BRD4 inhibitors kill ABC-DLBCL cells by targeting BTK*

FAN Zhi-wei，WU Yuan-yuan

（Laboratory of Basic Medicine，School of Medicine，Nantong University，Nantong 226001，China.E-mail：wyy84@ntu.edu.cn）

［ABSTRACT］AIM：To investigate the effect of bromodomain-containing protein 4（BRD4）inhibitors on the viability and apoptosis of activated B cell-like diffuse large B-cell lymphoma（ABC-DLBCL）cells and the molecular mechanism.METHODS：The ABC-DLBCL cells were treated with BRD4 inhibitors JQ1 and I-BET-762，and Bruton tyrosine kinase（BTK）inhibitor ibrutinib.The viability and death of the cells were determined by CCK-8 assay and PI staining，respectively.The mRNA levels of BTK，phospholipase Cγ（PLCγ），LYN，SYK，interleukin-6（IL-6），MYC，protein kinase Cβ（PKCβ），mucosa-associated lymphoid tissue lymphoma translocation protein 1（MALT1），MYC and RELA were detected by real-time PCR.The protein levels of BTK，PLCγ，MYC and RELA were determined by Western blot.Superenhancer around BTK gene was revealed by bioinformatics analysis.RESULTS：The ABC-DLBCL cells were sensitive to BRD4/super-enhancer inhibitors such as JQ1 and I-BET-762.Both JQ1 and I-BET-762 inhibited the chronic active B-cell receptor（BCR）/nuclear factor κB（NFκB）signaling through reducing the transcription of BTK，but they had minimal effect on other components in BCR/NFκB signaling.Interestingly，there was no super-enhancer around BTK gene，and the inhibitory effect of JQ1 was likely due to disruption of BRD4 binding within BTK gene.Inhibition of BRD4 had synergic effect with BTK inhibitor ibrutinib.Moreover，inhibition of BRD4 induced significant cell death in ibrutinib-resistant ABCDLBCL cells.CONCLUSION：Inhibitors of BRD4 induce ABC-DLBCL cell death via blocking BCR/NFκB signaling and has synergic effect with BTK inhibitor.Inhibition of BRD4 might be a promising strategy for treatment of ABC-DLBCL，especially ibrutinib-resistant ABC-DLBCL.

［KEY WORDS］Activated B cell-like diffuse large B-cell lymphoma；Bromodomain-containing protein 4；Bruton tyrosine kinase；Super-enhancers；BCR/NFκB signaling

Patients with diffuse large B-cell lymphoma（DLBCL）account for more than 30% of all lymphoma cases and thus DLBCL becomes the most common lymphoma［1-2］.The DLBCL incidence is growing，which ranks the 8th in tumor incidence in urban and the 10th in rural［3］.DLBCL is a heterogeneous disease，but based on molecular signature and morphologic description，it can be subdivided into two subtypes that display distinct response and prognosis to current standard chemoimmunotherapy［4-6］.One subtype is the germinal center Bcell-like DLBCL（GCB-DLBCL）originating from centroblasts in the dark zone and shows good prognosis［7-8］.However，another subtype called activated B-cell-like DLBCL（ABC-DLBCL）derived from activated B cells demonstrates poor prognosis.The 3-year overall survival rate of the patients with ABC-DLBCL is only about 45%［7-8］.In all patients with DLBCL，about 40% of patients belong to ABC subtype，which is a major barrier to cure DLBCL.It is clear that novel therapeutic targets for ABC-DLBCL are urgently needed［9］.

In the past two decades，genomic and biological investigations in ABC-DLBCL uncover that inappropriate nuclear factor κB（NFκB）activation is required for ABC-DLBCL survival and therefore becomes druggable molecular feature for ABC-DLBCL therapy［10-11］.From that time，different labs found that many molecules or pathways such as CARD11/MALT1/BCL10 complex，MYD88 signaling，CD79-dependent B-cell receptor（BCR）signaling and TNFAIP3 are frequently kidnapped in ABC-DLBCL to induce chronic NFκB activation［12-14］.To extirpate such aggressive ABC-DLBCL，multiple strategies targeting abnormal NFκB activation have been developed.Several of them are under extensive studying and demonstrate promise outcomes［15-16］.The Bruton tyrosine kinase（BTK）inhibitor，ibrutinib，is one of the most promising drug for ABC-DLBCL［17-19］.Based on the data from a phase 1/2 clinical trial，about 40% of patients with relapsed or refractory DLBCL response to ibrutinib［17］.According to this finding，a randomized phase III study of ibrutinib with R-CHOP treatment including 841 patients is ongoing and expected to collect primary outcome soon.However，a large part of relapsed or refractory patients with ABC-DLBCL（about 60%）are still unresponsive to ibrutinib.Furthermore，emerging resistant mechanisms that bypass therapeutic targetBTKare often developed［17］.Therefore，more powerful drugs that can extend the therapeutic window of ibrutinib or overcome ibrutinib-resistance are very helpful.

The bromodomain and extra terminal domain（BET）family contains 4 members including bromodomain-containing protein 2（BRD2），BRD3，BRD4 and BRDT.All the members of BET family are epigenetic readers to recognize acetylatedlysine residues.Among them，BRD4 is the most studied member and bromodomain inhibitors targeting BRD4 have great implications in many cancers［16，20-21］.Recently，a small percentage of enhancers enriched with histone H3 lysine 27 acetylation（H3K27Ac）modification and BRD4 binding are defined as super-enhancers，which are extremely sensitive to BRD4 inhibitors such as JQ1［16］.Recent works have demonstrated that DLBCL shows super-enhancer dependency and is sensitive to JQ1，a BRD4 as well as super-enhancers inhibitor［16，22］.More specifically，JQ1 could block oncogenic IκB kinase（IKKβ）activity in ABC-DLBCL and subsequently results in ABC-DLBCL cell death［16，23］.Furthermore，a high-throughput platform to screen drug-drug synergy has identified ibrutinib，a BTK kinase inhibitor，synergized strongly with JQ1 in killing of ABC-DLBCL cells［16］.However，the molecular target of JQ1 upstream of IKKβ is still unknown.It is also mysterious why JQ1 synergized with ibrutinib in ABC-DLBCL cells.

In this study，we found that JQ1 profoundly affectsBTKtranscript with minimal effect on other components in BCR/IKKs signalingin vitro.Interestingly，there is no conspicuous super-enhancer aroundBTKgene.Although JQ1 is not as much as powerful to induce cell death in ABC-DLBCL cells compared with ibrutinib，it efficiently induces cell death in ibrutinib-resistant ABCDLBCL cells such as SU-DHL-2.Our results highlight that BRD4 inhibitors play a critical role in killing ABCDLBCL cells，especially for ibrutinib-resistant ABCDLBCL cells.

MATERIALS AND METHODS

1 Antibodies and reagents

The BRD4 inhibitors JQ1（A1910）and I-BET-762（B1498）were bought from ApexBio.We obtained the BTK inhibitor ibrutinib（PCI-32765；S2680）from Selleck.All inhibitors were dissolved in dimethyl sulfoxide（DMSO）.The antibodies such as anti-BTK（56044S）and anti-PLCG2（3872S）were obtained from Cell Signaling Technology.The antibodies including anti-C-MYC（sc-40）and anti-RELA（sc-8008）were purchased from Santa Cruz Biotechnology.

2 Cell lines and cell culture

Human ABC-DLBCL cell lines，including HBL-1，OCI-Ly10 and SU-DHL-2，GCB-DLBCL cell line BJAB and Burkitt lymphoma cell line Raji were provided by the Chinese Academy of Science Cell Bank.All these cell lines were cultured in RPMI-1640 medium（Hy-Clone）.The RPMI-1640 medium was supplemented with 10%（V/V）of heat-inactivated fetal bovine serum（FBS），1×105U/L of penicillin and 100 mg/L of streptomycin.All above mentioned cells were kept in an incubator containing 5%of CO2at 37℃.

3 Cell viability assay

The viability of the cells was determined using commercial available kit called Cell Counting Kit-8（CCK-8）from Dojindo Laboratories.Cultured cells were harvested and suspended in culture medium at desired concentration.Cells were seeded into 96-well plates at 2×104per well in 200 μL volume.Cells were divided into control and inhibitor treatment groups.In control group，cell lines were incubated with vehicle（DMSO）.In JQ1 treatment groups，HBL-1 and Raji cells were incubated with JQ1 at 0.5 and 1 μmol/L，while Ly10 and BJAB cells were incubated with JQ1 at 0.1，0.2 and 0.5 μmol/L.In I-BET-762 treatment groups，HBL-1，Ly10 and SU-DHL-2 cells were incubated with I-BET-762 at 0.5，1 and 5 μmol/L.In JQ1 and/or ibrutinib treatment groups，SU-DHL-2 cells were incubated with JQ1（0.05，0.1，0.2 and 0.5 μmol/L），ibrutinib（0.005，0.01，0.02 and 0.05 μmol/L）alone or their combinations.At 37℃，cells were continually incubated for 48 h before 20 μL of CCK-8 was added to each well to examine the viability of the cells.In generally，after 2 h of incubation，the microplate reader was used to read the absorbance of medium at 450 nm.The absorbance of cell culture medium without cells was used as background to normalize the result.All CCK-8 assays were done in triplicate，each performed in 4 technical replicates.

4 Propidium iodide（PI）staining assay

Based on experimental requirement，cells were prepared and transferred to cell culture plates.Following designed protocol，HBL-1 cells were treated with vehicle（DMSO），JQ1（0.5 and 1 μmol/L）or I-BET-762（1 and 5 μmol/L）for 48 h，and SU-DHL-2 cells was treated with vehicle（DMSO）or I-BET-762（1 and 5 μmol/L）for 48 h.To identify dead cells，PI staining solution was added to cell culture medium at working concentration of 50 mg/L.The plates were incubated for 15 min at 37℃in the dark and then photographed using Leica microscope（DM3000B）.Unstained cells were used as a negative control.Each experiment was carried out in triplicate.

5 Real-time PCR

According to experimental design，cultured cells were prepared and treated with vehicle（DMSO），JQ1 or I-BET-762 at indicated concentrations for 24 h.The cells were harvested and washed by cold PBS for 3 times.One mL of Trizol（Invitrogen）was added to cell pellets.Following manufacturers′protocols，total RNA was extracted and dissolved in DEPC（diethylpyrocarbonate）-treated water.One μg of total RNA was used for reverse transcription which was achieved using RevertAid?First Strand cDNA Synthesis Kits（Fermentas）and oligo-（dT）primers.The cDNA library was diluted into 10 folds and then kept at 20℃until used for real-time PCR.The real-time PCR amplification was carried out in a total volume of 20 μL of mixture containing SYBR Premix Ex Taq?（TaKaRa）.The PCR conditions：95℃5 min；95℃10 s，60℃30 s.The expression level of GAPDH in each cDNA library was also examined for normalization.The 2-ΔΔCtmethod was used to calculate the relative mRNA expression.The heatmap was generated by Heatmap Illustrator 1.0.3.3 based on the relative mRNA expression.The sequences of the primers are listed in Table 1.Each experiment was carried out in triplicate.

Table 1.The sequences of the primers used in real-time PCR

6 Western blot

HBL-1 and SU-DHL-2 cells were treated with JQ1 or I-BET-762 at desired concentrations.Treated cells were washed with ice cold PBS twice and harvested.RIPA lysis Buffer（P0013B；Beyotime）was added into harvested cell pellets at 4℃for 20 min.The lysate was centrifuged at 12 000 r/min for 20 min and supernatants were transferred to a new tube.The transferred supernatants were mixed with 5×loading buffer and cooked at 100℃for 5 min.Samples were then subjected to SDS-PAGE and transferred to PVDF membranes.The protein containing PVDF membranes were blocked with 5% of milk for 2 h at room temperature，and then the PVDF membranes were washed with new blocking mile and incubated with appropriate diluted primary antibodies at 4℃for one night.After washing，PVDF membranes were further incubated with HRP-conjugated secondary antibodies for 1 h at room temperature.After additional 3 times of washing，the Chemical ECL（enhanced chemiluminescence）Western Blotting Substrate（Tanon）was added to PVDF membranes and the signals were visualized based on protocols.Each experiment was carried out in triplicate.

7 Statistical analysis

The identification of super-enhancers was based on published standard protocols，in which highly acetylated chromatin segments within 12.5 kb of one another can be stitched together［24］.All values were presented as mean±SD（standard deviation）.Statistical significance was determined by two-tailed Student′st-test（comparing two groups）or one-way ANOVA（comparing more than two groups）.AP-value＜0.05 was considered statistically significant.

RESULTS

1 BRD4 inhibitor JQ1 efficiently promoted ABCDLBCL cell death

The results of CCK-8 assay suggested that JQ1 inhibited Raji cell proliferation（Figure 1A）.Interestingly，we found that HBL-1 cells were much more sensitive to JQ1 compared with Raji cells（Figure 1A）.Next，we examined cell death by PI staining.JQ1 treatment induced obvious death of HBL-1 cells（Figure 1B）.We sought to assess the mRNA level of IL-6，a well-known inflammation cytokine that frequently regulated by NFκB signaling.The expression of IL-6 was significantly decreased after JQ1 treatment（Figure 1C）.To further confirm this finding，we treated another ABC-DLBCL cell line（Ly10）and a GCB-DLBCL cell line（BJAB）with JQ1 and examined cell viability by CCK-8 assay.Treatment with JQ1 promoted cell death in both Ly10 and BJAB cells，but Ly10 cells were more sensitive to JQ1 than BJAB cells（Figures 1D and 1E）.The above results revealed that JQ1 significantly promoted ABC-DLBCL cell death.

2 BRD4 inhibitor I-BET-762 also promoted ABCDLBCL cell death

To further demonstrate the importance of BRD4 inhibitors in ABC-DLBCL cells，we used another BRD4 inhibitor I-BET-762，which is already in clinical trials for NUT（nuclear protein in testis）midline carcinoma and hematologic malignancies.Consistent with the finding from JQ1，I-BET-762 treatment could significantly reduce cell viability both in Ly10 and HBL-1 cells（Figure 2A）.The result of PI staining clearly demonstrated that I-BET-762 could induce obvious cell death（Figure 2B）.Taken together，ABC-DLBCL cells are sensitive to BRD4 inhibition.

3 BRD4 inhibition targetd BTK in BCR/NFκB signaling

BCR/NFκB signaling is well established and key components are depicted in Figure 3A.To identify the target（s）of JQ1，we examined transcripts of all known key components in BCR/NFkB signaling using real-time PCR，and found that JQ1 treatment specifically decreased the mRNA level of BTK，while the other components were intact or showed minimal changes（Figure 3B）.Treatment with JQ1 almost completely blocked transcription ofBTKin HBL-1 cells（Figure 3B）.Consistently，JQ1 profoundly down-regulated the protein level of BTK（Figure 3C）.In contrast，JQ1 slightly upregulated PLCG2 protein level（Figure 3C）.We also used I-BET-762 to treat HBL-1 cells.I-BET-762 could significantly down-regulate the transcription ofBTKbut notPLCG2（Figure 3D）.Western blot results also showed that I-BET-762 dramatically down-regulated the protein level of BTK but not PLCG2（Figure 3E）.Together，BRD4 inhibits both mRNA and protein expression of BTK.

Figure 1.JQ1 efficiently promoted ABC-DLBCL cell death.A：CCK-8 assay was used to measure the viability of Raji and HBL-1 cells with or without JQ1 treatment；B：propidium iodide（PI）staining assay was used to examine the death of HBL-1 cells with or without JQ1 treatment（scale bar=100 μm）；C：real-time PCR was used to examine IL-6 mRNA expression in HBL-1 cells treated with or without JQ1；D：CCK-8 assay was used to measure the viability of Ly10 cells with or without JQ1 treatment；E：CCK-8 assay was used to measure the viability of BJAB cells with or without JQ1 treatment.Mean±SD.n=3.*P＜0.05，**P＜0.01 vs 0 μmol/L group.

Figure 2.I-BET-762 efficiently promoted ABC-DLBCL cell death.A：CCK-8 assay was used to measure the viability of HBL-1 and Ly10 cells with or without I-BET-762 treatment；B：PI staining assay was used to examine the death of HBL-1 cells with or without I-BET-762 treatment（scale bar=100 μm）.Mean±SD.n=3.**P＜0.01 vs 0 μmol/L group.

Figure 3.JQ1 and I-BET-762 specifically inhibited BTK expression in BCR/NFκB signaling.A：schematically presentation of key components in BCR/NFκB signaling；B：real-time PCR was used to examine the mRNA expression of key components in BCR/NFκB signaling in HBL-1 cells treated with or without JQ1，and the heatmap was generated based on the relative expression；C：the protein levels of BTK and PLCG2 were analyzed by immunoblotting in HBL-1 cells treated with or without JQ1；D：real-time PCR was used to examine the mRNA expression of BTK and PLCG2 in HBL-1 cells treated with or without I-BET-762；E：the protein levels of BTK and PLCG2 were analyzed by immunoblotting in HBL-1 cells treated with or without I-BET-762.Mean±SD.n=3.*P＜0.05，**P＜0.01 vs 0 μmol/L group.

4 JQ1-induced BTK down-regulation was independent on super-enhancers

In bioinformatics analysis of reported chromatin immunoprecipitation（ChIP）/H3K27Ac data in HBL-1 cells，we did not find super-enhancers aroundBTKgene unexpectedly（Figures 4A and 4B）.We did find super-enhancers aroundPLCG2，CD19andSYKgenes（Figure 4B）.In GCB-DLBCL cell line Ly1，there is no super-enhancer aroundBTKgene based on H3K27Ac modification and BRD4 binding（Figure 4C）.JQ1 treatment did not change the modification of H3K27Ac aroundBTKgene.Interestingly，JQ1 treatment almost completely inhibited the BRD4 binding withinBTKgene（Figure 4C）.These results indicated thatBTKtranscription was independent on super-enhancers.Taken together，JQ1-mediated down-regulation ofBTKexpression is independent on super-enhancers.

5 BRD4 inhibition enhanced ibrutinib-mediated cytotoxicity and overcame ibrutinib resistance

Here，we found BRD4 inhibitors also disrupted the function of BTK by down-regulating its expression（Figure 5A）.Meanwhile，we examined and compared the effect of JQ1 and BTK inhibitor ibrutinib by CCK-8.Ibrutinib markedly reduced HBL-1 cell viability at 5 nmol/L as expected（Figure 5B）.Moreover，JQ1 was not as efficient as ibrutinib（IC50=50 nmol/L），but increasing the dose of JQ1 enhanced its cytotoxicity（Figure 5B）.We found a significant synergic effect between JQ1 and ibrutinib［combination index（CI）value at ED50=0.05；Figure 5C］.Remarkably，JQ1 efficiently induced death of SU-DHL-2 cells，a MYD88-mutant ibrutinib-resistant cell line（Figure 5D）.To confirm this result，we used I-BET-762 to treat SU-DHL-2 cells.Consistently，I-BET-762 significantly induced death of SU-DHL-2 cells（Figure 5E）.The effect of IBET-762 on SU-DHL-2 cells was further verified by PI staining（Figure 5F）.Together，these results indicate that BRD4 inhibition has synergic effect with BTK inhibitor ibrutinib.Furthermore，BRD4 inhibition could overcome ibrutinib resistance.

Figure 4.JQ1 treatment disrupted BRD4 binding within BTK gene.A：schematically presentation of key components in BCR/NFκB signaling；B：distribution of ChIP sequencing/H3K27Ac signal at indicated genes in HBL-1 cells（the data was obtained from public dataset GSM1254196）；C：distribution of H3K27Ac modification and BRD4 binding at BTK gene in Ly1 cells treated with or without JQ1（the data was obtained from public datasets of ChIP sequencing，GSM1133650，GSM1133651，GSM1133646，and GSM1133647）.

6 BRD4 inhibition reduced MYC expression

We then examined several transcription factors that are important for transcription ofBTK.Interestingly，we observed reduced MYC protein level after JQ1 treatment both in HBL-1 and SU-DHL-2 cells（Figure 6A）.However，the expression of RELA was unaffected by JQ1 in these cells（Figure 6A）.To further confirm these results，we treated HBL-1 cells with JQ1 and the mRNA level of MYC was examined by real-time PCR.As shown in Figure 6B，inhibiting BRD4 greatly reduced the mRNA level of MYC.These results demonstrated that BRD4 inhibition greatly reduced transcription factor MYC but not RELA expression.

Figure 5.BRD4 inhibition promoted cytotoxicity of ibrutinib and overcame ibrutinib resistance.A：schematical presentation showing JQ1 and ibrutinib targeting BTK；B：CCK-8 assay was used to measure the viability of HBL-1 cells treated with different concentrations of ibrutinib or JQ1；C：CCK-8 assay was used to measure the viability of HBL-1 cells treated with indicated combinations of JQ1 and ibrutinib at different doses，and the synergic effect was calculated using CompuSyn software［the combination index（CI）value at ED50 is 0.05］；D：CCK-8 assay was used to measure the viability of SU-DHL-2 cells treated with JQ1 or ibrutinib；E：CCK-8 assay was used to measure the viability of SU-DHL-2 cells treated with IBET-762；F：PI staining assay was used to examine the death of SU-DHL-2 cells with or without I-BET-762 treatment（scale bar=100 μm）.Mean±SD.n=3.

Figure 6.BRD4 inhibition reduced MYC but not RELA expression.A and B：HBL-1（A）and SU-DHL-2（B）cells were treated with JQ1 at indicated doses for 24 h，and the protein levels of MYC and RELA were determined by Western blot；C：HBL-1 cells were treated with JQ1 or I-BET-762 at indicated concentrations，and the mRNA level of MYC was examined by real-time RT-PCR.Mean±SD.n=3.*P＜0.05 vs JQ1 and I-BET-762 0 μmol/L group.

DISCUSSION

ABC-DLBCL subtype is one of the most malignant lymphoma with poor outcomes to current immunochemotherapy［25-26］.The BTK inhibitor ibrutinib is one of the most promising clinical-testing drugs for such disease［25，27］.However，there is still a large part of patients with ABC-DLBCL that are unresponsive to ibrutinib［17，28］.Furthermore，acquired resistance often occurs after a period of treatment［29-30］.Therefore，identification of alternative treatment targeting BTK or other components in BCR/NFκB pathway could overcome resistance.In this study，we found that JQ1 specifically and efficiently blocked transcription ofBTK，and subsequently resulted in cytotoxicity on ABC-DLBCL cells.BRD4 inhibition not only had synergic effect with ibrutinib but also efficiently induced cell death even in ibrutinib-resistant cells.The effect of BRD4 inhibition on BTK expression was independent on super-enhancers aroundBTKgene.It is very likely due to the disruption

of BRD4 binding withinBTKgene，which needs to be addressed by additional experiments.Our results provide convincing evidence to show that JQ1 or other bromodomain inhibitors might be an alternative approach to target BTK in ABC-DLBCL cells.

It has been reported that ABC-DLBCL is sensitive to JQ1［16，22-23，31-33］.However，the molecular mechanism is still unclear.It is novelty demonstrated that JQ1 promotes ABC-DLBCL cell death by targeting BTK to disrupt the BCR/NFκB signaling.Previous studies have reported that JQ1 acts as a bromodomain inhibitor to specifically disrupt the function of super-enhancer，a subtype of enhancer extensively enriched with BRD4［24，32］.We therefore looked all potential super-enhancers around the genes encoding all key components in BCR/NFκB signaling through database analysis.We observed that several genes such asPLCG2had super-enhancers，but failed to find such elements aroundBTKgene.However，JQ1 treatment inhibited the expression of BTK but not PLCG2.There are two high convincing BRD4 binding sites withinBTKgene.The BRD4 binding at these two sites can be fully disrupted by BRD4 inhibitors.It suggests that these two binding sites might be important forBTKtranscription.Another possibility is that the transcription factor OCA-B regulatingBTKgene is maintained by super-enhancers.Therefore，whether BRD4 inhibition blocksBTKtranscription dependent on BRD4 binding withinBTKgene or through OCA-B deserves to be further investigated.

Cancer cells have evolved the ability to escape the killing of single agent［34-36］.One of the mechanisms is to up-regulate other components in single-agent targeting pathway［36］.Therefore，simultaneously targeting multiple targets or multiple mechanisms in one pathway can significantly reduce the chance to develop resistance to single agent.Besides JQ1，there are several other available inhibitors targeting BCR/NFκB signaling，such as BTK inhibitor ibrutinib or MALT1 inhibitor MI2.We found JQ1 had the synergic ability to promote cytotoxicity with ibrutinib，suggesting that the combination of multiple inhibitors in BCR/NFκB signaling has much strong ability to kill ABC-DLBCL cells.Moreover，JQ1 and I-BET-762 efficiently promoted cell death in ibrutinib-resistant cells.However，our results and conclusions are based onin vitrokilling.Cultured cells in dishes are different from cancer cellsin vivo.It is urgent to test the response of ibrutinib-resistant ABCDLBCL to JQ1 or I-BET-762 in animal models.These results indicate that combination of inhibitors targeting one pathway might overcome drug resistance.

Overall，we identified BTK is a target of BRD4 inhibitors in BCR/NFκB signaling，which explains previous finding that ABC-DLBCL cells are sensitive to BRD4 inhibition.JQ1 strongly synergizes with other inhibitors targeting BCR/NFκB signaling，such as BTK inhibitor ibrutinib.Our data also demonstrate that BRD4 inhibition serves as an alternative approach to target BTK kinase.Due to the fact that ibrutinib-resistant cells are also sensitive to BRD4 inhibitors，BRD4 inhibitors might have more broad effect than ibrutinib.However，further study should be conductedin vivoto verify the effect of BRD4 inhibitors on ABC-DLBCL cells.