Cutting Edge: The RNA-Binding Ewing Sarcoma Is a Novel Modulator of Lymphotoxin β Receptor Signaling

This information is current as Richard Virgen-Slane, Ricardo G. Correa, Parham of September 28, 2021. Ramezani-Rad, Seth Steen-Fuentes, Thiago Detanico, Michael J. DiCandido, Jun Li and Carl F. Ware J Immunol published online 22 January 2020 http://www.jimmunol.org/content/early/2020/01/17/jimmun

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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 © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 22, 2020, doi:10.4049/jimmunol.1901260

Cutting Edge: The RNA-Binding Protein Ewing Sarcoma Is a Novel Modulator of Lymphotoxin b Receptor Signaling Richard Virgen-Slane,* Ricardo G. Correa,* Parham Ramezani-Rad,† Seth Steen-Fuentes,* Thiago Detanico,* Michael J. DiCandido,‡ Jun Li,‡ and Carl F. Ware* Lymphotoxin b receptor (LTbR) signaling is crucial NF-kB–inducing kinase (NIK) is no longer targeted for for lymphoid tissue organogenesis and immune ho- proteasome-dependent degradation by TRAF3:TRAF2, lead- meostasis. To identify novel regulatory mechanisms ing to phosphorylation of NF-kB-2 (p100), which is processed for signaling, we implemented a two-step screen that into p52 in a proteasome-dependent manner. Collectively, this uses coexpression analysis of human fibroblasts under- pathway is known as the alternative (noncanonical) NF-kB Downloaded from going LTbR stimulation and affinity-purification mass pathway (reviewed in Ref. 2). b spectrometry for the LTbR signaling protein TNFR- Accumulating evidence shows that LT R also contributes to associated factor 3 (TRAF3). We identify Ewing autoimmune pathology (4), including the formation of ec- b topic clusters of organized lymphoid tissue that form at sites sarcoma (EWS) protein as a novel LT R signaling b component that associates with TRAF3 but not with of chronic inflammation. Additionally, stimulation of LT R in rheumatoid arthritis fibroblast-like synoviocytes amplifies TNFR-associated factor 2 (TRAF2). The EWS:TRAF3 http://www.jimmunol.org/ complex forms under unligated conditions that are dis- inflammation through the production of ICAM, IL-8, and CCL-2 (5, 6). Induction of a steroid-resistant inflammatory rupted following activation of the LTbR. We conclude phenotype in human lung epithelial cells is driven by stimu- that EWS limits expression of proinflammatory mole- lation of LTbR (7). Furthermore, blockade of LTbR signal- cules, GM-CSF, and ERK-2, promoting immune ing resolves the type I IFN signature in rheumatoid arthritis homeostasis. The Journal of Immunology,2020,204: patients (8). Understanding the determinants for LTbR sig- 000–000. naling, which distinguish its homeostatic functions from its roles in autoimmunity, may elucidate novel strategies for by guest on September 28, 2021 ymphotoxin b receptor (LTbR) is a member of the controlling autoimmune disease. TNFR superfamily (TNFRSF), which coordinates In this work, we implemented a two-step method for L activation for lymphoid tissue differentiation, identifying candidate regulators of the LTbR pathway. We homeostasis, and immune responses (reviewed in Ref. 1). first used transcriptomics with weighted correlation network Ligation of the LTbR by the cytokines TNF ligand super- analysis (WGCNA) to detect gene modules in normal human a b b family member 14 (LIGHT) or lymphotoxin- 1 2 induces dermal fibroblasts (NHDF) undergoing LT R stimulation. receptor clustering, which activates NF-kB transcription fac- To identify a potential link between modules that may be tors (reviewed in Ref. 2). For the classical NF-kB pathway, regulated by the LTbR pathway at the protein level, we fo- inhibitor of NF-kB kinase subunits a and b phosphorylate cused on signaling inhibitor TRAF3. With this strategy, we IkB, inducing its degradation, which allows the nuclear identify Ewing sarcoma (EWS; encoded by EWSR1)asa translocation of NF-kB subunits. Prolonged LTbR stimula- novel component of the LTbR pathway. Depletion of EWS tion also induces allosteric regulation of the TNF receptor– altered NF-kB responsiveness to LTbR stimulation, elevated associated factor (TRAF) 3:TRAF2 complex, leading to its expression of ERK-2 signaling protein, and caused aberrant autoubiquitination and degradation (3). In this scenario, induction of GM-CSF.

*Laboratory of Molecular Immunology, Infectious and Inflammatory Diseases Center, Address correspondence and reprint requests to Prof. Carl F. Ware, Infectious and Inflam- Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037; †National matory Diseases Center, Sanford Burnham Prebys Medical Discovery Institute, 10901 Cancer Institute–Designated Cancer Center, Sanford Burnham Prebys Medical Discov- North Torrey Pines Road, La Jolla, CA 92037. E-mail address: [email protected] ery Institute, La Jolla, CA 92037; and ‡Department of Immunology and Respiratory The online version of this article contains supplemental material. Disease Research, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT 06877 Abbreviations used in this article: AP-MS, affinity-purification mass spectrometry; BN, ORCIDs: 0000-0002-0011-3844 (R.V.-S.); 0000-0001-6940-7034 (R.G.C.); 0000- Bayesian network; EWS, Ewing sarcoma; HEK293T, human embryonic kidney 293T; 0001-7984-5548 (S.S.-F.); 0000-0002-5006-418X (C.F.W.). HVEM, herpesvirus entry mediator; LIGHT, TNF ligand superfamily member 14; Received for publication October 24, 2019. Accepted for publication December 22, LTbR, lymphotoxin b receptor; NHDF, normal human dermal fibroblast; p100, 2019. NF-kB-2; shCtrl, control shRNA; shEWS, shRNA-targeting EWS; shRNA, short hair- pin RNA; TNFRSF, TNFR superfamily; TRAF, TNF receptor–associated factor; This work was supported in part by National Institutes of Health (NIH) Grants WGCNA, weighted correlation network analysis. CA164679, AI067890, and CA177322 (awarded to C.F.W.). R.V.-S. is a recipient of NIH Rheumatic Diseases Training Grant T32AR064194. Additional funds were pro- Ó vided by the Jean Perkins Foundation and a research contract with Boehringer Copyright 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 Ingelheim.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901260 2 CUTTING EDGE: EWS MODULATES LTbR SIGNALING

Materials and Methods Abs and reagents The following were used for stimulation: LIGHT (664-LI; R&D Systems) and goat anti-human LTbR Ab (9). The following were use as detection Abs: from Cell Signaling Technology (EWS 11910, phospho–ERK-1/-2 9101, phospho-p38 9211), from Santa Cruz Biotechnology (EWS sc-28327 AF647 and TRAF3 sc-1828), from Thermo Fisher Scientific (TRAF3 12H13L59), from Abcam (p100 ab32859), and from Sigma-Aldrich (Actin A2228). Affinity-purification mass spectrometry and Bayesian network learning Proteomics was performed by the Sanford Burnham Prebys Proteomics Core. Nonspecific were removed using SAINTexpress (10) and CRAPome (11), discretized into three levels, and subjected to a Tabu Search with bootstrapping via bnlearn (M. Scutari, manuscript posted on arXiv). Inverse relationships were detected by Bayesian network BN inference using the gRain package in R (12). Expression plasmids EWS– was expressed from pcDNA3.1 EWS-myc-HIS (13), which was a gift from H. Gehring (plasmid number 46386; Addgene). For FLAG-tagged proteins, pcDNA3.1(+)-FLAG-TRAF3 and pcDNA3.1(+)-FLAG-TRAF2 Downloaded from were used (3). Human LTbR and herpesvirus entry mediator (HVEM) were expressed from pcDNA3.1 and pcDNA3 vectors, respectively. Protein analysis Tagged proteins were isolated from human embryonic kidney 293T (HEK293T) using Sigma-Aldrich A2220. Endogenous EWS was isolated from HeLa cells using Santa Cruz Biotechnology sc-398318 with Dynabead Protein G (Thermo http://www.jimmunol.org/ Fisher Scientific). Quantitative RT-PCR, secretion, and RNA interference GM-CSF expression was measured with primers (7) in reference to L32:forward,59-GGATCTGGCCCTTGAACCTT-39 and reverse, 59- GAAACTGGCGGAAACCCA-39. GM-CSF was measured using the Luminex platform. Standards were used with drlumi to estimate absolute protein levels (14). Plasmids expressing short hairpin RNA (shRNA)– targeting EWS (shEWS) A, shEWS B, or control shRNA (shCtrl) were purchased from Dharmacon (V3LHS_641851, V3LHS_641854, or RHS4346, by guest on September 28, 2021 respectively). For small interfering RNA, pools targeting EWS or Null were purchased from Dharmacon (L-005119-02 and D-001810-10-05). FIGURE 1. Proteomic and transcriptomic screens link EWSR1 to LTbR– Statistics and WGCNA TRAF3 pathway. (A) Top, The workflow used to detect hubs in NHDF b Differential expression analysis of nCounter Human Inflammation Panel undergoing active LT R signaling. Bottom, A dendogram showing modules (GSE110102 via https://www.ncbi.nlm.nih.gov/geo/) was carried out using detected by WGCNA. (B) Top, The workflow used to detect TRAF3 com- DESeq2 with Wald and Benjamini–Hochberg p value adjustment (cut off = 0.1) plexes. Bottom, A BN summarizing the detected complexes. Dashed edges (15). All statistical analysis for quantitative RT-PCR and Western blots was indicate protein complexes that are inversely related. done in R, using linear regression. RNA-sequencing data were normalized using rlog variance stabilization (15) and analyzed using the WGCNA package in R (16), with a soft-power threshold of seven (Supplemental Fig. 1A). Module names were then selected by the most significant en- LTbR signaling mediate this inhibitory role of TRAF3. richment term (ontology size cut off = 150) using the GOstats package in R (17). Thus, we carried out an affinity-purification mass spec- trometry (AP-MS) study that focused on identifying TRAF3 Results and Discussion complexes formed in the absence of LTbRsignaling.A Proteomic and transcriptomic screens link EWSR1 to TRAF3 variant with the F474E substitution (TRAF3-M; b LT R–TRAF3 pathway Supplemental Fig. 1C), which blocks binding to LTbR WGCNA calculates gene-gene relationships from global ex- (3), was included to find interactions that may be influenced pression data to segregate into functional groups called by receptor binding (Supplemental Fig. 1D). We filtered for modules (16). Each module has a “hub,” which is a gene with binary interactions (Supplemental Table I) and predicted high influence over other genes within its module (16). Be- distinct complexes using BN learning with Bayesian infer- cause of the importance of a hub for module function, we ence(Fig.1B).AtotalofninecandidateslinkedtoTRAF3 started our screen by identifying hubs in cells undergoing were detected. LTbR signaling (Fig. 1A, top). We detected a total of 39 We cross-referenced the 39 module hubs detected by candidate gene hubs (Supplemental Fig. 1B), representing the WGCNA with the nine TRAF3-binding proteins detected by 39 modules linked to LTbR signaling (Fig. 1A, bottom). our AP-MS study, identifying EWS as a candidate linked to the Next, we proceeded to identify candidates that were linked LTbR–TRAF3 pathway (Supplemental Fig. 1E). EWS is an to TRAF3. We focused on TRAF3 for two reasons. First, it is a RNA-binding protein that participates in microRNA pro- major inhibitor of LTbR signaling, and its depletion is con- duction (19, 20), splicing, and mRNA transport (reviewed in comitant with activation (18). Second, the TRAF3:TRAF2 Ref. 21). Indeed, the EWS:TRAF3 complex has been detected and TRAF3:NIK complexes formed in the absence of active by others using a yeast two-hybrid screen (22), which gave us The Journal of Immunology 3 confidence in our AP-MS results. However, the novel link between EWS and the LTbR–TRAF3 pathway required validation.

EWS disassociates from TRAF3 upon stimulation and is required for optimal responsiveness to LTbR activation We first proceeded to validate the TRAF3:EWS complex with biochemical methods. Indeed, using tagged proteins, we de- tected the association of EWS (endogenous and MYC-tagged) with FLAG–TRAF3 (Fig. 2A). In contrast, as predicted by the inverse relationship between EWS and TRAF2 in our BN (Fig. 1B), we could not detect the association of EWS with FLAG–TRAF2. These results verified our AP-MS data, dem- onstrating the specificity of the EWS:TRAF3 complex. Next, we proceeded to verify the association of TRAF3 with EWS using endogenous proteins isolated from HeLa cells in the presence or absence of LTbR signaling. For stimulation, we used recombinant LIGHT in place of agonistic anti-LTbR Downloaded from Ab to prevent interference with immunoprecipitation. Using this design, we detected peak association of TRAF3 with EWS (Fig. 2B) in unstimulated cells. Following LIGHT stimula- tion, we measured a significant decrease in the association of TRAF3 with EWS (Fig. 2C). One potential caveat to our experimental design is that LIGHT can also ligate HVEM, http://www.jimmunol.org/ which is the TNFRSF member with the highest shared se- quence homology with LTbR. However, LIGHT stimulation of HeLa cells signal through LTbR because of the lack of HVEM expression on the cell surface (23). Taken together, these results validate the association of EWS with TRAF3 using endogenous protein and demonstrate that this complex FIGURE 2. EWS disassociates from TRAF3 upon stimulation and is re- b A is influenced by LTbR signaling. quired for optimal responsiveness to LT R activation. ( ) EWS–MYC as- sociates with FLAG–TRAF3, but not FLAG–TRAF2 from HEK293T cells as

We evaluated the impact of EWS depletion on LTbR sig- by guest on September 28, 2021 k shown by Western blot. Endogenous EWS is indicated by an asterisk (*). Blot naling, using an NF- B luciferase reporter system in HEK293T representative of three experiments. (B) TRAF3 associates with EWS isolated (Fig. 2D). In this system, receptor clustering is induced via from HeLa cells at different times after LTbR stimulation (10 ng/ml overexpression, which is a common method used for studying recombinant LIGHT) as shown by Western blot. (C) Regression shows the TNFRSF member signaling in vitro (24). In control cells negative relationship between EWS-associated TRAF3 with time. Levels D transfected with an empty vector and shRNA-targeting EWS quantified from three experiments. ( ) A plot shows the impact of EWS k 3 depletion on NF-kB reporter activity in HEK293T cells transfected with (shEWS), we measured increased NF- B activity (p =2.17 b k 22 empty vector or vectors expressing LT R or HVEM. NF- B activity mea- 10 ) versus cells expressing shCtrl). This suggested that EWS sured as firefly luciferase activity, normalized to Renilla luciferase control from might have a suppressive role in unstimulated cells, which is two experiments. Significance codes are as follows: **p , 0.01, *p , 0.05, either directly or indirectly linked to the NF-kB pathway. In NS . 0.05. (E) Western blot shows the impact of EWS knockdown by contrast, we measured a 28% decrease of NF-kB activation by shRNA on p52 accumulation in NHDF. Stimulation was carried out using LTbR in cells expressing shEWS versus cells expressing shCtrl 2 mg/ml agonistic anti-human LTbR Ab. (F) Regression shows the impact of 2 (p =5.993 10 3), suggesting that EWS is required for peak EWS depletion on p52 accumulation quantified by Western blot from two k b k separate experiments. Error bars represent 95% confidence intervals. Positions NF- B activation by LT R. Interestingly, NF- B activity in- of m.w. markers are shown for all cropped blots. Significance codes are as duced by overexpression of HVEM did not differ between follows: ***p , 0.001, **p , 0.01, NS . 0.05. shCtrl and shEWS conditions. Although the differential im- pact of shEWS on LTbR and HVEM-mediated activation of NF-kB may not be directly comparable because of differences To determine whether EWS has a role in the alternative in peak activation. It is possible that these observations may be NF-kB pathway, we stimulated control and EWS-depleted due to signaling differences. For example, although both NHDF with agonistic anti-LTbR Ab and measured p52 ac- HVEM and LTbR can recruit TRAF2 and activate classical cumulation by Western blot (Fig. 2E). We compared the NF-kB activity, TRAF3 and the alternative NF-kBpathwaydo stimulation-dependent responsiveness of p52 levels for each not respond to HVEM signaling (25). Moreover, depletion of knockdown condition and included p100 levels as variable EWS also reduces NF-kB activity downstream of nucleotide- for normalization by multivariate linear regression (adjusted 2 binding oligomerization domain-containing protein 2 signaling R2 = 0.973; p = 2.96 3 10 11). In control cells (Fig. 2F), we (26), which also activates the alternative NF-kB pathway (27). measured increased p52 accumulation at 2 h post-LTbR 2 Taken together, the differences with HVEM and similarities stimulation (p = 7.21 3 10 3), which continued through to 2 with nucleotide-binding oligomerization domain-containing 6h(p = 1.28 3 10 4). In EWS-depleted cells (Fig. 2F), no 2 protein 2 signaling suggested that EWS influences activation significant change in p52 was detected (p = 1.4 3 10 1)at of the alternative NF-kBpathway. 2 h poststimulation. However, at 6 h, increased p52 was 4 CUTTING EDGE: EWS MODULATES LTbR SIGNALING

2 detected (p = 7.61 3 10 6), suggesting that activation is re- duced at early time points but recovers by 6 h. Interestingly, in EWS-depleted cells, a trend toward increased p52 levels 2 appeared in unstimulated cells (p = 2.2 3 10 1), which re- sembled the elevated basal NF-kB activity in HEK293T expressing shEWS (Fig. 2D). These observations, along with the apparent defects in early p100 processing, suggested that EWS maintains normal LTbR signaling, perhaps by sup- pressing signaling in unstimulated cells.

EWS limits GM-CSF and ERK-2 levels for LTbR pathway Although NF-kB is a critical part of the LTbR pathway, we expanded our study to explore other potential functions for EWS. Using a multiplex inflammation panel by NanoString, we assayed for differential expression of genes between control and EWS-depleted NHDF, stimulated with anti-LTbR Ab for 0, 2, and 6 h. Of the 255 genes assayed, 2 99 genes showed significant (adjusted p value , 1.0 3 10 1) Downloaded from differences because of knockdown or stimulation conditions (Supplemental Fig. 2A). NF-kB signaling genes TRAF2, RIPK2, and BIRC2 were significantly increased in EWS- depleted cells, whereas TNFAIP3 and NFKB1 were re- duced. However, these genes represented a minor fraction of the total number of genes measured and may not capture http://www.jimmunol.org/ the main effect of EWS depletion on signaling. To identify representative genes, we used hierarchal clus- tering on principal component analysis (Fig. 3A), which identified a group of genes (Fig. 3B) enriched in cells depleted of EWS (v test = 2.17 for shEWS). Pathway enrichment analysis predicted that these genes have functions in regulating signaling of ILs, MAPK targets/nuclear events mediated by

MAP kinases, and PID P38 ALPHA BETA DOWNSTREAM by guest on September 28, 2021 PATHWAY (Supplemental Fig. 2B). EWS depletion elevated the expression of ERK-2 (also known as MAPK1), a key sig- naling , which prompted us to measure active phos- phor–ERK-2 (Fig. 3C). Knockdown of EWS (Fig. 3D) 2 significantly increased phosphor–ERK-2 (p = 4.56 3 10 3), b FIGURE 3. EWS limits GM-CSF and ERK-2 levels for LTbR pathway in which was also induced by LT R stimulation at 15 min A 3 28 3 27 NHDF. ( ) Principal component analysis plot showing the grouping of (p = 1.23 10 ) and 30 min (p = 5.18 10 ). This samples using the normalized expression levels for all differentially expressed observation is in agreement with the increased ERK-2 detected genes. Ellipses represent 90% confidence. (B) A heatmap shows normalized by NanoString in EWS-depleted NHDF. In contrast, we did NanoString data (scaled by row) for genes enriched during EWS knockdown not detect any significant impact to phospho–ERK-1 levels conditions. (C) A representative Western blot shows the impact of EWS 2 (Fig. 3E) because of knockdown of EWS (p = 9.9 3 10 1), knockdown on the induction of phospho–ERK-1 and phospho–ERK-2 fol- 3 26 lowing LTbR stimulation of NHDF. Positions of m.w. markers are shown. despite significant induction at 15 min (p = 7.42 10 ) and D 3 25 b Regression shows the impact of EWS depletion on phospho–ERK-2 ( ) and 30 min (p = 2.46 10 ) post-LT R stimulation. Taken phospho–ERK-1 levels (E). Levels quantified from three experiments. (F) together, our observations suggested that EWS is required to Regression shows the impact of EWS depletion on GM-CSF (L32-normal- maintain normal levels of ERK-2 signaling protein. ized) transcript following LTbR stimulation. Levels quantified from three Because LTbR signaling is critical for shaping the local experiments. (G) Regression shows the impact of EWS depletion on the se- extracellular environment in lymphoid tissue, we were curious cretion of GM-CSF following LTbR stimulation. Levels quantified from two whether loss of EWS also results in a change to secreted experiments. Error bars represent 95% confidence intervals. Red asterisks on proteins. Indeed, the NanoString data shows that depletion heatmap indicate genes of interest selected for validation. Western blot images were cropped. Significance codes are as follows: ***p , 0.001, **p , 0.01, of EWS increased expression of GM-CSF in response to *p , 0.05, NS . 0.05. LTbR stimulation (Fig. 3B). To verify this observation, as showninFig.3F,wemeasuredtheexpressionofGM-CSF in 2 NHDF by quantitative RT-PCR. In control knockdown (p =2.613 10 3). These results validate the increased cells, GM-CSF was weakly induced at 2 h poststimulation GM-CSF mRNA levels detected in our NanoString data. 2 (p =1.473 10 5) and remained unchanged at 6 h. In To determine whether depletion of EWS also impacted the unstimulated cells, depletion of EWS significantly increased production of GM-CSF protein, we measured levels in su- 2 GM-CSF (p =1.153 10 4),whichwasunchangedat2h pernatants using the Luminex platform (Fig. 3G), which has poststimulation. At 6 h poststimulation, an additional in- minimum detectable dose of 0.155 pg/ml. In the supernatants crease to GM-CSF was measured in EWS-depleted NHDF of unstimulated control NHDF, we measured 4.4 pg/ml The Journal of Immunology 5

GM-CSF, which is similar to levels detected in bronchoalveolar 3. Sanjo, H., D. M. Zajonc, R. Braden, P. S. Norris, and C. F. Ware. 2010. Allosteric regulation of the ubiquitin:NIK and ubiquitin:TRAF3 E3 ligases by the lymphotoxin- lavage fluid isolated from idiopathic lung disease patients (28) beta receptor. J. Biol. Chem. 285: 17148–17155. or in the sera of burn patients (29). Following LTbR stim- 4. Browning, J. L. 2008. Inhibition of the lymphotoxin pathway as a therapy for ulation for 24 h, levels increased an additional 0.8 pg/ml autoimmune disease. Immunol. Rev. 223: 202–220. 22 5. Ishida, S., S. Yamane, T. Ochi, S. Nakano, T. Mori, T. Juji, N. Fukui, T. Itoh, and (p = 3.0 3 10 ). In the supernatants of unstimulated cells R. Suzuki. 2008. LIGHT induces cell proliferation and inflammatory responses of depleted of EWS using two different shRNAs, we measured rheumatoid arthritis synovial fibroblasts via lymphotoxin beta receptor. J. 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Wai Lin, John Sedy´, and Caroline Stienne for helpful Mol. Syst. Biol. 7: 536. discussions. 23. Cheung, T. C., K. Coppieters, H. Sanjo, L. M. Oborne, P. S. Norris, A. Coddington, S. W. Granger, D. Elewaut, and C. F. Ware. 2010. Polymorphic variants of LIGHT (TNF superfamily-14) alter receptor avidity and bioavailability. Disclosures J. Immunol. 185: 1949–1958. The authors disclose support for this project was provided in 24. Chang, Y. H., S. L. Hsieh, M. C. Chen, and W. W. Lin. 2002. Lymphotoxin beta receptor induces interleukin 8 gene expression via NF-kappaB and AP-1 activation. part by Boehringer Ingelheim, with a research contract to Exp. Cell Res. 278: 166–174. Sanford Burnham Prebys Medical Discovery Institute. C.F.W. 25. Cheung, T. C., M. W. Steinberg, L. M. Oborne, M. G. Macauley, S. Fukuyama, H. Sanjo, C. D’Souza, P. S. Norris, K. Pfeffer, K. M. Murphy, et al. 2009. Un- served as Principal Investigator of the contract and has no other conventional ligand activation of herpesvirus entry mediator signals cell survival. potential conflict of interest with Boehringer Ingelheim; [Published erratum appears in 2009 Proc. Natl. Acad. Sci. USA 106: 16535–16536.] Proc. Natl. Acad. Sci. USA 106: 6244–6249. M.J.D. and J.L. are employees of Boehringer Ingelheim. The 26. Warner, N., A. Burberry, L. Franchi, Y. G. Kim, C. McDonald, M. A. Sartor, and funder had the following involvement with the study: exper- G. Nu´n˜ez. 2013. A genome-wide siRNA screen reveals positive and negative reg- ulators of the NOD2 and NF-kB signaling pathways. Sci. Signal. 6: rs3. imental design, data acquisition, and analysis. The other au- 27. Pan, Q., V. Kravchenko, A. Katz, S. Huang, M. Ii, J. C. Mathison, K. Kobayashi, thors have no financial conflicts of interest. R. A. Flavell, R. D. Schreiber, D. Goeddel, and R. J. Ulevitch. 2006. 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30. Zhu, Y., G. Xu, Y. T. Yang, Z. Xu, X. Chen, B. Shi, D. Xie, Z. J. Lu, and P. Wang. 32. Carter, A. B., and G. W. Hunninghake. 2000. A constitutive active MEK --. ERK 2019. POSTAR2: deciphering the post-transcriptional regulatory logics. Nucleic pathway negatively regulates NF-kappa B-dependent gene expression by Acids Res. 47: D203–D211. modulating TATA-binding protein phosphorylation. J. Biol. Chem. 275: 31. Maeng, Y. S., J. K. Min, J. H. Kim, A. Yamagishi, N. Mochizuki, J. Y. Kwon, 27858–27864. Y. W. Park, Y. M. Kim, and Y. G. Kwon. 2006. ERK is an anti-inflammatory signal 33. Li, H., W. Watford, C. Li, A. Parmelee, M. A. Bryant, C. Deng, J. O’Shea, and that suppresses expression of NF-kappaB-dependent inflammatory genes by inhib- S. B. Lee. 2007. Ewing sarcoma gene EWS is essential for meiosis and B lymphocyte iting IKK activity in endothelial cells. Cell. Signal. 18: 994–1005. development. J. Clin. Invest. 117: 1314–1323. Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021 A B Total Module hub Genes Go Module Name Scale independence 0 ATP6V1A 86 module 0 1 DPPA3 4251 regulation of blood pressure 2 HPRT1 3635 DNA-dependent DNA replication 10 18 3 EWSR1 1945 mRNA transport 7 16 4 LTN1 918 phosphatidylinositol biosynthetic process 8 9 12 14 0.8 6 5 JMY 887 sterol metabolic process 5 6 ADAP1 827 RNA modification 20 7 LDAH 732 glycosyl compound metabolic process 0.6 4 8 MYL12A 558 protein targeting to ER 9 LRRC1 503 cellular response to xenobiotic stimulus 10 LINC00222 495 cellular response to xenobiotic stimulus 0.4 11 FAM3B 492 sensory perception of chemical stimulus 3 12 MIR6791 461 antibiotic metabolic process

0.2 13 CYP4V2 372 positive regulation of lymphocyte proliferation 2 14 FAM225B 341 cotranslational protein targeting to membrane 1 15 SAA2 261 myeloid leukocyte migration 0.0 16 PRODH 235 phospholipid transport 5 10 15 20 17 UBE4B 200 embryonic skeletal system development 18 NIPAL2 189 regulation of tube size Scale Free Topology Model Fit, signed R^2 Scale Free Topology 19 KRTAP5-AS1 184 endocrine system development Soft Threshold (power) 20 LOC101926935 151 artery morphogenesis 21 ARID3B 146 cellular response to type I interferon 22 LOC101927914 137 cardiac chamber morphogenesis C LTβ-R 23 TMEM132B 135 transition metal ion transport binding 24 RBBP5 129 plasma membrane organization 25 GRHL2 128 regulation of epithelial cell differentiation TRAF3 RING ZF ZF CC TRAF-N TRAF-C 26 PRKAG2-AS1 118 sperm motility 27 C19orf84 110 regulation of cardiac muscle cell membrane repolarization 28 PCSK5 100 transition metal ion homeostasis 29 LOC101927151 97 inorganic anion transport ● F474E 3 30 RYR2 90 interleukin-8 production D TFG 31 TIMM23B 89 regulation of synapse assembly ● 32 KLLN 78 cellular response to heat ● ● 2 ETV6 33 PHACTR3 74 lymphocyte activation involved in immune response 3 TFG 34 RIMBP3 70 midbrain development TRAF2 Groups 35 MEF2C-AS1 62 positive regulation of inflammatory response 1 ●● ● ● TRAF3−WT 2 ETV6 ● 36 SGMS2 54 positive regulation of cation channel activity DYRK1A ●● TRAF3−M 37 STMN4 32 negative regulation of anion channel activity

Dim2 (22.6%) ● 0 TRAF2● Groups 38 USB1 30 double-strand break repair via homologous recombination 1 ● ● DCAF7 ● ● ●● TRAF3−WT EWS ● Module Hubs ● ●USO1 DYRK1A● ● TRAF3−M E −1 ● ● ●

Dim2 (22.6%) ● ● 0 ● DDX17 ● ● ● ● DCAF7 ● RBM14 EWS ● TRAF3 PPIs ● ●USO1 ● −2.5 −1 ●0.0 ● 2.5 5.0 Blocked by F474E mutation Dim1 (55.1%)● ● DDX17 ● DPPA3, ADAP1, JMY, RIMBP3, FAM3B, HPRT1, MYL12A RBM14 FAM225B, MEF2C−AS1, LDAH, LTN1, MIR6791, LINC00222, CYP4V2 DYRK1A, TFG −2.5 0.0 2.5 5.0 ARID3B, LOC101927151, PRODH, NIPAL2, PHACTR3, LRRC1, SAA2 DCAF7, ETV6 EWSR1 Dim1 (55.1%) RBBP5, TMEM132B, KRTAP5−AS1, LOC101926935, GRHL2, UBE4B, RYR2 RBM14, DDX17 PCSK5, TIMM23B, ATP6V1A, LOC101927914, PRKAG2−AS1, KLLN, C19orf84 SGMS2, USB1, STMN4

USO1 TRAF2 TRAF3 PPIs enriched by F474E mutation

Supplemental Figure 1. Parameters for ‘Omics screen. (A) Plot shows parameters for soft-threshold selection. (B) Table

shows module annotations. (C) The F474E substitution site (grey arrow) is shown in a TRAF3 domain map. (D) PCA biplot

shows the relationship between TRAF3 binding proteins and experimental conditions. (E) Venn diagram shows the overlap

between hubs and TRAF3 binding proteins. A B shCtrl shEWS

0h 2h 6h 0h 2h 6h IFIT2 IFIT3 PTGFR TWIST2 IFIT1 2 IL6R TLR3 Expression) (Normalized MKNK1 STAT2 MAP3K1 GRB2 BCL6 1 FOS MAP2K6 GAPDH Z score C D shCtrl shEWS KEAP1 POSTAR Reported EWS− HSPB2 CREB1 0h 2h 6h 0h 2h 6h DDIT3 bound mRNAs SHC1 2 PGK1 0 RHOA

● ● TUBB ● ● ● TGFBR1 ● ● ● ● ● ● ● ● ● PLCB1 ● ● IRF1 0.4 ● ● ● ● ● ● ● ● ● ● ● ● ROCK2 ● ● CEBPB ● ● ● ● ● ● ● CFL1 ● NFE2L2 ● ● ● RAF1 LY96 ● 1 ● IL1R1 0.3 ● HPRT1 MASP1 −1 ● TCF4 CFD MAPK1 ● ● ERK1 ● ● CLTC ● ● ● ● HIF1A ● ● GNB1

● ● C3 0.2 ● ● RAC1 0 ● CSF1 MAPK14 ● MX2 ● HMGB1 STAT1 HMGN1 −2 ● C1R HMGB2 C1S 0.1 ● PTGS1

● −1 GNAQ ● BIRC2 ● ● PRKCA enrichment score MAPK8 SHC1 0.0 ● ● ● BCL6 ● ● TUBB ● MAPKAPK2 RHOA ● ● MEF2A MAP2K1 ● JUN −2 TCF4 −0.1 MAP3K9 HRAS MEF2D

ERK24 0 25 50 75 shCON_0hrs_1 shCON_0hrs_2 shCON_2hrs_1 shCON_2hrs_2 shCON_6hrs_1 shCON_6hrs_2 shEWS_0hrs_1 shEWS_0hrs_2 shEWS_2hrs_1 shEWS_2hrs_2 shEWS_6hrs_1 shEWS_6hrs_2 HMGB1 HMGN1 CFL1 rank HPRT1 RAF1 LTB4R2 MMP3 BCL2L1 Supplemental Figure 2. EWS influences LTβR signaling. (A) Heatmap of normalized PTGS1 ROCK2 HMGB2 PTGER3 ERK2 NanoString data. Colors indicate normalized expression levels after scaling by rows. A CLTC RAC1 GNB1 total of 99 genes are represented, which were identified by differential expression IL1B DAXX NFKB1 RELB CCL7 analysis using DESeq2 package. Genes showed differential expression due to CXCL5 IL15 CCL2 CXCL6 MYC stimulation and/or knockdown of EWS (adjusted p-value < 0.1). (B) Gene ontology GMCSF TRAF2 NR3C1 FLT1 MAPK8 enrichment analysis for mRNAs suppressed by EWS. (C) Geneset enrichment analysis BIRC2 RIPK2 RELA IL1RN identifies 52 EWS-bound mRNAs with elevated expression (D) in cells depleted of EWS IL6 IL11 MAFF MAFG (adjusted p-value = 0.007). CXCL3 PTGS2 CXCR4 MAFK TNFAIP3 CXCL1 IL8 CXCL2 JUN SMAD7 CD40 MEF2D MAP3K9 MEF2A MAPKAPK2 shCON_0hrs_1 shCON_0hrs_2 shCON_2hrs_1 shCON_2hrs_2 shCON_6hrs_1 shCON_6hrs_2 shEWS_0hrs_1 shEWS_0hrs_2 shEWS_2hrs_1 shEWS_2hrs_2 shEWS_6hrs_1 shEWS_6hrs_2