Abstract
TRAF3 is a versatile intracellular adapter protein with multiple context-specific roles. Uniquely in B cells, TRAF3 deficiency enhances survival and increases the risk of transformation, as loss of TRAF3 is observed in several types of B cell cancers. Here, we report a new mechanism for TRAF3 in the restraint of B cell survival. We found that TRAF3 deficiency was associated with induction of the pro-survival kinase Pim2 in mouse primary B cells and human malignant B cell lines. The increase in Pim2 was independent of NF-κB2 activation but was ameliorated with inhibition of STAT3 expression or function. TRAF3 deficiency also led to a Pim2-dependent increase in c-Myc protein levels and was associated with reduced c-Myc ubiquitination. TRAF3-deficient primary B cells were less sensitive to cell death induced by the Pim inhibitors SGI-1776 and TP-3654. Interestingly, human malignant B cell lines with low expression of TRAF3 were more sensitive to Pim inhibition-induced cell death. Combination treatment of TRAF3-deficient B cells and B cell tumor lines with c-Myc inhibitors enhanced their sensitivity to Pim inhibition, suggesting a possible therapeutic strategy. TRAF3 thus suppresses a Pim2-mediated B cell survival axis, which can be a potential target for treatment of B cell malignancies.
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Introduction
The adaptor signaling protein tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) displays cell and context-specific regulation of a variety of signaling pathways, with distinct consequences in different cell types1,2. Whole-organism Traf3 deletion in mice leads to neonatal death, demonstrating the critical roles played by TRAF3 in key biological functions3. When genetic loss of Traf3 is restricted to the mouse B cell lineage (B-Traf3−/− mice), these mice display a striking phenotype resulting from markedly enhanced B cell survival, including accumulation of B cells in all secondary lymphoid organs, as well as occurrence of spontaneous germinal centers and autoantibody production4,5. As B-Traf3−/− mice age, most develop B cell lymphoma, consistent with the prediction that their enhanced B cell survival allows accumulation of mutations that ultimately increase the likelihood of transformation6. Importantly, genetic loss of TRAF3 in humans is also associated with B cell malignancies. It has been reported that >15% of diffuse large B cell lymphomas (DLBCL) and ~20% of multiple myelomas contain loss and/or loss-of-function mutations in TRAF37,8,9,10. Additionally, TRAF3 deficiency in human B cell tumors may result from post-translational modification. We recently showed that the Epstein-Barr virus (EBV)-encoded oncogenic protein latent membrane protein 1 (LMP1), through its high-avidity binding of TRAF3, can sequester TRAF3 away from regulating other pro-survival pathways in the cell, producing a TRAF3-deficient phenotype in mouse and human DLBCL cell lines11. TRAF3 is therefore an important B cell tumor suppressor.
The TNF family member B cell activating factor (BAFF) is critical to the survival of normal B cells. A critical part of signaling through the BAFF receptor (BAFFR) is activation of the serine-threonine kinase NF-κB inducing kinase (NIK). NIK plays a key role in activation of the pro-survival non-canonical NF-κB2 pathway. TRAF3 normally restrains NIK activation, as part of a cytoplasmic complex of proteins that mediate NIK poly-ubiquitination and proteasome-mediated degradation. BAFFR signaling recruits this complex to the membrane, after which TRAF3 itself becomes poly-ubiquitinated and degraded12,13. B cells lacking TRAF3 no longer require BAFF signals to survive, and display constitutive nuclear localization of NF-κB2 factors p52 and RelB2. However, it was subsequently discovered that T cells, dendritic cells, and macrophages lacking TRAF3 also display constitutive NF-κB2 activation, but show no increases in cellular survival14,15. In B cells, degradation of TRAF3 is also neither sufficient nor required for the activation of the NF-κB2 pathway16. Thus, TRAF3 regulates B cell survival via multiple mechanisms, which include inhibition of transcription of the pro-survival protein Mcl-117, and restraint of B cell glucose metabolism18.
Prior gene expression analysis compared normal and TRAF3-deficient B cells versus WT and TRAF3-deficient T cells to identify TRAF3-regulated targets in B cells associated with their uniquely enhanced survival17. This analysis revealed an increase in the kinase proviral insertion in murine lymphoma (Pim) 2 in TRAF3−/− B cells. The Pims are a transcriptionally-regulated, constitutively active serine/threonine kinase family consisting of Pim1, Pim2 and Pim319. Pim2 is overexpressed in human MM cells and is required for their proliferation and survival20,21. Pim2 inhibition also promotes death in B lymphoma cells22.
We show here that loss of TRAF3 was sufficient to induce Pim2 up-regulation in B cells. This increase was not attenuated by deletion of NIK. TRAF3-deficient B cells were more susceptible to biochemical Pim2 inhibition and more resistant to inhibitors of the phosphatidyl inositol-3 kinase/protein kinase B (PI3K/Akt) pathway compared to wild type (WT) B cells. Low TRAF3 expression in human BCL and MM cell lines correlated with higher Pim2 protein levels and decreased susceptibility to Pim2 inhibitor-mediated cell death. Interestingly, B cell TRAF3 deficiency also resulted in Pim2-dependent elevation in c-Myc protein levels without a change in c-Myc mRNA; this was associated with decreased c-Myc poly-ubiquitination. B cells lacking TRAF3 were resistant to inhibition of c-Myc expression but were susceptible to inhibition of c-Myc activity. Inhibition of c-Myc also enhanced cell death in response to a Pim2 inhibitor. These findings identify Pim2 as an important mediator of enhanced survival in B cells that coordinates with several related pro-viability molecular mechanisms. Pim2-regulated pathways thus present a promising potential therapeutic target in TRAF3-deficient B cell malignancies.
Results
TRAF3-mediated regulation of B cell Pim2 expression
To determine the pathways involved in the hyper-survival of TRAF3-deficient B cells (TRAF3−/−), we performed a microarray analysis, comparing the gene expression of TRAF3−/− mouse B cells compared to those of wild type littermate controls (WT)17. We also compared gene expression in TRAF3−/− B cells versus TRAF3−/− T cells from conditional knockout mice, as these T cells do not have enhanced survival1,14. Pim2 gene expression was increased in TRAF3−/− B cells compared to either WT B cells or TRAF3−/− T cells. Confirming microarray data, TRAF3−/− B cells had 6-fold higher expression of Pim2 mRNA compared to WT B cells when examined by RT-PCR (Fig. 1a). Pim2 protein was also increased in TRAF3−/− compared to WT B cells (Fig. 1b). Interestingly, TRAF3 deficiency specifically regulated the Pim2 isoform, as expression of Pim1 and Pim3 was unchanged (Supplemental Fig. 1).
Our observations in mouse primary B cells led us to predict that TRAF3 protein levels in B cell tumors would impact their relative levels of Pim2 protein. We examined 3 human MM-derived cell lines (OPM2, LP1, and RPMI8226) and observed an inverse correlation between their relative TRAF3 and Pim2 protein levels (Fig. 1c). In DLBCL-derived human cell lines, OCI-Ly7 cells had undetectable TRAF3 protein and increased Pim2 expression compared to TRAF3-positive BJAB cells (Fig. 1d). Figure 1c,d were previously presented in the doctoral dissertation of N.M.23. Although we expect that there are multiple gene alterations in tumor cells that could impact Pim2 expression, our results indicate that TRAF3 likely serves as an important regulator to restrain Pim2 expression at both the mRNA and protein levels in normal and malignant B cells. This conclusion is strengthened by the recent complementary finding described in the Introduction that human BCL cell lines expressing LMP1, which avidly binds and sequesters TRAF3, also display a TRAF3-deficient phenotype, including elevated Pim2 protein10.
Effect of loss of TRAF3 on Pim2 target phosphorylation
Phosphorylation of the pro-apoptotic BAD protein at serine-112 by Pim2 inhibits cell death24. The kinase p70-S6 (p70-S6K), S6 ribosomal protein (S6), and 4E-BP1, involved in protein translation, are also Pim2 phosphorylation targets, and contribute to regulation of cell survival25,26. Increased expression of Pim2, which is a constitutively active kinase, within TRAF3−/− mouse B cells resulted in enhanced expression of its known targets BAD27, p70-S6K, 4E-BP1, and ribosomal protein S6 (Fig. 2a–e), as well as phosphorylated (active) forms of these proteins. In the case of 4EBP1 and S6, there was also a selective increase in the phosphorylated forms, above the increase in total amounts. These results indicate that the increased Pim2 protein is accompanied by increased Pim2 activity in TRAF3-deficient B cells.
Impact of TRAF3 and Pim2 upon c-Myc stability and levels
The transcription factor c-Myc has frequently been implicated in B cell oncogenesis28,29, and is a potential therapeutic target. Additionally, proviral tagging in myc transgenic mice revealed Pim2 as a collaborating oncogene driving lymphomagenesis30. Pim2 directly phosphorylates c-Myc on serine 329, leading to stabilization of c-Myc protein and enhancing cell transformation31. We thus investigated whether TRAF3-deficient B cells also display alterations in c-Myc expression and/or post-translational modification. TRAF3-deficient B cells had increased expression of c-Myc protein, without an increase in c-Myc mRNA (Fig. 3a,b). Inhibiting Pim2 expression with siRNA attenuated levels of c-Myc expression in TRAF3−/− B cells (Fig. 3c). Although inter-mouse variability in absolute levels of both Pim2 and c-Myc resulted in differences that together did not reach statistical significance, the clear downward trend in c-Myc when Pim2 expression was reduced was consistent in all samples. Stability of c-Myc protein is regulated by K48-linked poly-ubiquitination32. In the absence of TRAF3, the elevated c-Myc protein levels were associated with reduction in K48-linked poly-ubiquitination of c-Myc (Fig. 3d; previously presented in the doctoral dissertation of N.M.23). These findings indicate that loss of TRAF3 in B cells results in increased cellular c-Myc protein, which requires the enhanced Pim2 expression downstream of TRAF3 deficiency and is associated with reduced c-Myc K48-linked ubiquitination.
To determine whether this increase in Pim2 was B cell-specific we examined T cell isolated from T cell-specific TRAF3−/− mice14. There was no significant difference in Pim2 mRNA in TRAF3−/− and WT T cells in microarray analysis17. Interestingly, Pim2 protein expression was increased in TRAF3−/− T cells compared to WT T cells, although this difference was not statistically significant. There was no difference in c-Myc protein expression by T cells, related to TRAF3 status (Supplemental Fig. 2).
STAT3 dependence of increased Pim2 in TRAF3-deficient B cells
As mentioned above, TRAF3 promotes degradation of NIK. In the absence of TRAF3, NIK accumulates and promotes activation of the NF-κB2 pathway (reviewed in1). B cell-specific deletion of NIK substantially decreases overall B cell survival and inhibits NF-κB2 activation33. Because BAFF-mediated induction of Pim2 is NF-κB2-dependent34, we tested the requirement for NIK in driving increased Pim2 expression of TRAF3-deficient B cells. Interestingly, loss of NIK in TRAF3-deficient B cells did not affect the elevated Pim2 (Supplemental Fig. 3; previously presented in the doctoral dissertation of N.M.23). Previously, it has been shown that TRAF3-deficient B cells still show enhanced survival when NIK is deleted35, suggesting that there are NIK-independent, pro-survival pathways that are regulated by TRAF3.
STAT3 activation increases Pim2 expression in both hematological and solid tumors21,36,37. TRAF3 inhibits STAT3 activation downstream of the IL-6 receptor by recruiting the phosphatase PTPN2238. We thus investigated the relationship between increased expression of Pim2 in TRAF3−/− B cells and levels of STAT3 activation. After treatment with the STAT3 inhibitor Cucurbatacin I39, Pim2 protein in TRAF3−/− B cells decreased to WT levels (Fig. 4a). Partial inhibition of STAT3 expression with siRNA in TRAF3−/− B cells resulted in a similar decrease in Pim2 protein (Fig. 4b). Our earlier studies demonstrated that TRAF3 associates with the IL-6R following IL-6 binding, and recruits PTPN22 to this receptor to reduce IL-6-mediated STAT3 phosphorylation. However, IL-6 treatment did not further increase Pim2 levels in TRAF3−/− B cells (data not shown), indicating that TRAF3 does not regulate Pim2-mediated homeostatic survival via the IL-6R pathway. Our results show that TRAF3 deficiency sustains increased Pim2 expression downstream of STAT3 activation and independently of NF-κB2 activation.
Impact of TRAF3 status on susceptibility of B cells to Pim inhibitors
Pim2 is important for B cell survival, and has been suggested as a potential target for treatment of B cell malignancies22,27. To determine whether Pim2 inhibition induces death of TRAF3-deficient B cells, we tested B cell survival after 24 hr treatment with two different Pim kinase inhibitors: SGI-177640 and TP-365441. While isoform-restricted Pim2 inhibitors are not currently available, Supp. Fig. 1 indicates that the expression of other Pim isoforms is not increased in TRAF3-deficient B cells. Pim inhibition in both WT and TRAF3−/− B cells led to increased cell death, consistent with the importance of Pim2 in B cell survival (Fig. 5a,b). TRAF3−/− primary B cells were relatively more resistant to cell death mediated by both inhibitors, consistent with their elevated levels of Pim2 kinase.
We next wished to examine the relative sensitivity to Pim2 inhibition of human tumor-derived B cell lines with varying levels of TRAF3 protein (Fig. 1). While primary TRAF3−/− B cells with elevated Pim2 resisted such inhibition, it is well-documented that tumors can develop dependence upon abnormally elevated proteins that promote growth and survival, a phenomenon known as ‘oncogene addiction’42. Consistent with such dependence, the TRAF3low RPMI8226 MM cell line was more susceptible to Pim2 inhibition compared to higher TRAF3-expressing LP1 and OPM2 MM cell lines, and at lower inhibitor concentrations (Fig. 5c,d). TRAF3-deficient OCI-Ly7 BCL cells were also more susceptible to SGI-1776-induced death than TRAF3high BJAB BCL cells at intermediate inhibitor concentration (Fig. 5d).
Increased effect of combined inhibition of c-Myc and Pim2 on survival of TRAF3-deficient B cells
Inhibiting c-Myc expression is a potential therapeutic strategy to treat c-Myc-driven malignancies; c-Myc is the most frequently dysregulated gene in MM43,44. In DLBCL, 5% to 14% of tumors have c-Myc rearrangements or gene amplifications45. The drug JQ1 inhibits bromodomain-containing proteins, as these can promote c-Myc gene expression, and has shown promise in treatment of human MM and DLBCL46,47. To test whether targeting Myc can decrease the enhanced survival of TRAF3-deficient B cells, we treated WT or TRAF3−/− B cells with the c-Myc inhibitors JQ1 or 10058-F4, the latter being an inhibitor of c-Myc-mediated transcriptional activity48. Combination treatment with either of the c-Myc inhibitors enhanced killing by the Pim inhibitor SGI-1776 in both TRAF3−/− mouse primary B cells (Fig. 6a,b) and in the TRAF3-deficient human MM cell line RPMI8226 (Fig. 6c,d). As use of Pim inhibitors has been limited by their cardiotoxic effects at higher doses41, these results suggest that combined targeting of Pim2 and c-Myc expression may be beneficial, and allow lower doses of Pim inhibitors to be efficacious.
Discussion
After it was revealed that TRAF3 plays a powerful B cell-specific role in restraint of homeostatic survival2,4, it has become evident that this TRAF3 role renders it an important tumor suppressor for B cell malignancies, particularly MM and BCL9. Because a striking component of the phenotype of all TRAF3-deficient cell types is constitutive activation of the NF-κB2 pathway49, it has often been assumed that enhanced NF-κB2 activation is both necessary and sufficient to explain how TRAF3 exerts its inhibition of B cell survival. However, a number of subsequent studies provide evidence that additional TRAF3-regulated pro-survival pathways, independent of NF-κB2, make important contributions to how TRAF3 restrains B cell survival (reviewed in 1, 2). We have thus worked to identify such pathways, as their characterization not only enhances understanding of basic TRAF3 functions, but also has important implications for choosing the most effective therapies for B cell malignancies.
Our results presented here show that loss of B cell TRAF3 led to induction of the constitutively active pro-survival kinase Pim2, and enhanced expression and activation of its downstream targets BAD, p-70S6K, S6, and 4E-BP1 (Fig. 7). Interestingly, the Pim2 increase in TRAF3−/− B cells is not attenuated with loss of NIK, suggesting that in these cells Pim2 levels are enhanced in TRAF3-deficient B cells independently of NF-κB2 activation. Our results also further illustrate multiple ways in which TRAF3 deficiency can enable and enhance pathogenesis of B cell malignancies. NF-κB2 is required for homeostasis of WT B cells33, but in TRAF3-deficient B cells certain oncogenic programming can be maintained independently of NF-κB2. This could potentially allow TRAF3-deficient B cells to serve as a reservoir for tumor recurrence and resistance to proliferation-targeting chemotherapy, as well as accumulation of additional oncogenic mutations.
TRAF3-deficient B cells maintained their elevated Pim2 expression in the absence of NIK, the initiating kinase of the NF-κB2 pathway, so we sought other upstream pathways that mediate the Pim2 increase resulting from TRAF3 deficiency. We observed here constitutive phosphorylation of STAT3 in TRAF3-deficient B cells and show that inhibition of STAT3 expression or function in TRAF3-deficient B cells decreased Pim2 elevation. Thus, TRAF3 in normal B cells inhibits Pim2 expression at least in part by restraining activation of STAT3, and by doing so inhibits B cell homeostatic survival, independent of direct engagement of the IL-6R. Interestingly, STAT3 is frequently constitutively active in bone marrow B cells from MM patients50, and TRAF3 deficiency is considered an early event in MM oncogenesis51.
Alternations in function of the oncogene c-Myc have long been implicated as playing an important role in the pathogenesis of BCL and MM44. Additionally, increased expression of c-Myc protein is associated with a poorer prognosis in patients with DLBCL, independent of other prognostic factors45,52. A substantial proportion (29% to 47%) of DLBCL have high protein expression of c-Myc, which, interestingly, is higher than the incidence of c-Myc gene translocations45. This suggests that in some cases of DLBCL, c-Myc protein is increased through alternative mechanisms such as post-translational modifications, and not purely from genetic alterations. c-Myc has many transcriptional targets and wide-ranging impacts on cell proliferation and growth. It is thus intriguing that the increase in Pim2 mediated by loss of TRAF3 also impacted B cell levels of c-Myc. Unlike the mRNA-directed increase in Pim2, this elevation in c-Myc was accomplished at the level of protein regulation, by decreased K48-linked poly-ubiquitination of c-Myc in TRAF3-deficient B cells, resulting in greater c-Myc protein stability. The present results are the first demonstration to our knowledge of Pim2-mediated enhancement of B cell c-Myc mediated at the post-translational level. In addition, c-Myc has pro-apoptotic functions45. Additional pro-survival pathways mediated by increased Pim2 expression, such as inhibition of activity of the pro-apoptotic BAD protein, may help to counteract the pro-apoptotic functions of c-Myc in TRAF3-deficient B cells, allowing their markedly enhanced viability.
In contrast to TRAF3-deficient B cells, TRAF3-deficient T cells do not have increased viability and loss of TRAF3 has not been reported to be associated with T cell malignancies5. TRAF3−/− T cells do have constitutive NF-κB2 activation, a feature we and others have seen in all TRAF3-deficient cell types examined to date4,5,15. Here, we found that TRAF3−/− T cells had a modestly trending increase in Pim2 protein. TRAF3−/− T cells do not have an increase in STAT3 expression or activation, suggesting another mechanism is responsible for the increased Pim2 in these T cells53. One possible explanation is that TRAF3−/− mice have increased regulatory T cells (Tregs) compared to WT mice54 and Tregs have increased Pim2 expression compared to conventional T cells, due to increased FoxP3 expression55. It would be interesting to explore this pathway further in future studies.
Our findings are summarized in a proposed model for TRAF3-mediated regulation of Pim2 and c-Myc in B cells (Fig. 7). These results have implications for selection of the most efficacious pathway inhibitors for treatment of TRAF3-deficient B cell malignancies, whether resulting from alterations in TRAF3 genes or post-translational protein reduction. Although TRAF3 levels inversely correlated with Pim2 expression in tumor-derived B cells as well as their primary cell counterparts, low expression of TRAF3 increased susceptibility to Pim inhibition in human malignant B cells, in contrast to resistance to Pim inhibitors in primary B cells. This is consistent with ‘oncogene addiction’, wherein tumors can develop dependence upon abnormally elevated proteins that promote growth and survival42. Oncogene addiction has been demonstrated previously with the oncogenes c-Myc and BCR-ABL in leukemia56. TRAF3-deficient tumors may be more likely to become dependent on Pim2 activity than TRAF3-sufficient tumors. Thus, inhibiting Pim2 may be a strategy to selectively target TRAF3-deficient B cell tumors. The Pim inhibitor SGI-1776 failed in Phase I clinical trials due to a narrow therapeutic window caused by cardiac toxicity19. Here, we show that the addition of c-Myc inhibitors enhanced lower-dose SGI-1776-mediated killing of TRAF3−/− B cells; in the case of 10058-F4, even the lowest dose of SGI-1776 effectively killed 70–90% of TRAF3-deficient B cells when the two drugs were combined. Combined targeting of these two proteins may thus have beneficial effect in treatment of B cell malignancies deficient in TRAF3.
In both mouse and human B cells, loss of TRAF3 leads to an enhanced survival phenotype. The frequent association of TRAF3 loss in MM and BCL, as well as mechanisms of post-translational loss of TRAF3 that may impact an additional group of B cell malignancies, suggest that this loss predisposes B cells to transformation, oncogenesis, and possibly resistance to conventional chemotherapy, because TRAF3 deficiency selectively enhances B cell survival without altering proliferation4. Thus, agents of conventional chemotherapy, many of which enhance DNA damage, will not kill TRAF3-deficient B cells, but could leave them with additional transforming mutations. It is thus important to fully understand the multiple ways in which TRAF3 regulates B cell survival and how these pathways may interact to most effectively treat B cell malignancies.
Materials and Methods
Mice
Traf3flox/flox mice bred with Cd19-Cre (B-Traf3−/−) mice and extensively backcrossed onto the C57BL/6 background were previously described4. Traf3flox/flox mice bred with Cd4-Cre (B-Traf3−/−) mice and extensively backcrossed onto the C57BL/6 background were previously described14. Map14k−/− (also known as Nik−/−) mice were originally generated by Dr. Robert Schreiber (Washington University, St. Louis, MO)57 and provided by Dr. David Parker (Oregon Health Science University, Portland, OR). Mice of 2–4 months of age were used for all experiments. All mice were maintained under specific pathogen-free conditions and were used in accordance with National Institute of Health guidelines under an animal protocol approved by the Animal Care and Use Committee at the University of Iowa. Similar numbers of male and female mice were used in experiments presented here.
Mouse primary cell isolation and culture
Splenic B cells were purified by negative selection as previously reported4. Briefly, B cells were isolated by anti-CD43 Ab-mediated negative selection, using a magnetic bead kit (Miltenyi Biotec, Auburn, CA) or mouse B cell isolation kit (STEMCELL, Vancouver, Canada), according to the manufacturers’ protocols. Mouse splenic pan-T cells were purified by negative selection with a mouse T cell isolation kit (STEMCELL). Cells, were maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10 μM 2-β-mercaptoethanol (Sigma Aldrich, St. Louis, MO), 10% heat-inactivated FBS (Atlanta Biologicals, Atlanta, GA, USA), 2 mM L-glutamine (Life Technologies), and 100 U/ml penicillin-streptomycin antibiotics (Life Technologies). This is referred to as B cell medium (BCM10).
Cell lines
All cell lines were maintained in BCM10. Human MM-derived cell lines included OPM258, LP159, and RPMI822660. Human DLBCL lines used were BJAB61 and OCI-Ly762. The mouse B cell lymphoma line A20.2J63 was used in experiments in the supplemental material.
Antibodies and reagents
Antibodies (Abs) used in immunoblotting included anti-phospho-BAD (S112), anti-BAD, anti-phospho-p70S6K (T389), anti-p70S6K, anti-4E-BP1 (T37/46), anti-4E-BP1, and anti-c-Myc, and were purchased from Cell Signaling Technology (Danvers, MA). Anti-TRAF3 (H122) Ab was from Santa Cruz Biotechnology (Dallas, TX) and the anti-mouse actin Ab was from Sigma (St. Louis, MO). The anti-Pim2 and anti-β-actin Abs were from Thermo Fisher (Waltham, MA). Rabbit anti-K48 polyubiquitin Ab was purchased from Abcam (Cambridge, MA). HRP-conjugated goat-anti-mouse IgG, and goat-anti-rabbit Ig Abs were from Jackson ImmunoResearch Laboratories (West Grove, PA). Cucurbatacin I was purchased from Santa Cruz Biotechnology. SGI-1776 was graciously provided by Dr. David Bearss (University of Utah, Salt Lake City, UT) or purchased from ApexBio (Houston, TX). TP-3654 was also a gift from Dr. Bearss. 10058-F4 was from Tocris (Bristol, UK). JQ1 was purchased from Cayman Chemical (Ann Arbor, MI).
Viability assays
Splenic B cells were cultured at 0.5–1 × 106 per ml. Cells were stained with the DNA dye propidium iodide (PI) and the % live cells was determined by the % of the PI-negative population, analyzed by fluorescence detection, using the Accuri C6 Flow Cytometer (BD Bioscience, San Jose, CA). The data were analyzed with FlowJo software (FlowJo LLC, Ashland, OR).
Real-time PCR (RT-PCR)
RNA from mouse primary B cells was extracted with an RNeasy extraction kit (Qiagen, Gaithersburg, MD) and cDNA synthesized using SuperScript III polymerase (Invitrogen, Carlsbad, CA). RT-PCR was performed on an ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Grand Island, NY). Primers were purchased from Applied Biosystems (Mm00454579_m1 for Pim2, Mm00487804_m1 for c-Myc, Mm00435712_m1 for Pim1, Mm01308047_g1for Pim3, and Mm99999915_g1 for GAPDH). Expression data were analyzed using the comparative Ct method64 and normalized to GAPDH expression.
Immunoprecipitation
20–30 × 106 cells/sample were lysed with IP Lite lysis buffer (0.5% Triton X, 40 mM Tris, 100 mM NaCl, 1 mM CaCl2) containing EDTA-free Mini Complete protease inhibitor (Roche) and 100 ug/mL DNase I (Roche). For assaying K48 ubiquitination, lysis buffer was supplemented with 20 mM N-ethylmaleimide (Sigma). Lysates were incubated with an Ab against c-Myc overnight at 4 °C with constant agitation. The immune complex was precipitated with protein G Dynabeads (Life Technologies), washed and resuspended in SDS-PAGE loading buffer and heated to 95 °C for 10 min.
Transfection of mouse B cells with siRNA
B cells were transfected using electroporation following a protocol previously described for primary T cells65. The Pim2 and STAT3 siRNAs were purchased from Santa Cruz Biotechnology. 5–10 × 106 cells were resuspended in 100 μL 2S electroporation buffer (5 mM KCl, 15 mM MgCl2, 15 mM HEPES, 150 mM Na2HPO4/NaH2PO4 pH 7.2 and 50 mM sodium succinate). 300 pmol of siRNA was added and cells were electroporated using the Nucleofector II electroporation system (Lonza, Basel, Switzerland), with the X-001 program. Cells were resuspended in 20% FBS-supplemented BCM without antibiotics and analyzed 48 or 24 hrs later by WB.
Statistical analysis
Unpaired, two-tailed student’s t tests were used to evaluate differences for statistical significance for most experiments. A paired two-tailed student’s t test was used to evaluate statistical significance for paired sets of samples. An unpaired t test with Welch’s correction was to compare groups with unequal variances. The Mann-Whitney test was used for independent samples that are not normally distributed. A Wilcoxon signed rank test was used to assess statistical significance for paired samples that were not normally distributed. Statistical details are listed in the caption of each figure. Statistical analysis and graphs were prepared using GraphPad Prism (GraphPad Software, San Diego, CA). (NS = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
Change history
20 November 2019
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
References
Bishop, G. A. TRAF3 as a powerful and multitalented regulator of lymphocyte functions. J Leukoc Biol 100, 919–926, https://doi.org/10.1189/jlb.2MR0216-063R (2016).
Bishop, G. A., Stunz, L. L. & Hostager, B. S. TRAF3 as a multifaceted regulator of B lymphocyte survival and activation. Front Immunol 9, 2161, https://doi.org/10.3389/fimmu.2018.02161 (2018).
Xu, Y., Cheng, G. & Baltimore, D. Targeted disruption of TRAF3 leads to postnatal lethality and defective T-dependent immune responses. Immunity 5, 407–415 (1996).
Xie, P., Stunz, L. L., Larison, K. D., Yang, B. & Bishop, G. A. TRAF3 is a critical regulator of B cell homeostasis in secondary lymphoid organs. Immunity 27, 253–267, https://doi.org/10.1016/j.immuni.2007.07.012 (2007).
Gardam, S., Sierro, F., Basten, A., Mackay, F. & Brink, R. TRAF2 and TRAF3 signal adapters act cooperatively to control the maturation and survival signals delivered to B cells by the BAFF receptor. Immunity 28, 391–401, https://doi.org/10.1016/j.immuni.2008.01.009 (2008).
Moore, C. R. et al. Specific deletion of TRAF3 in B lymphocytes leads to B-lymphoma development in mice. Leukemia 26, 1122–1127, https://doi.org/10.1038/leu.2011.309 (2012).
Bushell, K. R. et al. Genetic inactivation of TRAF3 in canine and human B-cell lymphoma. Blood 125, 999–1005, https://doi.org/10.1182/blood-2014-10-602714 (2015).
Zhang, B. et al. An Oncogenic Role for Alternative NF-κB Signaling in DLBCL Revealed upon Deregulated BCL6 Expression. Cell Rep 11, 715–726, https://doi.org/10.1016/j.celrep.2015.03.059 (2015).
Zhu, S. et al. Genetic alterations of TRAF proteins in human cancers. Front Immunol 9, 2111, https://doi.org/10.3389/fimmu.2018.02111 (2018).
San Miguel, J. F. Introduction to a series of reviews on multiple myeloma. Blood 125, 3039–3040, https://doi.org/10.1182/blood-2015-01-613596 (2015).
Bangalore-Prakash, P. et al. The oncogenic membrane protein LMP1 sequesters TRAF3 in B-cell lymphoma cells to produce functional TRAF3 deficiency. Blood Adv 1, 2712–2723, https://doi.org/10.1182/bloodadvances.2017009670 (2017).
Mackay, F. & Schneider, P. Cracking the BAFF code. Nat Rev Immunol 9, 491–502, https://doi.org/10.1038/nri2572 (2009).
Woodland, R. T. et al. Multiple signaling pathways promote B lymphocyte stimulator dependent B-cell growth and survival. Blood 111, 750–760, https://doi.org/10.1182/blood-2007-03-077222 (2008).
Xie, P., Kraus, Z. J., Stunz, L. L., Liu, Y. & Bishop, G. A. TRAF3 is required for T cell-mediated immunity and TCR/CD28 signaling. J Immunol 186, 143–155, https://doi.org/10.4049/jimmunol.1000290 (2011).
Lalani, A. I. et al. Myeloid cell TRAF3 regulates immune responses and inhibits inflammation and tumor development in mice. J Immunol 194, 334–348, https://doi.org/10.4049/jimmunol.1401548 (2015).
Lin, W. W., Hildebrand, J. M. & Bishop, G. A. A complex relationship between TRAF3 and non-Canonical NF-κB2 activation in B lymphocytes. Front Immunol 4, 477, https://doi.org/10.3389/fimmu.2013.00477 (2013).
Mambetsariev, N. et al. Nuclear TRAF3 is a negative regulator of CREB in B cells. Proc Natl Acad Sci USA 113, 1032–1037, https://doi.org/10.1073/pnas.1514586113 (2016).
Mambetsariev, N., Lin, W. W., Wallis, A. M., Stunz, L. L. & Bishop, G. A. TRAF3 deficiency promotes metabolic reprogramming in B cells. Sci Rep 6, 35349, https://doi.org/10.1038/srep35349 (2016).
Mondello, P., Cuzzocrea, S. & Mian, M. Pim kinases in hematological malignancies: where are we now and where are we going? J Hematol Oncol 7, 95 (2014).
Lu, J. et al. Pim2 is required for maintaining multiple myeloma cell growth through modulating TSC2 phosphorylation. Blood 122, 1610–1620, https://doi.org/10.1182/blood-2013-01-481457 (2013).
Asano, J. et al. The serine/threonine kinase Pim-2 is a novel anti-apoptotic mediator in myeloma cells. Leukemia 25, 1182–1188, https://doi.org/10.1038/leu.2011.60 (2011).
Gomez-Abad, C. et al. PIM2 inhibition as a rational therapeutic approach in B-cell lymphoma. Blood 118, 5517–5527, https://doi.org/10.1182/blood-2011-03-344374 (2011).
Mambetsariev, N. Mechanisms of TRAF3 mediated regulation of B cell survival. Doctor of Philosophy thesis, University of Iowa (2018).
Yan, B. et al. The PIM-2 kinase phosphorylates BAD on serine 112 and reverses BAD-induced cell death. J Biol Chem 278, 45358–45367, https://doi.org/10.1074/jbc.M307933200 (2003).
Donahue, A. C. & Fruman, D. A. Proliferation and survival of activated B cells requires sustained antigen receptor engagement and phosphoinositide 3-kinase activation. J Immunol 170, 5851–5860 (2003).
Fox, C. J. et al. The serine/threonine kinase Pim-2 is a transcriptionally regulated apoptotic inhibitor. Genes Dev 17, 1841–1854 (2003).
Keane, N. A., Reidy, M., Natoni, A., Raab, M. S. & O’Dwyer, M. Targeting the Pim kinases in multiple myeloma. Blood Cancer J 5, e325, https://doi.org/10.1038/bcj.2015.46 (2015).
Ott, G., Rosenwald, A. & Campo, E. Understanding MYC-driven aggressive B-cell lymphomas: pathogenesis and classification. Hematology Am Soc Hematol Educ Program 2013, 575–583, https://doi.org/10.1182/asheducation-2013.1.575 (2013).
Holien, T., Vatsveen, T. K., Hella, H., Waage, A. & Sundan, A. Addiction to c-MYC in multiple myeloma. Blood 120, 2450–2453, https://doi.org/10.1182/blood-2011-08-371567 (2012).
Berns, A. et al. Identification and characterization of collaborating oncogenes in compound mutant mice. Cancer research 59, 1773s–1777s (1999).
Zhang, Y., Wang, Z., Li, X. & Magnuson, N. S. Pim kinase-dependent inhibition of c-Myc degradation. Oncogene 27, 4809–4819, https://doi.org/10.1038/onc.2008.123 (2008).
Yeh, E. et al. A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells. Nat Cell Biol 6, 308–318, https://doi.org/10.1038/ncb1110 (2004).
Brightbill, H. D. et al. Conditional Deletion of NF-κB-inducing kinase (NIK) in adult mice disrupts mature B cell survival and activation. J Immunol 195, 953–964, https://doi.org/10.4049/jimmunol.1401514 (2015).
Enzler, T. et al. Alternative and classical NF-κB signaling retain autoreactive B cells in the splenic marginal zone and result in lupus-like disease. Immunity 25, 403–415, https://doi.org/10.1016/j.immuni.2006.07.010 (2006).
Lin, W. W. TRAF3 regulates B cell survival and IL-6 receptor signaling. Doctor of Philosophy thesis, University of Iowa (2015).
Uddin, N. et al. Persistent activation of STAT3 by PIM2-driven positive feedback loop for epithelial-mesenchymal transition in breast cancer. Cancer Sci 106, 718–725, https://doi.org/10.1111/cas.12668 (2015).
Shirogane, T. et al. Synergistic roles for Pim-1 and c-Myc in STAT3-mediated cell cycle progression and antiapoptosis. Immunity 11, 709–719 (1999).
Lin, W. W. et al. The adaptor protein TRAF3 inhibits IL-6 receptor signaling in B cells to limit plasma cell development. Sci Signal 8, ra88, https://doi.org/10.1126/scisignal.aaa5157 (2015).
Blaskovich, M. A. et al. Discovery of JSI-124 (cucurbitacin I), a selective Janus kinase/signal transducer and activator of transcription 3 signaling pathway inhibitor with potent antitumor activity against human and murine cancer cells in mice. Cancer research 63, 1270–1279 (2003).
Chen, L. S., Redkar, S., Bearss, D., Wierda, W. G. & Gandhi, V. Pim kinase inhibitor, SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells. Blood 114, 4150–4157, https://doi.org/10.1182/blood-2009-03-212852 (2009).
Foulks, J. M. et al. A small-molecule inhibitor of PIM kinases as a potential treatment for urothelial carcinomas. Neoplasia 16, 403–412, https://doi.org/10.1016/j.neo.2014.05.004 (2014).
Weinstein, I. B. & Joe, A. Oncogene addiction. Cancer research 68, 3077-3080; discussion 3080, https://doi.org/10.1158/0008-5472.CAN-07-3293 (2008).
Mertz, J. A. et al. Targeting MYC dependence in cancer by inhibiting BET bromodomains. Proc Natl Acad Sci USA 108, 16669–16674, https://doi.org/10.1073/pnas.1108190108 (2011).
Affer, M. et al. Promiscuous MYC locus rearrangements hijack enhancers but mostly super-enhancers to dysregulate MYC expression in multiple myeloma. Leukemia 28, 1725–1735, https://doi.org/10.1038/leu.2014.70 (2014).
Karube, K. & Campo, E. MYC alterations in diffuse large B-cell lymphomas. Semin Hematol 52, 97–106, https://doi.org/10.1053/j.seminhematol.2015.01.009 (2015).
Delmore, J. E. et al. BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell 146, 904–917, https://doi.org/10.1016/j.cell.2011.08.017 (2011).
Trabucco, S. E. et al. Inhibition of bromodomain proteins for the treatment of human diffuse large B-cell lymphoma. Clin Cancer Res 21, 113–122, https://doi.org/10.1158/1078-0432.CCR-13-3346 (2015).
Sampson, V. B. et al. MicroRNA let-7a down-regulates MYC and reverts MYC-induced growth in Burkitt lymphoma cells. Cancer Res 67, 9762–9770, https://doi.org/10.1158/0008-5472.CAN-07-2462 (2007).
Staudt, L. M. Oncogenic activation of NF-κB. Cold Spring Harb Perspect Biol 2, a000109, https://doi.org/10.1101/cshperspect.a000109 (2010).
Bharti, A. C. et al. NF-κB and STAT3 are constitutively active in CD138+ cells derived from multiple myeloma patients, and suppression of these transcription factors leads to apoptosis. Blood 103, 3175–3184, https://doi.org/10.1182/blood-2003-06-2151 (2004).
Paludo, J. & Ansell, S. M. Advances in the understanding of IgM monoclonal gammopathy of undetermined significance. F1000Res 6, 2142, https://doi.org/10.12688/f1000research.12880.1 (2017).
Zhou, K. et al. C-MYC aberrations as prognostic factors in diffuse large B-cell lymphoma: a meta-analysis of epidemiological studies. PLoS One 9, e95020, https://doi.org/10.1371/journal.pone.0095020 (2014).
Wallis, A. M. TRAF3 as a regulator of T lymphocyte activation PhD (Doctor of Philosophy) thesis, University of Iowa (2017).
Yi, Z., Lin, W. W., Stunz, L. L. & Bishop, G. A. The adaptor TRAF3 restrains the lineage determination of thymic regulatory T cells by modulating signaling via the receptor for IL-2. Nat Immunol 15, 866–874, https://doi.org/10.1038/ni.2944 (2014).
Basu, S., Golovina, T., Mikheeva, T., June, C. H. & Riley, J. L. Cutting edge: Foxp3-mediated induction of pim 2 allows human T regulatory cells to preferentially expand in rapamycin. J Immunol 180, 5794–5798, https://doi.org/10.4049/jimmunol.180.9.5794 (2008).
Pagliarini, R., Shao, W. & Sellers, W. R. Oncogene addiction: pathways of therapeutic response, resistance, and road maps toward a cure. EMBO Rep 16, 280–296, https://doi.org/10.15252/embr.201439949 (2015).
Yin, L. et al. Defective lymphotoxin-beta receptor-induced NF-kappaB transcriptional activity in NIK-deficient mice. Science 291, 2162–2165, https://doi.org/10.1126/science.1058453 (2001).
Katagiri, S. et al. Two distinct human myeloma cell lines originating from one patient with myeloma. Int J Cancer 36, 241–246 (1985).
Pegoraro, L. et al. The human myeloma cell line LP-1: a versatile model in which to study early plasma-cell differentiation and c-myc activation. Blood 73, 1020–1027 (1989).
Moore, G. E. & Kitamura, H. Cell line derived from patient with myeloma. N Y State J Med 68, 2054–2060 (1968).
Menezes, J., Leibold, W., Klein, G. & Clements, G. Establishment and characterization of an Epstein-Barr virus (EBC)-negative lymphoblastoid B cell line (BJA-B) from an exceptional, EBV-genome-negative African Burkitt’s lymphoma. Biomedicine 22, 276–284 (1975).
Tweeddale, M. E. et al. The presence of clonogenic cells in high-grade malignant lymphoma: a prognostic factor. Blood 69, 1307–1314 (1987).
Jones, B., Tite, J. P. & Janeway, C. A. Jr. Different phenotypic variants of the mouse B cell tumor A20/2J are selected by antigen- and mitogen-triggered cytotoxicity of L3T4-positive, I-A-restricted T cell clones. J Immunol 136, 348–356 (1986).
Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−ΔΔC(T)) Method. Methods 25, 402–408, https://doi.org/10.1006/meth.2001.1262 (2001).
Chicaybam, L., Sodre, A. L., Curzio, B. A. & Bonamino, M. H. An efficient low cost method for gene transfer to T lymphocytes. PLoS One 8, e60298, https://doi.org/10.1371/journal.pone.0060298 (2013).
Acknowledgements
The authors acknowledge the services of the Flow Cytometry Core, which receives support from NIH P30 award CA086862. This work was supported by the Holden Comprehensive Cancer Center at The University of Iowa and its National Cancer Institute Award P30CA086862, NIH P50 grant CA97274, VA Merit Review I01 BX001702, and a Senior Research Career Scientist award from the Department of Veterans Affairs to Gail A. Bishop. A.L.W. received support from T32 awards GM007337, AI007485, and AI007533. N.M. received support from T32 AI07485. This material is based upon work supported in part by facilities and equipment provided by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development. Some of the experiments reported here (Figures 1C, D and 3D) were presented in the doctoral dissertation of N.M. The authors are grateful to Drs. Bruce Hostager and Jon C. Houtman for critical review of the manuscript.
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Conceptualization, A.L.W., N.M., W.W.L. and G.A.B.; Investigation, A.L.W., N.M. and W.W.L.; Methodology, A.L.W., N.M., W.W.L. and L.L.S.; Writing – Original Draft, A.L.W., N.M. and G.A.B.; Writing – Review & Editing, A.L.W., N.M., W.W.L., L.L.S. and G.A.B.; Resources, G.A.B.; Supervision, G.A.B.; Funding Acquisition, G.A.B. All authors reviewed the manuscript.
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Whillock, A.L., Mambetsariev, N., Lin, W.W. et al. TRAF3 regulates the oncogenic proteins Pim2 and c-Myc to restrain survival in normal and malignant B cells. Sci Rep 9, 12884 (2019). https://doi.org/10.1038/s41598-019-49390-9
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DOI: https://doi.org/10.1038/s41598-019-49390-9
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