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. 2014 Apr;4(4):480-93.
doi: 10.1158/2159-8290.CD-13-0915. Epub 2014 Feb 3.

Essential role of the linear ubiquitin chain assembly complex in lymphoma revealed by rare germline polymorphisms

Yibin Yang  1 , Roland Schmitz, Joseph Mitala, Amanda Whiting, Wenming Xiao, Michele Ceribelli, George W Wright, Hong Zhao, Yandan Yang, Weihong Xu, Andreas Rosenwald, German Ott, Randy D Gascoyne, Joseph M Connors, Lisa M Rimsza, Elias Campo, Elaine S Jaffe, Jan Delabie, Erlend B Smeland, Rita M Braziel, Raymond R Tubbs, James R Cook, Dennis D Weisenburger, Wing C Chan, Adrian Wiestner, Michael J Kruhlak, Kazuhiro Iwai, Federico Bernal, Louis M Staudt
Affiliations

Essential role of the linear ubiquitin chain assembly complex in lymphoma revealed by rare germline polymorphisms

Yibin Yang et al. Cancer Discov. 2014 Apr.

Abstract

Constitutive activation of NF-κB is a hallmark of the activated B cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), owing to upstream signals from the B-cell receptor (BCR) and MYD88 pathways. The linear polyubiquitin chain assembly complex (LUBAC) attaches linear polyubiquitin chains to IκB kinase-γ, a necessary event in some pathways that engage NF-κB. Two germline polymorphisms affecting the LUBAC subunit RNF31 are rare among healthy individuals (∼1%) but enriched in ABC DLBCL (7. 8%). These polymorphisms alter RNF31 α-helices that mediate binding to the LUBAC subunit RBCK1, thereby increasing RNF31-RBCK1 association, LUBAC enzymatic activity, and NF-κB engagement VSports手机版. In the BCR pathway, LUBAC associates with the CARD11-MALT1-BCL10 adapter complex and is required for ABC DLBCL viability. A stapled RNF31 α-helical peptide based on the ABC DLBCL-associated Q622L polymorphism inhibited RNF31-RBCK1 binding, decreased NF-κB activation, and killed ABC DLBCL cells, credentialing this protein-protein interface as a therapeutic target. .

Significance: We provide genetic, biochemical, and functional evidence that the LUBAC ubiquitin ligase is a therapeutic target in ABC DLBCL, the DLBCL subtype that is most refractory to current therapy. More generally, our findings highlight the role of rare germline-encoded protein variants in cancer pathogenesis. V体育安卓版.

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Figures

Figure 1
Figure 1. Enrichment of two rare SNPs among ABC DLBCL tumors
a, Amino acid sequence (based on accession NP_060469) of a region of the UBA domain of RNF31 showing the residues altered by two SNPs, Q584H and Q622L, and the number and type of lymphoma biopsies in which they were identified. b, Location of RNF31 SNPs in two views of the three-dimensional structure of the RNF31 UBA domain. c, Frequencies of RNF31 SNPs in biopsy samples from different lymphoma subtypes.
Figure 2
Figure 2. LUBAC is essential for NF-κB activity in ABC DLBCL
a, RNF31 and SHARPIN shRNAs are selectively toxic for ABC DLBCL lines. Shown is the fraction of viable GFP+, shRNA-expressing cells relative to the total live cell fraction at the indicated times following shRNA induction, normalized to the day 0 values. b, Lysates were prepared using 1% SDS from HBL1 ABC DLBCL cells expressing the indicated shRNAs, diluted and then subjected to anti-NEMO immunoprecipitation, followed by immunoblotting for indicated proteins (left). Relative NEMO ubiquitination signal intensity was determined by densitometric analysis (middle). Lysates were additionally analyzed by immunoblotting for the indicated proteins (right). c, Relative IκBα-luciferase reporter activity in TMD8 ABC DLBCL cells induced for various days to express the indicated shRNAs. A specific IKKβ inhibitor (MLN120B; 10 μM) was included as a positive control. d, Nuclear fractions prepared from HBL1 cells expressing the indicated shRNAs were assayed for NF-κB p65 DNA-binding activity by ELISA. e, Relative activity of an NF-κB-dependent luciferase reporter in the ABC DLBCL lines expressing the indicated shRNAs. All error bars denote SEM of triplicates.
Figure 3
Figure 3. LUBAC involves in CBM complex mediated NF-κB activation in ABC DLBCL
a, ABC DLBCL lines engineered to express myc epitope-tagged RNF31 were immunoprecipitated using antibodies to IRAK1 or MALT1, or control IgG (ctrl.) and were analyzed by immunoblotting for the indicated proteins. b, HBL1 cells were engineered to express the indicated shRNAs, treated with MALT1 inhibitor Z-VRPR-fmk (75 μM) for 24 hours or left untreated. Whole cells lysate was immunoblotted for indicated proteins. MALT1-dependent A20 cleavage products are indicated. c, HBL1 cells expressing the indicated shRNAs were activated by anti-IgM treatment (10 μg/ml) for indicated times and analyzed by immunoblotting for the indicated proteins. d, Viability of ABC DLBCL lines expressing control or RNF31 shRNAs were treated with DMSO, ibrutinib (1 nM) or lenalidomide (2 μM) and analyzed by FACS for viable GFP+/shRNA-expressing cells over a time course.
Figure 4
Figure 4. RNF31 SNPs promote NF-κB activity in ABC DLBCL
a, NF-κB-driven luciferase reporter activity in HBL1 and TMD8 lines engineered to express the indicated myc epitope-tagged RNF31 isoforms. b, Control or RNF31 shRNAs were inducibly expressed in HBL1 cells that had been transduced with rescue vectors expressing RNF31 isoforms or with an empty vector. Indicated mRNA expression was quantified by Q-PCR and normalized to β2-microglobulin mRNA levels. c, Relative IκBα-luciferase reporter activity was measured in TMD8 cells induced to express the indicated myc-tagged RNF31 isoforms. d, Cells prepared as in Fig. 4b were analyzed by immunoblotting for the indicated proteins. e, Nuclear NF-κB p65 DNA-binding activity was determined by ELISA in HBL1 cells selected for expression of the indicated myc epitope-tagged RNF31 isoforms. f, Flow cytometry histograms of the expression of the NF-κB target gene CD83 in BJAB cells expressing vector only or the indicated exogenous RNF31 isoforms, with and without anti-IgM stimulation. Right panel: summary of CD83 expression in BJAB cells expressing vector only or RNF31 isoforms, either in unstimulated cells or anti-IgM stimulated cells. g, HBL1 cells engineered to express indicated myc epitope-tagged RNF31 isoforms were analyzed by immunoblotting for the indicated proteins (top). The relative A20 cleavage signal intensity was determined by densitometric analysis (bottom). h, Cells prepared as in Fig. 4g were subject to anti-MALT1immunoprecipitation. Immunoprecipitated proteins or whole cell lysates were analyzed by immunoblotting for the indicated proteins. All error bars denote SEM of triplicates.
Figure 5
Figure 5. Gain-of-function conferred by RNF31 SNPs
a, Control or RNF31 shRNAs were inducibly expressed in HBL1 cells that had been transduced with rescue vectors expressing RNF31 isoforms or with an empty vector. Dox induced cells were lysed in 1% SDS, diluted and then subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting for indicated proteins (top). The relative NEMO linear ubiquitination signal intensity was determined by densitometric analysis (bottom). Also shown are immunoblots for the indicated proteins in whole cell lysates from the same cells. b, HBL1 cells engineered to express indicated myc epitope-tagged RNF31 isoforms were subject to anti-myc immunoprecipitation followed by elution with myc peptides. The elutions were examined in an E3 ligase activity ELISA assay (top) or by immunoblotting for the indicated proteins. c, Protein prepared as in Fig. 5b were used in an in vitro ubiqutination assay, the products of which were analyzed by immunoblotting for the indicated proteins. d, Anti-myc immunoprecipitates prepared as in Fig. 5b or whole cell lysates were analyzed by immunoblotting for the indicated proteins. e, Densitometic quantititation of co-immunoprecipitation experiments demonstrating the association of RNF31 isoforms and RBCK as in panel 5d. All error bars denote SEM of replicate experiments (n=3 for all experiments except 5e (n=11).
Figure 6
Figure 6. Targeting RBCK1-RNF31 interface using stapled α-helical RNF31 peptides
a, Schematic and sequences of RNF31 stapled peptides. Asterisks indicate the location of the hydrocarbon crosslinker. b, Circular dichroism spectroscopy of RNF31 peptides. Dips in the curves at 205 and 225 nm are indicative of α-helical structure. c, Fluorescence polarization assay of of FITC–labeled RNF31 peptides (14.1 nM) binding to recombinant RBCK1. d, Competitive fluorescence polarization assay in which binding FITC– RNF31 N-Q622L (14.1 nM) to RBCK1 (0.7 μM) was inhibited by the indicated concentrations of unlabeled peptides.
Figure 7
Figure 7. Biological effects of RNF31 stapled peptides in ABC DLBCL
a, NF-κB-dependent luciferase reporter activity in HBL1 cells treated for 2 days with DMSO or the indicated peptides. Data are normalized to DMSO-treated cells. b, IκBα-luciferase reporter activity in TMD8 cells treated for 3 days with DMSO or the indicated peptides. Data are normalized to DMSO-treated cells. c, Viability of DLBCL lines treated with stapled RNF31 Q622L peptide at the indicated concentrations +/− ibrutinib (0.5 nM) for 3 days, normalized to DMSO-treated cells. d, HBL1 cells prepared as in Fig. 4b were exposed to etoposide (5 μM) or DMSO for 1.5 hours. Whole cells lysates was blotted for the indicated proteins. e, Viability of TMD8 cells treated for 4 days with DMSO or the indicated peptides at various concentrations +/− etoposide (100 nM), normalized to viability of DMSO-treated cells. Neg. ctrl.: negative control stapled peptide. All error bars denote SEM of triplicates.
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