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Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common form of malignant lymphoma and may arise de novo, or through transformation from a pre-existing low-grade B cell lymphoma such as follicular lymphoma (FL). However, the post-translational mechanisms and deregulated pathways underlying the pathogenesis of disease evolution are not fully understood. Methods: We employed integrated functional and structural genomics and mass spectrometry (MS)-driven proteomics which implicated a possible novel tumor suppressor role for a conserved E3 ubiquitin ligase FBXO45 in DLBCL pathogenesis. We generated conditional knockout mice targeting loss of Fbxo45 in germinal center (GC) B-cells using the Cg1-Cre-loxP system and an assortment of CRISPR-mediated knockouts of FBXO45 in B cell lymphoma cells (FL518, BJAB, U2932). We engineered B cell lines (BJAB, U2932) to inducibly express FLAG-tagged FBXO45 to identify candidate substrates of FBXO45 using liquid chromatography-tandem MS. In vitro biochemical and in vivo studies using a variety of genetically-modified lines in xenograft studies in immunodeficient mice were performed to validate observations from proteogenomic studies. Whole genome sequencing (WGS) and genomic copy number studies were interrogated to investigate structural alterations targeting FBXO45 in primary human lymphoma samples. Results: Conditional targeting of Fbxo45 in GCB-cells in transgenic mice resulted in abnormal germinal center formation with increased number and size of germinal centers. Strikingly, targeted deletion of Fbxo45 in GCB-cells resulted in spontaneous B cell lymphomas with (22/22);100%) penetrance and none of the wild-type (WT) littermates (0/20; 0%) developed lymphoma at 24 months. Macroscopic examination revealed large tumor masses, splenomegaly, and lymphadenopathy at different anatomic locations including ileocecal junction, mesenteric, retroperitoneal and cervical lymph nodes and thymus. Next generation sequencing of immunoglobulin heavy chain genes revealed monoclonal or oligoclonal B cell populations. Using proteomic analysis of affinity-purified FBXO45-immunocomplexes and differential whole proteome analysis from GCB-cells of Fbxo45 wt/wt vs Fbxo45 fl/fl mice, we discovered that FBXO45 targets the RHO guanine exchange factor GEF-H1 for ubiquitin-mediated proteasomal degradation. FBXO45 exclusively interacts with GEF H1 among 8 F-box proteins investigated and silencing of FBXO45 using three independent shRNA and CRISPR-Cas9-mediated knockouts in B-cell lymphoma cell lines promotes RHOA and MAPK activation, B cell growth and enhances proliferation. GEF-H1 is stabilized by FBXO45 depletion and GEF-H1 ubiquitination by FBXO45 requires phosphorylation of GEF-H1. Importantly, FBXO45 depletion and expression of a GEF-H1 mutant that is unable to bind FBXO45 results in GEF-H1 stabilization, promotes hyperactivated RHO and MAPK signaling and B-cell oncogenicity in vitro and in vivo. Notably, this phenotype is reverted by co-silencing of GEF-H1. Inducible ectopic expression of FBXO45 triggers accelerated turnover of GEF H1 and decreased RHOA signaling. Genomic analyses revealed recurrent loss targeting FBXO45 in transformed DLBCL (25%), de novo DLBCL (6.6%) and FL (2.3%). In keeping with our observation of prolonged hyperactivation of pERK1/2 consequent to FBXO45 ablation, in vitro and in vivo studies using B-cell lymphoma cell lines and xenografts demonstrated increased sensitivity to pharmacologic blockade with the MAP2K1/2 (ERK1/2) inhibitor Trametinib. Conclusions: Our findings define a novel FBXO45-GEF-H1-MAPK signalling axis, which plays an important role in DLBCL pathogenesis. Our studies carry implications for potential exploitation of this pathway for targeted therapies. Disclosures Siebert: AstraZeneca: Speakers Bureau. Lim: EUSA Pharma: Honoraria.

The comprehensive genetic alterations underlying the pathogenesis of T-cell prolymphocytic leukemia (T-PLL) are unknown. To address this, we performed whole-genome sequencing (WGS), whole-exome sequencing (WES), high-resolution copy-number analysis, and Sanger resequencing of a large cohort of T-PLL. WGS and WES identified novel mutations in recurrently altered genes not previously implicated in T-PLL including EZH2, FBXW10, and CHEK2. Strikingly, WGS and/or WES showed largely mutually exclusive mutations affecting IL2RG, JAK1, JAK3, or STAT5B in 38 of 50 T-PLL genomes (76.0%). Notably, gain-of-function IL2RG mutations are novel and have not been reported in any form of cancer. Further, high-frequency mutations in STAT5B have not been previously reported in T-PLL. Functionally, IL2RG-JAK1-JAK3-STAT5B mutations led to signal transducer and activator of transcription 5 (STAT5) hyperactivation, transformed Ba/F3 cells resulting in cytokine-independent growth, and/or enhanced colony formation in Jurkat T cells. Importantly, primary T-PLL cells exhibited constitutive activation of STAT5, and targeted pharmacologic inhibition of STAT5 with pimozide induced apoptosis in primary T-PLL cells. These results for the first time provide a portrait of the mutational landscape of T-PLL and implicate deregulation of DNA repair and epigenetic modulators as well as high-frequency mutational activation of the IL2RG-JAK1-JAK3-STAT5B axis in the pathogenesis of T-PLL. These findings offer opportunities for novel targeted therapies in this aggressive leukemia.

EZH2 (enhancer of zeste homolog 2) is a critical enzymatic subunit of the polycomb repressive complex 2 (PRC2), which trimethylates histone H3 (H3K27) to mediate gene repression. Somatic mutations, overexpression and hyperactivation of EZH2 have been implicated in the pathogenesis of several forms of cancer. In particular, recurrent gain-of-function mutations targeting EZH2 Y641 occur most frequently in follicular lymphoma and aggressive diffuse large B-cell lymphoma and are associated with H3K27me3 hyperactivation, which contributes to lymphoma pathogenesis. However, the post-translational mechanisms of EZH2 regulation are not completely understood. Here we show that EZH2 is a novel interactor and substrate of the SCF E3 ubiquitin ligase β-TrCP (FBXW1). β-TrCP ubiquitinates EZH2 and Jak2-mediated phosphorylation on Y641 directs β-TrCP-mediated EZH2 degradation. RNA interference-mediated silencing of β-TrCP or inhibition of Jak2 results in EZH2 stabilization with attendant increase in H3K27 trimethylation activity. Importantly, the EZH2(Y641) mutants recurrently implicated in lymphoma pathogenesis are unable to bind β-TrCP. Further, endogenous EZH2(Y641) mutants in lymphoma cells exhibit increased EZH2 stability and H3K27me3 hyperactivity. Our studies demonstrate that β-TrCP has an important role in controlling H3K27 trimethylation activity and lymphoma pathogenesis by targeting EZH2 for degradation.

Sézary syndrome (SS) is an aggressive leukaemia of mature T cells with poor prognosis and limited options for targeted therapies. The comprehensive genetic alterations underlying the pathogenesis of SS are unknown. Here we integrate whole-genome sequencing (n=6), whole-exome sequencing (n=66) and array comparative genomic hybridization-based copy-number analysis (n=80) of primary SS samples. We identify previously unknown recurrent loss-of-function aberrations targeting members of the chromatin remodelling/histone modification and trithorax families, including ARID1A in which functional loss from nonsense and frameshift mutations and/or targeted deletions is observed in 40.3% of SS genomes. We also identify recurrent gain-of-function mutations targeting PLCG1 (9%) and JAK1, JAK3, STAT3 and STAT5B (JAK/STAT total ∼11%). Functional studies reveal sensitivity of JAK1-mutated primary SS cells to JAK inhibitor treatment. These results highlight the complex genomic landscape of SS and a role for inhibition of JAK/STAT pathways for the treatment of SS., Sézary syndrome is a T cell malignancy that has been poorly characterized at the genome level. In this study, Kiel et al. perform whole-genome analyses and identify mutations in the JAK–STAT pathway and show that primary cells are sensitive to JAK inhibitors.

Prostate apoptosis response protein 4 (Par-4) also known as PRKC apoptosis WT1 regulator is a tumor suppressor that selectively induces apoptosis in cancer cells. However, its post-translational regulation by ubiquitin-mediated proteolysis and the cellular machinery that is responsible for its proteasomal degradation are unknown. Using immunopurification and an unbiased mass spectrometry-based approach, we show that Par-4 interacts with the SPRY-domain containing E3 ubiquitin ligase Fbxo45 through a short consensus sequence motif. Fbxo45 interacts with Par-4 in the cytoplasm and mediates its ubiquitylation and proteasomal degradation. Fbxo45 silencing results in stabilization of Par-4 with increased apoptosis. Importantly, a Par-4 mutant that is unable to bind Fbxo45 is stabilized and further enhances staurosporine-induced apoptosis. Co-expression of Fbxo45 with Par-4 protects cancer cells against Par-4-induced apoptosis. Our studies reveal that Fbxo45 is the substrate-receptor subunit of a functional E3 ligase for Par-4 that has a critical role in cancer cell survival.

A conserved aspartic acid residue in the third transmembrane region of many of the G protein-coupled receptors has been shown to play a role in ligand binding. In the case of endothelin receptors, however, a lysine residue replaces this conserved aspartic acid residue. To access the importance of this residue in ligand binding, we have replaced it with an aspartic acid in the rat endothelin type B (ETb) receptor by PCR mediated mutagenesis. The binding characteristics and functional properties of both the wild type and mutant receptors were determined in COS-7 cells transiently expressing the cloned receptor cDNAs. Using 125I-ET-1 as the radioactive peptide ligand in displacement binding studies, the wild type receptor displayed a typical non-isopeptide-selective binding profile with similar IC50 values (0.2-0.6 nM) for all three endothelin peptides (ET-1, ET-2, and ET-3) and sarafotoxin 6c (SRTX 6c). Interestingly, the mutant receptor showed an increase in IC50 values for ET-1 (5 nM), ET-2 (27 nM), and ET-3 (127 nM) but displayed a much larger increase in IC50 value for SRTX 6c (> 10 uM). The lysine mutant receptor still elicited full inositol phosphate (IP) turnover responses in the presence of saturating concentrations of endothelins (10 nM of ET-1, 100 nM of ET-2, or 1 uM of ET-3), indicating that the mutation (K181D) did not affect the coupling of mutant receptor to the appropriate G protein. These results demonstrate that lysine-181 on the receptor is important for binding ET peptides; however, it is required for binding the ETb selective agonist-SRTX 6c.

The GABA(B) receptor is a member of the "family 3" G protein coupled receptors. The GABA(B) receptors modulate activity inwardly rectifying potassium channels and high voltage activated calcium channels. The GABA(B) receptors require heterodimerization between two subunits, GABA(B1) and GABA(B2), for functional expression. A robust functional calcium cell line was developed that contained both the human truncated GABA(B(1b)) and human truncated GABA(B(2)) receptors. The cell line was analyzed and sorted using beta-lactamase as a reporter. Single cell clones were sorted and isolated using flow cytometry based on high beta-lactamase expression. The single cell clones were further tested in a 384-well calcium mobilization assay using the Fluo-4 AM calcium indicator on the fluorescent imaging plate reader system (FLIPR). Twenty-seven clones were grown up from single cell collections and 10 clones demonstrated a high response to GABA stimulation. The 10 clones were re-evaluated based on agonist dose response and EC(50). Clone-16 was identified and utilized in high throughput screening (HTS) assay development. Using sorting and beta-lactamase as a reporter, we were able to develop a robust, functional cell-based, GABA(B), calcium mobilization assay. The cell line described here can be used for high throughput FLIPR screening and also to compare and rank the potency and selectivity of agonists, antagonists and potentiators of the GABA(B) receptor.

Introduction: Enhancer of zeste homologue 2 (EZH2) is a critical enzymatic subunit of the polycomb repressive complex 2 (PRC2) which trimethylates histone H3 (H3K27) to mediate gene repression. EZH2 is aberrantly activated and overexpressed in several hematologic malignancies and is associated with aggressive clinical behavior. In particular, recurrent mutations targeting Y641 of EZH2 are common in germinal center derived lymphomas. Critically, the cellular machinery and mechanisms that regulate EZH2 by post-translational modification are not well understood. We have previously shown that β-transducin repeat containing protein (βTrCP) is the substrate specific adaptor for ubiquitin mediated degradation of EZH2, however the degron motif by which βTrCP recognizes EZH2 is unknown. In order to better understand the post-translational regulation of EZH2, we sought to identify the non-canonical degron motif that regulates EZH2 stability. Methods: βTrCP recognizes a phosphorylated consensus degron motif DpSG(X)2-5pS in its substrates. To investigate the post-translational modifications regulating the half-life of EZH2 protein we generated a series of truncation and site directed mutants of βTrCP including deletion of 7th WD repeat domain and mutation of R474A. The afore mentioned βTrCP mutants were assessed for their interaction with EZH2. Having established the non-canonical degron (DpS601KNVpS605) in EZH2, to further characterize the role of this degron in EZH2-βTrCP interaction we generated several EZH2 mutants including site-directed deletion of putative degron motif (DSKNVS) or alanine substitution mutations of critical serine residues S601A and S605A alone or in combination. Using wild-type as well as EZH2 mutants we investigated their ability to interact with βTrCP and consequent lysine (K48) linked polyubiquitination on EZH2 using co-immunoprecipitation and western blotting techniques. Further we characterized the impact of the identified degron in EZH2 stability using cycloheximide chase mediated protein turnover analysis of wild type and EZH2 degron deletion and serine (S601A/S605A) substitution mutants. Moreover, we investigated the impact of increased stability of EZH2 on its H3K27 trimethylation activity using wild type and degron deletion and serine substitution mutants by western blot analysis. We also investigated the impact of increased stability of EZH2 via degron modification by analyzing its downstream target p21. Results: Extensive interaction between substrates and R474 and Y488 residues in the 7th WD repeat domain of βTrCP is critical for stable binding and this is mediated via the conserved degron motif. In co-immunoprecipitation experiments, the mutation of R474A or deletion of 7th WD repeat domain in βTrCP abrogated the EZH2-βTrCP interaction. Further, the deletion of the identified degron DSKNVS in EZH2 or substitution of critical serine residues residing within this degron abrogated EZH2-βTrCP interaction and consequent lysine K48-linked polyubiquitination and proteasomal degradation. Half-life measurements by cycloheximide chase experiments demonstrated that the degron deletion or substitution mutations exhibit increased protein stability as compared to wild type EZH2. Motif analysis revealed that the novel EZH2 degron harbors the GSK3β recognition and phosphorylation motif (DpSKNVpS) involved in the phosphorylation of several known βTrCP substrates. Therefore, we examined the involvement of GSK3β in EZH2-βTrCP interaction. Pharmacologic inhibition of GSK3β compromised EZH2- βTrCP interaction in a dose dependent manner. Further, deletion of the degron or alanine substitution mutation of the critical serine residues (S601A and S605A) within the degron increased H3K27 trimethylation activity of EZH2 and consequently increased its repression on its downstream target p21. Conclusion: In the present study, we identify the presence of a novel non-canonical degron and GSK3β-mediated phosphorylation of the site that regulates EZH2 stability and activity. Our studies demonstrate that βTrCP/GSK3β axis plays an important role in controlling H3K27 trimethylation activity by targeting EZH2 for degradation. We propose that this newly identified mechanism might help in designing novel therapeutic approaches for clinical management of hematologic malignancies driven by aberrant activity of EZH2. Disclosures No relevant conflicts of interest to declare.

Introduction Sezary Syndrome (SS) is an aggressive mature T-cell neoplasm characterized by erythroderma, generalized lymphadenopathy and circulating T-cells. SS cells are characterized by complex karyotypes with numerous structural alterations and copy-number variants. Previous studies have implicated disruption and/or haploinsufficiency of TP53, CDKN2A, RB1 and PTEN in SS. Nevertheless, the comprehensive genetic alterations underlying the pathogenesis of SS are unknown. In this study, we used an integrated genomic sequencing approach to characterize the genetic basis of SS. Methods SS cases that fulfilled established diagnostic criteria including characteristic cytologic, immunophenotypic and karyotypic features were analyzed. We performed genomic analysis on 84 primary SS cases using whole genome sequencing (WGS; n = 6), high-resolution array comparative genome hybridization (aCGH using NimbleGen 270K Feature Array; n = 80) and whole exome sequencing (WES) using Illumina TruSeq Exome Capture (n = 66). We filtered this combined dataset to identify 463 genes within regions of recurrent copy-number variations that were also impacted by protein coding mutations. We focused on 16 of these genes and confirmed 75 mutations in these genes by Sanger sequencing. For 35 of these mutations, normal tissue was available to confirm somatic acquisition. Results WGS revealed a complex structural genome with numerous regions of chromothrypsis and aneuploidy with over 1,000 inter- and intra-chromosomal translocations in the 6 SS genomes but did not reveal recurrent structural alterations. Array CGH identified many aneuploidies reported previously including 8+, 10-, 17p-/iso17 and 19q-. These results were corroborated by aCGH analysis which also revealed recurrent genomic losses at 9p21.3 (CDKN2A), 10p11.22 (PTEN) and 13q14.2 (RB1). Importantly, exome sequencing further identified several novel somatic, deleterious mutations in these three genes. Strikingly, in addition to confirming loss of function alterations of genes previously implicated in SS pathogenesis, we identified genetic alterations in genes involved in epigenetic modification including chromatin/histone modification (including ARID1A, ARID5B, MLL, MLL2, MLL4, SETD1A, SETD1B, SETD6) and DNA methylation (including NCOR1, TET1, TET2, DNMT3A, DNMT3B) not previously implicated in SS pathogenesis. Among the most frequent and most restricted regions of recurrent deletion was the 1p36.11 region. Of the 18 genes within this region, ARID1A showed the highest frequency of disruption (27/80 showing deletion; 7/66 showing novel mutations including a frameshift mutation at residue 1449 within the GR binding domain of ARID1A). Of the SS genomes for which both WES and aCGH analysis were performed, 25/62 (40.3%) showed disruption of ARID1A either by deletion or mutation or both. Moreover ARID5B was also targeted by deletions of region 10q21.2 in 23/80 genomes and 3/66 SS genomes showed mutations. We also identified a narrow region of recurrent aneuploidy affecting the SWI/SNF family-member of SMARC genes known to interact with ARID-family SMARCC1at 3p21.31. This gene was recurrently targeted by a deletion in 17/80 (21.3%) SS genomes by highly restricted deletional events ( Altogether, deletions and/or mutations in ARID1A, ARID5B or SMARCC1 were identified in 38 of 62 (61.3%) of SS genomes. Further analysis revealed that other members of the ARID- and SMARC-families, ARID1B, ARID2 and ARID4A, were mutated. Moreover, ARID3A and SMARCA4 (both located on 19p) were targeted by recurrent deletions in 29/80 and 18/80 SS genomes, respectively. Altogether, deletions and/or mutations in at least one of the ARID- or SMARC-family genes were identified in 91.9% of SS. Conclusion This study is the first to comprehensively profile the genomic landscape in SS. Integration of high-resolution aCGH with mutational analysis reveals alterations in epigenetic regulators through loss-of-function lesions in ARID- and SMARC-family genes in over 90% of SS. The results of our study offer further insights into the pathogenesis of SS and provide support for use of drugs targeting deregulated chromatin remodeling pathways in the treatment of SS patients. Disclosures No relevant conflicts of interest to declare.

EZH2 is a critical enzymatic subunit of the PRC2 which trimethylates histone H3 (H3K27) to mediate gene repression. While overexpression of EZH2 and its increased H3K27 methylation have generally been associated with hematologic malignancies and solid tumors, recurrent gain of function somatic mutations in EZH2 have been recently reported in germinal center B-cell (GCB) derived lymphomas. These mutations, which replace a single tyrosine in the SET domain of the EZH2 protein (Y641), occur in 7.2% of FLs (follicular lymphoma) and 21.7% of GCB DLBCL (diffuse large B-cell lymphoma) and are absent from ABC DLBCLs. Consequently, B-cell lymphoma cell lines and lymphoma primary samples harboring heterozygous EZH2Y641mutations exhibit increased levels of histone H3 Lys27 trimethylation (H3K27me3) and promote a lymphoproliferative phenotype with expansion of the GC B cell compartment. Although extensive studies have been conducted to understand the role of polycomb group of proteins in cancer pathogenesis, few studies have been aimed at understanding the post-translational regulation of these proteins. Skp1/Cullin1/F- box protein (SCF) ubiquitin ligase complexes are the largest family of multicomponent ring E3 ubiquitin ligases that control the degradation of many important regulatory proteins and have been implicated in the regulation of epigenetic regulators. However, the mechanism of EZH2 degradation and its implication in PRC2 mediated methyl transferase activity in a cancer-specific context is not fully understood. Cullin-ring ubiquitin ligases (CRLs) form the largest known class of multicomponent E3 ubiqutin ligase family in eukaryotes and target a wide array of substrates involved in several biological processes. To investigate a CRL-mediated basis for post-translational EZH2 regulation, we screened a panel of cullin family E3 ligases for interaction with EZH2 and discovered that EZH2 was immunoprecipitated by cullin1 exclusively among all other cullins (cul2,3,4a,4b,5 and 7). We engineered and expressed a cullin1 dominant negative protein which resulted in EZH2 accumulation in a dose dependent manner. Using co-immunoprecipitation approach, we screened 8 different F box proteins and observed that SCF E3 ubiquitin ligase β-TrCP (FBXW1) uniquely and specifically interacted with EZH2. Further, RNAi-mediated silencing of β-TrCP resulted in EZH2 stabilization with attendant increase in H3K27 trimethylation activity. Importantly, the Y641 mutants recurrently identified and relevant in lymphoma pathogenesis were unable to bind β-TrCP. Further, cycloheximide chase experiments showed that Y641 mutants endogenously expressed in lymphoma-derived cells exhibit increased EZH2 stability as well as enhanced H3K27 trimethylation activity. Our studies demonstrate that β-TrCP plays an important role in controlling H3K27 trimethylation activity via an unusual recognition mechanism targeting EZH2 for proteasomal degradation. Our findings delineate a novel β-TrCP/EZH2 axis requiring an intact Y641 residue for EZH2 regulation by β-TrCP. Oncogenic mutations in Y641 in GCB-cell derived lymphomas confer increased stability to EZH2 and render it resistant to β-TrCP-mediated degradation. This newly identified mechanism might help in design potential novel therapeutic approaches for clinical management of cancer potentially driven by aberrant accumulation/function of EZH2. Disclosures: No relevant conflicts of interest to declare.

In the G protein-coupled receptor family, a highly conserved aspartic acid located within the third transmembrane domain has been shown to be involved in ligand binding. Within the endothelin (ET) peptide receptor family, this aspartic acid has been replaced by a lysine. To assess the importance of this residue in ET binding, the lysine (position 181) of rat ET type B receptor was replaced by an aspartic acid. The effects on ligand binding and phosphoinositide turnover of both the wild-type and K181D mutant receptors were examined using transient receptor expression in COS-7 cells. Using [125I]ET-1 as the radioactive peptide ligand in displacement binding studies, the wild-type receptor displayed a typical non-isopeptide-selective binding profile with similar IC50 values (0.2-0.6 nM) for all three ET peptides (ET-1, ET-2, and ET-3). The mutant receptor showed an increase in IC50 values for ET-1 (5 nM), ET-2 (27 nM), and ET-3 (127 nM). The K181D mutant receptor still elicited full inositol phosphate (IP) accumulation responses in the presence of saturating concentrations of ETs (10 nM of ET-1, 100 nM of ET-2, or 1 microM of ET-3), indicating that the mutation did not affect G protein coupling.