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A. Uchida et al.
immunophenotypic, and genetic features.1,4 Gene expres- sion signatures have stratified DLBCL into germinal center B-cell (GCB)-like, activated B-cell (ABC)-like, and other subtypes, each of which results from different pathogenic mechanisms.1,5,6 DH-HGBL cases with DLBCL morpholo- gy frequently result in disastrous consequences in spite of showing the GCB phenotype, which is regarded as a rela- tively favorable marker for survival.1,2,4 Thus, to be DHL and DPL (DH-DPL) seems to have a negative impact on survival, especially in GCB-like DLBCL cases.1-3
MYC is a powerful transcriptional activator, target genes of which are associated with cell proliferation, DNA repli- cation, protein synthesis, and cell metabolism, and its over- expression is a hallmark of tumor aggressivity.7,8 In con- trast, BCL2 is the first identified anti-apoptotic regulator that contributes to the survival of lymphoma cells.9,10 Dysregulation of both genes likely generates aggressive lymphoma cells showing a fast growth rate and resistance to apoptotic stimuli. Clinically, DH-DPL has a poor prog- nosis when treated with the standard rituximab-combined cyclophosphamide, doxorubicin, vincristine, and pred- nisone (R-CHOP) regimen, with a median survival of around 20 months.2,11 Until now, optimal therapeutic strategies against DH-DPL remain to be determined.
Recent reports suggest that targeting MYC and BCL2 may be a promising strategy to control DH-DPL.12-15 BRD4, a member of the bromodomain and extra-termi- nal domain (BET) family, is considered to be a conven- ient target for MYC-driven lymphomas.16,17 BET family proteins recognize acetylated chromatin and act as tran- scription co-factors.18 BRD4 is upregulated in DLBCL and Burkitt lymphoma cells, and its inhibition leads to a strong downregulation of MYC and its regulating genes, resulting in suppression of their cell growth.16,17 Meanwhile, the selective BCL2 inhibitor venetoclax demonstrated excellent antitumor effects in chronic lym- phocytic leukemia.19,20 BCL2 and its family proteins func- tion as inhibitors and activators of the intrinsic apoptotic pathway at the mitochondrial membrane level.10,21 They contain at least one of four BCL2 homology (BH) domains (BH1-4) and are classified into three groups based on their structure and function: i.e., the pro-sur- vival proteins (BCL2, BCL-xL, MCL1, BFL1, and BCLw) sequester the pro-apoptotic BH3-only proteins (BID, BIM, BAD, NOXA, PUMA, BMF, HRK, and BIK), which in turn activate the pore-forming proteins (BAX and BAK).10,21 Oligomerization of BAX/BAK permeabilizes the mitochondrial membrane, resulting in cytochrome c release and apoptosis.10,21 The BH3 mimetic venetoclax binds to the BH3 domain of BCL2, releases BH3-only proteins, and induces apoptosis.10,21 Although short expo- sure to venetoclax can trigger significant antitumor effects in DLBCL cells,12-15,19,22-24 this drug’s clinical efficacy in DLBCL is less promising,25 probably because the apop- totic sensitivity to venetoclax is influenced not only by total amounts of BCL2, but also by its phosphorylation status, especially at serine 70 (Ser70), and the further presence of other pro-survival proteins.14,15,22-24,26-28 Among the pro-survival proteins, MCL1 is considered the major determinant of resistance to venetoclax.22-24,28 Therefore, the therapeutic application of venetoclax to DH-DPL needs further investigation. In this study, we examined the apoptotic sensitivity of GCB-like DLBCL cells to the BRD4 inhibitor JQ-1 and BH3 mimetics, focusing on the association of BCL2 with MCL1.
Methods
Reagents
The BCL2 inhibitor venetoclax (Selleck Chemicals, Houston, TX, USA), MCL1 inhibitor S63845 (ApexBio, Houston, TX, USA), BCL-xL inhibitor A-1155463 (Selleck Chemicals), and BRD4 inhibitor JQ-1 (Sigma-Aldrich, St. Louis, MO, USA) were dis- solved with dimethyl sulfoxide (DMSO; Nacalai Tesque, Kyoto, Japan) and added to the culture medium.
Cell lines
We used four GCB-like DLBCL cell lines: BJAB,29 SU-DHL10 (ATCC, Manassas, VA, USA), Karpas231 (ECACC, Salisbury, UK), and OCI-Ly8 (kindly provided by Dr. Masao Seto, Kurume University, Kurume, Japan). They were maintained as described previously.29
Fluorescence in situ hybridization analyses
Fluorescence in situ hybridization analyses were performed using a Vysis LSI MYC dual-color, break-apart rearrangement probe, a Vysis LSI IGH/MYC/CEP8 tri-color dual-fusion probe, and a Vysis LSI IGH/BCL2 dual-color, dual-fusion translocation
probe (Abbott Molecular, Des Plaines, IL, USA).
Clinical samples and immunohistochemistry
Clinical samples were obtained at biopsy performed for initial diagnosis at our institution between September 2012 and October 2018. Diagnoses were made according to the current WHO classi- fication,1 and GCB-like DLBCL was defined using the Hans crite- ria.30 A bone marrow specimen from one patient (UPN3) was sub- jected to in vitro susceptibility testing using venetoclax after having obtained written informed consent. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded specimens using standard techniques. The primary antibodies used are listed in the Online Supplementary Methods. The percentages of stained lym- phoma cells were evaluated by visual estimation and recorded in 10% increments by at least two observers. The study protocol was approved by the Ethics Review Board of St. Marianna University.
Cell proliferation and annexin V-binding assays
We performed direct cell counting using a trypan blue-exclusion test (Thermo Fisher Scientific, Carlsbad, CA, USA) and annexin-V binding/7-amino-actinomycin D (7-AAD) rejection assays (Beckman Coulter, Brea, CA, USA) with a FACSCalibur flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA). Acquired data were analyzed using FlowJo software (BD Bioscience).
Western blot analysis
Cell lysates were prepared as described previously.29 Equal amounts of protein (30 μg/well) were separated on a discontinu- ous sodium dodecyl sulfate-10% polyacrylamide gel and blotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The primary antibodies used are listed in the Online Supplementary Methods. Antibody signals were visualized using Western blue (Promega, Madison, WI, USA).
Immunoprecipitation
Whole cell lysate (100 μg of total protein) was added to 1/200 volume of each rabbit antibody and adjusted to 680 μL of volume with the lysis buffer. The antibodies used are listed in the Online Supplementary Methods. Rabbit serum-saturated protein G- Sepharose beads (20 μL) (GE Health Care, Uppsala, Sweden) were added to each lysate, and immunoprecipitation was performed for 1 h while rotating at 4oC. After incubation, the beads were
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haematologica | 2019; 104(7)