Germline Polymorphisms in RNF31 Regulate Linear Ubiquitination And

VIEWS

IN THE SPOTLIGHT Germline Polymorphisms in RNF31 Regulate Linear Ubiquitination and Oncogenic Signaling Paolo Grumati and Ivan Dikic Summary: A new study proposes that the cancerous behavior of B cells in a subtype of diffuse large B-cell lymphoma is caused by excessive activity of the linear ubiquitin chain assembly complex (LUBAC) complex, which underlies abnormal NF-κB signaling. Two rare germline single-nucleotide polymorphisms in the RNF31 gene have been identiﬁ ed as being responsible. The use of a small inhibiting peptide may downregulate the abnormal LUBAC activity and counteract neoplastic cell growth. Cancer Discov; 4(4); 394–6. ©2014 AACR.

See related article by Yang et al., p. 480 (3).

Single-nucleotide polymorphisms (SNP) add the spice to adjacent ubiquitin (7 ). Recently, SHARPIN was identifi ed as the genomic soup (1 ). In addition to factors such as environ- a third component of active LUBAC (8–10 ). Linear ubiquitin mental and societal infl uences, these small variations in the chains are the only known products of LUBAC and have an DNA sequence are important drivers for individuality and important role as regulators of cellular signaling pathways diversity ( 2 ). They also affect our susceptibility to diseases that control innate immunity and infl ammation through and modulate response to therapy. Many human diseases NF-κB activation ( 5–10 ). Although their role in pathologies result from SNPs, and SNPs have been extremely useful as and diseases is still poorly understood, LUBAC is undoubt- markers in the hunt for disease-associated genes in genome- edly involved in infl ammatory diseases and cancer. Therefore, wide association studies. In addition, the analysis of SNPs the development of new drugs targeted to LUBAC compo- and the availability of tailored genotyping platforms have nents may prove useful for therapeutic purposes. also had an enormous impact on personalized medicine and In their article, Yang and colleagues ( 3 ) established a clear may well revolutionize the fi eld. However, not all polymor- causative link between LUBAC and the pathogenesis of ABC phisms in the human genome have been discovered nor their DLBCL. In these cells, constitutive activation of the NF-κB relevance understood; in this respect, the genome is still a pathway plays an important role in their cancerous behavior. treasure trove of discoveries. In fact, the malignancy of ABC DLBCL is a consequence of The feelings of Yang and colleagues (3 ) must have been the persistent activation of the B-cell receptor (BCR) and similar when they realized that two missense germline SNPs MYD88 signaling, with 20% of oncogenic cells presenting in exon 10 of the RNF31 gene are enriched in patients mutations in the immunoreceptor tyrosine-based activation with the activated B cell–like (ABC) subtype of diffuse large motifs (ITAM) of the BCR subunits CD79B and CD79A and B-cell lymphoma (DLBCL), as they describe in this issue of 10% presenting mutations in CARD11 . CARD11 forms a Cancer Discovery. multiprotein complex with MALT1 and BCL10, determining, RNF31 is a RING fi nger protein, also known as HOIL-1 through the activation of IκB kinase, the activation of the interacting protein [HOIP; belonging to the RING–IBR– classical NF-κB signaling. Moreover, a somatic gain-of-func- RING (RBR) protein family ( 4, 5 )]. The interaction of RNF31 tion mutation of MYD88 is also responsible for the activation and RBCK1 occurs through binding of the ubiquitin-associ- of the NF-κB pathway in a large percentage of ABC DLBCL ated (UBA) domain of RNF31 with the ubiquitin-like (UBL) lymphoma cells (11 ). domain of RBCK1 (6 ). The specifi c binding between these Yang and colleagues (3) performed a genetic screen on ABC two domains is critical for the formation of the linear ubiq- DLBCL patient biopsies, focusing on genes coding for the uitin chain assembly complex (LUBAC). This complex has LUBAC complex proteins, and identifi ed two recurrent mis- the unique ability to catalyze the formation of linear pep- sense mutations, which were previously annotated as rare SNPs. tide bonds between the amino-terminal methionine of one These genetic variations result in a change of glutamine 584 to ubiquitin molecule and the carboxyl-terminal glycine of the histidine (Q584H) and glutamine 622 to leucine (Q622L) in the UBA domain of RNF31. The UBA is a highly conserved region responsible for the interaction of RNF31 with RBCK1 and consequent LUBAC activation ( 6 ). The two SNPs resulted Authors’ Affi liation: Institute of Biochemistry II, Goethe University, School of Medicine, Frankfurt am Main, Germany in a stronger interaction between RNF31 and RBCK1, with a consequent increase in E3 ligase activity of the complex. The Corresponding Author: Ivan Dikic, Institute of Biochemistry II, Goethe Uni- versity School of Medicine, University Hospital, Bldg 75, Theodor-Stern- exact mechanism by which the two SNPs affect the complex Kai 7, D-60590 Frankfurt am Main, Germany. Phone: 49-69-6301-83647; formation is still unclear, but the analysis of their position in Fax: 49-69-6301-5577; E-mail: [email protected] the crystal structure suggests a signifi cant modifi cation in the doi: 10.1158/2159-8290.CD-14-0177 structure of RNF31. Both mutations are located at peculiar ©2014 American Association for Cancer Research. sites: Q622L is set in an unusual bent α-helical region and

394 | CANCER DISCOVERYAPRIL 2014 www.aacrjournals.org

CBM Figure 1. In ABC DLBCL lymphoma cells, complex two SNPs in the UBA domain of RNF31 lead to a stronger interaction with RBCK1. The direct consequence is an increased activity of the LUBAC complex. Excessive linear ubiquitin chain signaling inﬂ uences the NF-κB pathway, Ub which is hyperactivated, and is responsible for Q584H Q622L Ub the cancer cell malignancy. Treatment with a UBL Ub Ub Ub small-stapled peptide (N-Q622L) diminishes the Ub RNF31–RBCK1 interaction and restores a normal Ub UBA Ub interaction between the LUBAC components. Ub Ub After peptide exposure, ABC DLBCL cells show a reduced level of NF-κB signaling and are more UBL NF-κB sensitized to chemotherapy. oncogenic RBCK1 signaling NEMO/IKKγ

Ub Ub Ub RNF31 Ub SHARPIN Ub

CBM complex

Q584H Q622L Ub

UBL Ub N-Q622L UBA Inhibition of NF-κB Ub signaling UBL Ub

RBCK1 NEMO/IKKγ

RNF31 SHARPIN

Q584L in the α6 helix. In both cases, the alteration generates Although the authors did not provide a clear molecular picture a modifi cation in the protein conformation of RNF31, which to explain the recruitment of A20 to the CBM complex, they could be responsible for a stronger electrostatic interaction did propose two interesting theories. One possibility is that with RBCK1. The natural consequence of this stronger link is the recruitment of A20 to the CBM complex is determined by increased linear ubiquitination by LUBAC, which determines ubiquitination of the receptor complex proteins by LUBAC. the persistent activation of NF-κB signaling. The consequence of increased avidity of the CBM complex for The authors elegantly demonstrated the direct involvement A20 results in its cleavage mediated by MALT1 and CARD11. of LUBAC in the regulation of NF-κB signaling in ABC DLBCL An alternative explanation is that LUBAC-mediated ubiquiti- cells. RNF31 and SHARPIN keep the NF-κB pathway active by nation of the CBM complex increases the proteolytic activity maintaining constitutively linear ubiquitination of the IKKγ/ of MALT1 by a yet-unidentifi ed mechanism. NEMO subunit. The RNF31 SNPs affect NF-κB activity on These new fi ndings implicate LUBAC in the regulation of multiple levels. In ABC DLBCL cells, mutant RNF31 increased oncogenic signaling and open new avenues to therapeuti- the expression of the two well-known NF-κB target genes NFK- cally targeting the cancerous pathology. In their article, the BIA and IRF4 , and at the same time positively stimulated inhib- authors adopted an interesting strategy for modulation of itor of IκB kinase (IKK) activity favoring nuclear NF-κB p65 LUBAC activity. Using “hydrocarbon stapling,” which stabi- DNA binding activity. Moreover, the abnormal LUBAC activity lizes peptides forming α-helical structures, they were able to contributes to the increased linear ubiquination of NEMO, generate a peptide, RNF31 N-Q622L, with increased α-helical stimulating recruitment of A20 to the CARMA1–BCL10– character. This “stapled” peptide interacted more with RBCK1 MALT1 (CBM) complex, leading to its consequent cleavage. and outcompeted the wild-type RNF31 peptide in preventing

APRIL 2014CANCER DISCOVERY | 395

Downloaded from cancerdiscovery.aacrjournals.org on September 27, 2021. © 2014 American Association for Cancer Research. VIEWS endogenous LUBAC formation in ABC DLBCL cells. Treat- REFERENCES ment with the peptide resulted in reduced activity of the NF-κB pathway, which is the main survival pathway for the 1. Sachidanandam R , Weissman D , Schmidt SC , Kakol JM , Stein LD , Marth G , et al. A map of human genome sequence variation contain- lymphoma cells, and increased the effect of chemotherapeutic ing 1.42 million single nucleotide polymorphisms . Nature 2001 ; drugs (Fig. 1). Additional analysis using in vivo ABC DLBCL 409: 928 – 33 . cell grafting would be extremely useful to evaluate the qual- 2. Barreiro LB , Laval G , Quach H , Patin E , Quintana-Murci L . Natura l ity and the safety of this approach. Moreover, as the authors selection has driven population differentiation in modern humans . discussed, it is important to emphasize that LUBAC does not Nat Genet 2008 ; 40 : 340 – 5 . affect all signaling events in the NF-κB pathway. Drugs inhib- 3. Yang Y , Schmitz R , Mitala J , Whiting A , Xiao W , Ceribelli M, et al. iting LUBAC could have different effects, other than IKKβ, so Essential role of the linear ubiquitin chain assembly complex in lymphoma revealed by rare germline polymorphisms. Cancer Discov their applications could be even more interesting and diverse. 2014 ; 4 :480–93 . The overall data provided in the article by Yang and col- 4. Kirisako T , Kamei K , Murata S , Kato M , Fukumoto H , Kanie M , e t leagues (3) lead to new and exciting questions about the role al. A ubiquitin ligase complex assembles linear polyubiquitin chains . of LUBAC in the regulation of the NF-κB pathway through EMBO J 2006 ; 25 : 4877 – 87 . signaling by the CBM complex. Moreover, the development 5. Walczak H , Iwai K , Dikic I . Generation and physiological roles of of stapled peptides opens interesting avenues for the possible linear ubiquitin chains. BMC Biol 2012 ; 10 : 23 . 6. Tokunaga F , Sakata SI , Saeki Y , Satomi Y , Kirisako T , Kamei K , et al. use of small-molecule inhibitors to counteract the oncogenic Involvement of linear polyubiquitylation of NEMO in NF-kappaB role of E3 ligases in various malignancies. Of note, this is the activation. Nat Cell Biol 2009 ; 11 : 123 – 32 . fi rst time that a therapeutic treatment targeting the LUBAC 7. Stieglitz B , Rana RR , Koliopoulos MG , Morris-Davies AC , Schaeffer complex has been proposed and could have additional impor- V , Christodoulou E , et al. Structural basis for ligase-specifi c conjuga- tant clinical applications. The regulatory role of LUBAC on tion of linear ubiquitin chains by HOIP. Nature 2013 ; 503 : 422 – 6 . BCR and Toll-like receptor signaling also makes this complex 8. Tokunaga F , Nakagawa T , Nakahara M , Saeki Y , Taniguchi M , Sakata SI , et al. SHARPIN is a component of the NF-κB–activating linear an attractive pharmacologic target for infl ammatory diseases. ubiquitin chain assembly complex . Nature 2011 ; 471 : 633 – 6 . Last but not least, this study shows that the continued inves- 9. Ikeda F , Deribe YL , Skånland SS , Stieglitz B , Grabbe C , Franz-Wachtel tigation of SNPs may lead to valuable insights in the patho- M , et al. SHARPIN forms a linear ubiquitin ligase complex regulating genesis of cancer and other genetically modulated diseases, NF-κB activity and apoptosis. Nature 2011 ; 471 : 637 – 41 . and has the potential to drive the fi eld forward. 10. Gerlach B , Cordier SM , Schmukle AC , Emmerich CH , Rieser E , Haas TL , et al. Linear ubiquitination prevents infl ammation and regulates Disclosure of Potential Confl icts of Interest immune signalling. Nature 2011 ; 471 : 591 – 6 . 11. Hailfi nger S , Lenz G , Ngo V , Posvitz-Fejfar A , Rebeaud F , Guzzardi No potential confl icts of interest were disclosed. M , et al. Essential role of MALT1 protease activity in activated B cell–like diffuse large B-cell lymphoma. Proc Natl Acad Sci U S A Published online April 4, 2014. 2009 ; 106 : 19946 – 51 .

396 | CANCER DISCOVERYAPRIL 2014 www.aacrjournals.org

Downloaded from cancerdiscovery.aacrjournals.org on September 27, 2021. © 2014 American Association for Cancer Research. Germline Polymorphisms in RNF31 Regulate Linear Ubiquitination and Oncogenic Signaling

Paolo Grumati and Ivan Dikic

Cancer Discovery 2014;4:394-396.

Updated version Access the most recent version of this article at: http://cancerdiscovery.aacrjournals.org/content/4/4/394

Cited articles This article cites 11 articles, 3 of which you can access for free at: http://cancerdiscovery.aacrjournals.org/content/4/4/394.full#ref-list-1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerdiscovery.aacrjournals.org/content/4/4/394. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.