Epigenetic enzymes that add or remove histone post-translational modifications are essential for cellular identity and frequently mutated in cancer. For example, in B-cell lymphomas, change-of-function mutations in EZH2, the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), result in increased H3K27me3 levels and transcriptional silencing of key genes required for B-cell differentiation. However, the efficacy of EZH2 inhibitors in clinical trials has been limited by acquired resistance. Understanding the mechanisms of resistance to EZH2 inhibitors is crucial for improving treatment outcomes. Genome-wide CRISPR screens were performed in an EZH2-mutant B-cell lymphoma cell line treated with and without the FDA approved EZH2 inhibitor Tazemetostat. Flow cytometry-based GFP reporter assays using CRISPR sgRNAs, and viability assays using NSD2 PROTAC degraders, were employed to validate resistance mechanisms. Co-immunoprecipitation and mass spectrometry (IP-MS) of endogenous NSD2 was performed to identify potential protein interactors. Chromatin binding and histone post-translational modifications were mapped using quantitative CUT&RUN. RNA-seq was performed to identify transcriptional changes upon NSD2 loss. A genome-wide CRISPR screen of EZH2-mutant lymphomas revealed that loss of either NSD1 or NSD2 confers resistance to EZH2 inhibition. This was validated in independent cell competition assays on an extended cohort of B-cell lymphoma cell lines. Furthermore, PROTAC mediated degradation of NSD2 also renders EZH2-mutant lymphomas resistant to EZH2 inhibition. While endogenous IP-MS of NSD2 indicates it functions in isolation, its loss in EZH2-mutant B-cell lymphoma cells leads to genome-wide decreases in its H3K36me2 modification, as expected. These global decreases in H3K36me2 led to consequent increases in the genome-wide binding of PRC2. The consequent increased deposition of PRC2 H3K27 methylations led to focal reductions in H3K27ac at enhancer elements. Furthermore, NSD2 binding increased at active enhancer regions in EZH2 inhibitor treated cells. Taken together, our results indicate that loss of NSD2 in B-cell lymphoma confers resistance to EZH2 inhibitors by altering the balance of repressive and active histone post translational modifications at key regulatory elements in the genome. We report a novel mechanism by which loss of NSD2 confers resistance to EZH2 inhibitors in EZH2-mutant B-cell lymphoma. Loss of NSD2 leads to impaired activation of critical enhancers required for response to EZH2 inhibitors. Our results indicate that EZH2 inhibitors should not be used to treat cancers with loss of function mutations in NSD1 or NSD2 and indicate NSD2 has a role in regulating enhancer functions. Daniel Angelov, Darragh Nimmo, Gráinne Holland, Molly Davies, Eugene Dillon, Eric Conway, Elisabeth Vandenberghe, Adrian P. Bracken. Loss of NSD2 induces acquired resistance to EZH2 inhibitors in B-cell lymphoma by disrupting enhancer function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3805.

The catalytic subunit of Polycomb Repressive Complex 2 (PRC2), EZH2, is recurrently mutated in 25% of diffuse large B-cell lymphomas (DLBCL), causing increased H3K27me3 and decreased H3K27me2 levels. EZH2 inhibitors provide clinical benefit, but resistance frequently develops, highlighting the need for alternative therapeutic targets. Here, we identify the PRC2 accessory protein AEBP2 as a specific genetic dependency in EZH2-mutant DLBCL. While AEBP2 acts through PRC2, its essential role is surprisingly independent of canonical H3K27me3-mediated gene silencing. Instead, AEBP2 functions within a PRC2.2 complex lacking JARID2, using its zinc-finger domains to sample intergenic chromatin to sustain H3K27me2. Notably, loss of AEBP2 or NSD2 caused contrasting changes in intergenic H3K27me2 levels, driving sensitivity or resistance to PRC2 inhibitors, respectively. Our findings identify AEBP2-PRC2.2-maintained intergenic H3K27me2 as a therapeutic vulnerability in EZH2-mutant DLBCL and highlight dysregulated H3K27me2 as an underappreciated form of PRC2 dysfunction in cancer, with important therapeutic implications. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Polycomb Repressive Complex 2 (PRC2) is an essential chromatin regulator responsible for mono-, di- and tri-methylating H3K27. Control of PRC2 activity is a critical process in development and disease, yet no inhibitory cofactor has been identified in somatic cells. Here, we show that the alternative isoforms of its accessory subunit AEBP2, namely AEBP2S (short) and AEBP2L (long), perform opposite functions in modulating PRC2 activity. Contrary to prior assumptions that AEBP2 enhances PRC2 function, we find that the widely expressed AEBP2L isoform inhibits it. AEBP2L is expressed throughout embryogenesis and adulthood and inhibits PRC2 DNA binding, histone methyltransferase activity, and binding to target genes. In contrast, AEBP2S, expressed during early embryogenesis, promotes PRC2 DNA-binding activity and is essential for de novo repression of target genes during the transition from naïve to primed pluripotency. Mechanistically, through high-resolution cryo-EM and mutagenesis, we show that the recently evolved, negatively charged N-terminal region of AEBP2L inhibits PRC2. We propose a scenario in which the N-terminus of AEBP2L arose in vertebrates to restrain PRC2 activity in somatic cells.

Introduction EZH2 is the catalytic subunit of Polycomb Repressive Complex 2 (PRC2) and plays a central role in B-cell lymphopoiesis. EZH2 is an oncogene in B-cell lymphoma with recurrent change-of-function hotspot mutations causing decreased levels of di-methylation of H3K27 (H3K27me2), coupled with increased levels of H3K27me3. Although enzymatic inhibitors of EZH2 are entering the clinic for the treatment of selected patients with B-cell lymphoma, their utility is limited by disease progression due to acquired resistance. Overcoming acquired resistance to EZH2 inhibitors will assist in their application in the clinic and improve patient outcomes. Materials and Methods To explore the mechanistic basis of acqured resistance to EZH2 inhibitors, a genome-wide CRISPR screen was performed in B-cell lymphoma cell lines treated either with or without Tazemetostat, an FDA approved EZH2 inhibitor drug. An additional, more focussed CRISPR tiling screen was performed to identify the specific domains within EZH2 and its 10 associated PRC2 proteins that are functionally essential in B-cell lymphoma. Subsequent validation work using small guide RNAs (sgRNAs) and short hairpin RNAs (shRNAs) were employed to validate both novel genetic dependencies, such as AEBP2, and genes that conferred resistance, such as NSD2. Immunoprecipitation coupled with mass spectrometry (IP-MS) of endogenous PRC2 was performed to profile the forms and compositions of PRC2 in lymphoma cells. Chromatin Immunopreciptiation (ChIP-seq) and CUT&RUN-seq of histone post-translational modifications and PRC2 components, as well as RNA-seq, were performed to understand the mechanisms of action of AEBP2-PRC2.2 in lymphoma cells. Results The genome-wide CRISPR screen identified AEBP2 as a gene whose loss sensitises lymphoma cells to treatment with EZH2 inhibitor Tazemetostat, while NSD2 was identified as a gene whose loss confers resistance to Tazemetostat. Both NSD2 knockout and EZH2 inhibitor resistant mutant lymphoma cells were sensitive to AEBP2 depletion, indicating that AEBP2 is a candidate target for overcoming acquired resistance. A PRC2-tiling CRISPR screen identified AEBP2's Zinc finger domains, which interact with chromatin, and its PRC2 interaction domain as being vital for its function. IP-MS established that AEBP2 forms part of a novel form of PRC2.2 that lacks JARID2. In contrast with loss of EZH2, loss of AEBP2 leads to increased genome-wide levels of H3K27me3 with distinct effects on gene transcription. We explore these mechanisms, including an apparent role of AEBP2-PRC2.2 in promoting H3K27me2 deposition at intergenic regions. Conclusion We identified a novel AEBP2-containing PRC2.2 subcomplex, which lacks JARID2, as a genetic dependency in B-cell lymphoma. The loss of AEBP2-PRC2.2 overcomes acquired resistance to Tazemetostat. We propose a mechanism whereby loss of AEBP2-PRC2.2 results in genome-wide disruption of H3K27 methylation marks with adverse effects on cellular proliferation. These data indicate that pharmacological inhibitors or degraders of AEBP2 could be a promising strategy to treat germinal centre lymphomas and overcome resistance to EZH2 inhibitors.

Abstract Introduction The characterization of the mutational landscape of human cancers over the past 30 years has resulted in the rise of targeted cancer therapies. This era of precision oncology aims to enable cancer patients to receive therapies that are specific, efficacious, and result in fewer toxicities. A major challenge however is the emergence of drug resistance. Predicting resistance mechanisms using in vitro models can help to plan rational clinical trials using combination approaches and avoiding treating specific subtypes of cancers that are unlikely to respond. Here, we apply CRISPR screens in non-Hodgkin lymphoma (NHL) to predict resistance mechanisms and genetic synergies to EZH2 inhibitor (EZH2i) treatment. Materials and Methods Genome-wide CRISPR-Cas9 screens were performed in EZH2 ‘change-of-function’ mutant and EZH2 wild-type patient-derived NHL cell lines, treated with either EZH2i or vehicle control. Validatory cell competition assays were then performed using individual CRISPR sgRNAs targeting specific genes of interest, including AEBP2 and NSD2. These sgRNAs were tagged with a green fluorescence protein enabling the identification of the knock-out population using flow cytometry. An additional CRISPR screen using a PRC2 ‘tiled’ approach was also performed in the NHL cell lines to elucidate the essential domains within PRC2 proteins. Clonal knock out cells were generated using CRISPR and CUT&RUN experiments were performed for histone post-translational modifications in the knock-out cells to understand the mechanisms of resistance. Lastly, Quant-seq experiments were undertaken in the clonal knock out cells to characterise the transcriptional changes underlying the mechanism of resistance and sensitization observed with specific genetic knock outs. Results and Discussion We identified the NSD2 gene as a ‘resistor’ gene to EZH2i treatment, as sgRNAs targeting NSD2 were more abundant in the EZH2i treated cells compared to control cells. Conversely, the PRC2 accessory component AEBP2 was identified as a ‘sensitizer’ gene in this context. Cell competition assays using several independent sgRNAs to target the NSD2 and AEBP2 genes validated these findings. The EZH2i resistance phenotype was replicated using an orthogonal approach of pharmacological NSD2 degradation. These results point to the possibility of acquired loss of function mutations in NSD2 resulting in resistance to EZH2i in vivo. Finally, we explored the changes in transcriptional programs and H3K36 and H3K27 methylations across the genome. We propose a model for how NSD2 and AEBP2 loss confer resistance and sensitivity, respectively, to EZH2i in NHL cells. Conclusion We identified NSD2 loss as a potential mechanism by which NHL cells can acquire resistance to therapeutic EZH2i. We propose a potential solution of combining EZH2i treatment with targeted inhibition of AEBP2, thus preventing the occurrence of EZH2i resistance. Our work suggests that pharmacological inhibitors or degraders of AEBP2 should be prioritised for development. Citation Format: Daniel I Angelov, James Nolan, Grainne Holland, David Reck, Darragh Nimmo, Craig Monger, Elisabeth Vandenberghe, Adrian P Bracken. B-cell lymphoma CRISPR screens: NSD2 loss confers EZH2 inhibition resistance, while AEBP2 is a specific genetic dependency [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P10.

Raw data used to plot survival graphs

A 33-year-old Romanian woman presented with a one day history of "cola-coloured" urine with associated fevers. She reported generalised malaise, arthralgia and reduced appetite for 24 h. The patient had a history of beta-thalassemia trait. She was not taking any regularly prescribed or over-the-counter medications and had no known allergies. Scleral icterus was noted on examination. There was no pathological lymphadenopathy or organomegaly. Her laboratory investigations were as follows; haemoglobin 3.6 g/dL, mean corpuscular volume 99.2 fl, platelets 219 × 109/L, white cell count 13.4 × 109/L, reticulocytes 351 × 109/L, direct antiglobulin test – negative, total bilirubin 88 umol/L, C-reactive protein 100 mg/L, lactate dehydrogenase 1250 u/L (upper limit 240 u/L) and haptoglobin 0.41 umol/L.

Preeclampsia complicates up to 8% of pregnancies and is a leading cause of fetomaternal morbidity andmortality. Treatment options are limited, with supportive care and delivery of the placenta representing the cornerstone of current management strategies. Derangements in blood coagulation are wellrecognised in this disorder and appear to favour an increased risk of venous thromboembolism among affected women. This risk appears to be most significant in the postpartum period. The mechanisms underlying this increased thrombosis risk remain to be fully elucidated although increased expression of procoagulant factors, endothelial dysfunction, attenuation of endogenous anticoagulant activity and increased platelet activity have been implicated in the prothrombotic tendency. Preeclampsia is also occasionally complicated by life-threatening haemorrhagic events and current evidence suggests that in some severe manifestations of this disease a coagulopathy with a clinical bleeding tendency may be the predominant haemostatic abnormality. Identifying affected women at significant risk of thrombosis and managing the competing thrombotic and haemorrhagic risks continue to be a significant clinical challenge. Derangements in blood coagulation are also implicated in the pathogenesis of preeclampsia; however, the role of antiplatelet or anticoagulant drugs in the prevention and treatment of this disorder remains a source of considerable debate. In addition, the potential role of specific haemostatic markers as diagnostic or screening tools for preeclampsia has also yet to be determined. Further characterisation of the underlying molecular mechanisms would likely be of major translational relevance and could provide insights into the pathogenesis of this disease as well as the associated haemostatic dysfunction.

A 22-year-old female presented the Emergency Department on the 14th March 2020 with a 3 day history of cough, pyrexia, sore throat, chills and rigors. Dyspnoea, myalgia, anosmia did not feature at presentation. The patient had a history of early-stage unfavourable-risk, classical Hodgkin Lymphoma (HL) for which she was diagnosed in May 2017. She underwent two cycles of adriamycin/bleomycin/vinblastine/dacarbazine (ABVD) with escalation to bleomycin/etoposide/adriamycin/cyclophosphamide/vincristine/procarbazine/prednisolone (escBEACOPP) for four cycles due to a suboptimal response on interval positron emission tomography (PET) scan.

Introduction Smoldering Myeloma (SMM) is a heterogeneous asymptomatic stage between Monoclonal Gammopathy of Unknown Significance (MGUS) and Multiple Myeloma (MM). Risk of progression to MM is 10% in the first 5 years following diagnosis and greatly diminishes thereafter (Kyle et al NEJM 2007). Early intervention in SMM patients at high risk of progression extends survival (Mateos et al NEJM 2013). The IMWG 2014 Updated Criteria establish a subset of SMM patients who are at ultra-high risk for progression to MM within 2 years, and therefore merit treatment (Rajkumar et al Lancet Oncol 2014). Analyses of the genetic and molecular landscape of SMM to date report a near identical picture to MM, however most patients in these studies progress rapidly and are therefore not representative of the entire group. We performed a comprehensive analysis of SMM and MGUS patients to determine markers of high risk of progression that could identify patients to benefit from early treatment Methods MGUS not progressing after at least 10 years of follow up and SMM in which follow up data were available were extracted from the Plasma Cell Dyscrasia Biobank. A clinically applicable Custom Capture MM-specific sequencing platform was developed for detection of the most frequently mutated pathways in MM based on analysis of CoMMpass dataset. Coding exons of actionable genes, clinically relevant copy number abnormalities, and regions surrounding IgH (0.5Mb), IgK (0.1Mb), IgL (0.1Mb) and MYC loci (1.6Mb) to identify relevant structural variants (SVs) were included, with combined design 2.2Mb. 12 samples were pre-pooled before capture, and 2 captures were sequenced per lane of Illumina HiSeq4000. Paired-end 150bp reads were mapped to hg19 using BWA-MEM. Single nucleotide variants (SNVs) and small INDELs in capture regions were identified using the GenomeGPS analytic pipeline following Broad GATK variant discovery practices. Copy number variants (CNVs) were identified by patternCNV. SVs of translocations, inversions, large INDELs, and segmental duplications were called by the SnowShoes-SV algorithm developed in-house. False positive SVs, polymorphic SVs, and other artefacts were filtered out using in-house normal SV database. Results We identified and sequenced 128 patients including 32 MGUS patients not progressing after 10 years. Of 96 SMM patients included 36 had not progressed to SMM after minimum follow up of 5 years, while 37 and 23 progressed to MM in less than 2 years and between 2-5 years, respectively. The genetic subtype of each patient was determined and verified by clinical FISH. Proportions in each genetic subgroup in MM and SMM/MGUS were similar, indicating that these are primary genetic lesions occurring early in MM pathogenesis. Median SMM time to progression (TTP) was 46 months. As in other series, HRD with IGH translocation, and t(4;14) predicted shorter TTP. Analysis of CoMMpass dataset found frequent MYC SV (38%) in untreated MM with higher frequency in HRD versus NHRD MM: 53% versus 28% (Misund, ASH 2016). No MYC SV were detected in MGUS cohort, SMM non-progressors at >5 years or SMM progressing between 2-5 years. By contrast, MYC SV were detected in 49% SMM that progressed within 2 years, 55% in HRD and 41% NHRD. SMM with MYC SV had a significantly shorter median TTP compared to patients without MYC SV (11.5 months vs 61 month; p /=60% and FLC ratio 100) confirm MYC SV as an independent variable for progression to MM (hazard ratio=7, 95% confidence interval 3.6-13.7, p=0.00001). RAS and NFKB pathway mutations were observed with similar frequencies in MM and SMM progressing within 5 years of diagnosis, but with lower frequency in those not progressing by 5 years follow up, and were not observed in the MGUS cohort. A trend toward shorter TTP was observed in patients with RAS pathway mutations but did not reach statistical significance. Conclusion In conclusion, we describe MYC translocations as a genetic marker of and likely cause of progression to MM that are absent in MGUS and SMM with TTP >2 years. In contrast MM and SMM early progressors (TTP Download : Download high-res image (98KB) Download : Download full-size image Figure . Disclosures Kumar: Skyline: Honoraria; Celgene, Millennium/Takeda, Onyx, AbbVie, Janssen, Sanofi, Novartis, Amgen, Genentech, Merck, Oncopeptides, Roche, Skyline Diagnostics: Research Funding; Celgene, Millennium, BMS, Onyx, Janssen, Noxxon, AbbVie, Amgen, Merck, Oncopeptides, Skyline Diagnostics, Takeda: Consultancy. Dispenzieri: Celgene, Millenium, Pfizer, Janssen: Research Funding. Fonseca: Novartis: Consultancy; Merck: Consultancy; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; AMGEN: Consultancy; Celgene Corporation: Consultancy, Research Funding; Sanofi: Consultancy; Mayo Clinic & Dr Fonseca: Patents & Royalties: Prognostication of myeloma via FISH, ~$2000/year; Jansen: Consultancy; Bristol-Myers Squibb: Consultancy; Bayer: Consultancy; Pharmacyclics: Consultancy; Takeda: Consultancy. Stewart: Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Roche: Consultancy; Amgen: Consultancy.

Elevated platelet counts are frequently encountered in hospital medicine and arise from both physiological and pathological mechanisms. Thrombocytosis may be secondary, reflecting an inflammatory state, iron deficiency, recent surgery or point towards an underlying neoplasm. Thrombocytosis may be the presenting sign of solid tumours and haematological conditions. The discovery of the activating mutations affecting thrombopoiesis led to greater understanding of the pathobiology of essential thrombocythaemia and other myeloproliferative neoplasms. The investigation of suspected primary thrombocytosis has evolved to include testing for these disease-associated mutations. Therapy for patients with essential thrombocythaemia aims to reduce their risk of thrombotic complications by addressing cardiovascular risk factors, and using antiplatelet agents and, in selected patients, cytoreductive therapy. This article provides a logical approach to distinguishing reactive or secondary thrombocytosis from thrombocytosis associated with an underlying myeloproliferative neoplasm and gives an overview of the management of essential thrombocythaemia.

N ‐Methyl‐3‐phenyl‐norbornan‐2‐amine ( N ‐methyl‐3‐phenylbicyclo[2.2.1]heptan‐2‐amine, Camfetamine ™ ) is available from a number of online legal highs/research chemicals' vendors. Although it was developed as an analeptic by Merck in the early 1960s, it was never commercialized. However, the Association of Independent Research Chemical Retailers (AIRCR), an umbrella organization for a number of online vendors, has redeveloped it for use as a recreational drug. N ‐Methyl‐3‐phenyl‐norbornan‐2‐amine is closely related to fencamfamine which has been widely used as a central nervous system (CNS) stimulant and appetite suppressant. In this paper we describe the synthesis of N ‐methyl‐3‐phenyl‐norbornan‐2‐amine, its characterization and interpretations of its electron impact, and electrospray ionization mass spectra. Copyright © 2012 John Wiley & Sons, Ltd.