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Offprint requests to: Toshikage Nagao, Department of Hematology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 14F, M&D Tower, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
Department of Hematology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, JapanDepartment of Laboratory Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
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 [
]. 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) [
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 [
]. 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 [
]. 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 [
]. 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 [
] 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 [
]. 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 [
]. 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 [
] 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 [
]. 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
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.
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.
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 [
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 [
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.
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. [
] 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).
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.
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.
T cells, some B cells
Germinal center B cells
B cells or activated T cells
Plasmacytes, B cells, T cells
B cells or activated T cells
Light Ig chains
Light 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)/47, idem, −Y/49, idem, +mar/ (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 [
]. 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) [
], 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) [
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.
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.
]. 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.
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.
]. 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 [
]) 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 [
] 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) [
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 [
]. 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 [
], 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 [
], showed relatively reduced phosphorylation at S866/870 despite its higher expression compared with that of p105 (Figure 5A).
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 [
], 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) [
]. 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.
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 [
]. 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 [
], 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 [
]. 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 [
]. 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 [
]. 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 [
]. 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.
] 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.
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.
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.
TN and KoYo contributed to the project's conception and the design of the experiments. TN and KoYo performed most of the experiments and analyzed the results. DS analyzed whole-exome sequencing data. YM, MS, AN, KuYa, and KoYa also performed experiments and analyzed the results. TN wrote the paper with contributions from all of the other co-authors. All authors reviewed the manuscript.