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Loss of KLHL6 promotes diffuse large B-cell lymphoma growth and survival by stabilizing the mRNA decay factor roquin2

Abstract

Kelch-like protein 6 (KLHL6) is an uncharacterized gene mutated in diffuse large B-cell lymphoma (DLBCL). Here we report that KLHL6 assembles with cullin3 to form a functional cullin–RING ubiquitin ligase. Mutations in KLHL6 inhibit its ligase activity by disrupting the interaction with cullin3. Loss of KLHL6 favours DLBCL growth and survival both in vitro and in xenograft models. We further established that the mRNA decay factor roquin2 is a substrate of KLHL6. Degradation of roquin2 is dependent on B-cell receptor activation, and requires the integrity of the Tyr691 residue in roquin2 that is essential for its interaction with KLHL6. A non-degradable roquin2(Y691F) mutant requires its RNA-binding ability to phenocopy the effect of KLHL6 loss. Stabilization of roquin2 promotes mRNA decay of the tumour suppressor and NF-κB pathway inhibitor, tumour necrosis factor-α-inducible gene 3. Collectively, our findings uncover the tumour suppressing mechanism of KLHL6.

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Fig. 1: KLHL6 mutations in human DLBCL abolish its catalytic function as cullin3–RING–ligase.
Fig. 2: KLHL6 interacts and promotes ubiquitylation and degradation of roquin2.
Fig. 3: KLHL6 functions as a tumour suppressor in ABC-DLBCL by regulating its growth and survival.
Fig. 4: A non-degradable roquin2 mutant phenocopies loss of KLHL6.
Fig. 5: KLHL6 is a BCR/NF-κB target gene that links roquin2 degradation to BCR signaling.
Fig. 6: Stabilization of roquin2 downregulates BCR responsive genes.
Fig. 7: The KLHL6–roquin2 axis controls NF-κB activation.

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Acknowledgements

We thank C. Vinuesa for providing roquin cDNAs, A. Thomas-Tikhonenko, L. Pasqualucci and Y. Yang for providing DLBCL cell lines, M. Pagano for providing FBP cDNAs, B. Kim for helping with the 3D Matrigel colony formation assay; R. Saffie, D. Brady, E. Witze and R. Greenberg for critically reading the manuscript. This work was supported in part by grant R00-CA166181-04, R01-CA207513-01 from the National Cancer Institute and Gilead Sciences Research Scholars Program in Hematology/Oncology to L.B. R.B. is supported by an NIH Innovator Award (DP2MH107055), the Searle Scholars Program (15-SSP-102), the March of Dimes Foundation (1-FY-15-344) and the W.W. Smith Charitable Trust (C1404).

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Authors

Contributions

L.B. conceived, directed the project and oversaw the results. J.C. designed and performed most experiments. K.L. helped J.C. with experiments in Figs. 1i,3b,d,4h,6a,7h,j and Supplementary Figs. 3b,4h. K.I. helped J.C. with experiments in Fig. 6b. R.B. helped with the bioinformatics analysis of RNA-seq data. A.S., L.F. and M.P.W. performed the mass spectrometry analysis of the KLHL6 complex purified by L.B. M.R.G. and S.T. helped with Figs. 1a,b and 3a,7g and Supplementary Figs. 1a,b,7e,f. L.B. and J.C. wrote the manuscript.

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Correspondence to Luca Busino.

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Integrated supplementary information

Supplementary Figure 1 Copy number and transcriptional analysis of KLHL6 in primary DLBCLs.

a, The DNA copy number of chromosome 3 is shown. DNA copy number data from high-resolution SNP microarray analysis of 609 primary DLBCL tumours were utilized form a previously published study26. The position of KLHL6 is annotated for 21 DLBCL tumours with copy number <1.8. KLHL6 loss is detectable in 3.4% of patients. b, A row-normalized heat map is shown for probe sets corresponding to KLHL6. Gene expression microarray from 249 tumours with matched DNA copy number data were obtained from a previously published study26. The data are annotated for the cell of origin subtype and DNA copy loss of KLHL6 as shown in a. 6% of DLBCL cases with expression ≤1 s.d. below the mean are indicated.

Supplementary Figure 2 KLHL6 WT, but not KLHL6 BTB-domain mutant, promotes roquin2 ubiquitylation and degradation.

a, Immunoblot analysis from immunoprecipitated endogenous KLHL6 in U2932 cells. IgG antibody immunoprecipitates = negative control. A representative blot from two independent experiments is shown. *Non-specific band. b, Immunoblot analysis from immunoprecipitated Flag–KLHL6 wild-type (WT) and mutants in HEK293T cells. EV, empty vector. c, Quantification of (x axis) and KLHL6 (y axis) protein levels in each DLBCL cell line. n = 11 DLBCL cell lines. r, Pearson correlation coefficient (95% confidence interval). d, Quantification of roquin1 and roquin2 immunoblots. Relative intensities were plotted over time for OCI-LY10 (left panel) and U2932 (right panel) (mean ± s.d., n = 3 independent experiments, two-way ANOVA, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; n.s., not significant). e, Immunoblot analysis of whole-cell lysates from HEK293T (TET)-OFF cells transduced with retroviruses encoding a doxycycline (DOX) inducible expression of Flag-tagged KLHL6 carrying a hygromycin cassette (top panel). DOX was added and/or washed at the indicated times. Bottom panel shows the immunoblot analysis of the indicated proteins in HBL1 cells transduced with lentiviruses encoding a doxycycline (DOX) inducible expression of KLHL6 wild-type (WT) carrying a puromycin cassette. The cells were treated with DOX for 12 h. f, Immunoblot analysis of whole-cell lysates from OCI-LY8 cells transduced with retroviruses encoding empty vector (EV), KLHL6 wild-type (WT) or BTB-domain mutants (L65P, S94I and F97L) carrying a puromycin cassette. g, Immunoblot analysis of whole-cell lysates from HEK293T cells transduced with lentiviruses encoding a doxycline (DOX) inducible expression of KLHL6 carrying a puromycin cassette and infected with lentiviruses encoding empty vector(EV) or KLHL6 BTB-domain mutants (L65P and S94I) carrying a GFP marker. The cells were treated with DOX for 12 h. h, Immunoblot analysis of immunoprecipitated endogenous roquin2 in U2932 KLHL6+/+ and KLHL6−/− (clone-derived) cells treated with or without MG132 for 6 h. A representative blot from two independent experiments is shown. Unprocessed original scans of immunoblots for a,b,eh are shown in Supplementary Fig. 8, and statistical source data for c,d and exact P values for d can be found in Supplementary Table 6. Unless otherwise noted, immunoblots are representative of three independent experiments.

Supplementary Figure 3 Loss of KLHL6 promotes proliferation of ABC-DLBCL cells.

a, Immunoblot analysis of whole-cell lysates from U2932-Cas9 cells infected with lentiviruses encoding scrambled gRNA or gRNAs against KLHL6 exon 1 carrying a puromycin cassette. b, Left panel shows a representative image of U2932 cell colonies expressing indicated gRNAs and plated into a matrigel. After 14 days, the matrigel was dissolved and recovered. Cells were counted and plotted as shown on the right panel (mean ± s.d., n = 4 independent experiments, one-way ANOVA, ****P ≤ 0.0001). Scale bar, 150 μm. c, Immunoblot analysis of whole-cell lysates from U2932 cells infected with lentiviruses encoding the indicated shRNAs carrying a puromycin cassette. d, MTS assay for U2932 cells infected with lentiviruses encoding the indicated shRNAs and grown in media containing 1 μg ml−1 of F(ab′)2–IgM. Values were normalized to the shCTRL cells at time 0 h and set to 100% (mean ± s.d., n = 3 independent experiments, two-way ANOVA; ***P ≤ 0.001, ****P ≤ 0.0001). e, Immunoblot analysis of whole-cell lysates from OCI-LY10 cells infected with lentiviruses encoding indicated shRNAs carrying a puromycin cassette. f, Left panel shows a representative image of OCI-LY10 cell colonies infected with indicated shRNAs and plated in Matrigel. After 14 days, the Matrigel was dissolved and recovered. Cells were counted and plotted as shown on the right panel (mean ± s.d., n = 4 independent experiments, one-way ANOVA, ****P ≤ 0.0001). Scale bar, 150 μm. g, Gating strategy for Fig. 4c. The box indicates the stained cells. Unprocessed original scans of immunoblots for a,c,e are shown in Supplementary Fig. 8, and statistical source data and exact P values for b,d,f can be found in Supplementary Table 6. Unless otherwise noted, immunoblots are representative of three independent experiments.

Supplementary Figure 4 The roquin2(Y691F) mutant is resistant to KLHL6-mediated degradation.

a, Schematic representation of the sequence of the biotinylated roquin2 peptides. b, A pull-down assay using the indicated amount of biotinylated roquin2 peptides incubated with cell extracts from HEK293T cells stably expressing KLHL6. AP, Affinity purification. c,d, Same as in b except that Flag-tagged in vitro translated proteins as indicated were used instead of cell extracts. Immunoblot analysis for the indicated proteins was performed using Flag antibody. e, Quantification of immunoblots from BJAB cells stably expressing roquin2(WT) or roquin2(Y691F). Relative intensities were plotted over time under cycloheximide (CHX) treatment (mean ± s.d., n = 3 independent experiments, two-way ANOVA, *P ≤ 0.05; **P ≤ 0.01). f, Immunoblot analysis of whole-cell lysates from HEK2932T cells stably expressing KLHL6(WT) or KLHL6(L65P) carrying hygromycin cassette and further infected with retroviruses encoding an empty vector (EV), roquin2(WT) or roquin2(Y691F) carrying a puromycin cassette. A low exposure (l.e.) and high exposure (h.e.) are shown. g, Immunoblot analysis for indicated amounts of recombinant roquin2 (set as the standard) along with whole-cell lysates from U2932 stably expressing HA–roquin2(WT) and HA–roquin2(Y691F) (left panel) or U2932 KLHL6+/+ and KLHL6−/− cells (clone-derived) (middle panel). Right panel shows intensity of quantified roquin2 bands compared to the standard. A representative blot from one experiment is shown. h, Flow cytometry analysis of GFP+-live OCI-LY10 KLHL6+/+ and KLHL6−/− (clone-derived) cells infected with lentiviruses encoding the indicated shRNAs carrying a GFP marker. Cells were grown in media containing 2 μg ml−1 of F(ab′)2–IgM and normalized to the shCTRL cells set as 100% (mean ± s.d., n = 3 independent experiments, two-way ANOVA, **P ≤ 0.01; ****P ≤ 0.0001). i, Immunoblot analysis of whole-cell lysates from GFP-sorted U2932 (left panel) and OCI-LY10 (right panel) KLHL6+/+ and KLHL6−/− (clone-derived) infected with lentiviruses encoding the indicated shRNAs carrying a GFP marker. A representative blot from two independent experiments is shown. Unprocessed original scans of immunoblots for bd,f,g,i are shown in Supplementary Fig. 8, and source data for g and statistical source data and exact P values for e,h can be found in Supplementary Table 6. Unless otherwise noted, immunoblots are representative of three independent experiments.

Supplementary Figure 5 roquin 2 is degraded upon BCR stimulation in a KLHL6-dependent manner.

a, Flow cytometry analysis of a panel of human DLBCLs, as indicated, stained with anti-IgM or anti-IgG antibody to detect surface expression. A darker curve indicates a positive signal. A representative image from two independent experiments is shown. b, MTS assay for U2932 cells KLHL6+/+ and KLHL6−/− treated with increasing amounts of ibrutinib for 48 h. Values were normalized to the non-treated cells and set as 100% (mean ± s.d., n = 3 independent experiments, two-way ANOVA; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). c, Immunoblot analysis of whole-cell lysates from U2932 cells electroporated with a siRNA scramble (siCTRL) or siRNA targeting KLHL6 (siKLHL6) and treated with increasing concentrations of F(ab′)2–IgM for 6 h. d, Immunoblot analysis of whole-cell lysates from OCI-LY10 cells electroporated with siRNA scramble (siCTRL) or siRNA targeting KLHL6 (siKLHL6) and treated with 10 μg ml−1 F(ab′)2–IgM for the indicated times. e, Gating strategy for a. The box indicates the stained cells. Unprocessed original scans of immunoblots for c,d are shown in Supplementary Fig. 8, and statistical source data and exact P values for b can be found in Supplementary Table 6. Unless otherwise noted, immunoblots are representative of three independent experiments.

Supplementary Figure 6 Analysis of transcripts deregulated by non-degradable roquin2(Y691F) mutant.

a, Gene ontology (GO) analysis of genes regulated by the non-degradable roquin2(Y691F) mutant. Bar plot for the −log10 of the P value of the top 10 enriched GO terms of genes regulated by roquin2(Y691F) as determined by the hypergeometric distribution. −reg., negative regulation; +reg., positive regulation. Statistical analysis and genes list are provided in Supplementary Table 4. b, Ranking of roquin2-regulated genes by the percentage of genetic alteration in human DLBCLs from the TCGA database along with the base mean expression from the RNA-seq analysis. The cut-off was set at 6%. 'Yes' indicates transcripts that are dependent on the ROQ domain of roquin2; 'No' indicates transcripts that are not dependent. (c) Level of indicated mRNAs analysed by quantitative PCR from U2932 cells stably expressing HA–roquin2(WT), HA–roquin2(Y691F) or HA–roquin2(Y691FΔROQ) and treated with 10 μg ml−1 of F(ab′)2–IgM for 12 h. The value for each PCR product present in HA–roquin2(WT) cells was set as 100%. Rescued transcripts are defined as ones whose levels reach at least 70% of the roquin2(WT) control (indicated by the dashed line) (mean ± s.d., n = 3 independent experiments). Source data for c can be found in Supplementary Table 6.

Supplementary Figure 7 KLHL6 regulates TNFAIP3 levels in a roquin2-dependent manner.

a, Immunoblot analysis of whole-cell lysates from GFP-sorted U2932 KLHL6+/+, KLHL6−/− or KLHL6−/− cells infected with lentiviruses encoding the indicated shRNAs carrying a GFP marker and treated with 10 μg ml−1 of F(ab′)2–IgM for 6 h. b, Immunoblot analysis of whole-cell lysates from GFP-sorted U2932 KLHL6−/− (clone-derived) cells infected with retroviruses encoding empty vector (EV) or KLHL6(WT) carrying a GFP marker and treated with 10 μg ml−1 of F(ab′)2–IgM for 6 h. c, Same as in b except that cells were treated with 10 μg ml−1 F(ab′)2–IgM for the indicated times (min). d, GFP-sorted U2932 KLHL6+/+, KLHL6−/− or KLHL6−/− cells infected with lentiviruses encoding the indicated shRNAs carrying a GFP marker were fractionated into cytoplasmic and nuclear extracts and analysed by immunoblotting for the indicated proteins. e, A heat map showing the presence of biallelic deletion (dark blue), monoallelic deletion (light blue) and monoallelic mutation (green) of TNFAIP3 in DLBCL tumours sequenced at UNMC6 and DCI7 (n = 1,175). Deleterious mutations of KLHL6 BTB-domain are shown in these same cases. Based on DCI database, exclusivity analysis of KLHL6 and TNFAIP3 mutations is provided in Supplementary Table 5 (n = 1,001). f, A heat map showing tumour gene alterations matched with gene expression profiling data available at UNMC. Single sample gene set enrichment analysis (GSEA) was utilized to infer NF-κB activity via expression of target gene sets from the molecular signatures database (NFkB_Q and NFkB_C)26,59. The enrichment score is displayed as a row-normalized heat map. Unprocessed original scans of immunoblots for ad are shown in Supplementary Fig. 8. Unless otherwise noted, immunoblots are representative of three independent experiments.

Supplementary Figure 8

Unprocessed blots.

Supplementary information

Supplementary Information

Supplementary Figures 1–8 and Supplementary Table legends

Reporting Summary

Supplementary Table 1

Supplementary Table 2

Proteomic analysis of KLHL6 complex

Supplementary Table 3

Transcriptomic analysis of roquin2-regulated genes

Supplementary Table 4

GO enrichments of roquin2-regulated genes

Supplementary Table 5

Mutually exclusive analysis of KLHL6 BTB-mutations in DLBCL

Supplementary Table 6

Statistical Source Data

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Choi, J., Lee, K., Ingvarsdottir, K. et al. Loss of KLHL6 promotes diffuse large B-cell lymphoma growth and survival by stabilizing the mRNA decay factor roquin2. Nat Cell Biol 20, 586–596 (2018). https://doi.org/10.1038/s41556-018-0084-5

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