination of HDAC and BCL2 inhibitors for treating patients with DLBCL could be beneficial. References Adams CM , Hiebert SW , Eischen CM , 2016 . 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Drugs Today , 53 ( 3 ): 167 - 176 . https://doi.org/10.1358/dot.2017.53.3.2595452 https://doi.org/10.1358/dot.2017.53.3.2595452 Chidamide tablets: HDAC inhibition to treat lymphoma Yuan XG , Huang YR , Yu T , et al. , 2019 . Chidamide, a histone deacetylase inhibitor, induces growth arrest and apoptosis in multiple myeloma cells in a caspase-dependent manner . Oncol Lett , 18 ( 1 ): 411 - 419 . https://doi.org/10.3892/ol.2019.10301 https://doi.org/10.3892/ol.2019.10301 Chidamide, a histone deacetylase inhibitor, induces growth arrest and apoptosis in multiple myeloma cells in a caspase-dependent manner Cancan LUO performed research design, the experimental research, and data analysis, wrote and edited the manuscript. Tiantian YU performed the experimental research and data analysis. Ken H. YOUNG performed the research design and data analysist. Li YU performed research design and data analysis, wrote and edited the manuscript. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and securityof the data. This work was supported by the National Natural Science Foundation of China (Nos. 81460030 and 81770221). 10.1631/jzus.B2200016 1673-1581（2022）08-0666-16 This work was supported by the National Natural Science Foundation of China (Nos. 81460030 and 81770221). 2022-01-11 2022-04-10 2022-08-15 2022-12-27 2022-06-02 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2022 浙江大学出版社 Cancan LUO, Tiantian YU, Ken H. YOUNG, and Li YU declare that they have no conflict of interest. This study was carried out in accordance with the recommendations of the Ethics Committee of the Second Affiliated Hospital of Nanchang University. The protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 8 23