Elevated Choline Kinase α−Mediated Choline Metabolism Supports the Prolonged Survival of TRAF3-Deficient B Lymphocytes This information is current as Samantha Gokhale, Wenyun Lu, Sining Zhu, Yingying Liu, of October 3, 2021. Ronald P. Hart, Joshua D. Rabinowitz and Ping Xie J Immunol published online 11 December 2019 http://www.jimmunol.org/content/early/2019/12/10/jimmun ol.1900658 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2019/12/10/jimmunol.190065 Material 8.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 3, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 11, 2019, doi:10.4049/jimmunol.1900658 The Journal of Immunology Elevated Choline Kinase a–Mediated Choline Metabolism Supports the Prolonged Survival of TRAF3-Deficient B Lymphocytes Samantha Gokhale,*,† Wenyun Lu,‡,x,{ Sining Zhu,*,† Yingying Liu,* Ronald P. Hart,*,{,‖ Joshua D. Rabinowitz,‡,x,{ and Ping Xie*,{ Specific deletion of the tumor suppressor TRAF3 from B lymphocytes in mice leads to the prolonged survival of mature B cells and expanded B cell compartments in secondary lymphoid organs. In the current study, we investigated the metabolic basis of TRAF3- mediated regulation of B cell survival by employing metabolomic, lipidomic, and transcriptomic analyses. We compared the polar metabolites, lipids, and metabolic enzymes of resting splenic B cells purified from young adult B cell–specific Traf32/2 and littermate control mice. We found that multiple metabolites, lipids, and enzymes regulated by TRAF3 in B cells are clustered in the choline metabolic pathway. Using stable isotope labeling, we demonstrated that phosphocholine and phosphatidylcholine Downloaded from biosynthesis was markedly elevated in Traf32/2 mouse B cells and decreased in TRAF3-reconstituted human multiple myeloma cells. Furthermore, pharmacological inhibition of choline kinase a, an enzyme that catalyzes phosphocholine synthesis and was strikingly increased in Traf32/2 B cells, substantially reversed the survival phenotype of Traf32/2 B cells both in vitro and in vivo. Taken together, our results indicate that enhanced phosphocholine and phosphatidylcholine synthesis supports the prolonged survival of Traf32/2 B lymphocytes. Our findings suggest that TRAF3-regulated choline metabolism has diagnostic and thera- peutic value for B cell malignancies with TRAF3 deletions or relevant mutations. The Journal of Immunology, 2020, 204: 000– http://www.jimmunol.org/ 000. berrant B cell survival is an important contributing factor TRAF3, a member of the TRAF family, regulates the signal to the pathogenesis of B cell malignancies, which transduction pathways of a diverse array of immune receptors, A comprise .50% of blood cancers and .80% of lym- including the TNFR superfamily, TLRs, NOD-like receptors, RIG- phomas (1–3). We and others have recently identified TNFR- I-like receptors, and cytokine receptors (10, 18, 19). Specifically in associated factor (TRAF) 3, a cytoplasmic adaptor protein, as a B lymphocytes, TRAF3 directly binds to two receptors pivotal critical regulator of cell survival and tumor suppressor in mature for B cell physiology, the BAFFR and CD40, which are required by guest on October 3, 2021 B lymphocytes (4–9). Deletions and inactivating mutations of the for B cell survival and activation, respectively (20, 21). Specific TRAF3 gene are some of the most frequently occurring genetic deletion of the Traf3 gene in B lymphocytes results in severe alterations in a variety of human B cell malignancies (9, 10), in- peripheral B cell hyperplasia in mice due to the prolonged survival cluding multiple myeloma (MM, 17%) (6, 11), gastric marginal of mature B cells independent of the principle B cell survival zone lymphoma (MZL, 21%), splenic MZL (10%) (12, 13), dif- factor BAFF (4, 5). This effect of TRAF3 deficiency in B cells fuse large B cell lymphoma (DLBCL, 14%) (14), B cell chronic eventually leads to spontaneous development of splenic MZL and lymphocytic leukemia (13%) (15), Hodgkin lymphoma (HL, 15%) B1 lymphomas at high incidence by 18 mo of age (8). These (16), and Waldenstro¨m’s macroglobulinemia (5%) (17). in vivo findings are consistent with the frequent deletions and *Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854; Address correspondence and reprint requests to Dr. Ping Xie, Department †Graduate Program in Cellular and Molecular Pharmacology, Rutgers University, Piscat- of Cell Biology and Neuroscience, Rutgers University, 604 Allison Road, away, NJ 08854; ‡Department of Chemistry, Princeton University, Princeton, NJ 08544; Nelson Laboratory Room B336, Piscataway, NJ 08854. E-mail address: xLewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544; [email protected] {Rutgers Cancer Institute of New Jersey, New Brunswick, NJ 08901; and ‖W.M. Keck The online version of this article contains supplemental material. Center for Collaborative Neuroscience, Rutgers University, Piscataway, NJ 08854 Abbreviations used in this article: AEC, adenylate energy charge; B-Traf32/2, ORCIDs: 0000-0003-0554-099X (S.G.); 0000-0003-4836-8712 (R.P.H.); 0000-0002- 2 2 B cell–specific Traf3 / ;Chka,Chokinasea; Cho, choline; DAG, diacylglycerol; 1247-4727 (J.D.R.). Dgka, diacylglycerol kinase a; DLBCL, diffuse large B cell lymphoma; Etn, Received for publication June 12, 2019. Accepted for publication November 13, ethanolamine; Faah, fatty acid amide hydrolase; Gdpd3, glycerophosphodiester 2019. phosphodiesterase domain containing 3; G6P, glucose-6-phosphate; HL, Hodgkin lymphoma; LC, liquid chromatography; LC-MS, liquid chromatography–mass This work was supported by National Institutes of Health (NIH) Grant R01 CA158402 spectrometry; LMC, littermate control; Lpcat, lysophosphatidylcholine acyltrans- (to P.X.), Department of Defense Grant W81XWH-13-1-0242 (to P.X.), a Pilot Award ferase; MAG, monoacylglycerol; MM, multiple myeloma; MZL, marginal zone of the Cancer Institute of New Jersey through Grant Number P30CA072720 from the lymphoma; NF-kB2, noncanonical NF-kB; NIK, NF-kB–inducing kinase; PC, National Cancer Institute (NCI) (to P.X. and J.D.R.), and a Busch Biomedical Grant (to phosphatidylcholine; P-Cho, phosphocholine; P-DMEtn, phosphodimethylethanolamine; P.X.); supported in part by grants from NCI R50 CA211437 (to W.L.) and NIH R01 PPP, pentose phosphate pathway; TRAF, TNFR-associated factor. ES026057 (to R.P.H.). The metabolomic and lipidomic analyses were supported by the Rutgers Cancer Institute of New Jersey Metabolomics Shared Resource and FACS Ó analyses were supported by the Flow Cytometry Core Facility with funding from Copyright 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 NCI–Cancer Center Support Grant P30CA072720. The microarray data presented in this article have been submitted to the National Institutes of Health Gene Expression Omnibus database (https://www.ncbi.nlm.nih. gov/geo/) under the accession number GSE113920. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900658 2 TRAF3 REGULATES CHOLINE METABOLISM IN B CELLS inactivating mutations of the TRAF3 gene identified in human (Manassas, VA) (30). All mouse and human B lymphoma cell lines B cell neoplasms, demonstrating the tumor suppressive role of were cultured as previously described (30). TRAF3 in mature B lymphocytes. Reagents and Abs The signal transduction pathway underlying TRAF3-mediated regulation of B cell survival has been elucidated in previous Tissue culture supplements, including stock solutions of sodium pyruvate, L-glutamine, nonessential amino acids, and HEPES (pH 7.55), were from studies. It was found that, in the absence of stimulation, TRAF3 2 Invitrogen (Carlsbad, CA). Trimethyl-D9-Cho ([ H]9-Cho) was purchased constitutively binds to NF-kB–inducing kinase (NIK) (the up- from Cambridge Isotope Laboratories (Tewksbury, MA). MN58B and stream kinase of the noncanonical NF-kB [NF-kB2] pathway) and RSM932A were obtained from Aobious (Gloucester, MA). Abs against TRAF2, whereas TRAF2 also constitutively associates with Chka were from Abcam (Cambridge, MA). Polyclonal rabbit Abs to cIAP1/2 (18, 22). In this complex, cIAP1/2 induces K48-linked TRAF3 (H122) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-actin Ab was from Chemicon (Temecula, CA). HRP- polyubiquitination of NIK, thereby targeting NIK for proteasomal labeled secondary Abs were from Jackson ImmunoResearch Labora- degradation and thus inhibiting NF-kB2 activation (23–26). Upon tories (West Grove, PA). BAFF or CD154 stimulation, trimerized BAFFR or CD40 recruits TRAF3, TRAF2, and cIAP1/2 to the plasma membrane, releasing Splenic B cell purification, culture, and stimulation NIK from the TRAF3–TRAF2–cIAP1/2