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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.

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.