TRAF3, a cytoplasmic adaptor protein, was recently identified as a candidate tumor suppressor in a range of human B-cell lineage neoplasms. Homozygous deletions and inactivating mutations of the Traf3 gene were detected in non-Hodgkin lymphoma, including splenic marginal zone lymphoma (MZL), B-cell chronic lymphocytic leukemia, mantle cell lymphoma, as well as multiple myeloma and Waldenström's macroglobulinemia.1-4 However, whether TRAF3 deletions or mutations have a causative role in B lymphomagenesis awaits further in vivo study using TRAF3-knockout mice.
We have recently generated a conditional loss-of-function allele of the Traf3 gene that allows specific deletion of TRAF3 in B lymphocytes in mice (B-TRAF3–/– mice).5 These mice exhibit greatly expanded B-cell populations in secondary lymphoid organs, due to remarkably prolonged, BAFF-independent survival of mature B cells, a finding subsequently confirmed by Gardam et al.6 TRAF3 and TRAF2 assemble to form a regulatory complex with cIAP1/2 and NIK to inhibit the NF-κB2-signaling pathway.7,8 These observations define a central role of the TRAF3-NIK-NF-κB2 axis in regulating B-cell survival. Prolonged survival of B cells is a known predisposing factor to the development of autoimmunity and B lymphoma.9,10 Indeed, aging B-TRAF3–/– mice developed high levels of serum autoantibodies to double-stranded DNA as well as immune-complex glomerulonephritis.5 Autoimmunity itself is recognized as a risk factor of B lymphoma in patients with systemic lupus erythematosus or Sjogren's syndrome.11 Hence, B-TRAF3–/– mice may be predisposed to B-cell malignancies. Here we report that B-TRAF3–/– mice spontaneously developed clonal splenic MZL or B1a lymphomas by the age of 18 months.
We first noticed that B-TRAF3–/– mice have markedly decreased survival rate at ages >9 months (Figure 1a). We examined tissues of 50 B-TRAF3–/– mice 9-18 months of age, 18 that were necropsied when moribund and 32 that had no external signs of disease at the time of study. All 18 moribund B-TRAF3–/– mice and 20 out of the other 32 (62.5%) had splenic B lymphoma (Supplementary Table 1). Gross observations and microscopic studies also revealed involvement by lymphoma of other tissues including bone marrow, cervical and mesenteric lymph nodes, kidney, lung and liver as well as ascites (Figures 1b and c and Supplementary Figure S1, and Supplementary Materials and Methods). Based on criteria of the Bethesda classification of lymphoid neoplasms,12 the tumors of B-TRAF3–/– mice were splenic MZL, including both high-grade MZL with the cytology of centroblastic diffuse large B-cell lymphomas and low-grade MZL with features of normal MZ B cells.
We next used flow cytometry to characterize the immunophenotypic features of tumor cells present in spleen, bone marrow and ascites (Figure 1d and Supplementary Figure S2). Flow cytometric data of 21 individual mice are summarized in Supplementary Table 2. In keeping with the histologic diagnoses, the immunophenotypic profiles of the tumors were characteristic of mature B-cell lymphomas. With one exception, all tests for canonical mature B-cell markers–B220, CD19, IgM, IgD and Igκ or Igλ–were positive to varying extents. One exceptional case (27-9) was IgG+IgM–IgD– with 20% CD138+. The immunophenotypic profiles of nine examined lymphomas resembled that of B1a cells whereas two additional cases resembled that of B1b cells. Out of the remaining 10 cases, 5 were CD5 CD11b–CD21–CD23+, resembling normal MZ B cells. Thus, over half (11/21) of TRAF3–/– B lymphomas originated from B1 B cells, and another 23% (5/21) originated from MZ B cells. This suggests that many cases diagnosed histologically as centroblastic diffuse large B-cell lymphomas were actually high-grade splenic MZL.
We further performed Southern blot analyses of the IgH gene of primary tumor samples from 21 individual B-TRAF3–/– mice to determine their clonality. The results (Figure 1e) revealed the presence of one or several non-germline bands in each of the DNAs examined, indicating monoclonal or oligoclonal expansions of malignant B cells. DNA prepared from two or three tissues of the same mice often exhibited rearranged bands of similar size, indicating metastatic spread of the same clone.
These observations prompted us to seek more direct evidence for malignant transformation by intraperitoneal transfer of tumor cells with known clonal populations to immunodeficient NOD SCID mice. At 2-3 months after transfer, recipient mice were found to have lymphoma infiltrates in multiple tissues. The transplanted tumors had the same immuno phenotypic features as the donor population (Figure 1f). Southern blot analyses revealed that one or two clonal populations from primary TRAF3–/– B lymphomas were propagated in NOD SCID recipient mice (Figure 1g). Thus, the spontaneous B lymphomas that developed in B-TRAF3–/– mice are clonal, malignant and transplantable.
To gain additional information about the origin, partitioning and history of malignant B cells, we cloned and sequenced the IgH V(D)J regions of lymphomas from 11 B-TRAF3–/– mice (Table 1). The IgH V region sequences of 10 of the 11 examined mice did not exhibit significant somatic hypermutation, suggesting that they were not derived from germinal center-passaged cells but rather from naive pre-germinal center B cells, MZ B cells or B1 cells. Interestingly, all of the major VH sequences identified in TRAF3–/– B lymphomas belonged to one of four VH families, VH1 (J558), VH3 (VH36-60), VH5 (VH7183) and VH12. These gene families are frequently used by MZ, B1, or autoreactive B cells. Thus, IgH V(D)J sequences, together with the histopathological and immunophenotypic data, allow us to classify B lymphomas developed in most B-TRAF3–/– mice as splenic MZL or B1 lymphomas.
Table 1.
Mouse | Tissue | Bands on Southerna | Heavy-chain variable region |
DHe | JH | Frequency | SHM | |||
---|---|---|---|---|---|---|---|---|---|---|
DNAb | AAb | GLc | VHd | |||||||
6983-2 | Spleen | 1 dominant | 98.6 | 99.1 | VH36-60.a2.90 | VH36-60 | DSP2.2 | JH2 | 11/20 | No |
1 minor | 98.9 | 97.2 | J558.37.127 | J558 | DSP2.8 | JH2 | 5/20 | No | ||
99.7 | 99.1 | VHD6.96 (or VH7183.a4.6) | VHD6 | DST4.3 | JH2 | 4/20 | No | |||
7041-10 | Spleen | 1 dominant | 98.6 | 99.1 | VH36-60.a2.90 | VH36-60 | DSP2.5 | JH2 | 18/20 | No |
99.3 | 99.2 | VH7183.a19.31 | VH7183 | DSP2.11 | JH3 | 2/20 | No | |||
7060-8 | Spleen | 1 dominant | 99.0 | 98.2 | VH7183.a25.43 (or VH283) | VH7183 | DST4.3 | JH4 | 8/19 | No |
98.6 | 99.1 | VH36-60.a2.90 | VH36-60 | DSP2.9 | JH2 | 11/19 | No | |||
Ascites | 1 dominant (same) | 99.0 | 98.2 | VH7183.a25.43 (or VH283) | VH7183 | DST4.3 | JH4 | 11/18 | No | |
98.6 | 99.1 | VH36-60.a2.90 | VH36-60 | DSP2.9 | JH2 | 6/18 | No | |||
7140-7 | Spleen | 1 dominant | 97.9 | 96.6 | J558.22 | J558 | DSP2.2 | JH3 | 9/17 | No |
99.7 | 99.1 | VH98-3G (VH7183.a21.35) | VH98-3G | DSP2.6 | JH3 | 5/17 | No | |||
99.3 | 98.3 | V11 (VHS107.a3.106) | V11 | DSP2.2 | JH2 | 3/17 | No | |||
7140-3 | Spleen | 1 dominant | 97.9 | 97.5 | J558.17 | J558 | DSP2.2 | JH4 | 7/18 | No |
99.7 | 100 | VH7183.a47.76 | VH7183 | DFL16.2 | JH3 | 4/18 | No | |||
Ascites | Not determined | 99.0 | 99.1 | VH12.a3.101 | VH12 | DSP2.8 | JH1 | 12/19 | No | |
98.6 | 98.3 | J558.17 | J558 | DSP2.2 | JH4 | 3/19 | No | |||
7079-8 | Ascites | 2 equimolar | 99.7 | 99.1 | VH7183.a2.3 (7183.2.3) | VH7183 | DSP2.11 | JH3 | 18/21 | No |
95.6 | 94.2 | V98-3G | VH98-3G | DSP2.9 | JH4 | 2/21 | No | |||
5-5 | Spleen | 2 equimolar | 99.7 | 99.1 | VH98-3G (VH7183.a21.35) | VH98-3G | DSP2.9 | JH1 | 15/20 | No |
99.3 | 98.3 | VS107.a3.106 | VS107 | DFL16.1 | JH4 | 3/20 | No | |||
7079-4 | Ascites | 2 equimolar | 98.0 | 98.3 | VH7183.a19.31 | VH7183 | DQ52 | JH2 | 4/22 | No |
99.7 | 99.1 | V98-3G (VH7183.a21.35) | VH98-3G | DSP2.x | JH2 | 4/22 | No | |||
100 | 100 | VH7183.a47.76 | VH7183 | DSP2.7 | JH2 | 3/22 | No | |||
99.7 | 99.1 | VH7183.a30.50 | VH7183 | DST4.2 | JH3 | 2/22 | No | |||
33-3 | Spleen | 2 equimolar dominant | 97.6 | 96.7 | VH7183.a19.31 | VH7183 | DFL16.1 | JH4 | 8/23 | No |
2 equimolar minor | 99.0 | 99.1 | VOx-1 | Vox | DSP2.7 | JH4 | 4/23 | No | ||
100 | 100 | VH7183.a47.76 | VH7183 | DQ52 | JH4 | 3/23 | No | |||
99.7 | 99.2 | VH7183.a7.10 | VH7183 | DSP2.2 | JH2 | 2/23 | No | |||
115-6 | Spleen | Not determined | 93.0 | 87.5 | J558.39.129 | J558 | DSP2.9 | JH2 | 21/21 | Yes |
Ascites | Not determined | 90.9 | 88.9 | J558.39.129 | J558 | DSP2.9 | JH2 | 8/19 | Yes | |
99.7 | 99.1 | VHD6.96 | VHD6 | DSP2.2 | JH1 | 5/19 | No | |||
99.2 | 98.2 | VHF102 | 2,36,48 | DST4.3 | JH4 | 3/19 | No | |||
105-8 | Spleen | 2 dominant | 98.6 | 98.3 | J558.17 | J558 | DSP2.8 | JH4 | 8/22 | No |
100 | 100 | VH7183.a47.76 | VH7183 | DSP2.2 | JH4 | 6/22 | No | |||
Ascites | 2 dominant | 98.6 | 100 | J558.17 | J558 | DFL16.2 | JH4 | 10/21 | No | |
99.3 | 98.2 | J558.39.129 | J558 | DSP2.9 | JH2 | 5/21 | No | |||
100 | 100 | VH7183.a47.76 | VH7183 | DFL16.2 | JH4 | 3/21 | No |
Abbreviations: AA, amino acid; GL, germline sequences; SHM, somatic hypermutation.
The IgH VDJ regions of primary TRAF3–/– B lymphomas were cloned by reverse transcription and PCR using primers VH consensus (5′-GTGCAGCTGGTGGAG TCTGG-3′) and C-μ4(5′-CCTGGATGACTTCAGTGTTGTTCTG-3′). The high-fidelity polymerase, Pfu UltraII, was used in the amplification reaction. PCR products were subsequently subcloned into pBlueScript vector. Mini-prep DNAs of about 20 clones of each B-lymphoma sample were sequenced using the primer C-μSq (5′-CCA-CCA-GAT-TCT-TAT-CAG-ACA-GGG-3′).
Clonal IgH rearrangements identified by Southern blot hybridization (Figure 1e).
Percent homology between tumor DNA and amino acid sequences and germline sequences.
Closest matched GL gene.
VH family.
DH segment.
We previously showed constitutive activation of NF-κB2 and decreased nuclear translocation of PKCδ, but normal CD40-induced NF-κB1 activation in premalignant TRAF3–/– B cells.5 Here, we extended our analyses of these pathways to lymphomas of B-TRAF3–/– mice. We found that the elevated nuclear levels of the NF-κB2 subunits, p52 and RelB, and the decreased nuclear levels of PKCδ were also features of primary B lymphomas (Figure 2a). Interestingly, TRAF3–/– B lymphomas also had moderately increased nuclear levels of the NF-κB1. Hence, the signaling landscape of TRAF3–/– B lymphomas is characterized by constitutive activation of both the NF-κB2 and NF-κB1 (albeit to a lesser extent) pathways as well as reduced nuclear translocation of PKCδ.
To test whether aberrant NF-κB activation pathways could serve as therapeutic targets for B-cell neoplasms associated with TRAF3 inactivation, we evaluated the effects on cell viability of drugs that modulate NF-κB activation using primary B-lymphoma cells purified from B-TRAF3–/– mice. We used an inhibitor of IKK2, BMS-345541,13 and an inhibitor of NF-κB, oridonin.14 The effects of these agents were compared with the activities of drugs used clinically to treat B lymphomas or leukemias–vincristine, all trans-retinoic acid, doxorubicin and cyclophosphamide. We found that oridonin exhibited potent dose-dependent tumoricidal activity on primary lymphoma cells, whereas BMS-345541 and the four clinical drugs were inactive (Figures 2b and c and Supplementary Figure S3).
To understand the mechanisms of oridonin, we determined the levels of NF-κB2 and NF-κB1 subunits in cytosolic and nuclear extracts of primary B lymphomas. The levels of activated NF-κB2 and NF-κB1 in the nucleus were markedly reduced in B lymphoma cells treated with oridonin (Figure 2d). Furthermore, nuclear translocation of PKCδ, or activation of ERK, p38, JNK, and AKT was unaffected by oridonin (Figure 2d and data not shown). Thus, the potent tumoricidal effects of oridonin can be ascribed to its activity in inhibiting the activation of both NF-κB2 and NF-κB1, suggesting that oridonin or closely related agents should be considered as new candidates for the treatment of B-cell neoplasms characterized by genetic/epigenetic inactivation of TRAF3.
We next sought to specifically decrease the NF-κB2 level using lentiviral shRNA vectors to evaluate the role of constitutive NF-κB2 activation in TRAF3–/– B lymphomagenesis. We screened 4 NF-κB2 shRNA lentiviral vectors. The NF-κB2 shRNA 653 and 1226 knocked down both p100 and p52 NF-κB2 proteins to ~95% and ~75% reduction, respectively. We subsequently used these shRNA lentiviruses to transduce the TRAF3–/– B-lymphoma cell line 27-9.5.3 generated in this study (Supplementary Materials and Methods). Interestingly, both NF-κB2 shRNA 653 and 1226 inhibited the proliferation and induced apoptosis in 27-9.5.3 cells. Importantly, the potency of the two shRNAs in knocking down NF-κB2 protein levels correlated with their ability in inhibiting the proliferation and inducing apoptosis in TRAF3–/– B-lymphoma cells (Figure 3). Although confirmation using additional TRAF3–/– B-lymphoma lines is necessary, our data suggest that constitutive NF-κB2 activation is one major oncogenic pathway in TRAF3–/– B cells.
Studies that identified TRAF3 deletions and mutations in B-cell chronic lymphocytic leukemia, MZL, mantle cell lymphoma, Waldenström's macroglobulinemia and multiple myeloma suggest potential tumor-suppressive function of TRAF3.1-4 Paradoxically, transgenic mice overexpressing TRAF3 in B cells display autoimmune disease, systemic inflammation and are predisposed to cancers.15 One possibility raised by Zapata et al.15 is that TRAF3 may promote plasma cell differentiation. Alternatively, the unexpected phenotype of TRAF3-transgenic mice may potentially relate to the site of transgene insertion in the genome that may affect the expression or function of additional important gene(s), considering that only a single transgenic founder line was examined in detail.15 Here, the spontaneous, highly penetrant development of B lymphomas in B-TRAF3–/– mice provides conclusive evidence that Traf3 is a tumor-suppressor gene in B cells.
Collectively, data presented in the present study indicate that B-TRAF3–/– mice closely model human splenic MZL and B-cell chronic lymphocytic leukemia. Using B lymphoma cells derived from B-TRAF3–/– mice as model systems, we demonstrated that oridonin and NF-κB2 shRNAs have therapeutic potential. Our findings suggest that restoration of TRAF3 protein or its downstream signaling pathways represents important therapeutic avenues of B lymphomas. In this context, B-TRAF3–/– mice provide a useful tool for developing and testing therapeutic drugs for the treatment of human B-cell neoplasms involving TRAF3 inactivation or relevant genetic/epigenetic alterations.
Supplementary Material
ACKNOWLEDGEMENTS
We are grateful to Dr Janet Hartley for expert advice on mouse B-lymphoma dissection, and Dr Hongsheng Wang for expert advice on IgH VDJ cloning as well as for the critical review of the manuscript. We also thank Jessica Kim, Anano Zangaladze and Will Meng for providing technical assistance to this study. This study was supported by a seed grant from the New Jersey Commission on Cancer Research (10-1066-CCR-EO, P Xie), a Busch Biomedical Grant (P Xie), and the Arthur Herrmann Endowed Cancer Research Fund (P Xie), and in part by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases (H Morse III). The FACS analyses described in this paper were supported by the Flow Cytometry Core Facility of The Cancer Institute of New Jersey (P30CA072720).
Footnotes
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)
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