Signaling mechanisms of bortezomib in TRAF3-deficient mouse B lymphoma and human multiple myeloma cells.

Bortezomib, a clinical drug for multiple myeloma (MM) and mantle cell lymphoma, exhibits complex mechanisms of action, which vary depending on the cancer type and the critical genetic alterations of each cancer. Here we investigated the signaling mechanisms of bortezomib in mouse B lymphoma and human MM cells deficient in a new tumor suppressor gene, TRAF3. We found that bortezomib consistently induced up-regulation of the cell cycle inhibitor p21(WAF1) and the pro-apoptotic protein Noxa as well as cleavage of the anti-apoptotic protein Mcl-1. Interestingly, bortezomib induced the activation of NF-κB1 and the accumulation of the oncoprotein c-Myc, but inhibited the activation of NF-κB2. Furthermore, we demonstrated that oridonin (an inhibitor of NF-κB1 and NF-κB2) or AD 198 (a drug targeting c-Myc) drastically potentiated the anti-cancer effects of bortezomib in TRAF3-deficient malignant B cells. Taken together, our findings increase the understanding of the mechanisms of action of bortezomib, which would aid the design of novel bortezomib-based combination therapies. Our results also provide a rationale for clinical evaluation of the combinations of bortezomib and oridonin (or other inhibitors of NF-κB1/2) or AD 198 (or other drugs targeting c-Myc) in the treatment of lymphoma and MM, especially in patients containing TRAF3 deletions or relevant mutations.

Targeting TRAF3 Downstream Signaling Pathways in B cell Neoplasms.

B cell neoplasms comprise >50% of blood cancers. However, many types of B cell malignancies remain incurable. Identification and validation of novel genetic risk factors and oncogenic signaling pathways are imperative for the development of new therapeutic strategies. We and others recently identified TRAF3, a cytoplasmic adaptor protein, as a novel tumor suppressor in B lymphocytes. We found that TRAF3 inactivation results in prolonged survival of mature B cells, which eventually leads to spontaneous development of B lymphomas in mice. Corroborating our findings, TRAF3 deletions and inactivating mutations frequently occur in human B cell chronic lymphocytic leukemia, splenic marginal zone lymphoma, mantle cell lymphoma, multiple myeloma, Waldenström's macroglobulinemia, and Hodgkin lymphoma. In this context, we have been investigating TRAF3 signaling mechanisms in B cells, and are developing new therapeutic strategies to target TRAF3 downstream signaling pathways in B cell neoplasms. Here we discuss our new translational data that demonstrate the therapeutic potential of targeting TRAF3 downstream signaling pathways in B lymphoma and multiple myeloma.

Myeloid cell TRAF3 regulates immune responses and inhibits inflammation and tumor development in mice.

Myeloid cells, including granulocytes, monocytes, macrophages, and dendritic cells, are crucial players in innate immunity and inflammation. These cells constitutively or inducibly express a number of receptors of the TNFR and TLR families, whose signals are transduced by TNFR-associated factor (TRAF) molecules. In vitro studies showed that TRAF3 is required for TLR-induced type I IFN production, but the in vivo function of TRAF3 in myeloid cells remains unknown. In this article, we report the generation and characterization of myeloid cell-specific TRAF3-deficient (M-TRAF3(-/-)) mice, which allowed us to gain insights into the in vivo functions of TRAF3 in myeloid cells. We found that TRAF3 ablation did not affect the maturation or homeostasis of myeloid cells in young adult mice, even though TRAF3-deficient macrophages and neutrophils exhibited constitutive NF-κB2 activation. However, in response to injections with LPS (a bacterial mimic) or polyinosinic-polycytidylic acid (a viral mimic), M-TRAF3(-/-) mice exhibited an altered profile of cytokine production. M-TRAF3(-/-) mice immunized with T cell-independent and -dependent Ags displayed elevated T cell-independent IgG3 and T cell-dependent IgG2b responses. Interestingly, 15- to 22-mo-old M-TRAF3(-/-) mice spontaneously developed chronic inflammation or tumors, often affecting multiple organs. Taken together, our findings indicate that TRAF3 expressed in myeloid cells regulates immune responses in myeloid cells and acts to inhibit inflammation and tumor development in mice.

Transcriptomic profiling of splenic B lymphomas spontaneously developed in B cell-specific TRAF3-deficient mice.

TRAF3, a critical regulator of B cell survival, was recently recognized as a tumor suppressor gene in B lymphocytes. Specific deletion of TRAF3 from B lymphocytes leads to spontaneous development of marginal zone lymphomas (MZL) or B1 lymphomas in mice. To identify novel oncogenes and tumor suppressive genes involved in malignant transformation of TRAF3-deficient B cells, we performed a microarray analysis to identify genes differentially expressed in TRAF3-/- mouse splenic B lymphomas. We have identified 160 up-regulated genes and 244 down-regulated genes in TRAF3-/- B lymphomas as compared to littermate control splenocytes. Here we describe the samples, quality control assessment, as well as the data analysis methods in detail for the transcriptomic profiling study. Data are archived at NIH GEO with accession number GSE48818.

Mutated in colorectal cancer (MCC) is a novel oncogene in B lymphocytes.

BACKGROUND: Identification of novel genetic risk factors is imperative for a better understanding of B lymphomagenesis and for the development of novel therapeutic strategies. TRAF3, a critical regulator of B cell survival, was recently recognized as a tumor suppressor gene in B lymphocytes. The present study aimed to identify novel oncogenes involved in malignant transformation of TRAF3-deficient B cells. METHODS: We used microarray analysis to identify genes differentially expressed in TRAF3-/- mouse splenic B lymphomas. We employed lentiviral vector-mediated knockdown or overexpression to manipulate gene expression in human multiple myeloma (MM) cell lines. We analyzed cell apoptosis and proliferation using flow cytometry, and performed biochemical studies to investigate signaling mechanisms. To delineate protein-protein interactions, we applied affinity purification followed by mass spectrometry-based sequencing. RESULTS: We identified mutated in colorectal cancer (MCC) as a gene strikingly up-regulated in TRAF3-deficient mouse B lymphomas and human MM cell lines. Aberrant up-regulation of MCC also occurs in a variety of primary human B cell malignancies, including non-Hodgkin lymphoma (NHL) and MM. In contrast, MCC expression was not detected in normal or premalignant TRAF3-/- B cells even after treatment with B cell stimuli, suggesting that aberrant up-regulation of MCC is specifically associated with malignant transformation of B cells. In elucidating the functional roles of MCC in malignant B cells, we found that lentiviral shRNA vector-mediated knockdown of MCC induced apoptosis and inhibited proliferation in human MM cells. Experiments of knockdown and overexpression of MCC allowed us to identify several downstream targets of MCC in human MM cells, including phospho-ERK, c-Myc, p27, cyclin B1, Mcl-1, caspases 8 and 3. Furthermore, we identified 365 proteins (including 326 novel MCC-interactors) in the MCC interactome, among which PARP1 and PHB2 were two hubs of MCC signaling pathways in human MM cells. CONCLUSIONS: Our results indicate that in sharp contrast to its tumor suppressive role in colorectal cancer, MCC functions as an oncogene in B cells. Our findings suggest that MCC may serve as a diagnostic marker and therapeutic target in B cell malignancies, including NHL and MM.

Expression and function of a novel isoform of Sox5 in malignant B cells.

Using a mouse model with the tumor suppressor TRAF3 deleted from B cells, we identified Sox5 as a gene strikingly up-regulated in B lymphomas. Sox5 proteins were not detected in normal or premalignant TRAF3(-/-) B cells even after treatment with B cell stimuli. The Sox5 expressed in TRAF3(-/-) B lymphomas represents a novel isoform of Sox5, and was localized in the nucleus of malignant B cells. Overexpression of Sox5 inhibited cell cycle progression, and up-regulated the protein levels of p27 and ß-catenin in human multiple myeloma cells. Together, our findings indicate that Sox5 regulates the proliferation of malignant B cells.

N-benzyladriamycin-14-valerate (AD 198) exhibits potent anti-tumor activity on TRAF3-deficient mouse B lymphoma and human multiple myeloma.

BACKGROUND: TRAF3, a new tumor suppressor identified in human non-Hodgkin lymphoma (NHL) and multiple myeloma (MM), induces PKCδ nuclear translocation in B cells. The present study aimed to evaluate the therapeutic potential of two PKCδ activators, N-Benzyladriamycin-14-valerate (AD 198) and ingenol-3-angelate (PEP005), on NHL and MM. METHODS: In vitro anti-tumor activities of AD 198 and PEP005 were determined using TRAF3-/- mouse B lymphoma and human patient-derived MM cell lines as model systems. In vivo therapeutic effects of AD 198 were assessed using NOD SCID mice transplanted with TRAF3-/- mouse B lymphoma cells. Biochemical studies were performed to investigate signaling mechanisms induced by AD 198 or PEP005, including subcellular translocation of PKCδ. RESULTS: We found that AD 198 exhibited potent in vitro and in vivo anti-tumor activity on TRAF3-/- tumor B cells, while PEP005 displayed contradictory anti- or pro-tumor activities on different cell lines. Detailed mechanistic investigation revealed that AD 198 did not affect PKCδ nuclear translocation, but strikingly suppressed c-Myc expression and inhibited the phosphorylation of ERK, p38 and JNK in TRAF3-/- tumor B cells. In contrast, PEP005 activated multiple signaling pathways in these cells, including PKCδ, PKCα, PKCε, NF-κB1, ERK, JNK, and Akt. Additionally, AD198 also potently inhibited the proliferation/survival and suppressed c-Myc expression in TRAF3-sufficient mouse and human B lymphoma cell lines. Furthermore, we found that reconstitution of c-Myc expression conferred partial resistance to the anti-proliferative/apoptosis-inducing effects of AD198 in human MM cells. CONCLUSIONS: AD 198 and PEP005 have differential effects on malignant B cells through distinct biochemical mechanisms. Our findings uncovered a novel, PKCδ-independent mechanism of the anti-tumor effects of AD 198, and suggest that AD 198 has therapeutic potential for the treatment of NHL and MM involving TRAF3 inactivation or c-Myc up-regulation.

Roles of the TRAF2/3 binding site in differential B cell signaling by CD40 and its viral oncogenic mimic, LMP1.

The EBV protein, latent membrane protein 1 (LMP1), is a functional mimic of the cellular receptor CD40, but signals to B lymphocytes in an amplified and sustained manner compared with CD40. LMP1 contributes to the development of B cell lymphoma in immunosuppressed patients, and may exacerbate flares of certain autoimmune diseases. The cytoplasmic domain of LMP1 binds the signaling adaptor TRAF2 with lower avidity than the cytoplasmic domain of CD40, and TRAF2 is needed for CD40-mediated degradation of TRAFs 2 and 3. LMP1 doesn't induce TRAF degradation, and employs TRAF3 as a positive mediator of cell signaling, whereas CD40 signals are inhibited by TRAF3. We thus tested the hypothesis that relative affinity for TRAF2, and/or distinct sequence differences in the TRAF2/3 binding sites of CD40 vs LMP1, controls the disparate ways in which CD40 and LMP1 use TRAFs 2 and 3, and their distinct signaling characteristics. CD40 and LMP1 mutants in which the TRAF binding site sequences were swapped were examined, testing TRAF binding and degradation, and induction of B cell activation. Results revealed that TRAF binding affinity and TRAF binding site sequence dictate a distinct subset of CD40 vs LMP1 signaling properties. Examination of TRAF binding, degradation, cytokine production, IgM secretion, and the activation of c-Jun kinase and NF-kappaB revealed that some events are dictated by TRAF binding site sequences, others are partially regulated, and still others are independent of the TRAF binding site sequence.

TRAF proteins in CD40 signaling.

The tumor necrosis factor receptor (TNFR) superfamily molecule CD40 is expressed by a wide variety of cell types following activation signals, and constitutively on B lymphocytes, macrophages, and dendritic cells. CD40 signals to cells stimulate kinase activation, gene expression, production of a antibody and a variety of cytokines, expression or upregulation of surface molecules, and protection or promotion of apoptosis. Initial steps in CD40-mediated signal cascades involve the interactions of CD40 with various members of the TNFR-associated factor (TRAF) family of cytoplasmic proteins. This review summarizes current understanding of the nature of these interactions, and how they induce and regulate CD40 functions.

Cooperation between TNF receptor-associated factors 1 and 2 in CD40 signaling.

TNFR-associated factor 1 (TRAF1) is unique among the TRAF family, lacking most zinc-binding features, and showing marked up-regulation following activation signals. However, the biological roles that TRAF1 plays in immune cell signaling have been elusive, with many reports assigning contradictory roles to TRAF1. The overlapping binding site for TRAFs 1, 2, and 3 on many TNFR superfamily molecules, together with the early lethality of mice deficient in TRAFs 2 and 3, has complicated the quest for a clear understanding of the functions of TRAF1. Using a new method for gene targeting by homologous recombination in somatic cells, we produced and studied signaling by CD40 and its viral oncogenic mimic, latent membrane protein 1 (LMP1) in mouse B cell lines lacking TRAF1, TRAF2, or both TRAFs. Results indicate that TRAFs 1 and 2 cooperate in CD40-mediated activation of the B cell lines, with a dual deficiency leading to a markedly greater loss of function than that of either TRAF alone. In the absence of TRAF1, an increased amount of TRAF2 was recruited to lipid rafts, and subsequently, more robust degradation of TRAF2 and TRAF3 was induced in response to CD40 signaling. In contrast, LMP1 did not require either TRAFs 1 or 2 to induce activation. Taken together, our findings indicate that TRAF1 and TRAF2 cooperate in CD40 but not LMP1 signaling and suggest that cellular levels of TRAF1 may play an important role in modulating the degradation of TRAF2 and TRAF3 in response to signals from the TNFR superfamily.

Differential regulation of CD40-mediated TNF receptor-associated factor degradation in B lymphocytes.

Engagement of CD40 on murine B cells by its ligand CD154 induces the binding of TNFR-associated factors (TRAFs) 1, 2, 3, and 6, followed by the rapid degradation of TRAFs 2 and 3. TRAF degradation occurs in response to signaling by other TNFR superfamily members, and is likely to be a normal regulatory component of signaling by this receptor family. In this study, we found that receptor-induced TRAF degradation limits TRAF2-dependent CD40 signals to murine B cells. However, TRAFs 1 and 6 are not degraded in response to CD40 engagement, despite their association with CD40. To better understand the mechanisms underlying differential TRAF degradation, mixed protein domain TRAF chimeras were analyzed in murine B cells. Chimeras containing the TRAF2 zinc (Zn) domains induced effective degradation, if attached to a TRAF domain that binds to the PXQXT motif of CD40. However, the Zn domains of TRAF3 and TRAF6 could not induce degradation in response to CD40, regardless of the TRAF domains to which they were attached. Our data indicate that TRAF2 serves as the master regulator of TRAF degradation in response to CD40 signaling, and this function is dependent upon both the TRAF Zn domains and receptor binding position.