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Loss of KLHL6 promotes diffuse large B-cell lymphoma growth and survival by stabilizing the mRNA decay factor Roquin2

Kelch-like protein 6 (KLHL6) is an uncharacterized gene mutated in diffuse large B-cell lymphoma (DLBCL). We report that KLHL6 assembles with CULLIN3 to form a functional CULLIN-Ring ubiquitin ligase. Mutations of KLHL6 inhibit its ligase activity by disrupting the interaction with CULLIN3. Loss of KLHL6 favors DLBCL growth and survival both in vitro and in xenograft models. We further established the mRNA decay factor Roquin2 as a substrate of KLHL6. Degradation of Roquin2 is dependent on B-cell receptor activation, and requires the integrity of the tyrosine 691 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 tumor suppressor and NF-κB pathway inhibitor, tumor necrosis factor-α-inducible gene 3 (TNFAIP3). Collectively, our findings uncover the tumor suppressing mechanism of KLHL6.


Introduction
B-cell cancers hijack protein ubiquitylation and degradation to promote growth and survival, as shown by the successful use of a proteasome inhibitor (bortezomib) and E3 ubiquitin ligase inhibitor (lenalinomide) in multiple myeloma (MM) and mantle cell lymphoma 1 . Despite the significant progress, much remains to be explored in the field of ubiquitin and molecular mechanisms of tumorigenesis.
Kelch-like protein 6 (KLHL6) is a member of the bric-a-brac/tramtrack/broad-complex (BTB) domain family of proteins with a lymphoid tissue-restricted expression pattern 2,3 . Whole-genome and exome sequencing have revealed cancer-associated mutations of the KLHL6 gene in B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL) 4-7 ; however, the relevance of these mutations as well as the molecular function of KLHL6 is currently unknown.
DLBCL is the most common type of lymphoid malignancies with two distinct molecular subtypes: activated B cell-like (ABC) and germinal center B cell-like (GCB) lymphoma 8,9 . ABC-DLBCLs depend on hyper-activation of the inhibitor of IκB kinase (IKK) and the NF-κB transcription factor program for their proliferation and survival 10,11 . This is evidenced by frequent mutations in the BCR pathway, including activating mutations of positive (CD79A/B and CARD11 12,13 ) and inactivating mutations of negative (TNFAIP3 [12][13][14][15] ) NF-κB regulators. How KLHL6 contributes to the pathology of human DLBCL and whether it influences NF-κB activation are currently unknown.
Regulatory networks that promote cancer progression modulate gene expression at the level of mRNA stability 16,17 . The RNA-binding proteins RC3H1 and RC3H2 (from now on Roquin1 and Roquin2) promote mRNA decay via recognition of stem-loop motifs in the 3′ untranslated region (UTR) of target mRNAs 18,19 . Through this recognition, Roquins recruit the CCR4-CAF1-NOT complex, leading to mRNA deadenylation and subsequent destabilization [18][19][20][21][22] . In T-cells, Roquin proteins contribute to immune homeostasis by promoting decay of Inducible T-cell Costimulator (ICOS). However, the role of Roquin in B-cell cancers has not been investigated 18,23 .
Here, we demonstrate that KLHL6 is an E3 ligase for Roquin2. Cancer-associated mutations of KLHL6 inhibit its ubiquitin ligase activity and inactivation or loss of KLHL6 in ABC-DLBCL promotes cancer cell growth and survival through stabilization of Roquin2 and subsequent decay of the TNFAIP3 mRNA. This study shows how ABC-DLBCL cells hijack experiments confirmed Roquin2, but not Roquin1, as a KLHL6 interactor (Fig. 2c). This complex was also detectable at an endogenous level in DLBCL cells (Fig. S2a). Importantly, interaction between KLHL6 and Roquin2 required the intact Kelch domain in KLHL6 (Fig.  S2b), further supporting substrate-like interaction 28 .
In DLBCL cell lines, abundance of KLHL6 and Roquin2 displayed an inverse correlation ( Fig. 2d and S2c). To investigate whether KLHL6 controls Roquin2 protein levels, we assessed Roquin2 protein turnover upon knockdown or knockout of KLHL6 in OCI-LY10 and U2932 cells, respectively. In both cases, down-regulation of KLHL6 significantly extended the half-life of Roquin2, but not that of Roquin1 (Fig. 2e, 2f and S2d).
Next, we utilized B-cell lymphoma cell lines harboring endogenous KLHL6 mutations. VAL cells harbor two BTB-mutations: N60T and T72R (Fig. 2g). Binding analysis revealed that only the KLHL6(T72R) mutant lost interaction with CULLIN3 (Fig. 2g), while KLHL6(N60T) mutant did not. Knockdown of KLHL6 in VAL cells did not result in Roquin2 accumulation (Fig. 2h), whereas re-expression of KLHL6 induced Roquin2 downregulation (Fig. 2j). In VAL cells, expression of KLHL6 at the mRNA level was comparatively low (Fig. 2i). This suggests that VAL cells display one KLHL6 allele inactivated by a mutation in the BTB-domain and an additional down-regulation of KLHL6 mRNA. In contrast, mutations of KLHL6 in SUDHL10 cells had no impact on KLHL6 function (Fig. 2g, 2h 2i, and 2j).

KLHL6 functions as a tumor suppressor in ABC-DLBCL by regulating its growth and survival
Although mutations of KLHL6 occur at a similar rate in GCB-and ABC-DLBCL (Fig. 1B), low KLHL6 expression correlated with a significantly poorer survival in ABC-DLBCL patients (Fig. 3a) as previously reported 29,30 .
To assess the biological effect of KLHL6 loss in ABC-DLBCL lines, we infected Cas9expressing U2932, OCI-LY10 and TMD8 cells with lentiviruses encoding three different gRNAs targeting the KLHL6 gene locus. Ablation of KLHL6 resulted in an increase in cellular proliferation and a decrease in apoptosis ( Fig. 3b and 3c). This effect was confirmed in 3D cultures as measured by a larger number and size of colonies ( Fig. S3a and S3b). To rule out the possible off-target effects of gRNAs, we utilized shRNA-mediated knockdown of KLHL6 in U2932 and OCI-LY10 and observed similar results ( Fig. S3c-f).
To investigate the role of cancer mutations in cell growth, we re-expressed KLHL6(WT) or KLHL6 BTB-domain mutants or an empty vector (EV) in a clonally-derived U2932 KLHL6 −/− cell line (Fig. 3d). While re-expression of KLHL6(WT) decreased cell proliferation, expression of KLHL6 BTB-domain mutants phenocopied loss of KLHL6, confirming these mutations as loss of function. In xenograft models, expression of KLHL6(WT) decreased tumor burden while expression of KLHL6(S94I) displayed a similar tumor burden compared to KLHL6 −/− cells (EV) (Fig. 3e).

A non-degradable Roquin2 mutant phenocopies loss of KLHL6
We mapped the KLHL6 binding motif in Roquin2. By performing mutagenesis experiments, we identified a region in Roquin2 between amino acids 640 and 700 as necessary for interaction with KLHL6 ( Fig. 4a and 4b). More refined deletions narrowed the interaction domain between amino acids 690 and 695 (Fig. 4c). Alanine scanning mutagenesis of the individual residues surrounding the 691-704 region revealed that a conserved tyrosine residue, in position 691, was required for KLHL6-Roquin2 interaction (Fig. 4d). In vitro binding assays confirmed that a Roquin2 peptide from 686 to 700 directly interacts with KLHL6 ( Fig. S4a-d). Mutations of tyrosine 691 into alanine or phenylalanine impaired the ability of Roquin2 to co-immunoprecipitate with KLHL6 in vivo and in vitro ( Fig. 4e and S4c), suggesting that the integrity of the tyrosine hydroxyl group is critical for KLHL6 interaction.
Next, we analyzed the effect of Roquin2 stabilization on DLBCL growth. Expression of the non-degradable Roquin2(Y691F) mutant increased tumor burden as monitored by tumor volume and weight at the experimental endpoint (Fig. 4g). This effect was not an artifact of overexpression because the levels of Roquin2(Y691F) were comparable to those of endogenous Roquin2 in KLHL6 −/− cells (Fig. S4g). Moreover, knockdown of Roquin2 impaired the cell growth advantage of KLHL6 −/− U2932 and OCI-LY10 cells (Fig. 4h, and S4h and S4i). Importantly, loss of Roquin2 increased toxicity of KLHL6 −/− cells preferentially (Fig. S4h), suggesting that loss of KLHL6 promotes cell proliferation in a Roquin2-dependent manner.

KLHL6 is a BCR/NF-κB target gene that links Roquin2 degradation to BCR signaling
KLHL6 is a member of the B-cell Receptor (BCR)-signalosome 31 and is induced upon antigen stimulation in the germinal center 3 . Thus, we investigated whether mRNA and protein levels of KLHL6 and Roquin2 were affected by BCR stimulation. First, we found ABC-DLBCL cells predominantly expressed an IgM-BCR as opposed to GCB-DLBCL cells, which are positive for IgG-BCR (Fig. S5a) 32 . Then, we analyzed levels of KLHL6 and Roquin2 in IgM-positive ABC-DLBCL cell lines (U2932, OCI-LY10 and HBL1) (Fig 5a). BCR stimulation using the fragment affinity-purified antibody F(ab′)2-IgM induced upregulation of KLHL6 and a corresponding down-regulation of Roquin2 protein levels in OCI-LY10 and U2932, but not in HBL1 [cell line with low KLHL6 for both mRNA and protein levels (Fig. 5a)].
To demonstrate that BCR-dependent down-regulation of Roquin2 protein depends on CRLs, we pre-treated U2932 cells with MLN4924, a NEDD8-activating enzyme (NAE) inhibitor that blocks Cullin neddylation 33 . MLN4924 treatment rescued Roquin2 down-regulation induced by BCR-crosslinking, suggesting that a functional CRL-complex is required to promote Roquin2 degradation (Fig. 5b). Notably, BCR stimulation induced KLHL6 upregulation both at transcriptional and protein levels (Fig. 5a, 5b and 5c).
Correspondingly, treatment of cells with an IKK or a BTK inhibitor (ibrutinib) 13 , resulted in a down-regulation of KLHL6 both at mRNA and protein levels (Fig. 5e). Interestingly, a reduced sensitivity of KLHL6 −/− cells to ibrutinib was observed (Fig. S5b).

Stabilization of Roquin2 down-regulate BCR responsive genes
To investigate whether the pro-proliferative effect of the non-degradable Roquin2(Y691F) mutant depends on its RNA binding ability, we generated a double mutant Roquin2(Y691FΔROQ) lacking the ROQ domain. Notably, deletion of the ROQ domain abolished the growth advantage induced by Roquin2(Y691F) expression (Fig. 6a).
Next, we investigated whether misregulation of Roquin2 degradation would result in a deregulation of the BCR transcriptional program. We measured differential gene expression in U2932 cells expressing Roquin2(WT) or Roquin2(Y691F) upon BCR ligation via RNAseq ( Fig. 6b and Supplementary Table 3). Pairwise comparison revealed that 133 mRNAs were significantly down-regulated in Roquin2(Y691F) expressing cells as compared to Roquin2(WT). 64 of these 133 mRNAs overlapped with BCR-responsive genes defined as those with at least a two-fold up-regulation in expression upon BCR stimulation in cells expressing Roquin2(WT) (Fig. 6b and 6c). These 64 genes represented BCR responsive genes that failed to be up-regulated upon BCR stimulation in presence of a non-degradable Roquin2 mutant.
Gene ontology (GO) enrichment analysis ( Fig. S6a and Supplementary Table 4) revealed Roquin2 regulated genes are involved in immune and inflammatory responses and implicated as regulators of the NF-κB pathway and lymphoid tumor suppressors (e.g., TNF, NFKBIE, TNFAIP3, LTA, TNFRSF14) 14,[35][36][37][38] . To identify targets relevant to DLBCL biology, we ranked these genes by the percentage of genetic alterations in human DLBCL (TCGA, http://cancergenome.nih.gov/) and the base mean expression in our RNA-Seq analysis ( Fig. S6b and Supplementary Table 3). We validated the top 11 candidates in a secondary screen and found 7 of them were dependent on a functional ROQ domain ( Fig.  S6b and c).
Amongst these targets, we focused on tumor necrosis factor-α-inducible gene 3 (TNFAIP3) because it is frequently inactivated in ABC-DLBCLs 12-15 and is a direct target of the Roquin proteins 19 . We found that TNFAIP3 was up-regulated upon BCR stimulation consistent with its function as a negative regulator of the NF-κB program (Fig. 6d) 39 . This response was abolished in cells expressing Roquin2(Y691F), suggesting that stabilization of Roquin2 contributes to a reduction of TNFAIP3 mRNA levels during BCR signaling. This effect was rescued partially in cells expressing Roquin2(Y691FΔROQ) as a build-up of TNFAIP3 mRNA levels was observed. Notably, other NF-κB target genes (NFKBIE and LTA) and the tumor suppressor gene TNFRSF14 displayed a similar pattern.
Lastly, we investigated whether the KLHL6-Roquin2 axis directly controls TNFAIP3 mRNA stability. Ablation of KLHL6 shortened the half-life of TNFAIP3, which was partially rescued by concomitant ablation of Roquin2 (Fig. 6e). Re-expression of KLHL6(WT) in VAL, which carries endogenous BTB-mutations, also increased the half-life of TNFAIP3 ( Fig. 6e).

KLHL6-Roquin2 axis controls NF-κB activation
Since KLHL6 regulates TNFAIP3 mRNA levels, we hypothesized that loss of KLHL6 would lead to increased NF-κB activation in ABC-DLBCL. First, we investigated whether the TNFAIP3 transcriptional changes would reflect similar changes in protein levels. BCR dependent degradation of Roquin2(WT) inversely correlated with the up-regulation of TNFAIP3 levels, which was reduced in cells stably expressing Roquin2(Y691F) (Fig. 7a). Correspondingly, TNFAIP3 protein levels were down-regulated in KLHL6 −/− cells, both at steady state and in response to BCR stimulation (Fig. 7b). Knockdown of Roquin2 in U2932 KLHL6 −/− cells rescued TNFAIP3 levels similar to those of KLHL6 +/+ cells (Fig. S7a).
Next, we investigated whether loss of KLHL6 resulted in increased IKK activation. The amount of IκBα phosphorylation upon BCR stimulation was higher in KLHL6 −/− cells, suggesting increased IKK activity (Fig. 7d). Importantly, the increase in phosphorylation was reduced by re-expression of KLHL6(WT) (Fig. S7c). We also detected an increase in the nuclear translocation of the NF-κB transcriptional factors in KLHL6 −/− cells (Fig. 7e), which was partially mitigated by the concomitant knockdown of Roquin2 (Fig. S7d).
Moreover, ablation of KLHL6 increased RelA DNA-binding at the NFKBIA promoter, an effect reversed by simultaneous knockdown of Roquin2 (Fig. 7f).
We analyzed the mutual exclusivity of BTB-associated KLHL6 mutations with TNAFAIP3 alterations in DLBCL patients. Deleterious KLHL6 BTB-mutations showed no overlap with TNFAIP3 biallelic deletion or mutation ( Fig. 7g and S7e). Using a weighted test 40 , we found exclusivity with a trend toward significance (P-value 0.085, Supplementary Table 5). Instead, 4 BTB-mutations had a coincident monoallelic deletion in TNFAIP3, suggesting that these mutations might confer additional NF-κB activation when only one TNFAIP3 allele is lost. Interestingly, tumour cells from patients with KLHL6 mutations revealed a higher NF-κB activity, although this signature was not only confined to KLHL6 mutated cases (Fig. S7f).
To investigate whether the tumor suppressor role of KLHL6 would be diminished in TNFAIP3-null ABC-DLBCLs, we ablated KLHL6 in RCK8 cells 14  Furthermore, we identified Roquin2 as the bona fide substrate of KLHL6. KLHL6 specifically binds, ubiquitylates and triggers Roquin2 (but not Roquin1) degradation in a BCR-dependent manner. Expression of a non-degradable Roquin2(Y691F) mutant phenocopies loss of KLHL6 and concomitant ablation of Roquin2 in KLHL6 −/− cells results in an inhibition of cell proliferation. This pro-proliferative effect depends on the ability of Roquin2 to bind RNA [18][19][20][21][22] . Although Roquin1 and Roquin2 are genetically redundant in the T-cell compartment 21 , no studies have assessed the functional redundancy in B-cell cancers. Transgenic mice overexpressing Roquin2 or the non-degradable mutant in the GC might be helpful to model the oncogenic role of Roquin2 in DLBCL in vivo.
TNFAIP3 is a relevant target of Roquin2-mediated mRNA decay 19 . In ABC-DLBCL, BCRsignaling triggers transcription of KLHL6 and degradation of Roquin2, thus releasing TNFAIP3 from mRNA decay. We speculate that KLHL6 inhibits TNFAIP3 decay via Roquin2 degradation contributing in establishing a negative feedback loop to terminate the NF-κB signaling (Fig. 7k). Thus, loss of KLHL6 function promotes BCR-dependent activation of NF-κB in ABC-DLBCL. Correspondingly, mutations of KLHL6 correlate with high NFκB signatures and could serve as a marker of resistance to NFκB pathway targeting drugs such as Ibrutinib 13 , MLN4924 33 or bortezomib 1 .
TNFAIP3 genetic mutations and deletions are frequently observed in ABC-DLBCLs [12][13][14][15] . Functional reconstitution of TNFAIP3 in TNFAIP3-null DLBCL causes apoptosis and growth arrest, supporting a tumor suppressor role in DLBCL 44,45 . Interestingly, cooccurrence of KLHL6 BTB-mutations and TNFAIP3 biallelic deletion or mutations was rarely observed in human DLBCL, suggesting that these two genes may have similar downstream components (i.e. IKK activation). A partial overlap between KLHL6 BTBmutations and TNFAIP3 monoallelic deletion further points to a possible synergy towards NF-κB activation. On the other hand, neither Roquin2 amplification nor sequence alteration of TNFAIP3 mRNA at the Roquin2 binding site has been observed in DLBCLs. This suggests that the tumor suppressor mechanism of KLHL6 might extend beyond Roquin2/ TNFAIP3 deregulation. It is possible that KLHL6 might be involved in other biological processes such as cell adhesion, migration or immune surveillance in patients.
Much remains in evaluating the functional impact of the KLHL6-Roquin2 axis in GCB-DLBCLs, where unknown KLHL6 substrates or different Roquin2 mRNA targets can contribute to the proliferation and survival of GCB-DLBCL cells. It is also worth noting that KLHL6 mutations are not observable at high frequency in non-B-cell cancers, suggesting a possibly differential role for KLHL6 in a different genetic and cellular context.
Biochemical methods-Extract preparation, immunoprecipitation, and immunoblotting, fractionation of DLBCLs and chromatin immunoprecipitation were carried out according to ref 46 . Bands quantification was performed using ImageJ software and plotted using nonlinear-fit curve in Prism. All antibodies were used at a dilution of 1:1000 unless specified. The following antibodies were used . KLHL6, Roquin1/2, TNFAIP3, CULLIN1, CULLIN3, p100/p52, p105/p50 and RelA antibodies were validated in our lab utilizing RNAi as well as overexpression, and were also validated by manufacturer. All other primary antibodies were validated on manufacturer datasheets.
Xenotransplantation experiments-All animal work was performed following the ethical guidelines and protocols approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. NOD/SCID/IL2Rγ−/− (NSG) mice were purchased from the Jackson Laboratory. Six to eight-week-old NSG mice received subcutaneous (s.c.) flank injections of 1×10 7 U2932 KLHL6 −/− (clone-derived) cells reexpressed either with empty vector, KLHL6(WT), or KLHL6(S94I)), and 1×10 7 U2932 cells infected with retroviruses encoding HA-Roquin2(WT) or HA-Roquin2(Y691F) in 100μl sterile PBS. Tumor burden was monitored weekly by palpation and eye inspection. Tumor volume calculated by caliper measurement. Tumor weight was analyzed on the excised tumors at the experimental endpoint using an analytical scale. After about one month, tumor volume and weight were measured.
In vivo ubiquitylation assay-U2932 KLHL6 +/+ and KLHL6 −/− (clone-derived) cells were treated with or without MG132 for 6hrs and lysed in 1% SDS. Lysates were then diluted to 0.1% SDS in NP-40 buffer and immunoprecipated (IP) with a polyclonal antibody against Roquin2. The immunocomplexes were subjected to SDS-PAGE and analyzed by immunoblot.

In vitro binding assays-For in vitro binding assays, in vitro-translated FLAG-tagged
KLHL6 was added to lysis buffer (50 mM Tris-HCl pH 7.5, 250 mM NaCl 0.1% Triton X-100, 1 mM EGTA) with indicated amounts of Roquin2 peptides. Anti-streptavidin resin was then added to the lysis buffer and incubated at 4°C for 2hrs. Samples were washed three times with the lysis buffer, and complexes were eluted in Laemmli buffer. For the use of HEK293T cells stably expressing KLHL6(WT), cells were lysed with the lysis buffer and anti-streptavidin resin was directly added to the whole cell lysates with the peptides.

Transient transfections and retrovirus-mediated gene transfer-HEK293T cells
were transfected using polyethylenimine (PEI). For retrovirus and lenvirius production, GP-293 packaging cells (Clontech) or pCMV-DeltaR8.2 were used respectively. The viruscontaining medium was collected after forty-eight hours of transfection and supplemented with 10 μg/ml polybrene (Sigma). Then, cells were spin-infected at 1800rpm for 30 minutes with the viral supernatant for six hours to overnight. For RCK8, cells were electroporated using Neon transfection system according to manufacture's protocol with LentiCRISPRv2 vector carrying a GFP marker. Transfected cells were sorted by GFP. siRNA oligos transfection was performed with Neon transfection system.

Generating clonal KLHL6 −/− cell lines-U2932
or OCI-LY10 Cas9-expressing cells were infected with lentiviruses encoding gRNAs targeting the KLHL6 exon1. Upon infection and puromycin selection, cells were plated in a 96 well plate at a concentration of 0.5 cells/well. Single clones were screened for KLHL6 expression by immunoblotting.
Plasmids-Human Roquin1 and Roquin2 cDNA were kindly provided by Dr. Carola Vinuesa and subcloned into pcDNA3.1-FLAG, pcDNA3.1-HA or pcDNA3.1-FLAG-Streptavidin. Human KLHL6 cDNA was purchased from Dharmacon and subcloned into pcDNA3.1-FLAG, pcDNA3.1-FLAG-Streptavidin, pREV-TRE. C-terminal deletion mutants and point mutants were generated by utilizing the QuikChange Site-directed Mutagenesis kit (Stratagene), and N-terminal deletion mutants were generated by standard PCR methods. For retrovirus production, cDNAs encoding FLAG-tagged or HA-tagged KLHL6 and KLHL6 mutants were subcloned into the retroviral vector MIGR1 and pREV-TRE vector. cDNAs encoding FLAG-HA-tagged or untagged Roquin2 and Roquin2 mutants were subcloned into the retroviral vector pBabe Puro or pMSCV. Lentivirus encoding shRNAs targeting human KLHL6 were subcloned in pSicoR-Puro. Lentivirus encoding shRNAs targeting human Roquin2 were subcloned in pSicoR-GFP.
KLHL6 cDNA was clone into pTRIPZ using AgeI and HpaI. Cell Proliferation Assay-2 × 10 3 cells were plated for MTS assay according to manufacturers' protocols.
Flow cytometry-Flow cytometry was performed on Attune NxT Flow Cytometer using FITC for GFP-expressing cells, Alexa-680 AnnexinV to detect apoptosis, FITC to detect IgM or APC to detect IgG staining. All stainings were performed according to manufacturers' protocols. For investigating shRNA or gRNA effects on survival, 5×10 5 cells were spin-infected in a 24-well plate with 1000 μl lentivirus containing medium in the presence of polybrene(8μg/ml). Media was changed after the spin-infection and the number of infected cells was determined on day 2 when GFP was fully expressed in all infected cells. The number of viable GFP positive cells on day 2 was set to 100% to normalize for transduction efficiency and every consecutive assessment was calculated in relation to day 2. When indicated, AnnexinV positive cells were gated on GFP positive cells (see gating strategy in Supplementary Figures 3 and 5).
MudPIT analysis-TCA-precipitated proteins were urea-denatured, reduced, alkylated and digested with endoproteinase Lys-C (Roche), followed by modified trypsin (Roche), as described 47,48 . Peptide mixtures were loaded onto 100-μm fused silica microcapillary columns packed with 5-μm C18 reverse phase (Aqua, Phenomenex), strong cation exchange particles (Luna, Phenomenex), and reverse phase 49 . Loaded microcapillary columns were placed in-line with a Quaternary Agilent 1100 series HPLC pump and a LTQ linear ion trap mass spectrometer equipped with a nano-LC electrospray ionization source (Thermo Scientific). Fully automated 10-step MudPIT runs were carried out on the electrosprayed peptides, as described 47 . Tandem mass (MS/MS) spectra were interpreted using SEQUEST 50 against a database of 61,318 sequences, consisting of 30,449 non-redundant human proteins (downloaded from NCBI on 2012-08-27, 160 usual contaminants (such as human keratins, IgGs and proteolytic enzymes), and, to estimate false discovery rates, 30,659 randomized amino-acid sequences derived from each non-redundant protein entry. Peptide/spectrum matches were sorted and selected using DTASelect with the following criteria set: spectra/peptide matches were only retained if they had a DeltCn of at least 0.08 and a minimum XCorr of 1.8 for singly-, 2.0 for doubly-, and 3.0 for triply-charged spectra. In addition, peptides had to be fully tryptic and at least seven amino acids long. Combining all runs, proteins had to be detected by at least two such peptides, or one peptide with two independent spectra. Under these criteria the final FDRs at the protein and spectral levels were 2.1%±0.3 and 0.94% ± 0.03, respectively. Peptide hits from multiple runs were compared using CONTRAST 51 . To estimate relative protein levels, Normalized Spectral Abundance Factors (NSAFs) were calculated for each detected protein, as described 52,53,54 .

Analysis of KLHL6 expression in DLBCL patients-Raw
DNA copy number data from high resolution single nucleotide polymorphism (SNP) microarray analysis of 609 primary DLBCL tumors were utilized form a previously published study 26 . The data were visualized using the integrative genomics viewer (IGV) 55 . Cases were sorted according to their KLHL6 copy number status, and those with copy number <1.8 were classified as possessing a deletion, according to previously described criteria 56 .
Gene expression microarray from 249 tumors with matched DNA copy number data were obtained from a previously published study 26 . Their cell of origin subtype was determined using the Wright algorithm 57 , as previously reported 56 . Row normalized heatmaps for 4 probe sets corresponding to KLHL6 were sorted according to their average expression, and significant reduction in KLHL6 expression defined as being 1 standard deviation below the mean.
Raw cel files for publicly available Affymetrix U133 plus 2.0 gene expression microarray data for diffuse large B-cell lymphoma tumors (GSE10846, GSE34171, GSE31312) were obtained from the gene expression omnibus. Data were RMA normalized using the ExpressionFileCreator module of GenePattern 58 . Scores to categorize diffuse large B-cell lymphoma tumors by cell of origin subtype were calculated according to the Wright algorithm 57 , Intensities from the 4 probes for KLHL6 (1555275_a_at, 1560396_at, 1560397_s_at, 228167_at) were averaged for use in the survival analysis. Cases were dichotomized into being above or below the median expression level of KLHL6 expression within each dataset to avoid confounding batch effects. For NF-κB signatures, Affymetrix U133 plus 2 gene expression microarrays were performed on 84 matched DLBCL tumors 26,59 . Raw cel files were RMA normalized with median scaling using the ExpressionFileCreator module of GenePattern 58 . Sample-level enrichment of NF-κB target genes was calculated using the single sample gene set enrichment analysis 60 and the c3 TFT gene set database of mSigDB 61 .
RNA-seq-Total RNA was extracted from U2932 cells using RNeasy Mini Kit (QIAGEN, #74104) and polyA+ transcripts isolated with oligo (dT) 25 -conjugated magnetic Dynabeads (Thermo Fisher). Strand specific RNA-seq libraries were prepared following a published protocol 62 . Briefly, RNA was chemically fragmented in first strand buffer, converted to cDNA using SuperScript® III reverse transcriptase (Invitrogen), end-repaired, A-tailed and ligated to custom-designed universal adapters using an end-repair mix, klenow fragment, and T4 DNA ligase (all from Enzymatics). After ligation, adapters were removed by SPRI purification using SPRIselect beads (Beckman coulter) and amplified with Q5 Hot Start DNA polymerase (New England Biolabs) while introducing custom dual indexes. Three biological replicates were sequenced on a NextSeq 500 (Illumina) at a depth of at least 2×10 7 reads each. Reads were mapped and analyzed with a custom bioinformatic pipeline based on STAR 63 , SAMTOOLS 64 , and the R packages DEGseq 65 and DEseq2 66 . We used human genome version GRCh38 and gene annotations from the ENSEMBL release 83. GO analyses were performed using version 6.8 of the DAVID web server 67,68 .
CHIP-seq-The data discussed in this publication were obtained from the NCBI's Gene Expression Omnibus 34 and are accessible through GEO at Series accession numbers GSE55105. FASTQs were downloaded and mapped to hg19 with bowtie2 (v2.1.0). Genome browser tracks were generated using custom scripts. When available, biological replicate were merged by taking the mean of the reads density at each position. The data were visualized using the integrative genomics viewer (IGV) 55 .
Statistics and Reproducibility-All graphs show mean values with error bars signifying standard deviation (s.d.) as indicated in the figure legends. Exact P values for each experiment are provided in Supplementary Table 6. Unless otherwise noted, all immunoblots were successfully repeated at least three times. One-tailed t-test was performed for Fig. 3e (right panel) and two-tailed t-test for Fig 4g. Other analyses performed were either one-way (Fig. 3c, 3e (left panel), 5e, 6a, 7i, 7j, S3b, S3f) or two-way ANOVA (Fig. 1i, 3b, 3d, 4g, 4h, 6e, 7h, S2d, S3d, S4e, S4h, S5b) as indicated in the figure legends. DEseq2 was performed for RNA-seq analysis in Fig. 6b  Raw data from independent experiments with n<5 can be found in the Statistical source data (Supplementary Table 6). Unprocessed immunoblots are provided in Supplementary Fig. 8.
All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.      qPCR. The value for the PCR product from U2932 was set as 1. A representative graph from two independent experiments is shown.     Supplementary Fig.  8, and source data for (f) and statistical source data for (h,i,j) and exact P values for (j) can be found in Supplementary Table 6. Unless otherwise noted, immunoblots are representative of three independent experiments.