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Establishment and characterization of a new activated B-cell-like DLBCL cell line, TMD12

Open AccessPublished:September 30, 2022DOI:https://doi.org/10.1016/j.exphem.2022.09.005

      HIGHLIGHTS

      • A novel diffuse large B-cell lymphoma (DLBCL) cell line, designated as TMD12, was established and characterized.
      • TMD12 cells displayed constitutive activation of the nuclear factor κB pathway because of mutations in MYD88 and CD79B, indicating an activated B-cell-like (ABC) subtype.
      • Oncogenic activation of the tumor progression locus 2-p105 pathway might be involved in the tumorigenesis of ABC-DLBCL.
      We report the establishment of a novel activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) cell line, designated as TMD12, from a patient with highly refractory DLBCL. ABC-DLBCL is a subtype with a relatively unfavorable prognosis that was originally categorized using gene expression profiling according to its cell of origin. TMD12 cells were isolated from the pleural effusion of the patient at relapse and passaged continuously in vitro for >4 years. The cells displayed cluster of differentiation (CD)19, CD20, CD22, CD38, human leukocyte antigen-DR isotype, and κ positivity and CD5, CD10, CD23, and λ negativity, as detected using flow cytometric analysis. The chromosomal karyotypic analysis, including the spectral karyotyping method, confirmed t(1;19)(q21:q13.1), del(6q23), gain of chromosome 18, and other abnormalities. Mutation analyses, including whole-exome sequencing, revealed that TMD12 cells harbored mutations in MYD88 and CD79B, indicating an ABC subtype. TMD12 cells exhibited chronic active B-cell receptor signaling and constitutive activation of the nuclear factor κB pathway, which is typically associated with sensitivity to a specific Bruton tyrosine kinase inhibitor, ibrutinib. Intriguingly, TMD12 cells displayed moderate resistance to ibrutinib and lacked activation of Janus kinase/signal transducers and activators of transcription 3 signaling, another hallmark of this DLBCL subtype. Treatment with an inhibitor against tumor progression locus 2 (TPL2), a multifunctional intracellular kinase that is activated particularly downstream of Toll-like receptors or MYD88 and IκB kinase α/β (IKKα/β), suppressed the proliferation of TMD12 cells, implying the possible involvement of the TPL2-p105 pathway in the tumorigenesis of ABC-DLBCL. Because only a limited number of ABC-DLBCL cell lines are currently available, TMD12 cells might provide a useful tool in the search for novel druggable targets for this intractable lymphoma.
      Diffuse large B-cell lymphoma (DLBCL) is an essentially heterogeneous subgroup of aggressive B-cell lymphomas. To categorize the entity based on the “cell of origin” (COO), gene expression profiling studies have classified DLBCL into 3 subtypes, with different clinical outcomes: activated B-cell-like (ABC) subtype, germinal center B-cell-like (GCB) subtype, and unclassified subtype [
      • Alizadeh AA
      • Eisen MB
      • Davis RE
      • et al.
      Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.
      ,
      • Rosenwald A
      • Wright G
      • Chan WC
      • et al.
      The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.
      ]. Patients with ABC-DLBCL exhibit a relatively refractory clinical course compared with others when treated with the standard regimen of rituximab-cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) [
      • Alizadeh AA
      • Eisen MB
      • Davis RE
      • et al.
      Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.
      ,
      • Rosenwald A
      • Wright G
      • Chan WC
      • et al.
      The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.
      ].
      The tumorigenesis of ABC-DLBCL is characterized by a constitutively activated nuclear factor kB (NF-κB) pathway, which can attribute to gain-of-function mutations in the components of B-cell receptor (BCR) signaling [
      • Davis RE
      • Ngo VN
      • Lenz G
      • et al.
      Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.
      ], such as CD79A/B and CARD11, or those of Toll-like receptor (TLR) signaling, including MYD88 [
      • Ngo VN
      • Young RM
      • Schmitz R
      • et al.
      Oncogenically active MYD88 mutations in human lymphoma.
      ], which is implicated in sensitivity to Bruton tyrosine kinase (BTK) inhibitors [
      • Wilson WH
      • Young RM
      • Schmitz R
      • et al.
      Targeting B cell receptor signaling with ibrutinib in diffuse large B cell lymphoma.
      ]. Activation of the Janus kinase 1-signal transducers and activators of transcription 3 (STAT3) pathway has been reported to contribute to the survival of ABC-DLBCL cells partly through the epigenetic regulation of genes, including IRF4, MYD88, and MYC [
      • Rui L
      • Drennan AC
      • Ceribelli M
      • et al.
      Epigenetic gene regulation by Janus kinase 1 in diffuse large B-cell lymphoma.
      ], whereas the activation of STAT3 impacts the regulation of the expression of c-MYC or p27 [
      • Ding BB
      • Yu JJ
      • Yu RY
      • et al.
      Constitutively activated STAT3 promotes cell proliferation and survival in the activated B-cell subtype of diffuse large B-cell lymphomas.
      ]. Similarly, Src family kinases (SFKs) function as drivers of activation of spleen tyrosine kinase, which has been reported to propagate BCR signaling in MYD88-mutated ABC-DLBCL [
      • Munshi M
      • Liu X
      • Kofides A
      • et al.
      A new role for the SRC family kinase HCK as a driver of SYK activation in MYD88 mutated lymphomas.
      ].
      These pathways are reinforced mutually and associated with the expression of antiapoptotic molecules and chemoresistance, leading to an intractable clinical course [
      • Scuto A
      • Kujawski M
      • Kowolik C
      • et al.
      STAT3 inhibition is a therapeutic strategy for ABC-like diffuse large B-cell lymphoma.
      ,
      • Ok CY
      • Chen J
      • Xu-Monette ZY
      • et al.
      Clinical implications of phosphorylated STAT3 expression in de novo diffuse large B-cell lymphoma.
      ]. In 2018, the formation of a multiprotein supercomplex called My-T-BCR, consisting of MYD88, TLR9, and BCR subunits (cluster of differentiation [CD]79A/B), was reported to predict the sensitivity to BTK inhibitors in ABC-DLBCL cells [
      • Phelan JD
      • Young RM
      • Webster DE
      • et al.
      A multiprotein supercomplex controlling oncogenic signalling in lymphoma.
      ]. By colocalizing with mammalian target of rapamycin (mTOR) on endosomes, My-T-BCR drives prosurvival NF-κB and mTOR signaling cooperatively, providing a rationale for synergistic toxicity of inhibitors of BCR and phosphatidylinositol-3-kinase (PI3K)-Akt-mTOR signaling [
      • Phelan JD
      • Young RM
      • Webster DE
      • et al.
      A multiprotein supercomplex controlling oncogenic signalling in lymphoma.
      ]. More recently, because of advances in next-generation sequencing, Wright et al. [
      • Wright GW
      • Huang DW
      • Phelan JD
      • et al.
      A probabilistic classification tool for genetic subtypes of diffuse large B cell lymphoma with therapeutic implications.
      ] developed a new algorithm based on genetics to categorize DLBCL into 6 subtypes: MCD (including MYD88L265P and CD79B mutations), BN2 (including BCL6 translocations and NOTCH2 mutations), N1 (including NOTCH1 mutations), A53 (aneuploid with TP53 inactivation), ST2 (including SGK1 and TET2 mutations), and EZB (including EZH2 mutations and BCL2 translocations).
      Cell lines derived from clinical samples could contribute to various preclinical research studies, such as testing sensitivity to specific inhibitors. However, given that a limited number of ABC-DLBCL cell lines are available currently, the establishment of as many of these lines as possible is desired to encompass this disparate entity, which may contribute to the development of lymphomagenesis-oriented strategies.
      We report the establishment of a new lymphoma cell line that we designated as TMD12, which was obtained from a patient with highly refractory non-GCB-DLBCL. Based on mutation status, i.e., harboring MYD88L265P and CD79BY196N, TMD12 cells appear to be classified into the ABC subtype in the COO-based categorization system and as the MCD subtype in a recent genetics-based classification system [
      • Wright GW
      • Huang DW
      • Phelan JD
      • et al.
      A probabilistic classification tool for genetic subtypes of diffuse large B cell lymphoma with therapeutic implications.
      ]. We evaluated TMD12 in comparison with TMD8, a well-described ABC-DLBCL cell line with equivalent mutations, also established at our institute [
      • Tohda S
      • Sato T
      • Kogoshi H
      • Fu L
      • Sakano S
      • Nara N.
      Establishment of a novel B-cell lymphoma cell line with suppressed growth by gamma-secretase inhibitors.
      ]. TMD12 could provide a valuable tool to obtain novel insights into the lymphomagenesis of ABC-DLBCL.
      Because ABC-DLBCL is driven by IκB kinase α/β (IKKα/β) downstream of aberrant BCR or TLR signaling and resultant constitutive activation of NF-κB, the molecules associated with these pathways have received considerable attention in efforts to identify therapeutic targets [
      • Jost PJ
      • Ruland J.
      Aberrant NF-kB signaling in lymphoma: mechanisms, consequences, and therapeutic implications.
      ,
      • Nagel D
      • Vincendeau M
      • Eitelhuber AC
      • Krappmann D.
      Mechanisms and consequences of constitutive NF-kB activation in B-cell lymphoid malignancies.
      ]. Therefore, we focused on tumor progression locus 2 (TPL2), also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8), a cytoplasmic Ser or Thr protein kinase that was initially identified as a proto-oncogene [
      • Miyoshi J
      • Higashi T
      • Mukai H
      • Ohuchi T
      • Kakunaga T.
      Structure and transforming potential of the human cot oncogene encoding a putative protein kinase.
      ]. However, it was reported to be multifunctional in the context of inflammation, infection, and immune response [
      • Njunge LW
      • Estania AP
      • Guo Y
      • Liu W
      • Yang L.
      Tumor progression locus 2 (TPL2) in tumor-promoting inflammation, tumorigenesis and tumor immunity.
      ].
      Intriguingly, TPL2 was activated downstream of IKKα/β following the stimulation of TLRs or MYD88 by immune responses [
      • Xu D
      • Matsumoto ML
      • McKenzie BS
      • Zarrin AA.
      TPL2 kinase action and control of inflammation.
      ]. TPL2 formed a signaling complex with NF-κB1, p105, and A20-binding inhibitor of NF-κB 2 (ABIN2) [
      • Njunge LW
      • Estania AP
      • Guo Y
      • Liu W
      • Yang L.
      Tumor progression locus 2 (TPL2) in tumor-promoting inflammation, tumorigenesis and tumor immunity.
      ] for inactivation or stabilization. Once p105 is phosphorylated at S932 by IKKα/β, TPL2 is released from the complex to be activated via phosphorylation at T290, whereas p105 is subjected to processing into p50 through proteasomal degradation, consequently forming a heterodimer with p65, leading to the nuclear translocation of NF-κB [
      • Gantke T
      • Sriskantharajah S
      • Sadowski M
      • Ley SC.
      IkB kinase regulation of the TPL-2/ERK MAPK pathway.
      ,
      • Concetti J
      • Wilson CL.
      NFKB1 and cancer: friend or foe?.
      ]. Then, disengaged or activated TPL2 functions as MAP3K and subsequently activates the mitogen-activated protein kinase (MAPK) pathway [
      • Lee HW
      • Choi HY
      • Joo KM
      • Nam DH.
      Tumor progression locus 2 (Tpl2) kinase as a novel therapeutic target for cancer: double-sided effects of Tpl2 on cancer.
      ] in immune cells, including macrophages and TLR4-activated B cells (known as a COO of ABC-DLBCL) [
      • Banerjee A
      • Grumont R
      • Gugasyan R
      • White C
      • Strasser A
      • Gerondakis S.
      NF-kB1 and c-Rel cooperate to promote the survival of TLR4-activated B cells by neutralizing Bim via distinct mechanisms.
      ]. TPL2 has also been reported to play oncogenic roles in various tumors, including hematopoietic malignancies [
      • Njunge LW
      • Estania AP
      • Guo Y
      • Liu W
      • Yang L.
      Tumor progression locus 2 (TPL2) in tumor-promoting inflammation, tumorigenesis and tumor immunity.
      ]. In this regard, the involvement of TPL2 remains to be investigated in B-cell lymphomas, except for Epstein-Barr virus (EBV)-associated lymphoproliferative disorders (LPDs) [
      • Voigt S
      • Sterz KR
      • Giehler F
      • et al.
      A central role of IKK2 and TPL2 in JNK activation and viral B-cell transformation.
      ]. Intriguingly, we observed that inhibition of TPL2 affected the proliferation of ABC-DLBCL cells compared with that of GCB-DLBCL cells, implying subtype-specific roles of TPL2. In the present article, in addition to well-characterized oncogenic pathways, we pictured an unrecognized role of the TPL2-p105 axis in lymphomagenesis using the newly established cell line, TMD12, as a platform for ABC-DLBCL.

      MATERIALS AND METHODS

      Case Report

      A 68-year-old man was referred to our hospital in August 2016 with complaints, including double vision and a tumor on the inferior aspect of his right eyelid. The tumor's invasion was widespread in the right paranasal sinuses, and a biopsy of the lesion led to the diagnosis of DLBCL (non-GCB type). Brain magnetic resonance imaging and a cerebrospinal fluid study confirmed the involvement of the central nervous system, and 18F-fluorodeoxyglucose positron emission tomography-computed tomography (PET-CT) revealed bilateral neck lymphadenopathy, suggesting clinical stage IV. The study was approved by the ethical committee of Tokyo Medical and Dental University. Written informed consent was obtained from patient in compliance with the Declaration of Helsinki.
      A total of 6 courses of a rituximab-cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) regimen combined with 3 doses of intrathecal chemotherapy and 2 courses of rituximab with high-dose methotrexate were administered. Although the patient's complete metabolic remission was confirmed using PET-CT, the lymphoma relapsed to intrathoracic lymph nodes and bilateral pleurae in January 2018, and the retention of pleural effusion progressed rapidly. The relapse was confirmed using cytology and flow cytometric analyses of the pleural effusion (described as PE-1 or 2 in Figure 1), displaying the proliferation of clonal B lymphocytes. A G-banding analysis revealed complex aberrations, including t(1;19)(q21;p13), add(6)(q21), and trisomy 18. Although 2 different salvage regimens were attempted, the patient died of exacerbation of the disease in April 2018. Figure 1 summarizes the patient's clinical course, including chemotherapies, change in tumor burden in response to the treatments, and key images. Written informed consent was obtained from the patient and his family for the publication of this case report and accompanying images.
      Figure 1
      Figure 1The patient's clinical course. The tumor burden of lymphoma in response to adopted chemotherapy regimens is shown where images of 18F-FDG PET-CT at diagnosis, remission, and relapse are displayed. A chest CT image at relapse, indicating massive retention of PE as involvement of the relapsed lymphoma, is also presented. Clinical samples from the PE containing lymphoma cells were sampled twice after relapse (PE-1 in January and PE-2 in April 2018). DEX=Dexamethasone; GCD=gemcitabine, carboplatin, and dexamethasone; IT=intrathecal chemotherapy; R+HD-MTX=rituximab, and high-dose methotrexate; R-CEPP=rituximab, cyclophosphamide, etoposide, procarbazine, and prednisolone; R-CHOP=rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone.

      Cell Isolation or Culture

      Lymphoma cells from the pleural effusion (PE-2 in Figure 1) drained via thoracentesis from the patient as a palliative treatment in April 2018 were isolated as nucleated cells using the Ficoll cell separation protocol and subjected to cell culture in RPMI 1640 medium containing 10% fetal bovine serum and antibiotics without additional growth factors or stimulatory cytokines.

      Reagents, Antibodies, and Cell Lines

      The reagents, antibodies, and cell lines are described in Supplementary Data.

      Cell Growth and Viability Assay

      We assessed cell proliferation and viability by counting the numbers of viable and nonviable cells using the trypan blue dye exclusion method. The number of viable cells was also assessed using the cell counting kit-8 (CCK-8) assay (Dojindo, Mashiki, Japan) according to the manufacturer's instructions. All of the data shown are representative of experiments repeated at least 3 times.

      Flow Cytometric Analysis

      The original lymphoma cells from the patient's pleural effusion and TMD12 cells were centrifuged and subjected to an immune-phenotypic analysis using flow cytometry (FCM) performed at SRL Inc. (Hachioji, Tokyo). The panel of monoclonal antibodies used in the assay included those specific for CD45, CD2, CD3, CD4, CD5, CD8, CD10, CD11c, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD34, CD38, CD56, an epitope of CD20 (FMC-7), human leukocyte antigen-DR isotype (HLA-DR), κ-chain, and λ-chain.

      Whole-Exome Sequencing Analysis and Detection of Copy Number Variants

      Whole-exome sequencing (WES) was performed using SureSelect Human All Exon V6 (Agilent Technologies, Santa Clara, California) on NovaSeq 6000 (Illumina, San Diego, California) at Rhelixa Inc. (Tokyo, Japan). The analysis pipeline for variant discovery has been described previously [
      • Sadato D
      • Hirama C
      • Kaiho-Soma A
      • et al.
      Archival bone marrow smears are useful in targeted next-generation sequencing for diagnosing myeloid neoplasms.
      ]. Finally, the detected mutations were manually curated by experts in genetics of hematologic malignancies. Copy number variants (CNVs) identified in TMD12 cells were analyzed using a CNV kit [
      • Talevich E
      • Shain AH
      • Botton T
      • Bastian BC.
      CNVkit: genome-wide copy number detection and visualization from targeted DNA sequencing.
      ] in comparison with normal control samples.

      Immunoblotting

      For immunoblotting experiments, the cells were lysed in a lysis buffer containing 1% Triton X-100, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM ethylenediaminetetra-acetic acid, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 μg/mL each of aprotinin and leupeptin at 4°C for 15 min. The cell lysates were subjected to an immunoblot analysis essentially as described previously [
      • Nogami A
      • Oshikawa G
      • Okada K
      • et al.
      FLT3-ITD confers resistance to the PI3K/Akt pathway inhibitors by protecting the mTOR/4EBP1/Mcl-1 pathway through STAT5 activation in acute myeloid leukemia.
      ].
      The procedures of cytogenetic analysis, G-banding, and spectral karyotyping (SKY) analysis; DNA extraction and direct sequence analysis; detection of apoptotic cells and cell cycle analysis using FCM; and reverse transcription (RT)-polymerase chain reaction (PCR) and quantitative RT-PCR (qRT-PCR) are described in Supplementary Data.

      RESULTS

      Pathologic and Immunohistochemical Findings of the Case

      Pathologic findings at the patient's initial diagnosis (paranasal lesion) revealed that the tumor cells were large and showed diffuse infiltration in the sampled tissue. Immunohistochemical staining revealed that the tumor cells were positive for CD20, CD79a, and BCL2; partially positive for CD138; and negative for CD5 and Epstein-Barr encoding region-in situ hybridization (ISH), leading to the diagnosis of DLBCL. The Ki-67 index was up to 70%, indicating a high cell proliferation rate. Based on the classification by Hans et al. [
      • Hans CP
      • Weisenburger DD
      • Greiner TC
      • et al.
      Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray.
      ] and because of being negative for CD10, partially positive for BCL6, and positive for multiple myeloma 1/interferon regulatory factor 4 (MUM1/RF4), the patient's tumor was classified as a non-GCB (suggesting ABC) subtype (Figure 2).
      Figure 2
      Figure 2Histopathologic findings at diagnosis. At diagnosis, hematoxylin and eosin staining of the tumor tissue was sampled from the lesion in the right paranasal sinuses. Microscopically, the lesion was composed of a monotonous, diffuse infiltrate of abnormal lymphoid cells at low (A, × 40) and high magnification (B, × 400). Immunohistochemically, the neoplastic cells were strongly immune reactive to (C) cytoplasmic CD20 and (D) CD79a as well as (G) nuclear BCL2 (not shown) and MUM1. The lesion was partially positive for (H) BCL6 and (I) CD138 and negative for (E) CD10 and (C) CD5. (J) The proliferation rate was high, represented by a Ki-67 labeling index of up to 70%. Appropriately controlled EBER studies using ISH showed no nuclear staining of EBV-infected cells (not shown).

      Establishment of the TMD12 Cell Line

      TMD12 cells proliferated stably at a doubling time of approximately 24 hours and showed nonadherent growth with slight cellular clump formation (Figure 3A, bottom). The cells have been passaged continuously for >4 years as of this writing and were confirmed to grow again after a conventional freeze-thaw procedure, verifying the establishment of the new cell line.
      Figure 3
      Figure 3Morphologic, cytogenetic, and genetic analyses of TMD12 cells. (A) The morphologic appearance of TMD12 cells. Top: May-Grünwald Giemsa staining of a cytospin preparation of TMD12 cells (original magnification, × 1,000). Scale bar = 10 μm. Bottom: morphology of TMD12 cells as shown by phase-contrast microscopy (original magnification, × 1,000). Scale bar = 10 μm. (B) FCM analysis of TMD12 cells by CD45 gating (dot plots) after confirmation of the establishment of the cell line. The antibodies used for the analyses are indicated. (C) Chromosomal analyses of TMD12 cells. A G-band karyotype of TMD12 cells in culture. (D) Mutation analyses of key driver molecules in ABC-DLBCL. Left: Direct sequence analysis of the MYD88 gene (exon 5) obtained by PCR using genomic DNA extracted from TMD12 cells. Nucleotide sequences around the codon coding for L265 in normal MYD88 or P265 in the MYD88 mutant are shown with the mutated nucleotide and amino acid sequences indicated in red. Right: direct sequence analysis of the CD79B gene (exon 5) obtained in the same way as described above. Nucleotide sequences around the codon coding for Y196 in normal CD79B or N196 in the CD79B mutant are shown with the mutated nucleotide and amino acid sequences indicated in red.

      Morphologic, Immune-Phenotypic, Cytogenetic, and Genetic Characterization of TMD12 Cells and the Patient's Lymphoma Cells

      TMD12 cells were observed as abnormal, irregular-shaped lymphoid cells composed of medium-to-large-sized cells, as revealed by May-Grünwald-Giemsa staining of a cytospin preparation. The cells showed irregular or enlarged nuclei with 1–4 distinct nucleoli. Multinucleated cells were sporadically observed. The TMD12 cells also showed a distinct perinuclear halo and abundant basophilic cytoplasm with occasional vacuoles (Figure 3A, top). Phase-contrast microscopy revealed that the shapes of the lymphoma cells were irregular or round (Figure 3A, bottom). The FCM analysis findings are shown as dot plots (Figure 3B) and summarized in Table 1 in comparison with the original lymphoma cells (PE-2 in Figure 1).
      Table 1Flow cytometric findings of TMD12 cells and the patient's lymphoma cells
      The fluorescence intensity was classified into 4 groups (−: positive cells < 20%; +: 20% < positive cells < 50%; ++: 50% < positive cells < 90%; +++: 90% < positive cells) according to the percentage of CD45-gated cells expressing the indicated markers with the indicated descriptions.
      MarkersDescriptionPE-2TMD12
      CD5T cells, some B cells
      CD10Germinal center B cells
      CD19B cells++++++
      CD20B cells+++++
      CD21B cells+++
      CD22B cells++++++
      CD23B-cell subset
      CD30B cells or activated T cells
      CD38Plasmacytes, B cells, T cells++
      FMC7B-cell subset+
      HLA-DRB cells or activated T cells++++++
      κLight Ig chains++++++
      λLight Ig chains
      FMC-7= an epitope of CD20; Ig=immunoglobulin.
      a The fluorescence intensity was classified into 4 groups (−: positive cells < 20%; +: 20% < positive cells < 50%; ++: 50% < positive cells < 90%; +++: 90% < positive cells) according to the percentage of CD45-gated cells expressing the indicated markers with the indicated descriptions.
      TMD12 cells were positive for CD19, CD20, CD21, CD22, HLA-DR, and κ-chain but negative for CD5, CD10, CD23, and λ-chain, displaying a similarity with the original cells, except for the additional expressions of CD38 and FMC-7. A conventional chromosomal analysis of TMD12 cells demonstrated a complex karyotype: 48, XY, t(1;19)(q21;q13;1), t(5;15)(q32;q24), add(6)(q13), del(7)(p?), der(15)t(5;15), +18, +18, del(19)(p?), del(20)(q11.2q13.1)[11]/47, idem, −Y[4]/49, idem, +mar[3]/[18] (Figure 3C). Supplementary Table E1 summarizes the comparison of TMD12 cells and clinical samples. The karyotype of TMD12 cells shared majority of the aberrations with cells of the original lymphoma, characterized by t(1;19), t(5;15), add(6q), and +18. The SKY analysis confirmed reciprocal translocations for t(1;19) and t(5;15) as well as some other findings (Supplementary Figure E1 and Supplementary Table E1). These results indicated that the TMD12 cell line was derived from the original case. Of note, 6q23 (TNFAIP3 locus, coding A20) was lacking monoallelically among them, which was confirmed using the CNV analysis of TMD12 cells, indicating the loss of chromosome 6q (Supplementary Figure E2).
      To identify the subtype-defining genetic features of TMD12 in DLBCL, the WES analysis was performed. As shown in Table 2, TMD12 has mutations in MYD88 (L265P) and CD79B (Y196N) as well as well-described somatic hypermutation-related genes, including PIM1 and IGLL5 [
      • Chen F
      • Zhang Y
      • Creighton CJ.
      Systematic identification of non-coding somatic single nucleotide variants associated with altered transcription and DNA methylation in adult and pediatric cancers.
      ]. The direct sequencing analysis confirmed the presence of the MYD88L265P and CD79BY196N mutations (Figure 4D). Because the combination of MYD88L265P and CD79BY196 is observed exclusively in the MCD subtype (inherently comprised in ABC-DLBCL) [
      • Wright GW
      • Huang DW
      • Phelan JD
      • et al.
      A probabilistic classification tool for genetic subtypes of diffuse large B cell lymphoma with therapeutic implications.
      ,
      • Chen R
      • Zhou Wang L
      • Zhu L
      • Ye X.
      MYD88L265P and CD79B double mutations type (MCD type) of diffuse large B-cell lymphoma: mechanism, clinical characteristics, and targeted therapy.
      ], these results strongly supported the categorization of TMD12 into this subtype. Our comparison of the mutation status of TMD12 with other well-characterized ABC-DLBCL cell lines revealed that TMD12 cells have mutations equivalent to those of the cell lines TMD8 and HBL1 (Supplementary Table E2) [
      • Tohda S
      • Sato T
      • Kogoshi H
      • Fu L
      • Sakano S
      • Nara N.
      Establishment of a novel B-cell lymphoma cell line with suppressed growth by gamma-secretase inhibitors.
      ,
      • Ferch U
      • Kloo B
      • Gewies A
      • et al.
      Inhibition of MALT1 protease activity is selectively toxic for activated B cell-like diffuse large B cell lymphoma cells.
      ,
      • Compagno M
      • Lim WK
      • Grunn A
      • et al.
      Mutations of multiple genes cause deregulation of NF-kB in diffuse large B-cell lymphoma.
      ]. Intriguingly, the WES analysis also identified PRDM1E97E mutation at the end of the exon, which may affect the process of splicing, with 6q21 (PRDM1 locus) deletion [
      • Xia Y
      • Xu-Monette ZY
      • Tzankov A
      • et al.
      Loss of PRDM1/BLIMP-1 function contributes to poor prognosis of activated B-cell-like diffuse large B-cell lymphoma.
      ] in the CNV analysis (Supplementary Figure E2). In addition, XPO1E571K mutation was also identified (Table 2), which has been reported in association with primary mediastinal large B-cell lymphoma [
      • Mine S
      • Hishima T
      • Suganuma A
      • et al.
      Interleukin-6-dependent growth in a newly established plasmablastic lymphoma cell line and its therapeutic targets.
      ].
      Table 2Mutations detected in TMD12 cells using whole-exome sequencing
      Mutations identified in the genes reported to be implicated in the development of malignant lymphoma were listed. The class of mutations was documented as follows: driver mutation, gain-of-oncogenic-function; loss-of-tumor-suppressor-activity mutants, hypermutation related; mutants of non-IG somatic hypermutation targets; VUS, variant of unknown significance but detected in lymphoma-associated genes.
      ClassGeneChrCoordinateVariantNucleic acid changeAmino acid changeVAFRead depth
      Driver mutationMYD88338182641T>T/Cc.818T>Cp.Leu273Pro
      Also described as Leu265Pro.
      66.32380
      CD79B1762006799A>A/Tc.589T>Ap.Tyr197Asn
      Also described as Tyr196Asn.
      39.39132
      XPO1261719472C>C/Tc.1711G>Ap.Glu571Lys68.06310
      PIM1637139268G>G/Ac.880+1G>A59.72288
      Hypermutation relatedPIM1637138355C>C/Gc.277C>Gp.Leu93Val40.66396
      PIM1637139039C>C/Gc.652C>Gp.Gln218Glu48.26547
      IGLL52223230279C>C/Gc.46C>Gp.Leu16Val46.21132
      IGLL52223230328C>C/Tc.95C>Tp.Ala32Val52.03123
      IGLL52223230399G>G/Ac.166G>Ap.Val56Ile44.4481
      IGLL52223230403G>G/Ac.170G>Ap.Gly57Glu56.182
      IGLL52223230415C>C/Tc.182C>Tp.Ser61Phe41.2580
      IGLL52223235947C>G/Gc.274C>Gp.Pro92Ala100142
      VUSHIST1H1E626157109C>C/Tc.491C>Tp.Ala164Val41.4682
      HIST1H1E626157204C>C/Tc.586C>Tp.Pro196Ser44.379
      PRDM16106536324G>A/Ac.291G>Ap. =10097
      DTX112113496202C>C/Tc.205C>Tp.Leu69Phe30.5572
      Chr=Chromosome; VAF=variant allele frequency.
      a Mutations identified in the genes reported to be implicated in the development of malignant lymphoma were listed. The class of mutations was documented as follows: driver mutation, gain-of-oncogenic-function; loss-of-tumor-suppressor-activity mutants, hypermutation related; mutants of non-IG somatic hypermutation targets; VUS, variant of unknown significance but detected in lymphoma-associated genes.
      b Also described as Leu265Pro.
      c Also described as Tyr196Asn.
      Figure 4
      Figure 4Compared with TMD8 cells, TMD12 cells exhibit relatively lower sensitivity to IBR, dasatinib, and idelalisib. (A) Cell growth of TMD8 (left) and TMD12 (right) cells under IBR treatment. TMD8 or TMD12 cells were cultured with dimethyl sulfoxide (DMSO) as the control or with the indicated concentration of IBR for the indicated number of days. Viable cell numbers were counted and plotted. (B) Cell viability and apoptosis assay for TMD8 and TMD12 cells under IBR treatment. Left: cell viability assay for IBR-treated TMD8 or TMD12 cells using the CCK-8 assay. TMD8 and TMD12 cells (as indicated) were incubated with DMSO as the control or with the indicated concentrations of IBR for 72 hours. The cells were then subjected to a colorimetric assay. Each column represents the mean of triplicate assays and is expressed as the percentage of the cell numbers of the control. Error bars: SE. The 1-way analysis of variance and Dunnett post hoc tests were performed to calculate differences between means. ***p < 0.001 vs. control. Right: annexin V-PI apoptosis assay for IBR-treated TMD8 or TMD12 cells. TMD8 and TMD12 cells (as indicated) were incubated with DMSO as the control or 100 nM IBR for 48 hours. The cells were then then double stained with annexin V-fluorescein isothiocyanate (FITC) and PI, followed by an FCM analysis for annexin V-positive apoptotic cells. The percentages of apoptotic cells in each experiment are shown. Each column represents the mean of triplicate assays; error bars = SE. p < 0.05, differences between means using unpaired 2-tailed Student t test. *p < 0.05 vs. control cells. (C) Immunoblot analysis of IBR-treated TMD8 or TMD12 cells. TMD8 and TMD12 cells were treated for 16 hours with the indicated concentrations of IBR or left untreated as the control. The cells were then harvested, lysed, and subjected to the immunoblot analysis. The transferred membrane was repeatedly immunoblotted with specific antibodies against the indicated proteins in the panel: STAT3-Y705-P, phospho-Y705-STAT3; IKKα/β-S176/180-P, phosphor-S176/180-IKKα/β; p65-S536-P, phosphor-S536-NF-κB p65; p105-S932-P, phospho-S932-NF-κB1 p105; Cl. Casp3, cleaved caspase 3. (D) Cell viability and apoptosis assay results for TMD8 and TMD12 cells under treatment with dasatinib or idelalisib. Left: TMD8 and TMD12 cells were incubated for 24 hours with DMSO (control), the indicated concentration of dasatinb (SFK inhibitor), or idelalisib (PI3Kδ inhibitor) and then subjected to a colorimetric assay. Each column: the mean of triplicate assays expressed as a percentage of the control; error bars = SE. Right: TMD8 and TMD12 cells were incubated for 48 hours with DMSO (control), 100 nM dasatinib, or 1 μM idelalisib. The cells were then double stained with annexin V-FITC and PI, followed by an FCM analysis for annexin V-positive apoptotic cells. The percentages of apoptotic cells in each experiment are shown. Each column represents the mean of triplicate assays; error bars = SE. The 1-way analysis of variance and Dunnett post hoc tests were used to calculate differences between means. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control. (E) Immunoblot analysis for TMD8 and TMD12 cells under treatment with dasatinib or idelalisib. TMD8 and TMD12 cells were treated for 16 hours with the indicated concentrations of dasatinib, and ideralisib or left untreated as indicated. The cells were then harvested, lysed, and subjected to the immunoblot analysis. The transferred membrane was repeatedly immunoblotted with specific antibodies against indicated proteins in the panel: Akt-S473-P, phospho-S473-Akt, mTOR-S2448-P, phospho-S2448-mTOR.

      Sensitivity of TMD12 Cells to Specific Inhibitors for Signaling Molecules Involved in the Tumorigenesis of ABC-DLBCL

      Next, to uncover signaling pathways activated in TMD12 cells, we tested the sensitivity of TMD12 cells to various inhibitors targeting molecules specifically involved in ABC-DLBCL [
      • Pasqualucci L
      • Dalla-Favera R.
      Genetics of diffuse large B-cell lymphoma.
      ]. The respective IC50 values, inhibitors, and their targets are summarized in Table 3. Supplementary Figure E3 provides the dose-response curves with half-maximal inhibitory concentration (IC50) for these inhibitors. As expected, TMD12 cells showed significant sensitivity to all inhibitors, except lenalidomide.
      Table 3IC50 values for the inhibitor concentration versus the survival of TMD12 cells
      The IC50 value represents the minimal drug concentration required for 50% inhibition in vitro. The sensitivity of TMD12 cells to the various inhibitors reported to be effective against ABC-DLBCL cells was evaluated by cell growth inhibition (IC50), as shown in Supplementary Figure E3.
      InhibitorsTargetsIC50 value
      BortezomibProteasome0.93ng/mL
      DasatinibSRC108.13nM
      FostamatinibSYK0.59μM
      IbrutinibBTK0.25μM
      IdelalisibPI3Kδ2.93μM
      LenalidomideIKZF1/3 etc.NAμM
      RapamycinmTORC10.23μM
      IKZF1/3=ikaros zinc finger protein family 1/3; NA=not available.
      a The IC50 value represents the minimal drug concentration required for 50% inhibition in vitro. The sensitivity of TMD12 cells to the various inhibitors reported to be effective against ABC-DLBCL cells was evaluated by cell growth inhibition (IC50), as shown in Supplementary Figure E3.
      DLBCL cells with both MYD88 and CD79B mutations reportedly acquired sensitivity to specific BTK inhibitors, including ibrutinib (IBR) [
      • Phelan JD
      • Young RM
      • Webster DE
      • et al.
      A multiprotein supercomplex controlling oncogenic signalling in lymphoma.
      ], unless the DLBCL cells had an activating mutation in CARD11 or inactivation of A20 (coded by TNFAIP3) [
      • Ondrisova L
      • Mraz M.
      Genetic and non-genetic mechanisms of resistance to BCR signaling inhibitors in B cell malignancies.
      ]. Accordingly, we observed that the proliferation of TMD12 and TMD8 cells was suppressed by IBR treatment in a concentration-dependent manner. However, TMD12 cells showed moderate resistance to IBR compared with TMD8 cells (Figure 4A). To further test this result, we performed the CCK-8 cell proliferation assay and annexin-V-propidium iodide (PI) apoptosis assay, and as shown in Figure 4B, similar trends were observed, showing that TMD12 cells were less sensitive to IBR than TMD8 cells despite the equivalent mutation status. Consistent with these results, in the immunoblot analysis (Figure 4C), IBR treatment of TMD8 cells elicited the dephosphorylation of IKKα/β as well as downregulation of both c-MYC and IRF4 in a concentration-dependent manner, resulting in cell apoptosis, which was confirmed based on caspase 3 cleavage. TMD12 cells were less affected by IBR in terms of apoptosis induction, although we observed c-MYC downregulation at a level comparable with that of observed in TMD8 cells.
      Notably, TMD12 cells exhibited more enhanced activation of the NF-κB pathway, as evidenced by increased phosphorylation of IKKα/β at S176/180 and p65 at S536 compared with TMD8 cells. On the other hand, the expression and phosphorylation of STAT3 were surprisingly lacking in TMD12 cells (Figure 4C). The augmented activity of the NF-κB pathway and resistance to IBR in TMD12 cells implied the existence of other machinery that may be associated with NF-κB activation. It is also noteworthy that the phosphorylation of p105 (a precursor of p50 activated downstream of IKKα/β [
      • Concetti J
      • Wilson CL.
      NFKB1 and cancer: friend or foe?.
      ]) and the expression of IRF4 seemed to be correlated with the survival and proliferation of TMD12 cells, whereas the survival and proliferation of TMD8 cells were more dependent on c-MYC expression and STAT3 activation compared with those of TMD12 cells (Figure 4C).
      The other pathways include the SFK pathway, which promotes aberrant BCR signaling in MYD88-mutated ABC-DLBCL [
      • Munshi M
      • Liu X
      • Kofides A
      • et al.
      A new role for the SRC family kinase HCK as a driver of SYK activation in MYD88 mutated lymphomas.
      ], and PI3K-Akt-mTOR signaling, which functionally coordinates with the My-T-BCR supercomplex, as mentioned earlier [
      • Phelan JD
      • Young RM
      • Webster DE
      • et al.
      A multiprotein supercomplex controlling oncogenic signalling in lymphoma.
      ]. In parallel with an earlier study [
      • Davis RE
      • Ngo VN
      • Lenz G
      • et al.
      Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.
      ] and as indicated by the IC50 values (Table 3 and Supplementary Figure E3), both dasatinib (an SFK inhibitor) [
      • Scuoppo C
      • Wang J
      • Persaud M
      • et al.
      Repurposing dasatinib for diffuse large B cell lymphoma.
      ] and idelalisib (a PI3Kδ inhibitor) [
      • Yahiaoui A
      • Meadows SA
      • Sorensen RA
      • et al.
      PI3Kδ inhibitor idelalisib in combination with BTK inhibitor ONO/GS-4059 in diffuse large B cell lymphoma with acquired resistance to PI3Kδ and BTK inhibitors.
      ] significantly suppressed the growth and induced the apoptosis of TMD8 cells, as shown by the CCK-8 viability assay (Figure 4D, left) and the annexin-V-PI apoptosis assay (Figure 4D, right), in a concentration-dependent manner. However, they did so to only a limited degree in TMD12 cells. Accordingly, the immunoblot analyses revealed that these inhibitors consequently downregulated c-MYC to induce apoptosis in TMD8 cells, confirmed based on caspase 3 cleavage (Figure 4E). In contrast, the sensitivity of TMD12 cells to these inhibitors was relatively limited, and the survival of TMD12 cells was consistent with the phosphorylation status of p105 and IKKα/β and the stability of IRF4, with a possible association with lenalidomide resistance (Figure 4E and Table 3) [
      • Zhang LH
      • Kosek J
      • Wang M
      • Heise C
      • Schafer PH
      • Chopra R.
      Lenalidomide efficacy in activated B-cell-like subtype diffuse large B-cell lymphoma is dependent upon IRF4 and cereblon expression.
      ].

      TPL2 Protein Expression in B-Cell Malignancies and Its Function in ABC-DLBCL

      To decipher the potential dependence of the ABC phenotype on p105 signaling, we next focused on TPL2, the intracellular kinase mentioned earlier. It is activated downstream of IKKα/β, especially in response to stimulation of MYD88-dependent TLRs, such as TLR9 and TLR4, in immune responses, leading to coordination with NF-κB signaling via p105 [
      • Yang HT
      • Papoutsopoulou S
      • Belich M
      • et al.
      Coordinate regulation of TPL-2 and NF-kB signaling in macrophages by NF-kB1 p105.
      ]. The significance of each signaling pathway in the development of certain lymphoma subtypes may mirror their distinct functions in normal counterparts. Thus, it is possible to speculate the involvement of TPL2 in the oncogenesis of MYD88-mutated B-cell lymphomas, the COO of which is suspected to be activated B cells expressing several TLRs.
      Figure 5A depicts the results of the immunoblot analysis, showing T290 phosphorylation or the expression of TPL2 and other subtype-characterizing molecules in B-cell malignancy cell lines or clinical samples from patients with DLBCL. Consistent with an earlier report [
      • Hebron E
      • Hope C
      • Kim J
      • et al.
      MAP3K8 kinase regulates myeloma growth by cell-autonomous and non-autonomous mechanisms involving myeloma-associated monocytes/macrophages.
      ], TPL2 was widely expressed in all tumors and phosphorylated at T290 (Figure 5A). As expected, the expression or activation of other molecules, such as NF-κB components, IRF4, and c-MYC, was context dependent. Lymphoma cells with the ABC phenotype showed enhanced phosphorylation of p105 or p65 and increased expression of IRF4, whereas cells with the GCB phenotype did not. In addition, the expression level of c-MYC differs among B-cell tumors. These results implied a link between activation of the TPL2-p105 pathway and upregulation of IRF4 (Figure 5A). Interestingly, NF-κB2 p100, which has been reported to be more closely associated with the ABC phenotype and IRF4 compared with p105 [
      • Guo X
      • Koff JL
      • Moffitt AB
      • et al.
      Molecular impact of selective NFKB1 and NFKB2 signaling on DLBCL phenotype.
      ], showed relatively reduced phosphorylation at S866/870 despite its higher expression compared with that of p105 (Figure 5A).
      Figure 5
      Figure 5TPL2 and its involvement in ABC-DLBCL tumorigenesis. (A) Immunoblot analysis; the TPL2 protein expression in B-cell malignancy cell lines and clinical lymphoma samples. As indicated, lymphoma cells isolated from clinical samples from patients diagnosed with non-GCB-DLBCL (case 1) or GCB-DLBCL (case 2) were lysed and subjected to an immunoblot analysis. The equivalent number of untreated tumor cells of the B-cell malignancy cell lines TMD8, TMD12, BJAB, Raji, and RPMI8226 were lysed and subjected to an immunoblot analysis. Both TMD8 and TMD12 cells have an ABC-DLBCL phenotype (described as ABC), whereas BJAB cells have the GCB-DLBCL phenotype (GCB). The cell line Raji is derived from Burkitt lymphoma, and RPMI8226 is derived from multiple myeloma. The transferred membrane was repeatedly immunoblotted with specific antibodies against the proteins indicated in the panel. Dotted and solid arrows indicate the levels of blotted protein bands of p105 and p100, respectively. TPL2-T290-P, phosphor-T290-TPL2; p100-S866/870-P, phospho-S866/870-NF-κB2 p100. (B) Specific TPL2 inhibition affected the cell growth and viability of ABC-DLBCL cells compared with those of GCB-DLBCL cells. Top: TMD12 cells, TMD8 cells (both ABC phenotype), and BJAB cells (GCB phenotype) were incubated with dimethyl sulfoxide (DMSO) as the control or the indicated concentrations of a specific TPL2 inhibitor for the indicated number of days. The number of viable cells was counted and plotted. Error bars = the SE obtained from 3 independent assays. Bottom: TMD12, TMD8, and BJAB cells were incubated with DMSO as a control or the indicated concentration of the specific TPL2 inhibitor for 24 or 48 hours. The cells were then subjected to a colorimetric assay. Each column represents the mean of triplicate assays (error bars = SE) and is expressed as a percentage to the control. The 1-way analysis of variance and Dunnett post hoc tests were performed to calculate differences between means; *p < 0.05, **p < 0.01, ***p < 0.001 vs. control. (C) Immunoblot analysis of TMD12 and TMD8 cells under treatment with the specific TPL2 inhibitor. The cells were incubated in the presence or absence of the TPL2 inhibitor at the indicated concentrations for 22 hours. The cells were then harvested, lysed, and subjected to an immunoblot analysis. The transferred membrane was repeatedly immunoblotted with specific antibodies against the proteins indicated in the panel. JNK-T183/Y185-P, phospho-T183/Y185-JNK (SAPK). (D) Cell cycle analysis of TMD12 cells under treatment with the specific TPL2 inhibitor. TMD12 cells were incubated with DMSO as the control or the TPL2 inhibitor at 5 μM for 0, 4, 8, or 16 hours. The cells were then harvested and stained with a PI-based buffer for the cell cycle analysis. The cellular DNA contents were analyzed using FCM. The percentages of cells in the G1/S phase were recorded at each time point after treatment, as indicated, and are shown as columns (error bars = SE) obtained from 3 independent assays. *p < 0.05 vs. control by unpaired 2-tailed Student t test. (E) Quantitative qPCR for the genes in TMD12 cells affected by the treatment with the specific TPL2 inhibitor (TPL2-I). TMD12 cells were treated with DMSO as the control or the TPL2 inhibitor at 2.5 μM for 12 hours and then harvested, and messenger RNA (mRNA) was extracted for complementary DNA synthesis. The mRNA expression levels of the MYC, CDKN1B, and CCNE genes relative to that of the TUBB gene as a reference were analyzed using qRT-PCR and are shown as columns (error bars = SE) obtained from 3 independent assays. *p < 0.05, **p < 0.01 vs. control, using unpaired 2-tailed Student t test. (F) A schematic model of intracellular signaling mechanisms by which the TPL2-p105 axis is involved in the tumorigenesis of ABC-DLBCL with aberrant TLRs-MYD88 signaling, resulting in constitutive NF-κB activation. Specific inhibitors are underlined. Stars indicate recurrent mutations. CBM=CBM complex consists of CARD11, BCL10, and MALT1; TF=transcription factor.
      To evaluate the differential function of TPL2-p105 in ABC-DLBCL, we treated DLBCL cells with a specific TPL2 inhibitor (Tpl2 Kinase Inhibitor II, CAS 1186649-59-1, a potent adenosine triphosphate-competitive, small-molecule TPL2 inhibitor) and assessed its effect on cell growth and viability. Intriguingly, the inhibitor suppressed the growth of TMD12 and TMD8 cells (ABC) with similar intensity but not of BJAB cells (GCB) (Figure 5B, top). The CCK-8 assay revealed the same tendency (Figure 5B, bottom), suggesting an ABC subtype-specific role of TPL2.
      These findings suggested that the TPL2-p105 pathway is implicated in the tumorigenesis of DLBCL, preferentially in the ABC subtype. To elucidate the mechanism by which the inhibition of TPL2 regulates cell proliferation, we performed the immunoblot analysis and investigated the effect of the TPL2 inhibitor on the molecules downstream of TPL2-p105. As expected, the pharmacologic inhibition of TPL2 induced the dephosphorylation of p105 (at S932), p65, and c-Jun N-terminal kinase (JNK), which is a well-described substrate of TPL2 [
      • Njunge LW
      • Estania AP
      • Guo Y
      • Liu W
      • Yang L.
      Tumor progression locus 2 (TPL2) in tumor-promoting inflammation, tumorigenesis and tumor immunity.
      ], and it induced the downregulation of c-MYC in TMD12 and TMD8 cells (Figure 5C). Consistently, the cell cycle analysis revealed that treatment with the TPL2 inhibitor induced cell cycle arrest at G1 in TMD12 cells in a time-dependent manner (Figure 5D), indicating a role of TPL2 in regulating cell cycle progression.
      Accordingly, the qPCR analysis showed that the inhibition of TPL2 induced the downregulation of MYC and CCNE and upregulation of CDKN1B (coding for p27), a predominant negative regulator of cell cycle progression in non-GCB-DLBCL (Figure 5E) [
      • Pasanen AK
      • Haapasaari KM
      • Peltonen J
      • et al.
      Cell cycle regulation score predicts relapse-free survival in non-germinal centre diffuse large B-cell lymphoma patients treated by means of immunochemotherapy.
      ]. Taking together, we speculated that the p105-TPL2 axis regulates cell cycle progression in ABC-DLBCL via the activation of NF-κB and a MAPK cascade (a schematic model is presented in Figure 5F). To the best of our knowledge, the present study is the first to imply the roles of the TPL2-p105 pathway in the tumorigenesis of ABC-DLBCL. However, the specificity of the involvement of TPL2-p105 in ABC-DLBCL or other TLR-MYD88-driven lymphomas requires a detailed investigation in future studies.

      DISCUSSION

      We described the establishment of a new ABC-DLBCL cell line, designated as TMD12, from a patient with refractory non-GCB-DLBCL. TMD12 cells were characterized by markedly enhanced activation of the NF-κB pathway because of mutations in MYD88 and CD79B featuring a COO-based ABC subtype and an MCD subtype based on a recent genetics-based classification system [
      • Wright GW
      • Huang DW
      • Phelan JD
      • et al.
      A probabilistic classification tool for genetic subtypes of diffuse large B cell lymphoma with therapeutic implications.
      ,
      • Chen R
      • Zhou Wang L
      • Zhu L
      • Ye X.
      MYD88L265P and CD79B double mutations type (MCD type) of diffuse large B-cell lymphoma: mechanism, clinical characteristics, and targeted therapy.
      ], as is also true of TMD8 [
      • Tohda S
      • Sato T
      • Kogoshi H
      • Fu L
      • Sakano S
      • Nara N.
      Establishment of a novel B-cell lymphoma cell line with suppressed growth by gamma-secretase inhibitors.
      ]. It is noteworthy that the CNV analysis of TMD12 (Supplementary Figure E2) showed the gain of chromosome 3p (MYD88 locus) and loss of chromosome 6q (loci of TNFAIP3 and PRDM1). In addition, the activation of STAT3, another hallmark of this subtype [
      • Ding BB
      • Yu JJ
      • Yu RY
      • et al.
      Constitutively activated STAT3 promotes cell proliferation and survival in the activated B-cell subtype of diffuse large B-cell lymphomas.
      ,
      • Scuto A
      • Kujawski M
      • Kowolik C
      • et al.
      STAT3 inhibition is a therapeutic strategy for ABC-like diffuse large B-cell lymphoma.
      ,
      • Ok CY
      • Chen J
      • Xu-Monette ZY
      • et al.
      Clinical implications of phosphorylated STAT3 expression in de novo diffuse large B-cell lymphoma.
      ], was unexpectedly absent in TMD12 cells, implying the heterogeneity of the entity. In this context, the establishment of as many cell lines as possible with different phenotypes but a shared genetic background could facilitate comprehensive and unbiased preclinical studies of ABC-DLBCL.
      The specific BTK inhibitor IBR has shown remarkable therapeutic potential for this DLBCL subtype, particularly when it harbors both MYD88 and CD79B mutations in vitro [
      • Phelan JD
      • Young RM
      • Webster DE
      • et al.
      A multiprotein supercomplex controlling oncogenic signalling in lymphoma.
      ] and in vivo, as reported very recently [
      • Wilson WH
      • Wright GW
      • Hodkinson B
      • et al.
      Effect of ibrutinib with R-CHOP chemotherapy in genetic subtypes of DLBCL.
      ]. This underlines the critical role of aberrant BCR signaling accompanied by BTK activation in the tumorigenesis of ABC-DLBCL [
      • Phelan JD
      • Young RM
      • Webster DE
      • et al.
      A multiprotein supercomplex controlling oncogenic signalling in lymphoma.
      ]. In this regard, because TMD12 cells harbor MYD88L265P and CD79BY196N (Figure 3D), we assumed that TMD12 cells would respond to IBR. However, unexpectedly, TMD12 cells displayed resistance to IBR compared with TMD8 cells (Figure 4A–C) through, as yet, undefined mechanisms.
      We suspect that dysfunction of A20, a negative regulator of the NF-kB pathway, coded by TNFAIP3 [
      • Honma K
      • Tsuzuki S
      • Nakagawa M
      • et al.
      TNFAIP3/A20 functions as a novel tumor suppressor gene in several subtypes of non-Hodgkin lymphomas.
      ,
      • Kato M
      • Sanada M
      • Kato I
      • et al.
      Frequent inactivation of A20 in B-cell lymphomas.
      ], or the deletion of 6q23 (TNFAIP3 locus) might be partly responsible for our present findings [
      • Thelander EF
      • Ichimura K
      • Corcoran M
      • et al.
      Characterization of 6q deletions in mature B cell lymphomas and childhood acute lymphoblastic leukemia.
      ]. As shown in the results (Figure 3C, Supplementary Table E1, and Supplementary Figure E2) indicating heterozygous 6q23 deletion, the dysfunction of A20 might potentiate IBR resistance in TMD12 cells. In addition, the survival and proliferation of TMD12 cells were still dependent on the activation of IKKα/β and its downstream NF-κB pathway even under treatment with IBR, which could mechanically diminish IKKα/β phosphorylation (Figure 4A–C), as in TMD8 cells. Interestingly, similar results were observed when TMD12 cells were treated with inhibitors of SFKs or PI3K-Akt-mTOR (Figure 4D, E). These results suggested the involvement of ≥1 other pathways reinforcing IKKα/β activation, by which the effect of IBR could be alleviated.
      In light of this, we focused on TPL2-p105 signaling because it is activated by IKKα/β, particularly under the activation of TLR-MYD88 signaling in immune responses [
      • Njunge LW
      • Estania AP
      • Guo Y
      • Liu W
      • Yang L.
      Tumor progression locus 2 (TPL2) in tumor-promoting inflammation, tumorigenesis and tumor immunity.
      ]. Of note, in TLR4-activated B cells, TPL2 has been reported to play a role in the regulation of cell survival and proliferation [
      • Banerjee A
      • Grumont R
      • Gugasyan R
      • White C
      • Strasser A
      • Gerondakis S.
      NF-kB1 and c-Rel cooperate to promote the survival of TLR4-activated B cells by neutralizing Bim via distinct mechanisms.
      ]. As expected, a specific TPL2 inhibitor suppressed the proliferation of ABC-DLBCL cells (TMD8 and TMD12 cells), presumably through the induction of cell cycle arrest at G1 (Figure 5D) via upregulation of CDKN1B (coding p27) and downregulation of CCNE (coding cyclin E) (Figure 5E). Conversely, the inhibitor exerted only a limited effect on the BJAB cells, which display the GCB phenotype lacking NF-κB activity (Figure 5B). Intriguingly, the TPL2 inhibitor also induced the dephosphorylation of IKKα/β (Figure 5C), an upstream molecule of the TPL2-p105-ABIN2 complex, suggesting its physiologic function in the regulation of the canonical NF-κB pathway itself, possibly through ABIN2 [
      • Verstrepen L
      • Carpentier I
      • Verhelst K
      • Beyaert R.
      ABINs: A20 binding inhibitors of NF-kB and apoptosis signaling.
      ], known as a negative regulator of NF-κB. These results might indicate a selective role of the TPL2-p105 pathway in the tumorigenesis of ABC-DLBCL but not in that of GCB-DLBCL.
      Of note, an earlier report suggested that the function of p105 affects the prognosis of patients with DLBCL via regulation of plasma interleukin 6 [
      • Giachelia M
      • Voso MT
      • Tisi MC
      • et al.
      Interleukin-6 plasma levels are modulated by a polymorphism in the NF-kB1 gene and are associated with outcome following rituximab-combined chemotherapy in diffuse large B-cell non-Hodgkin lymphoma.
      ]. In addition, mutations in ABIN1/2 as well as TNFAIP3 have been implicated in poor clinical outcomes in a subset of DLBCL [
      • Dong G
      • Chanudet E
      • Zeng N
      • et al.
      A20, ABIN-1/2, and CARD11 mutations and their prognostic value in gastrointestinal diffuse large B-cell lymphoma.
      ]. Together, these findings could shed light on the TPL2-p105 pathway as a potential therapeutic target in B-cell malignancies with MYD88 mutations such as ABC-DLBCL (Figure 5F). However, because these findings were obtained from experimental settings using a few immortalized cell lines and a limited number of clinical samples, the results must be interpreted with caution. Additionally, the result indicating the differential dependency of the ABC subtype on TPL2-p105 might have been caused by unrecognized off-target effects of the TPL2 inhibitor used, eliciting concurrent inhibition of other pathways that cannot be simply generalized to the ABC subtype.
      Contrary to our hypothesis, Guo et al. [
      • Guo X
      • Koff JL
      • Moffitt AB
      • et al.
      Molecular impact of selective NFKB1 and NFKB2 signaling on DLBCL phenotype.
      ] reported the dependency of ABC-DLBCL on the p100-noncanonical NF-κB pathway, particularly driven by MYD88L265P, rather than the p105 canonical pathway. Because the dependence of the ABC phenotype on p100 was not necessarily reproducible in our study (Figure 5A), gene expression analyses under the pharmacologic or genetic knockdown of TPL2-p105 signaling might be helpful for uncovering the precise functions of TPL2-p105 in the tumorigenesis of ABC-DLBCL.
      In conclusion, TMD12 was identified as a novel ABC-DLBCL cell line with nontypical activation of signaling pathways, implying novel therapeutic targets, such as the TPL2-p105 pathway associated with the COO of this subtype. TMD12 cells could provide a useful tool for basic research to explore the heterogeneous properties of ABC-DLBCL.

      Conflicts of Interest Disclosure

      The authors do not have any conflicts of interest to declare in relation to this work.

      Acknowledgments

      We thank Dr. S. Tohda for the generous gifts of experimental materials and Dr. K. Watanabe for the clinical care of the patient from whom the new cell line was derived. We thank KN International, Inc. for English language editing. The study was supported in part by a Grant-in-Aid for Scientific Research (C), from the Japan Society for the Promotion of Science KAKENHI, No. 19K08832.

      Data Availability Statement

      The data that support the findings of this study will be available from the corresponding author upon reasonable request.

      Appendix. Supplementary materials

      • Supplementary Figure E1 Additional cytogenetic analysis of TMD12 cells. The SKY analysis of TMD12 cells in representative metaphase revealed multiple chromosomal aberrations. The reversed 4′,6-diamidino-2-phenylindole -stained image is shown on the upper left, and the RGB display image is on the upper right. The full karyotype is shown in the lower panel, with each chromosome in its spectra-based classification color flanked by 4′,6-diamidino-2-phenylindole and RGB images.

      • Supplementary Figure E2 Gene copy number variations in TMD12 cells. The calculated copy numbers were plotted in order of chromosome number. The amplification regions (red colored) were as follows: chr3, 239350–49336097; chr5, 155756421–180899058; chr6, 41874513–58287733; and chr18, 47395–78005277. The deletion regions (blue colored) were as follows: chr6, 62390739–171055067; chr7, 6906965–57528764; and chr15, 82636531–102519089.

      • Supplementary Figure E3. IC50 values of specific inhibitors. TMD12 cells were cultured with the indicated concentrations of the following specific inhibitors for the indicated durations: (A) bortezomib, (B) dasatinib, (C) fostamatinib, (D) Ibrutinib, (E) Idelalisib, (F) lenalidomide, and (G) rapamycin. The dose-response curves with IC50 values with respect to the viable cell number under treatment with the respective inhibitors are shown. The relative cell numbers expressed as the percentage of cell numbers with no inhibitors from triplicate samples are plotted using 4-parameter logistic curves obtained using the ImageJ software (U.S. National Institute of Health, Bethesda, Maryland) using the calculated IC50 values shown. The calculated regression curve is represented by the line. The IC50 results are also summarized in Table 3. NA=Not available.

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