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Diffuse large B-cell lymphoma (DLBCL) is the most common non- Hodgkin lymphoma and is characterized by a remarkable heterogeneity with diverse variants that can be identified histologically and molecularly. Large-scale gene expression profiling studies have identified the germinal center B-cell (GCB-) and activated B-cell (ABC-) subtypes. Standard chemo-immunotherapy remains standard front-line therapy, curing approximately two thirds of patients. Patients with refractory disease or those who relapse after salvage treatment have an overall poor prognosis highlighting the need for novel therapeutic strategies. Transducin b-like protein 1 (TBL1) is an exchange adaptor protein encoded by the TBL1X gene and known to function as a master regulator of the Wnt signaling pathway by binding to β-CATENIN and promoting its downstream transcriptional program. Here, we show that, unlike normal B cells, DLBCL cells express abundant levels of TBL1 and its overexpression correlates with poor clinical outcome regardless of DLBCL molecular subtype. Genetic deletion of TBL1 and pharmacological approach using tegavivint, a first-in-class small molecule targeting TBL1 (Iterion Therapeutics), promotes DLBCL cell death in vitro and in vivo. Through an integrated genomic, biochemical, and pharmacologic analyses, we characterized a novel, β-CATENIN independent, post-transcriptional oncogenic function of TBL1 in DLBCL where TBL1 modulates the stability of key oncogenic proteins such as PLK1, MYC, and the autophagy regulatory protein BECLIN-1 through its interaction with a SKP1-CUL1-F-box (SCF) protein supercomplex. Collectively, our data provide the rationale for targeting TBL1 as a novel therapeutic strategy in DLBCL.

Maladaptive perfectionism and controlled motivation are vulnerability factors for burnout. This study examined the relationships between two aspects of perfectionism (high standards, discrepancy), four academic motivational orientations (intrinsic, identified, introjected, extrinsic), and academic burnout. The target population was 12th graders in South Korea, and a total of 950 participants were recruited using cluster sampling. Data were collected from three waves of longitudinal study. In particular, the mediating role of academic motivation (T2) in the link between perfectionism (T1) and academic burnout (T3) was tested using structural equation modelling. The results indicated that introjected motivation mediated the relationship between perfectionism and burnout. Specifically, both high standards and discrepancy were positively associated with introjected motivation, and in turn, introjected motivation was positively associated with burnout. The theoretical and practical implications of these findings are discussed.

Protein arginine methyltransferase-5 (PRMT5) is overexpressed in aggressive B-cell non-Hodgkin's lymphomas, including mantle cell lymphoma and diffuse large B-cell lymphoma, and supports constitutive expression of CYCLIN D1 and c-MYC. Here, we combined ChIP analysis with next-generation sequencing to identify microRNA (miRNA) genes that are targeted by PRMT5 in aggressive lymphoma cell lines. We identified enrichment of histone 3 dimethylation at Arg-8 (H3(Me2)R8) in the promoter regions of miR33b, miR96, and miR503. PRMT5 knockdown de-repressed transcription of all three miRNAs, accompanied by loss of recruitment of epigenetic repressor complexes containing PRMT5 and either histone deacetylase 2 (HDAC2) or HDAC3, enhanced binding of co-activator complexes containing p300 or CREB-binding protein (CBP), and increased acetylation of specific histones, including H2BK12, H3K9, H3K14, and H4K8 at the miRNA promoters. Re-expression of individual miRNAs in B-cell lymphoma cells down-regulated expression of PRMT5, CYCLIN D1, and c-MYC, which are all predicted targets of these miRNAs, and reduced lymphoma cell survival. Luciferase reporter assays with WT and mutant 3′UTRs of CYCLIN D1 and c-MYC mRNAs revealed that binding sites for miR33b, miR96, and miR503 are critical for translational regulation of the transcripts of these two genes. Our findings link altered PRMT5 expression to transcriptional silencing of tumor-suppressing miRNAs in lymphoma cells and reinforce PRMT5's relevance for promoting lymphoma cell growth and survival.

Background Although case reports have suggested that the erector spinae plane block (ESPB) may help analgesia for patients after breast surgery, no study to date has assessed its effectiveness. This retrospective observational study analyzed the analgesic effects of the ESPB after total mastectomy. Methods Forty-eight patients were divided into an ESPB group (n = 20) and a control group (n = 28). Twenty patients in the control group were selected by their propensity score matching the twenty patients in the ESPB group. Patients in the ESPB group were injected with 30 mL 0.375% ropivacaine, followed by catheter insertion for further injections of local anesthetics every 12 hours. Primarily, total fentanyl consumption was compared between the two groups during the first 24 hours postoperatively. Secondary outcomes included pain intensity levels (visual analogue scale) and incidence of postoperative nausea and vomiting (PONV). Results Median cumulative fentanyl consumption during the first 24 hours was significantly lower in the ESPB (33.0 μg; interquartile range [IQR], 27.0–69.5 μg) than in the control group (92.8 μg; IQR, 40.0–155.0 μg) (P = 0.004). Pain level in the early postoperative stage (

Epigenetic regulation by the type II protein arginine methyltransferase, PRMT5, plays an essential role in the control of cancer cell proliferation and tumorigenesis. In this report, we investigate the relationship between PRMT5 and WNT/B- CATENIN as well as AKT/GSK3B proliferative signaling in three different types of non-Hodgkin's lymphoma cell lines, clinical samples, and mouse primary lymphoma cells. We show that PRMT5 stimulates WNT/B-CATENIN signaling through direct epigenetic silencing of pathway antagonists, AXIN2 and WIF1, and indirect activation of AKT/GSK3 signaling.PRMT5 inhibition with either shRNA-mediated knockdown or a specific small molecule PRMT5 inhibitor, CMP-5, not only leads to derepression of WNT antagonists and decreased levels of active phospho-AKT (Thr-450 and Ser-473) and inactive phospho- GSK3B (Ser-9) but also results in decreased transcription of WNT/B-CATENIN target genes, CYCLIN D1, c-MYC, and SURVIVIN, and enhanced lymphoma cell death. Furthermore, PRMT5inhibition leads to reduced recruitment of co-activators CBP, p300, and MLL1, as well as enhanced recruitment of co-repressors HDAC2 and LSD1 to the WNT/B-CATENIN target gene promoters. These results indicate that PRMT5 governs expression of prosurvival genes by promoting WNT/B- CATENIN and AKT/GSK3 proliferative signaling and that its inhibition induces lymphoma cell death, which warrants further clinical evaluation. This work was supported by Leukemia and Lymphoma Society Award MCL7001-18 (to R. A. B.) and National Priorities Research Program Grant NPRP8-617-3-131 from the National Research Fund (a member of Qatar Foundation) (to S. S). Scopus

Non-Hodgkin lymphoma (NHL) is a heterogeneous group of blood cancers arising in lymphoid tissues that commonly effects both humans and dogs. Protein arginine methyltransferase 5 (PRMT5), an enzyme that catalyzes the symmetric di-methylation of arginine residues, is frequently overexpressed and dysregulated in both human solid and hematologic malignancies. In human lymphoma, PRMT5 is a known driver of malignant transformation and oncogenesis, however, the expression and role of PRMT5 in canine lymphoma has not been explored. To explore canine lymphoma as a useful comparison to human lymphoma while validating PRMT5 as a rational therapeutic target in both, we characterized expression patterns of PRMT5 in canine lymphoma tissue microarrays, primary lymphoid biopsies, and canine lymphoma-derived cell lines. The inhibition of PRMT5 led to growth suppression and induction of apoptosis, while selectively decreasing global marks of symmetric dimethylarginine (SDMA) and histone H4 arginine 3 symmetric dimethylation. We performed ATAC-sequencing and gene expression microarrays with pathway enrichment analysis to characterize genome-wide changes in chromatin accessibility and whole-transcriptome changes in canine lymphoma cells lines upon PRMT5 inhibition. This work validates PRMT5 as a promising therapeutic target for canine lymphoma and supports the continued use of the spontaneously occurring canine lymphoma model for the preclinical development of PRMT5 inhibitors for the treatment of human NHL.

Objectives This study aimed to investigate the epidemiology of intravenous midazolam-induced postoperative expressive aphasia (EA). Methods The incidence rate, risk ratio, and contributing factors to intravenous midazolam-induced postoperative EA were analyzed retrospectively in 6756 orthopedic patients. A telephone interview was conducted with patients with EA after surgery. Results Patients were allocated to either the midazolam group (n = 6178) or no-midazolam group (n = 578). Twelve patients developed EA in the midazolam group, with an incidence of 0.19%, and no patient developed EA in the no-midazolam group. The mean age of EA patients was 70 years, and 92% were women. Among them, 75% received general anesthesia, and the mean dose of midazolam was 1.8 mg. EA was reversed in nine of 12 (75%) patients within 4 minutes of flumazenil administration, and >60 minutes were required to reverse EA in the other three patients (25%). Conclusion Intravenous midazolam administration for preoperative sedation caused transient EA in 0.19% of patients, especially elderly women who received general anesthesia, and EA could be reversed by flumazenil.

Background: Postoperative urinary retention (POUR) after anesthesia and surgery is influenced by many factors, and its reported incidence rate varies widely. The aim of this study was to investigate the occurrence and risk factors for urinary retention following general anesthesia for endoscopic nasal surgery in male patients aged over 60 years. Methods: Retrospective review of medical records between January 2015 and December 2019 identified 253 subjects for inclusion in our study. Age, body mass index, history of diabetes/hypertension, American Society of Anesthesiologists classification, and urologic history were included as patient-related factors. Urologic history was subdivided into three groups according to history of benign prostate hyperplasia (BPH)/lower urinary tract symptoms (LUTS) and current medication. The following were analyzed as perioperative variables for the development of POUR: duration of anesthesia and surgery; amount of fluid administered; rate of fluid administration; intraoperative requirement for fentanyl, ephedrine, and dexamethasone; postoperative pain; and analgesic use. Preoperatively measured prostate size and uroflowmetry parameters of patients on medication for symptoms were compared according to the incidence of urinary retention.Results: Thirty-seven patients (15.7%) had urinary retention requiring catheterization. Among analyzed variables, only urologic history was identified as a predisposing factor. The incidence rate among patients without urologic issues was 5.9%. This compared to 19.8% among patients with a history of BPH/LUTS, which was not reduced by taking medical treatment. Among patients taking medication for symptoms, the maximal and average velocity of urine flow were significantly lower in subjects with POUR. Conclusions: General anesthesia for endoscopic nasal surgery is a potent trigger of urinary retention in male patients aged over 60 years. The urological history of the patient was the most important risk factor, and the occurrence of POUR appears to be affected by urinary conditions. The present study is helpful in understanding the occurrence of POUR following general anesthesia in elderly male patients.

Lipomas are the most common soft tissue lesions occurring in the salivary glands but have a very low incidence. Lipomas commonly occur in the parotid gland, and lipomas in the submandibular gland (SMG) are rare. Until recently, ordinary lipomas of the parotid gland and some variants of lipomas of the SMG have been reported. However, few reports of ordinary lipomas occurring within the SMG exist in the literature. We report an extremely rare case of ordinary lipoma within the right SMG of a 65-year-old man. The tumor measured a 2.0 × 1.8 × 2.7 cm, was a well-capsulated homogenous yellow mass, which was composed of mature adipose tissue. A partially mixed area with salivary gland tissue was observed. There has not been much research on lipomatous tumors from the SMG because of their rareness. Most lipomatous tumors in the parotid gland are known as ordinary lipomas, but more research is needed to determine whether they can be applied to the SMG. Thus, this report will be instrumental in the understanding of lipomatous tumors of the SMG.

Mantle cell lymphoma (MCL) is an incurable B cell malignancy, comprising 5% of non-Hodgkin lymphomas diagnosed annually. MCL is associated with a poor prognosis due to emergence of resistance to immuno-chemotherapy and targeted agents. The average overall survival of patients with MCL is 4-6 years and for the majority of patients who progress on targeted agents, survival remains at a dismal 3-8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated to improve treatment outcomes and quality of life. The type II protein arginine methyltransferase enzyme, PRMT5 is overexpressed and promotes growth and survival of MCL. Inhibition of PRMT5 with a novel, SAM-competitive class of inhibitors drives anti-tumor activity in MCL cell lines and patient derived xenograft (PDX) models derived from patients with relapse or refractory disease. Selective inhibition of PRMT5 with PRT-382 (Prelude Therapeutics) in these models and MCL cell lines leads to disruption of constitutive PI3K/AKT signaling, dephosphorylation and nuclear translocation of FOXO1, and enhanced recruitment of this tumor suppressor protein to target genes. By performing chromatin immunoprecipitation-sequencing (ChIP seq) analysis, we identified over 800 newly emerged FOXO1-bound genomic loci, including multiple pro-apoptotic BCL2 family proteins (BAX, BAK1, BIK, BBC3, BMF and NOXA1). FOXO1 localization and transcriptional differences were confirmed by ChIP PCR and RT-PCR respectively. Protein levels were measured with Western blotting. BAX was identified as the most common direct target of FOXO1-transriptional activity that was upregulated on both a transcript and protein level. This led us to hypothesize that PRMT5 inhibition could potentially drive a therapeutic vulnerability to the BCL-2 inhibitor venetoclax. Single agent and combination treatment with venetoclax and PRT382 was performed in nine MCL lines. Of the nine lines, four were considered relatively resistant to PRT-382 and five resistant to venetoclax. Synergy scores, determined from MTS assays, showed significant levels of synergy in the majority of MCL lines tested. CCMCL1 and UPN1, BCL-2 negative MCL lines, and Maver1, which is highly resistant to PRMT5i, were the only cell lines to not show synergy. The cell line with the highest levels of synergy, Z-138, expressed high levels of BCL-2 and is ibrutinib resistant. Overall, there was a strong positive correlation between BCL-2 expression and synergy score (r= -0.8956, p=0.0064). The synergy seen was confirmed to be through the intrinsic apoptotic pathway based on caspase activity. To determine a mechanism of action, BAX and BAK1 were knocked down in four cell lines, three that displayed synergy and one that was resistant. This suggests that BAX expression is essential for synergy between PRMT5 and BCL2 inhibition to occur. Knock down of BAK1, the other effector of the BCL2 family of proteins, did not show protection suggesting that BAX is necessary and sufficient for this therapeutic synergy to occur. We also determined that p53 status did not correlate to the response seen (p=0.477), supporting that this mechanism is occurring through FOXO1 transcriptional regulation. In vivo evaluation in two preclinical MCL models showed therapeutic synergy with combination venetoclax/PRT382 treatment. Mice were treated with sub-therapeutic doses of venetoclax and/or PRT382 and disease burden was assessed weekly via flow cytometry. Combination treatment with well-tolerated doses of venetoclax and PRMT5 inhibitors in the MCL in vivo models showed synergistic anti-tumor activity. Both PDX models showed an extension of life with combination treatment (P Disclosures Zhang: Prelude Therapeutics: Current Employment. Vaddi: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Elemento: AstraZeneca: Research Funding; Janssen: Research Funding; Johnson and Johnson: Research Funding; One Three Biotech: Consultancy, Other: Current equity holder; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Eli Lilly: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Owkin: Consultancy, Other: Current equity holder; Champions Oncology: Consultancy. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Paik: Forkhead BioTherapeutics: Research Funding. Baiocchi: Prelude Therapeutics: Consultancy; viracta: Consultancy, Current holder of stock options in a privately-held company; Codiak Biosciences: Research Funding; Atara Biotherapeutics: Consultancy.

Objectives: Postoperative urinary retention (POUR) is influenced by many factors, and its reported incidence rate varies widely. This study aimed to investigate the occurrence and risk factors for urinary retention following general anesthesia for endoscopic nasal surgery in male patients aged >60 years. Methods: A retrospective review of medical records between January 2015 and December 2019 identified 253 patients for inclusion in our study. Age, body mass index (BMI), a history of diabetes/hypertension, American Society of Anesthesiologists (ASA) classification, and urologic history were included as patient-related factors. Urologic history was subdivided into 3 groups according to history of benign prostate hyperplasia (BPH)/lower urinary tract symptoms (LUTS) and current medication. The following was analyzed as perioperative variables for POUR development: duration of anesthesia and surgery; amount of fluid administered; rate of fluid administration; intraoperative requirement for fentanyl, ephedrine, and dexamethasone; postoperative pain; and analgesic use. Preoperatively measured prostate size and uroflowmetry parameters of patients on medication for symptoms were compared according to the incidence of urinary retention. Results: Thirty-seven (15.7%) patients developed POUR. Age (71.4 vs 69.6 years), BMI (23.9 vs 24.9 kg/m2), a history of diabetes/hypertension, ASA classification, and perioperative variables were not significantly different between patients with and without POUR. Only urologic history was identified as a factor affecting the occurrence of POUR ( P = .03). The incidence rate among patients without urologic issues was 5.9%, whereas that among patients with BPH/LUTS history was 19.8%. Among patients taking medication for symptoms, the maximal and average velocity of urine flow were significantly lower in patients with POUR. Conclusions: General anesthesia for endoscopic nasal surgery may be a potent trigger for urinary retention in male patients aged >60 years. The patient’s urological history and urinary conditions appear to affect the occurrence of POUR.

Objectives To evaluate the critical quality attributes that might affect the stability of an infliximab biosimilar (SB2, Flixabi) when reconstituted or diluted and stored under refrigeration and at room temperature. Methods We largely adhered to the UK9s National Health Service guidance requirements for the design of a robust stability study and for robust testing methods. Protocol components included evaluation of visual appearance, chemical stability, physical stability, pH, particle sizes and biological activity. The stability of reconstituted SB2 was assessed for 60 days at 5°C and for 7 days at 25°C. Stability of diluted SB2 at concentrations that ranged from 240 mg/250 mL (3 mg/kg; 80 kg patient) to 400 mg/250 mL (5 mg/kg; 80 kg patient) was assessed for 7 days at both temperatures. Dilutions were made in polyethylene bags containing 0.9% NaCl. Forced degradation studies were conducted with SB2 and its reference product (USA-sourced and European Union-sourced Remicade). Stress conditions of heat or light occurred before product reconstitution. Results In a laboratory environment under aseptic conditions, stability acceptance criteria with regard to physicochemical and biological properties were met for all reconstituted and diluted SB2 samples for all time periods and temperatures assessed. After either heat or light stress, similar stability and biological activity were noted for SB2 and both reference products. Conclusions When prepared under aseptic conditions in accordance with the product9s Summary of Product Characteristics, exposed for prolonged periods at 5°C and 25°C and assessed with the described methods, SB2 appears to remain a stable monoclonal antibody maintaining its expected biological function.

Rationale: The most commonly used regional techniques for analgesia following laparotomy thoracic epidural analgesia and paravertebral blocks are technically difficult to perform and carry a risk of severe complications. Recently, the erector spinae plane block (ESPB) has been reported to effectively treat neuropathic pain. The ultrasound-guided ESPB is an easily performed fascial plane block that can provide sensory blockade from T2–4 to T12–L1. Moreover, the ESPB reportedly blocks both the ventral rami of spinal nerves and the rami communicants, which contain sympathetic nerve fibres, through spread into the thoracic paravertebral space. Patient concerns: We report the case of a 35-year-old female patient who underwent excision of a larger ovarian mass via laparotomy with a wide, midline incision from the xiphoid process to the pubic tubercle. Diagnoses: They were diagnosed with mucinous cystadenoma originated from the right ovary and fallopian tube, and a right oophorectomy and salpingectomy were performed. Interventions: The ESPB was performed for postoperative pain control at the level of the T8 transverse process. Postoperative multimodal analgesia was provided according to the acute pain service protocol of our hospital. The patient was prescribed oral acetaminophen 175 mg every 6 hours and intravenous patient-controlled analgesia (PCA) with fentanyl 7 μg/mL. A 1:1 mixture of 0.75% ropivacaine (20 mL) and saline (20 mL) with epinephrine (1: 200,000) was manually injected through the indwelling catheter every 8 hours (20 mL per side). Outcomes: The first demand dose of fentanyl was administered at 9 hours and 39 minutes after the surgery. There were no reported resting pain scores >4, nor were any rescue analgesics needed during the first 5 postoperative days. Lessons: The ESPB provided highly effective analgesia as a part of multimodal analgesia after laparotomy with a wide midline incision.

Introduction Stability information for the trastuzumab biosimilar SB3 is limited to 48 h at 2–8 °C for the reconstituted solution and 24 h at up to 30 °C for diluted solutions. Extended physicochemical stability and biological activity were assessed to evaluate the advanced preparation of reconstituted and diluted SB3. Methods Under controlled and aseptic conditions, the stability of reconstituted and diluted SB3 was evaluated using several assessments and according to the UK’s National Health Service guidance. Reconstituted SB3 was stored at 25 ± 2 °C with 60 ± 5% relative humidity for 3 days, and subsequently diluted SB3 (0.32–4 mg/mL) was stored in an infusion bag in the absence of light at 25 ± 2 °C with 60 ± 5% relative humidity for 28 days and 5 ± 3 °C for 28 days, respectively. Physicochemical stability (appearance, pH, protein concentration, size exclusion high-performance liquid chromatography, non-reducing capillary electrophoresis–sodium dodecyl sulfate, imaged capillary isoelectric focusing), biological activity (competitive inhibition binding assay to human epidermal growth factor receptor 2 by fluorescence resonance energy transfer, anti-proliferation assay), and properties with a potential safety impact (subvisible particulates, submicronic aggregation by dynamic light scattering) were determined. Results No physicochemical instability signs or biological activity changes were observed for either reconstituted or diluted SB3 up to 28 days; all stability acceptance criteria were met. No major change was noted in the proportion of molecular weight variants (high molecular weight impurity, total purity) or relative percentages of acidic, main, and basic charge isoforms of the protein. No increases in particulates or aggregates in terms of a potential safety impact were noted. Conclusion The physicochemical stability and biological activity of reconstituted and diluted SB3 are maintained for extended time periods beyond those denoted in the product labeling, which allows for advanced SB3 preparation and may reduce drug wastage and preparation time. Funding Samsung Bioepis Co., Ltd.

Rationale: Stylohyoid complex syndrome is characterized by various cervicopharyngeal symptoms related to the ossification and abnormality of the styloid process, stylohyoid ligament, and the lesser horn of the hyoid bone. Eagle syndrome is the most well-known of the spectra of these diseases. Although surgical treatment is considered effective, conservative treatment may be beneficial if symptoms arise because of inflammation of the soft tissues attached to the styloid process or hyoid bone. Patient concerns: A 68-year-old man presented with pain in the right side of the neck and odynophagia after trauma on his philtrum. He was diagnosed with Eagle syndrome elicited by a fracture from indirect trauma. Despite analgesic medication and physiotherapy, the pain had somewhat relieved but persisted for 1 year. Diagnosis: Computed tomography revealed complete ossification of the bilateral stylohyoid complex. A fracture was observed in the ampulla on the right side of the neck. One year later, the fracture resolved by complete union. Interventions: Ultrasonography was performed and abnormal ossification was observed on the right side of the neck. Five milligrams of dexamethasone at a concentration of 1 kg/m3 was slowly injected into the tender point under ultrasonographic guidance. Outcomes: The patient reported immediate reduction of pain and was satisfied with the resolution. No recurrence was observed during a 6-month follow-up period. Lessons: Although traumatic fracture of the ossified ligament elicited the syndrome, the results were satisfactory because the origin of the patient's pain was presumed to arise from inflammatory conditions. This case demonstrates that treatment with local steroid injection may be appropriate for patients who present with pain originating from muscles and ligaments.

Introduction: Post-translational histone modifications directly modify chromatin structure to influence a wide variety of cellular events including gene expression, DNA replication and repair, and cell cycle control. While histone lysine methylation can confer either transcriptionally active or repressive states, the symmetric dimethylation of arginine residues on histone tails is generally associated with transcriptional repression. Overexpression and dysregulation of PRMT5, the major type II protein arginine methyltransferase, has been shown to drive cellular proliferation and survival of multiple cancer types including mantle cell lymphoma (MCL), a subtype of non-Hodgkin lymphoma associated with poor prognosis. Our work has shown that PRMT5 drives the symmetric dimethylation of arginine (histones H3R8 and H4R3) and attenuates RB/E2F regulatory pathways leading to the expression and activation of the PRC2 lysine methylation programs. We have previously shown that PRMT5 promotes constitutive expression of the WNT/β-CATENIN target genes (MYC, CCND1, and SURVIVIN) via the epigenetic repression of AXIN2 and WIF1 regulatory genes. This PRMT5 mediated epigenetic program allows for constitutive activation of WNT and PI3/AKT downstream pathways relevant to MCL growth and survival. The inhibition of PRMT5 triggers histone deacetylation and H3K4me3 demethylation on promoters of β-CATENIN target genes. H3K4 is modified by several enzymes containing SET domains including the SETD7 and MLL1 proteins. We hypothesized that PRMT5 inhibition, through its ability to repress AKT phosphorylation, would regulate SETD7 and MLL1 activity and regulate downstream pathways relevant to lymphomagenesis. Methods: PRMT5 inhibition of patient-derived MCL cell lines and primary lymphoma tumor cells was achieved with sh-PRMT5 lentivirus (or sh-GFP control) and utilization of a selective small molecule, SAM-competitive PRMT5 inhibitor (PRT382). Gene and protein expression was monitored by reverse transcription (RT) real time PCR and western immunoblotting, respectively. Recruitment of target proteins to promoter regions was examined by ChIP-PCR assays. Interactions within the transcriptional complex were examined by co-immunoprecipitation. Cellular growth and apoptosis was assessed by proliferation assays and FACS analysis. Results: Inhibition of PRMT5 by shRNA-mediated knock down or treatment with PRT382 (Prelude Therapeutics) reduced H3K4me2 in MCL cells via indirect epigenetic repression of SETD7. ChIP-PCR studies showed PRMT5 to be recruited to the SETD7 promoter, suggesting that the activity of PRMT5 promoted the transcription of this lysine methyltransferase. Our findings show that reduced phosphor-AKT by PRMT5 inhibition inhibits dimerization of the MLL1 complex leading to dissociation of MLL1 from the transcriptional activation complex and decreased H3K4me1 and H3K4me3. PRMT5 inhibition led to dissociation of the BCL9- Pygopus-MLL1 transcriptional activating complex and to assembly of repressive LSD1 (the histone demethylase affecting H3K4me3)-HDAC2 (the histone lysine deacetylase) containing complexes. Furthermore, PRMT5 inhibition led to differentially enhanced recruitment of this repressive LSD1/HDAC2 complex and decreased H3K9Ac and H3K14Ac epigenetic marks on promoters of β-CATENIN target genes. PRMT5 inhibition regulates lysine methylation at H3K4 through epigenetic silencing of SETD7 and MLL1 activity, as well as histone H3 acetylation driven by histone acetyltransferase KAT2A. ChIP-Seq studies examining SETD7 and PRC2 recruitment in context of PRMT5 inhibition are currently underway. Conclusions: Our observations show that dysregulated PRMT5 activity acts as a master epigenetic regulator affecting arginine and lysine histone marks. PRMT5 can act directly to repress target tumor suppressor genes while simultaneously indirectly activating SETD7 and MLL1 to drive transcriptional activation of key oncogenes that promote the proliferation and survival of MCL. These results support the notion that inhibiting PRMT5 leads to erasure of repressive histone arginine and lysine marks and promote the restoration of normal growth and survival checkpoints in this disease. Disclosures Scherle: Prelude Therapeutics: Employment. Vaddi:Prelude Therapeutics: Employment. Baiocchi:Prelude: Consultancy.

Mantle cell lymphoma (MCL) is an incurable B cell malignancy, defined by the t(11;14) translocation and comprises 3-6% of non-Hodgkin lymphomas diagnosed annually. MCL is associated with a poor prognosis due to emergence of resistance to immuno-chemotherapy and targeted agents. Due to the late median age of diagnosis, aggressive chemotherapy and stem cell transplantation are often not realistic options. The average overall survival of patients with MCL is 5 years and for the majority of patients who progress on targeted agents like ibrutinib, survival remains at a dismal 3-8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated by elderly patients to improve treatment outcomes and quality of life. Our group has identified the type II protein arginine methyltransferase enzyme, PRMT5, to be dysregulated in MCL and to promote growth and survival by supporting the cell cycle, PRC2 activity, and signaling via the BCR and PI3K/AKT pathways. We have developed first-in-class selective inhibitors of PRMT5 and, in collaboration with Prelude Therapeutics, we have demonstrated that novel SAM-competitive PRMT5 inhibitors provide potent anti-tumor activity in aggressive preclinical models of human MCL. Selective inhibition of PRMT5 in these models and MCL cell lines leads to disruption of constitutive PI3K/AKT signaling, dephosphorylation and nuclear translocation of FOXO1, and enhanced recruitment of this tumor suppressor protein to chromatin. We identified 136 newly emerged FOXO1-bound genomic loci following 48 hours of PRMT5 inhibition in the CCMCL1 MCL line by performing chromatin immunoprecipitation-seq analysis. These genes were markedly upregulated in CCMCL1 cells treated with the PRMT5 inhibitor PRT382 as determined by RNA-seq analysis. Among those genes, we identified and confirmed FOXO1 recruitment to the promoter of BAX, a pro-apoptotic member of the BCL2 family of proteins. Treatment of MCL cell lines (Granta-519, CCMCL1, Z-138, and SEFA) with the selective PRMT5 inhibitor PRT382 (10, 100nM) led to upregulation of BAX protein levels and induction of programmed cell death as measured by annexin V/PI staining and flow cytometry. We hypothesized that induction of BAX would trigger a therapeutic vulnerability to the BCL2 inhibitor venetoclax, and that combination PRMT5/BCL2 inhibitor therapy would drive synergistic cell death in MCL. Single agent and combination treatment with venetoclax and PRT382 was performed in eight MCL lines including a new cell line generated from our ibrutinib-refractory PDX model (SEFA) and IC50 and synergy scores were calculated. The Z-138 line was most sensitive to venetoclax (IC501uM). All lines reached an IC50 20) in 4 lines and moderate synergy (scores 10-20) in 2 lines. The two lines with the highest levels of synergy, Z-138 and SEFA, express high levels of BCL-2 and are Ibrutinib resistant. Overall there was a strong positive correlation between BCL2 expression and synergy score (r=0.707), and no correlation between PRMT5 expression and synergy score (r=0.084). In vivo evaluation in two preclinical MCL models (Granta-519 NSG mouse flank and an ibrutinib-resistant MCL PDX) showed therapeutic synergy with combination venetoclax/PRT382 treatment. In both models, mice were treated with sub-therapeutic doses of venetoclax and/or PRT543 (Granta) or PRT382 (IR-MCL PDX) and tumor burden assessed weekly via flank mass measurement (Granta) or flow cytometry (IR-MCL-PDX). Combination treatment with well-tolerated doses of venetoclax and PRMT5 inhibitors in both MCL in vivo models showed synergistic anti-tumor activity without evidence of toxicity. This preclinical data provides mechanistic rationale while demonstrating therapeutic synergy and lack of toxicity in this preclinical study and justifies further consideration of this combination strategy targeting PRMT5 and BCL2 in MCL in the clinical setting. PRT543, a selective PRMT5 inhibitor, has been advanced into clinical studies for the treatment of patients with solid tumors and hematologic malignancies, including MCL (NCT03886831). Disclosures Zhang: Prelude Therapeutics: Employment. Vaddi:Prelude Therapeutics: Employment. Scherle:Prelude Therapeutics: Employment. Baiocchi:Prelude: Consultancy.

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy characterized by genetic dysregulation of cyclin D1 and activation of signaling pathways driving uncontrolled MCL cell proliferation and survival. Ibrutinib is an FDA-approved irreversible inhibitor of Bruton's tyrosine kinase (BTK), a downstream target of the B-cell receptor (BCR) pathway. While ibrutinib exhibits significant single-agent therapeutic activity in patients with relapsed/refractory MCL, the vast majority of MCL patients on ibrutinib progress with aggressive disease and short survival (3-8 mo). Although ~80% of chronic lymphocytic leukemia patients with acquired ibrutinib resistance have mutations in BTK and PLCγ2, this is uncommon in MCL suggesting alternative mechanisms driving this resistant phenotype. Understanding drug-resistance mechanisms and developing effective therapies for ibrutinib resistant (IR) MCL are urgently needed. The major type II protein arginine methyltransferase enzyme, PRMT5, catalyzes symmetric dimethylation of arginine residues on histone tails (H3R8 and H4R3) and other proteins. PRMT5 regulates a vast array of biologic functions including RNA processing, DNA damage response, signal transduction, and gene expression. Amplified PRMT5 activity drives the expression and activity of key oncogenes (MYC, CYCLIND1, NOTCH1) while silencing expression and activity of tumor suppressors (ST7, RBL2, and p53). Our group has shown PRMT5 is overexpressed and dysregulated in MCL and strategies aimed at selectively targeting PRMT5 show anti-tumor activity in preclinical lymphoma models. Here we describe the development of a novel patient derived xenograft (PDX) of IR-MCL and explore PRMT5 inhibition as an alternative therapeutic option to circumvent IR. Peripheral blood mononuclear cells from a 75 yo male patient diagnosed with acquired classic IR-MCL were engrafted intravenously into NSG mice. After 5 passages, all mice engrafted with 107 MCL cells developed histologically confirmed MCL infiltrating kidney, lymph nodes, bone marrow, spleen and peripheral blood. Circulating human CD5+/CD19+ cells were detectable and quantifiable by flow cytometry by day 21 post-engraftment. Karyotype analysis confirmed the hallmark t(11;14)(q13;q32) of MCL while retaining nearly all cytogenetic abnormalities present in the patient's primary tumor including a deletion of chromosome 9, associated with deletion of MTAP, a therapeutic vulnerability for PRMT5-targeted therapy. Whole exome sequencing confirmed genomic stability with successive passages. Ex vivo cytotoxicity assays and protein pathway analysis further confirmed resistance to ibrutinib (IC50 >1 µM) with maintained hyper-phosphorylation of AKT (Ser473) and ERK (Thr202/Tyr204). Western blot analysis showed elevated levels of c-MYC, CYCLIND1, BCL2, and pERK. After validation of circulating disease at day 25 post engraftment, mice were treated with either a novel small molecule inhibitor of PRMT5 (PRT382, 10 mg/kg orally 4 days on 3 days off) or ibrutinib (75 mg/kg administered in drinking water, n=5 mice per treatment group). Treatment of this PDX model with PRT382 resulted in significantly decreased disease burden and improved median survival compared to control animals from 48 to 83 days, respectively (p=0.0045). We found no significant difference in survival (p= 0.6540) or circulating disease burden with ibrutinib therapy compared to control group. The full BTK occupancy of ibrutinib treated mice was validated using fluorescence resonance energy transfer-based assay. Ex vivo PDX MCL cells from PRT382-treated mice showed loss of symmetric dimethyl arginine with preservation of asymmetric dimethyl arginine levels, reduced H4(Sme2)R3 epigenetic marks, and elevated levels of BCL2, MYC, and pAKT/pERK. We developed a cell line (SEFA) allowing for in vitro mechanistic studies. We are currently investigating potential mechanisms responsible for circumventing IR-MCL by integrating genome-wide changes to chromatin accessibility and whole transcriptome analysis. This IR-MCL PDX mouse model serves as a useful tool to investigate mechanisms of drug resistance, provides a platform to explore novel pre-clinical therapeutic strategies to circumvent IR and demonstrates the therapeutic activity of PRMT5 targeted therapy in this aggressive disease. Disclosures Byrd: Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Genentech: Research Funding; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; BeiGene: Research Funding; Genentech: Research Funding; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau. Vaddi:Prelude Therapeutics: Employment. Scherle:Prelude Therapeutics: Employment. Baiocchi:Prelude: Consultancy.

INTRODUCTION The objective of this study was to compare postoperative pain between single-port access total laparoscopic hysterectomy (SPA-TLH) using a transumbilical single-port system and conventional multi (three)-port access total laparoscopic hysterectomy (MPA-TLH). MATERIAL AND METHODS A randomized controlled trial was conducted on 60 women who underwent SPA-TLH and MPA-TLH for benign gynecologic diseases between March 2014 and January 2015. Patients were randomly assigned to undergo SPA-TLH (n = 30) or MPA-TLH (n = 30). The variables measured included surgical outcomes and postoperative pain at 30 min and 1, 12, 24, and 48 h after surgery, assessed by the visual analog scale, bolus requirement of intravenous patient-controlled analgesia, and additional analgesic use. RESULTS The two study groups did not differ in terms of patient demographics or surgical outcomes except for operative time. The SPA-TLH group had a longer operative time (p < 0.0001) compared with the MPA-TLH groups. There were no differences in pain scores between the two groups. The SPA-TLH group had significantly more intravenous analgesia requests during the 12-24 h after surgery (2.17 ± 3.05 vs. 0.79 ± 1.99; p = 0.047), more 24-48 h postoperative analgesics (0.21 ± 0.41 vs. 0.03 ± 0.19; p = 0.045), and more total additional analgesics (0.97 ± 0.94 vs. 0.45 ± 0.87; p = 0.034). CONCLUSION SPA-TLH was feasible compared with MPA-TLH but the SPA-TLH group had a longer operative time. Although there is no difference in pain based on the visual analog scale pain score, the SPA-TLH group required more analgesia to give the same postoperative pain control.

Non-Hodgkin lymphoma (NHL) represents approximately 4 percent of all cancer diagnoses in the United States, with diffuse large B-cell lymphoma (DLBCL) accounting for approximately 40 percent of all new cases. There are numerous histologic (GC, NGC) and genetic (double hit/expressor) subtypes of DLBCL and the overall outcome of patients who receive standard therapy is heterogeneous. Lymphoma is also a common, malignancy in dogs, and while initial responses to combination chemotherapy show clinical responses, remission time is short and cures rare. Protein arginine methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase (PRMT) enzyme capable of driving symmetric demethylation of arginine residues on histones (H3(me2)R8; H4(me2)R3) and other proteins such as P53, and the NFkB subunit p65. PRMT5 is overexpressed and dysregulated in both solid and hematologic tumors and plays a key role in the driver activity of MYC, CYCLIND1 and NOTCH in lymphoma models. Experimental therapeutic strategies aimed at targeting PRMT5 activity have led to numerous, highly selective inhibitors that are currently being translated into the clinic with several clinical trials underway or in development. Here, we characterized patterns of PRMT5 expression and correlated these with histologic subtype in canine lymphoma tissue microarrays (TMAs, n=337 lymphoma specimens). We characterized expression of PRMT5 and its symmetric dimethylation histone marks in three canine lymphoma-derived cell lines, CLBL-1, 17-71, and OSW. Treatment of PRMT5 with a highly selective small molecule PRMT5 inhibitor (CMP220) was performed to determine the dose-dependent effects (1nM-10uM range) on proliferation (MTS assay), viability (annexin V/PI flow cytometry) and biomarkers of PRMT5 activity (SDMA, ADMA, histone symmetric demethylation). CMP220 showed PRMT5 inhibitory activity that was highly selective in a methyltransferase screening assay with 37 enzymes/complexes. TMAs of all histologic subtypes of lymphoma showed over-expression of PRMT5 in 96% of specimens. Canine DLBCL showed variable over-expression in the cytoplasmic compartment (48.8% strong, 50.0% weak, n = 165) compared to negative or weak staining in normal and hyperplastic lymph nodes (n = 40). Nuclear staining was observed primarily in lymphomas of T cell lineage (PTCL, and pre T lymphoblastic lymphomas). Primary and cell line lymphoma samples showed PRMT5 over expression by Western blot and RT-PCR. PRMT5 inhibition showed a dose-dependent decrease in symmetric dimethylarginine (SDMA) and symmetric demethylation of histone H4 arginine-3 (H4(me2s)R3) with no effect on asymmetric dimethylarginine (ADMA). Interestingly, in contrast to PRMT5 inhibition in human lymphomas, symmetric demethylation of histone H3 arginine-8 (H3(me2s)R8) showed no change with PRMT5 inhibition. The PRMT5 small molecule inhibitor CMP220 inhibited growth of CLBL-1 (IC50 of 123.2 nM at 6d) and 17-71 (IC50 of 100 nM at 6d). The OSW PTCL cell line showed profound resistance to PRMT5 inhibition with little cell death observed at 10 uM. Ex-vivo canine patient samples showed suppression of growth in a time and dose-dependent fashion. We have demonstrated that PRMT5 is expressed in canine B cell primary lymphomas and cell lines and that PRMT5 inhibition leads to growth suppression and induction of apoptosis. We are currently exploring genome-wide recruitment of PRMT5 on chromatin and examining chromatin and whole transcriptome changes that occur in canine lymphoma cell lines treated with PRMT5 inhibitors. This data provides justification for incorporating the canine lymphoma model into the preclinical development of PRMT5 inhibitors. Disclosures No relevant conflicts of interest to declare.

We describe a case of acute lumbar epidural hematoma at the L2-3 level complicated by paraplegia, which occurred after coagulation disorder because of massive bleeding intraoperatively in cesarean section. The preoperative coagulation laboratory finding was in normal range and so we tried combined spinal epidural anesthesia. Uterine atony occurred in the operation, and there was persistant bleeding during and after the operation. After the operation, she complained of paresthesia on her both legs and was diagnosed with epidural hematoma (EDH) by radiologic examination. Emergency laminectomy on lumbar spine was carried out for hematoma evacuation and decompression of the epidural space at once. In our experience, massive bleeding during surgery may potentially increase the risk of EDH postoperatively.

Background Anesthesia methods and drugs affect postoperative nausea and vomiting. Propofol is known to have antiemetic effects. We compared the incidence of postoperative vomiting (POV) in children undergoing an adenotonsillectomy; anesthesia in one group was induced with propofol and maintained with sevoflurane and nitrous oxide, and the other group received total intravenous anesthesia (TIVA) with propofol-remifentanil. Methods Ninety children, ASA physical status I, were assigned randomly to one of two groups. In the PSN group, anesthesia was maintained with 2-3 vol% sevoflurane and 50% nitrous oxide. In the PR group, anesthesia was maintained with 10 mg/kg/h propofol and 0.25 µg/kg/min remifentanil. In both groups, anesthesia was induced with 0.5 µg/kg remifentanil and 2 mg/kg propofol. The incidence of POV and the need for rescue antiemetics were assessed in the postanesthesia care unit at 6, 12, and 24 hours postoperatively. Results The total incidence of POV was not significantly different between the groups; POV occurred in eight (17.7%) and three (6.7%) children in the PSN and PR groups, respectively. Postoperative frequency of retching in the recovery room was significantly higher in the PSN group, with four children (8.9%) in the PSN group compared to none (0%) in the PR group (P = 0.041). The frequency of POV 24 hrs after exiting the recovery room tended to be higher in the PSN group than the PR group, but no statistically significant difference was observed. Conclusions If the development of POV in the early anesthetic recovery phase of children undergoing adenotonsillectomy is adequately prevented, propofol-induced anesthesia maintained with sevoflurane-nitrous oxide is as safe as TIVA with propofol-remifentanil.

Epigenetic events that are essential drivers of lymphocyte transformation remain incompletely characterized. We used models of Epstein-Barr virus (EBV)–induced B-cell transformation to document the relevance of protein arginine methyltransferase 5 (PRMT5) to regulation of epigenetic-repressive marks during lymphomagenesis. EBV+ lymphomas and transformed cell lines exhibited abundant expression of PRMT5, a type II PRMT enzyme that promotes transcriptional silencing of target genes by methylating arginine residues on histone tails. PRMT5 expression was limited to EBV-transformed cells, not resting or activated B lymphocytes, validating it as an ideal therapeutic target. We developed a first-in-class, small-molecule PRMT5 inhibitor that blocked EBV-driven B-lymphocyte transformation and survival while leaving normal B cells unaffected. Inhibition of PRMT5 led to lost recruitment of a PRMT5/p65/HDAC3-repressive complex on the miR96 promoter, restored miR96 expression, and PRMT5 downregulation. RNA-sequencing and chromatin immunoprecipitation experiments identified several tumor suppressor genes, including the protein tyrosine phosphatase gene PTPROt, which became silenced during EBV-driven B-cell transformation. Enhanced PTPROt expression following PRMT5 inhibition led to dephosphorylation of kinases that regulate B-cell receptor signaling. We conclude that PRMT5 is critical to EBV-driven B-cell transformation and maintenance of the malignant phenotype, and that PRMT5 inhibition shows promise as a novel therapeutic approach for B-cell lymphomas. This work was supported by grants from the American Society of Hematology/European Hematology Association (L.Alinari), the Leukemia & Lymphoma Society Translational Research Project (LLS TRP) (R.A.B.), Friends of Jason Gould Foundation (R.A.B., P.L.S., J.T.P.), The Ohio State University Drug Development Institute (R.A.B., C.L.), National Institutes of Health, National Institute of Neurological Disorders and Stroke grant R21NS071346 (R.A.B., C.L.) and National Cancer Institute grant R01CA116093 (S.S., R.A.B.).

Introduction: It is well established that altered expression of oncogenes and epigenetic dysregulation of tumor suppressor and regulatory genes promotes lymphomagenesis. Over-expression of the type II protein arginine methyltransferase (PRMT5) enzyme has been associated with increased cell proliferation and survival in both solid and blood cancers. We have reported that PRMT5 is essential for EBV-driven B cell transformation and that it regulates the EZH2, EED and SUZ12, components of the Polycomb-repressive complex 2 (PRC2) complex via transcriptional silencing of RBL2and hyper-phosphorylation of RB1 in aggressive lymphomas. Here we report a mechanistic assessment of how PRMT5 over-expression supports the WNT/β-CATENIN pathway at multiple levels and drives oncogenic β-CATENIN target genes in lymphoma. Methods: PRMT5 inhibition of patient-derived lymphoma cell lines, primary lymphoma tumor cells and mouse primary Eμ-BRD2 transgenic lymphoma cells was achieved by infecting with sh-PRMT5 lentivirus (or sh-GFP control), a selective small molecule PRMT5 inhibitor (Alinari et al, Blood 2015, CMP5) or CRISPR/CAS9 PRMT5 deletion. Gene expression was monitored by immunoblotting and reverse transcription (RT) real time PCR. Recruitment of target proteins to promoter regions was examined by ChIP-PCR assays. To evaluate PRMT5 and WNT antagonist expression in NHL patient samples, primary tumor samples were collected from 4 patients with MCL. Cellular growth and apoptosis was assessed by MTS proliferation assay and FACS analysis. WIF1 protein detection in cell culture media was performed by ELISA. Results: PRMT5 regulated WNT/β-CATENIN signaling by direct transcriptional repression of AXIN2 and WIF1, two proteins that negatively regulate this pathway. PRMT5 inhibition with shRNA, CRISPR/CAS9 deletion, or CMP5 led to restored expression of AXIN2 and WIF1 transcript and protein that was associated with transcriptional repression of WNT/β-CATENIN target genes CYCLIN D1, MYC, and SURVIVIN. With PRMT5 inhibition, we observed differential enhanced recruitment of co-repressors LSD and HDAC2 and loss of associated epigenetic marks H3K4Me3 and H3K9Me (associated with the LSD demethylase) and H3K9Ac and H3K14Ac (associated with HDAC2) at each b-CATENIN target promoter. PRMT5 inhibition was found to reduce recruitment of co-activators CBP and MLL1 and respective epigenetic mark H3K4me3. The de-repression of AXIN2 and WIF1 was associated with loss of phospho-AKT (S450, S473) and down-stream survival pathways. Reduction in phospho-AKT was attributed to physical association between AXIN2 and the down-stream activity of secreted WIF1 in media of lymphoma cells treated with PRMT5 inhibition or CRISPR/CAS9 PRMT5 deletion. Furthermore, reduction of phospho-AKT prevented dimerization of MLL1 leading to dissociation of the BCL9-Pygopus TCF1/b-CATENIN transcriptional activation complex at MYC, CYCLIND1, and SURVIVIN promoters. Our observations show that PRMT5 is an important epigenetic regulator that governs the expression WNT/β-CATENIN-driven oncogenes c-MYC, CYCLIND D1 and SURVIVIN. PRMT5 inhibition restores regulation at several levels that converge on AKT signaling; (i) AXIN2-AKT interaction and (ii) WIF1 inhibition of WNT signaling. The restored regulation occurs via modulation of the downstream transcriptional machinery that is supported by constitutive AKT activity. This data identifies a novel pathway to interfere with WNT/β-CATENIN signaling and validates the driver role orchestrated by PRMT5 in lymphoma. Disclosures Baiocchi: Essanex: Research Funding.

Epigenetic regulation mediated by lysine- and arginine-specific enzymes plays an essential role in tumorigenesis, and enhanced expression of the type II protein arginine methyltransferase PRMT5 as well as the polycomb repressor complex PRC2 has been associated with increased cell proliferation and survival. Here, we show that PRMT5 is overexpressed in three different types of non-Hodgkin lymphoma cell lines and clinical samples as well as in mouse primary lymphoma cells and that it up-regulates PRC2 expression through inactivation of the retinoblastoma proteins RB1 and RBL2. Although PRMT5 epigenetically controls RBL2 expression, it indirectly promotes RB1 phosphorylation through enhanced cyclin D1 expression. Furthermore, we demonstrate that PRMT5 knockdown in non-Hodgkin lymphoma cell lines and mouse primary lymphoma cells leads to RBL2 derepression and RB1 reactivation, which in turn inhibit PRC2 expression and trigger derepression of its CASP10, DAP1, HOXA5, and HRK pro-apoptotic target genes. We also show that reduced PRMT5 expression leads to cyclin D1 transcriptional repression via loss of TP53K372 methylation, which results in decreased BCL3 expression and enhanced recruitment of NF-κB p52-HDAC1 repressor complexes to the cyclin D1 promoter. These findings indicate that PRMT5 is a master epigenetic regulator that governs expression of its own target genes and those regulated by PRC2 and that its inhibition could offer a promising therapeutic strategy for lymphoma patients.

Background:The fascia iliaca compartment block (FICB) provides an analgesic effect in patients with femur fractures. However, the postoperative pain after hip surgery is different from that after femur fracture, because of the difference in the degree and location of tissue trauma. Whether F

A 48-year-old woman with cystic fibrosis and a previous left pneumonectomy had surgery planned for single lung transplantation under general anesthesia. Due to progressive dyspnea and recurrent respiratory infection, she could not maintain her normal daily life without lung transplantation. The anesthetic management and surgical procedure was expected to be difficult because of the left mediastinal shift and an asymmetric thorax after the left pneumonectomy, but the single lung transplantation was successfully done under cardiopulmonary bypass.

Inhaled nitric oxide (NO) is occasionally used to treat hypoxemia for patients with acute respiratory distress syndrome (ARDS) in the intensive care unit (ICU). However, it is controversial whether or not to maintain inhalation of NO during general anesthesia because of complications, such as nitrogen dioxide (NO2) production, methemoglobinemia, and inhibition of platelet aggregation. In this case, a 67-year-old male fell from a roof and was brought to an emergency care center. During management, he vomited gastric contents and aspirated. In spite of tracheal intubation and mechanical ventilation with high oxygen therapy, the hypoxia did not improve. NO inhalation with mechanical ventilation was performed to treat hypoxemia due to ARDS in the ICU. We maintained the NO inhalation during the surgery for a hemoperitonium. The surgery was completed without intra-operative hemodynamic instability or any complications. (Korean J Anesthesiol 2010; 58: 485-489)

In this paper, an efficient switching pattern to equalize the size of transformer is proposed for a multi-level inverter using cascaded transformers. It bases on the prior selected harmonic elimination PWM (SHEPWM) method. Because the maximum magnetic flux imposed on each transformer becomes exactly equal each other, all transformers can be designed with the same size regardless of their position. Therefore, identical full-bridge inverter units can be utilized, the modularity can be more efficient and the manufacture can be easier

Introduction: Aggressive histologic subtypes of lymphoma such as mantle cell (MCL) and activated B cell (ABC) are considered incurable and affected patients often have a short median survival despite multimodal therapy. It is well established that altered expression of oncogenes and epigenetic dysregulation of tumor suppressor and regulatory genes promote cellular transformation of normal B cells into malignant lymphoma. Hypermethylation of histone proteins (H3R8 and H4R3) by the protein arginine methyltransferase 5 (PRMT5) enzyme has been documented in multiple cancer types and has been shown to promote tumor cell growth and survival. Importantly, PRMT5 over expression does not occur in normal B cells (resting or activated) and is only detected in malignant lymphoma cells. We have previously shown that PRMT5 regulates the Polycomb-repressive complex 2 (PRC2) complex including EZH2, a core histone-lysine N methyl transferase. EZH2 has tumor suppressor functions and has been shown to regulate WNT antagonist’s gene expression. WNT/β-CATENIN signaling pathway has been associated with increased cell proliferation and survival in various forms of cancers including lymphoma. Until recently, the role of PRMT5 in controlling WNT/β-CATENIN signaling has been unclear. We hypothesized that PRMT5, through its ability to repress EZH2 expression, would control WNT/β-CATENIN signaling and orchestrate downstream pathways that are relevant to lymphomagenesis. Methods: PRMT5 inhibition of patient-derived lymphoma cell lines, primary lymphoma tumor cells and mouse primary Eμ-BRD2 transgenic lymphoma cells by infecting with sh-PRMT5 lentivirus (or sh-GFP control) or a selective small molecule PRMT5 inhibitor (tool compound CMP5). Gene expression was monitored by immunoblotting and reverse transcription (RT) real time PCR. Recruitment of target proteins to promoter regions was examined by ChIP assays. To evaluate PRMT5 and WNT antagonist expression in NHL patient samples, primary tumor samples were collected from 4 patients with MCL. Cellular growth and apoptosis was assessed by proliferation assay and FACS analysis. Results: PRMT5 supports WNT/β-CATENIN activity by direct transcriptional repression of AXIN2 and WIF1 via a PRMT5-EZH2 repressor complex. PRMT5 indirectly supports EZH2 expression via inactivation of the RB-E2F pathway. AXIN2 and WIF1 are two proteins that negatively regulate WNT/bCATENIN. Additionally, PRMT5 inhibition with shRNA or CMP5 leads to repression of the WNT/β-CATENIN signaling pathway by allowing de-repression of AXIN2 and WIF1, leading to decreased nuclear phospho-b-CATENIN and decreased transcription of the target genes CYCLIN D1, c-MYC and SURVIVIN. Reduced nuclear localization of phospho-β-catenin (S675) led to differential enhanced recruitment of co-repressors LSD and HDAC2 (and loss of epigenetic marks H3K4Me3 and H3K9Me3) and loss of activating epigenetic marks H3K9Ac and H3K14Ac on CYCLIN D1, c-MYC and SURVIVIN promoters. We also found that PRMT5 regulates target gene repression in primary blastic variant MCL patient samples and mouse primary lymphoma tumor cells. Significance: Our observations show that PRMT5 is an important epigenetic regulator that governs the expression of its own target genes, the PRC2 program as well as regulating the WNT/β-CATENIN-driven pro-growth and survival genes c-MYC, CYCLIND D1 and SURVIVIN. These results, together with the prevalence of PRMT5 and EZH2 over expression and activation of WNT targets in multiple lymphoma histologic subtypes, suggests that inhibiting PRMT5 is likely to result in removal of repressive histone arginine and lysine marks and promote restoration of normal growth and survival checkpoints in malignant lymphomas. Disclosures No relevant conflicts of interest to declare.

A 40-year-old woman was referred to our hospital because of bitemporal hemianopsia at 23 weeks of gestation. A brain magnetic resonance imaging showed a pituitary tumor having suprasellar extension. At 30 weeks of gestation, she complained of rapidly deteriorating vision and bitemporal hemianopsia in both eyes and the ensuing radiological examination revealed increased tumor size, displaced tumor location and compressed optic chiasm. The cesarean section was performed at 31 weeks and 3 days of gestation and simultaneous surgical removal of pituitary tumor was carried out due to the risk of irreversible blindness. Anesthetic management for combined cesarean section and brain surgery can be more complex and challenging for anesthesiologists, and the aim was to achieve both the control of intracranial pressure and fetal well being at the same time. In this case, maternal outcome was somewhat improved after the procedure, and neonatal complications were not detected.

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We present a patient with intractable neuropathic pain because of idiopathic transverse myelitis unresponsive to medical treatment. After a successful trial of spinal cord stimulation, a permanent stimulator was implanted. Improvement was noted in visual analogue scale, medication usage and daily function. Spinal cord stimulation may offer a therapeutic option for patients with neuropathic pain resulting from transverse myelitis and should be considered when other treatments are failed.

A tracheoesophageal fistula (TEF) was detected in a woman who received chemotherapy for acute lymphoblastic leukemia. The fistula biopsy confirmed the aspergillus infection. A large fistula was located at the lateral wall of the carina involving the proximal left main bronchus, and the orifice of left main bronchus was almost completely obstructed by white mass-like plaque. Primary repair was planned using the right thoracotomy approach. We originally planned to selectively intubate the left lung with the aid of fiberoptic bronchoscope without success. Therefore, we selectively intubated the right lung. Hypoxemia developed during surgery and the level of oxygenation was improved by selectively intubating the left bronchus from the surgical field once the defect had been exposed. We review the ventilation technique and anesthetic problems encountered in patients with a large distal TEF.

Background: The relationship between the injection speed of a local anesthetic and the success rate of unilateral spinal anesthesia has been a controversial issue. The aim of this thesis was to identify any significant effects of the drug injection speed on the success rate of unilateral spinal anesthesia. Methods: Forty patients were randomly allocated into 2 groups, group R and S. The injection speed was 4 ml/min in group R (n = 20) and 1 ml/min in group S (n = 20). Hyperbaric 0.5% bupivacaine 10 mg was injected via a syringe pump. The drug was administered at the L3-4 intervertebral space with the patient in the lateral decubitus position, which was maintained for 20 minutes after the injection. A spinal sensory block was assessed by examining the temperature sensation using an alcohol-sponge. The motor block was evaluated using the modified Bromage scale and the dependent and non-dependent sides were compared. Results: Significant differences (P ＜ 0.05) were observed in the success rate of unilateral motor paralysis (45% in group R vs 90% in group S). There were no significant blood pressure differences between the two groups 5, 10, 15, 30 and 60 minutes after injecting the hyperbaric 0.5% bupivacaine. Conclusions: The injection speed of local anesthetics is one of the crucial factors for achieving a unilateral spinal anesthesia. Therefore, it is important to maintain a slow injection speed of a local anesthetic in unilateral spinal anesthesia.