Novel Insights into the Roles of Bcl-2 Homolog Nr-13 (vNr-13) Encoded by Herpesvirus of Turkeys in the Virus Replication Cycle, Mitochondrial Networks, and Apoptosis Inhibition

ABSTRACT The Bcl-2 (B cell lymphoma 2)-related protein Nr-13 plays a major role in the regulation of cell death in developing avian B cells. With over 65% sequence similarity to the chicken Nr-13, herpesvirus of turkeys (HVT) vNr-13, encoded by the HVT079 and HVT096 genes, is the first known alphaherpesvirus-encoded Bcl-2 homolog. HVT-infected cells were reported to be relatively more resistant to serum starvation, suggested that vNr-13 could be involved in protecting the cells. Here, we describe CRISPR/Cas9-based editing of exon 1 of the HVT079 and HVT096 genes from the HVT genome to generate the mutant HVT-ΔvNr-13 to gain insights into its functional roles. Overall, wild-type HVT and HVT-ΔvNr-13 showed similar growth kinetics; however, at early time points, HVT-ΔvNr-13 showed 1.3- to 1.7-fold-lower growth of cell-associated virus and 3- to 6.2-fold-lower growth of cell-free virus. In transfected cells, HVT vNr-13 showed a mainly diffuse cytoplasmic distribution with faint nuclear staining. Further, vNr-13 localized to the mitochondria and endoplasmic reticulum (ER) and disrupted mitochondrial network morphology in the transfected cells. In the wild-type HVT-infected cells, vNr-13 expression appeared to be directly involved in the disruption of the mitochondrial network, as the mitochondrial network morphology was substantially restored in the HVT-ΔvNr-13-infected cells. IncuCyte S3 real-time apoptosis monitoring demonstrated that vNr-13 is unequivocally involved in the apoptosis inhibition, and it is associated with an increase of PFU, especially under serum-free conditions in the later stages of the viral replication cycle. Furthermore, HVT blocks apoptosis in infected cells but activates apoptosis in noninfected bystander cells. IMPORTANCE B cell lymphoma 2 (Bcl-2) family proteins play important roles in regulating apoptosis during homeostasis, tissue development, and infectious diseases. Several viruses encode homologs of cellular Bcl-2-proteins (vBcl-2) to inhibit apoptosis, which enable them to replicate and persist in the infected cells and to evade/modulate the immune response of the host. Herpesvirus of turkeys (HVT) is a nonpathogenic alphaherpesvirus of turkeys and chickens that is widely used as a live vaccine against Marek’s disease and as recombinant vaccine viral vectors for protecting against multiple avian diseases. Identical copies of the HVT genes HVT079 and HVT096 encode the Bcl-2 homolog vNr-13. While previous studies have identified the potential ability of vNr-13 in inhibiting apoptosis induced by serum deprivation, there have been no detailed investigations on the functions of vNr-13. Using CRISPR/Cas9-based ablation of the vNr-13 gene, we demonstrated the roles of HVT vNr-13 in early stages of the viral replication cycle, mitochondrial morphology disruption, and apoptosis inhibition in later stages of viral replication.

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MicroRNA-181a Inhibits Activated B-Cell-Like Diffuse Large B-Cell Lymphoma Progression by Repressing CARD11

Journal of Oncology ◽

10.1155/2019/9832956 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Danxia Zhu ◽

Cheng Fang ◽

Wenting He ◽

Chen Wu ◽

Xiaodong Li ◽

...

Keyword(s):

B Cell ◽

Binding Site ◽

Cell Lines ◽

Expression Vector ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Wild Type ◽

Protein Levels ◽

Large B Cell Lymphoma ◽

Large B Cell

We investigated the role of miR-181a in diffuse large B-cell lymphoma (DLBCL) and its potential target genes. miR-181a levels were lower in activated B-cell- (ABC-) like DLBCL cells than that in germinal center B-cell- (GCB-) like DLBCL cells. Overexpression of miR-181a in ABC-like DLBCL cell lines (OCI-LY10 and U2932) resulted in G0/G1 cell cycle arrest, increased apoptosis, and decreased invasiveness. miRNA target prediction programs (miRanda, TargetScan, and miRDB) identified caspase recruitment domain-containing protein 11 (CARD11) as a putative miR-181a target. CARD11 mRNA and protein levels were higher in the ABC-like DLBCL than that in GCB-like DLBCL. Moreover, CARD11 mRNA and protein levels were downregulated in the OCI-LY10 and U2932 cell lines overexpressing miR-181a. Dual luciferase reporter assays confirmed the miR-181a binding site in the CARD11 3′UTR region. OCI-LY10 and U2932 cells transfected with a CARD11 expression vector encoding miR-181a with a mutated binding site showed higher CARD11 protein levels, cell viability, G2/M phase cells, and invasiveness compared to those transfected with a wild-type CARD11 expression vector. Nude mice xenografted with OCI-LY10 cells with overexpressed wild-type miR-181a generated smaller tumors compared to those with overexpressed mutated binding site of CARD11 3′UTR and miR-181a. These results indicate that miR-181a inhibits ABC-like DLBCL by repressing CARD11.

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Combined EZH2 and Bcl-2 inhibitors as precision therapy for genetically defined DLBCL subtypes

Blood Advances ◽

10.1182/bloodadvances.2020002580 ◽

2020 ◽

Vol 4 (20) ◽

pp. 5226-5231

Author(s):

Hanna Scholze ◽

Regan E. Stephenson ◽

Raymond Reynolds ◽

Shivem Shah ◽

Rishi Puri ◽

...

Keyword(s):

Cell Lymphoma ◽

B Cell Lymphoma ◽

Wild Type ◽

3 Dimensional ◽

Molecular Alterations ◽

Dual Targeting ◽

Large B Cell Lymphoma ◽

Short Course ◽

Precision Therapy

Abstract Molecular alterations in the histone methyltransferase EZH2 and the antiapoptotic protein Bcl-2 frequently co-occur in diffuse large B-cell lymphoma (DLBCL). Because DLBCL tumors with these characteristics are likely dependent on both oncogenes, dual targeting of EZH2 and Bcl-2 is a rational therapeutic approach. We hypothesized that EZH2 and Bcl-2 inhibition would be synergistic in DLBCL. To test this, we evaluated the EZH2 inhibitor tazemetostat and the Bcl-2 inhibitor venetoclax in DLBCL cells, 3-dimensional lymphoma organoids, and patient-derived xenografts (PDXs). We found that tazemetostat and venetoclax are synergistic in DLBCL cells and 3-dimensional lymphoma organoids that harbor an EZH2 mutation and an IGH/BCL2 translocation but not in wild-type cells. Tazemetostat treatment results in upregulation of proapoptotic Bcl-2 family members and priming of mitochondria to BH3-mediated apoptosis, which may sensitize cells to venetoclax. The combination of tazemetostat and venetoclax was also synergistic in vivo. In DLBCL PDXs, short-course combination therapy resulted in complete remissions that were durable over time and associated with superior overall survival compared with either drug alone.

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An Activating Mutation (Ser525Pro) within the Transactivation Domain of REL in Two Patients with Human B-Cell Lymphomas Enhances REL’s In Vitro Transforming Activity.

Blood ◽

10.1182/blood.v106.11.2617.2617 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2617-2617

Author(s):

Heiko Trautmann ◽

Daniel T. Starczynowski ◽

Christiane Pott ◽

Lana Harder ◽

Norbert Arnold ◽

...

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Transactivation Domain ◽

Wild Type ◽

Spleen Cells ◽

B Cell Lymphomas ◽

Chicken Spleen ◽

Human B Cell

Abstract REL/NF-κB transcription factors are implicated in the control of apoptosis and cell growth particular in hematopoetic lineages. The REL locus at chromosomal region 2p13–16 is frequently amplified in B-cell lymphomas including diffuse-large B-cell lymphoma (DLBCL) and may play a role in lymphomagenesis. Overexpression of wild-type REL can transform chicken lymphoid cells in culture, and several experimentally-generated mutations within the REL C-terminal transactivation domain (TAD) have been previously shown to enhance REL’s transforming ability. We analysed 83 B-cell lymphomas included in the ‘Deutsche Krebshilfe’ funded network „Molecular Mechanisms in Malignant Lymphoma“ for the presence of activating mutations in the coding region of REL. We performed a systematic dHPLC screening for mutation discovery and identified an identical point mutation in two human B-cell lymphomas (a t(14;18)-positive follicular lymphoma and a mediastinal B-cell lymphoma) that changes Ser525 to Pro within the REL TAD. In the mediastinal B-cell lymphoma, the mutation in REL was proven to be of germline origin. FISH showed an amplification of the REL locus in the tumor cells of this case. Quantitative allelic discrimination of S525P indicates that the mutant REL gene was over-represented in both cases. By in vitro experiments we could show that the S525P mutation enhances the in vitro transforming ability of REL in chicken spleen cells. In addition, REL-S525P differs from wild-type REL in its ability to activate certain κB site-containing reporter plasmids in transient transfection assays. In particular, REL-S525P has a reduced ability to activate the human manganese superoxide dismutase (MnSOD) promoter in A293 cells; however, the MnSOD protein is over-expressed in REL-S525P-transformed chicken spleen cells as compared to wild-type REL-transformed cells. Ser525 of REL falls within a sequence that is similar to other known phosphorylation sites of the IκB kinase, and REL-S525P shows a reduced ability to be phosphorylated by IKKα in vitro. The S525P mutation reduces IKKα- and TNFα-stimulated transactivation by REL, as measured in GAL4 reporter assays. Furthermore, REL-S525P-transformed chicken spleen cells are more resistant to TNFα-induced cell death than cells transformed by wild-type REL. These results represent the first identification of a tumor-derived activating mutation in the REL proto-oncogene, and they suggest that the S525P mutation contributes to the development of human B-cell lymphomas by altering REL’s ability to induce target gene expression by affecting an IKKα-regulated transactivation activity.

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Lymphoma-Associated Mutations of EZH2 Result In a Change-of-Function

Blood ◽

10.1182/blood.v116.21.707.707 ◽

2010 ◽

Vol 116 (21) ◽

pp. 707-707

Author(s):

Victoria M Richon ◽

Christopher J Sneeringer ◽

Margaret Porter Scott ◽

Kevin W Kuntz ◽

Sarah K Knutson ◽

...

Keyword(s):

Cell Lymphoma ◽

Catalytic Efficiency ◽

B Cell Lymphoma ◽

Wild Type ◽

Wild Type Enzyme ◽

Malignant Phenotype ◽

H3k27 Methylation ◽

Large B Cell Lymphoma ◽

Mutant Forms ◽

Large B Cell

Abstract Abstract 707 EZH2, the catalytic subunit of the polycomb repressive complex 2 (PRC2), catalyzes the mono- through tri-methylation of lysine 27 on histone H3 (H3K27). While overexpression of EZH2 and increased H3K27 methylation have generally been associated with both hematologic malignancies and solid tumors, inactivating somatic mutations of Tyr641 (Y641F, Y641N, Y641S and Y641H) of EZH2 were recently reported to be associated with follicular lymphoma (FL) and the GCB subtype of diffuse large B-cell lymphoma (DLBCL) (Morin, Nat Genet 2010; 42: 181). In all cases, occurrence of the mutant EZH2 gene was heterozygous, and expression of both wild type and mutant alleles was detected in the mutant samples profiled by transcriptome sequencing. Further, the mutant forms of EZH2 could be incorporated into the multi-protein PRC2 complex, but the resulting complexes lacked the ability to catalyze trimethylation of an unmethylated H3K27 peptide substrate. To explore further the role of EZH2 in lymphomagenesis, we have evaluated the catalytic activity of the mutant EZH2 proteins in greater detail. Recombinant PRC2 complexes were prepared with wild type and Tyr641 mutant EZH2 forms. As previously reported, the wild type enzyme demonstrated robust activity but none of the mutant enzymes displayed significant methyltransferase activity on an unmodified H3K27 peptide. We next evaluated the activity of the enzymes using native avian erythrocyte olignucleosomes as the substrate in the reaction. In contrast to the peptide result, we found that the wild type and all of the mutant enzymes were active methyltransferases against the native nucleosome substrate. Since native nucleosome represents an admixture of the unmodified and mono-, di- and tri-methylated H3K27 we next evaluated the activity of the wild type and mutant enzymes on unmodified, and mono- and di-methylated H3K27 peptide. We demonstrate that the wild type enzyme displays greatest catalytic efficiency (kcat/K) for the zero to mono-methylation reaction of H3K27, and diminished efficiency for subsequent (mono- to di- and di- to tri-methylation) reactions. In stark contrast, the disease-associated Y641 mutants display very limited ability to perform the first methylation reaction, but have enhanced catalytic efficiency for the subsequent reactions, relative to WT-enzyme. Catalytic coupling between the mutant EZH2 species and PRC2 complexes containing either wild type EZH2 or wild type EZH1 are predicted to augment H3K27 trimethylation and thus produce the malignant phenotype associated with mutant heterozygosity. To test this prediction, the level of H3K27 methylation was evaluated in lymphoma cell lines harboring only wild type EZH2 (OCI-LY-19) or heterozygous for EZH2 Y641N (DB, KARPAS and SU-DHL-6) or EZH2 Y641F (WSU-DLCL2) by immunoblotting. As predicted by simulations, the level of H3K27 trimethylation was elevated in all of the lymphoma cell lines harboring the mutant EZH2 relative the wild type. Additionally, we observe decreased H3K27 dimethylation and monomethylation in the cells harboring the mutated EZH2 relative to wild type enzyme; these reductions in di- and monomethylation are likewise consistent with expectations based on steady state kinetic simulations. The present results imply that the malignant phenotype of follicular lymphoma and diffuse large B cell lymphoma of the GCB subtype, associated with expression of mutant forms of EZH2, results from of an overall gain-of-function with respect to formation of the trimethylated form of H3K27. These data suggest that selective, small molecule inhibitors of EZH2 enzymatic function may form a rational underpinning for molecularly targeted therapeutics against mutant-harboring lymphomas and other malignancies in which EZH2 gain-of-function is pathogenic. Disclosures: Richon: Epizyme, Inc: Employment. Sneeringer:Epizyme: Employment. Porter Scott:Epizyme, Inc: Employment. Kuntz:Epizyme, Inc: Employment. Knutson:Epizyme, Inc.: Employment. Pollock:Epizyme, Inc: Employment. Copeland:Epizyme, Inc: Employment.

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Recurrent Mutations of the Exportin 1 Gene (XPO1) in Primary Mediastinal B-Cell Lymphoma: A Lysa Study

Blood ◽

10.1182/blood.v126.23.129.129 ◽

2015 ◽

Vol 126 (23) ◽

pp. 129-129 ◽

Cited By ~ 1

Author(s):

Fabrice Jardin ◽

Anais Pujals ◽

Laura Pelletier ◽

Elodie Bohers ◽

Vincent Camus ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Cell Lines ◽

Nuclear Export ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Wild Type ◽

U2os Cells ◽

Aggressive B Cell Lymphoma

Abstract Background and aim of the study Primary mediastinal B-cell lymphoma (PMBL) is an entity of aggressive B-cell lymphoma that is clinically and biologically distinct from the other molecular subtypes of diffuse large B-cell lymphoma (DLBCL). We recently detected by Whole exome sequencing a recurrent point mutation in the XPO1 (exportin 1) gene (also referred to as chromosome region maintenance 1; CRM1), which resulted in the Glu571Lys (p.E571K) missense substitution in 2 refractory/relapsed PMBL (Dubois et al., ICML 2015; Mareschal et al. AACR 2015). XPO1 is a member of the Karyopherin-b superfamily of nuclear transport proteins. XPO1 mediates the nuclear export of numerous RNAs and cellular regulatory proteins, including tumor suppressor proteins. This mutation is in the hydrophobic groove of XPO1 that binds to the leucine-rich nuclear export signal (NES) of cargo proteins. In this study, we investigated the prevalence, specificity, and biological / clinical relevance of XPO1 mutations in PMBL. Patients and methods High-throughput targeted or Sanger sequencing of 117 PMBL patients and 3 PMBL cell lines were performed. PMBL cases were defined either molecularly by gene expression profile (mPMBL cohort) or by standard histological method (hPMBL cohort) and enrolled in various LYSA (LYmphoma Study Association) clinical trials. To assess the frequency and specificity of XPO1 mutations, cases of classical Hodgkin lymphoma (cHL) and primary mediastinal grey zone lymphoma (MGZL) were analysed. Cell experiments were performed to assess the impact of the E571 mutation on the activity of selective inhibitor of nuclear export (SINE) molecules. Results XPO1 mutations were present in 28/117 (24%) PMBL cases but were rare in cHL cases (1/19, 5%) and absent from MGZL cases (0/20). A higher prevalence (50%) of the recurrent codon 571 variant (p.E571K) was observed in PMBL cases defined by gene expression profiling (n = 32), as compared to hPMBL cases (n = 85, 13%). No difference in age, International Prognostic Index (IPI) or bulky mass was observed between the PMBL patients harboring mutant and wild-type XPO1 in the overall cohort whereas a female predominance was noticed in the mPMBL cohort. Based on a median follow-up duration of 42 months, XPO1 mutant patients exhibited significantly decreased PFS (3y PFS = 74% [CI95% 55-100]) compared to wild-type patients (3y PFS = 94% [CI95% 83-100], p=0.049) in the mPMBL cohort. In 4/4 tested cases, the E571K variant was also detected in cell-free circulating plasmatic DNA, suggesting that the mutation can be used as a biomarker at the time of diagnosis and during follow-up. Importantly, the E571K variant was detected as a heterozygous mutation in MedB-1, a PMBL-derived cell line, whereas the two other PMBL cell lines tested, Karpas1106 and U-2940, did not display any variants in XPO1 exon 15. KPT-185, the SINE compound that blocks XPO1-dependent nuclear export, induced a dose-dependent decrease in cell proliferation and increased cell death in the PMBL cell lines harbouring wild type or mutated alleles. To test directly if XPO1 mutation from E571 to E571K alters XPO1 inhibition by SINE compounds, the mutated protein was tested in vitro. The E571XPO1 mutated allele was transiently transfected into osteosarcoma U2OS cells which stably express the fluorescently labelled XPO1 cargo REV. Cells were treated with the clinical SINE compound selinexor, which is currently in phase I/II clinical trials and nuclear localization of REV-GFP was analysed in red transfected cells. The results showed that the nuclear export of the mutated XPO1 protein was inhibited by selinexor similarly to the wild-type XPO1 protein (Figure 1). Conclusion Although the oncogenic properties of XPO1 mutations remain to be determined, their recurrent selection in PMBL strongly supports their involvement in the pathogenesis of this curable aggressive B-cell lymphoma. XPO1 mutations were primarily observed in young female patients who displayed a typical PMBL molecular signature. The E571K XPO1 mutation represents a novel hallmark of PMBL but does not seem to interfere with SINE activity. Rev-GFP (green fluorescent) expressing U2OS cells were transfected with wild type XPO1-RFP (red fluorescent protein), XPO1-C528S-RFP, XPO1-E571K-mCherry, and XPO1-E571G-mCherry. The cells were then treated with 1µM KPT-330 for 8 hours. Figure 1. Rev-GFP expressing U2OS cells transfected with XPO1 variants. Figure 1. Rev-GFP expressing U2OS cells transfected with XPO1 variants. Disclosures Landesman: Karyopharm Therapeutics: Employment. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Argueta:Karyopharm Therapeutics: Employment. Milpied:Celgene: Honoraria, Research Funding.

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Role of Genetic Polymorphisms on Response to R-Chopregimen in Diffuse Large B-Cell Lymphoma Patients: An Interim Analysis of a Multicenter Prospective Pharmacogenetic Study

Blood ◽

10.1182/blood.v126.23.2483.2483 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2483-2483

Author(s):

Luigi Rigacci ◽

Gabriele Perrone ◽

Stefania Nobili ◽

Sofia Kovalchuk ◽

Benedetta Puccini ◽

...

Keyword(s):

Interim Analysis ◽

Cell Lymphoma ◽

Risk Group ◽

B Cell Lymphoma ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Pharmacokinetics And Pharmacodynamics ◽

Bulky Disease ◽

Pathological Characteristics ◽

Large B Cell Lymphoma

Abstract Introduction: Standard chemotherapy represented by the R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) regimen is successful in about 60% of patients (pts) with diffuse large B-cell lymphoma (DLBCL). Pts who do not benefit from this treatment, due to the development of tumor drug resistance, have a very poor prognosis. Currently, knowledge on reasons of treatment related failures in DLBCL are scanty and predictive biomarker of response are largely unknown. We hypothesized that polymorphisms of gene involved in the pharmacokinetics and pharmacodynamics of drugs included in R-CHOP regimen may play a role in predicting the outcome in DLBCL pts.Thus, we designed a multicentre prospective pharmacogenetic trial aimed at identifying gene polymorphisms potentially predictive of drug efficacy/resistance in DLBCL pts treated with R-CHOP. An interim analysis on the first 80 enrolled ptswas planned and has been performed. Methods: The study included chemonaive DLBCL pts at various stages of disease candidate to an R-CHOP standard treatment. The Ethical Committee of each participating centre approved the pharmacogenetic protocol, and all pts signed a written informed consent. According to the aims of this interim analysis, the impact of single nucleotide polymorphisms (SNPs) on R-CHOP efficacy was evaluated by objective response (OR) rate at the end of treatment. The efficacy of R-CHOP was evaluated according to the Cheson criteria by performing standard hematochemical and instrumental (TC and FDFG-PET) tests and defining complete remission (CR), partial remission (PR), non response or progressive disease (PD). Genomic DNA wasextracted from peripheral blood of 80 pts. Twentysingle nucleotide polymorphisms (SNPs) from18candidate genes (ABCB1, ABCC1, ABCC2, ABCG2, CYBA, CYP2C9, FCGR2A, GSTP1, IL2, MLH1, NCF4, NQO1, NQO2, RAC2, TNF, TOP2A, TP53, TUBB)involved in pharmacokinetics and pharmacodynamics of R-CHOP (www.pharmgkb.org) have been analysed by a genotyping array based on Affimetrix methodology. Univariate analysis was performed to evaluate associations between polymorphisms and clinical/pathological characteristics or OR (Fisher exact test). Multivariate logistic regression analysis was performed to estimate adjusted odds ratios along with the corresponding 95% confidence intervals for the polymorphisms and OR. Results: Median age was 63 years. There were 37 men and 43 women. 47.5 % of pts were in stage I-II,52.5 % of pts in stage III-IV. 27.5% of ptshad bulky disease, 43.8 % of pts had involvement of extranodal site. 47.5% of pts had pathological LDH value. According to the revised IPI, 15 % pts were in the low risk group, 58.7 % in the intermediate risk group, and 26.3 % in the high risk group.Overall, 468 courses of R-CHOP had been administered (mean: 5.85 courses, range: 4-6). 81% of pts had CR to R-CHOP whereas the remaining showed PR (14%) or PD(5%). No statistically significant correlation was found between OR and clinical characteristics of pts.However, stage III-IV pts showed a worst OR than stage I-II pts (77% vs 87% of CR, respectively); pts with bulky disease had worst OR than non-bulky disease pts(73% vs 84.5% of CR, respectively); ptswith R-IPI 3-5 a worst OR than pts with R-IPI 0-2 (71.5% vs 85% of CR, respectively). Univariate and multivariateanalysis identified TOPOII rs13695as a predictor of OR (p=0.042). Pts with CT or TT genotypesshowed worst OR than CC wild-type homozygous pts (odds ratio 3.070, CI95% 1.113-13.457). Also, a statistical trend toward significance was observed for MLH1 rs1800734 polymorphism (p=0.062): ptswith homozygous genotype for the mutant allele showed a better OR than wild-type and heterozygous pt genotypes. Conclusions: No significant relationship between clinical/pathological characteristics and OR was observed. Our preliminary data show that SNPs affecting a gene involved in doxorubicin pharmacodynamics, i.e. the drug target TOPOII, as well asone of the major components of DNA mismatch repair, i.e. MLH1 gene,may predict response in DLBCL pts treated with R-CHOP. These preliminary results from the interim analysis are promising and warrant completion of pt accrual to reach the planned number of cases at the end of our study. Acknowledgments This work was supported by a grant from the Associazione Giacomo Onlus, Castiglioncello (LI), Italy to E.M. and Cassa di Risparmio di Firenze, Firenze, Italy to S.N. Disclosures No relevant conflicts of interest to declare.

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Human Cytomegalovirus pUS24 Is a Virion Protein That Functions Very Early in the Replication Cycle

Journal of Virology ◽

10.1128/jvi.00399-06 ◽

2006 ◽

Vol 80 (17) ◽

pp. 8371-8378 ◽

Cited By ~ 22

Author(s):

Xuyan Feng ◽

Jörg Schröer ◽

Dong Yu ◽

Thomas Shenk

Keyword(s):

Human Cytomegalovirus ◽

Mutant Virus ◽

Replication Cycle ◽

Immediate Early ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Virion Protein ◽

Viral Dna ◽

Infected Cells

ABSTRACT We have characterized the function of the human cytomegalovirus US24 gene, a US22 gene family member. Two US24-deficient mutants (BADinUS24 and BADsubUS24) exhibited a 20- to 30-fold growth defect, compared to their wild-type parent (BADwt), after infection at a relatively low (0.01 PFU/cell) or high (1 PFU/cell) input multiplicity. Representative virus-encoded proteins and viral DNA accumulated with normal kinetics to wild-type levels after infection with mutant virus when cells received equal numbers of mutant and wild-type infectious units. Further, the proteins were properly localized and no ultrastructural differences were found by electron microscopy in mutant-virus-infected cells compared to wild-type-virus-infected cells. However, virions produced by US24-deficient mutants had a 10-fold-higher genome-to-PFU ratio than wild-type virus. When infections were performed using equal numbers of input virus particles, the expression of immediate-early, early, and late viral proteins was substantially delayed and decreased in the absence of US24 protein. This delay is not due to inefficient virus entry, since two tegument proteins and viral DNA moved to the nucleus equally well in mutant- and wild-type-virus-infected cells. In summary, US24 is a virion protein and virions produced by US24-deficient viruses exhibit a block to the human cytomegalovirus replication cycle after viral DNA reaches the nucleus and before immediate-early mRNAs are transcribed.

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Clinical Significance of MYD88 Mutation in Patients with Diffuse Large Cell Lymphoma

Blood ◽

10.1182/blood.v126.23.3881.3881 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3881-3881 ◽

Cited By ~ 2

Author(s):

Ritsuko Seki ◽

Takashi Okamura ◽

Koteda Satoko ◽

Kuniki Kawaguchi ◽

Kei Noumura ◽

...

Keyword(s):

Lymph Nodes ◽

B Cell ◽

Clinical Significance ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Wild Type ◽

Probe System ◽

Allele Specific ◽

Myd88 L265p Mutation ◽

Myd88 L265p

Abstract Mutation of the MYD88 has recently been identified in activated B cell like diffuse large B cell lymphoma (DLBCL) and enhanced cell proliferation systems such as JAK-STAT and NF-kB signaling pathways. However, much remains unclear about its clinical significance. In this study, we developed a highly sensitive and an automatic method utilizing guanine-quenching probes (QP) to detect mutation and investigated the relationship between MYD88 L265P mutation and clinical significance. We amplify a DNA fragment including the mutation to intend for by PCR and associate it with Q-probe with complementary sequence, using the temperature that Q-probe dissociates varying according to a conformity degree of the complementarity sequence. We judge it by detecting the fluorescence to be provided by dissociation. Results were obtained from 1ul of DNA solution(10ng) within 90 min by the method. Detected mutations were identical between QP method and allele-specific PCR (AS-PCR).Eighty-nine patients with a diagnosis of de novo DLBCL made between 1999 and 2014, and treated with CHOP or R-CHOP therapy. We retrospectively analyzed the outcome of 89 patients (age range; 21-88 and 59% were female). The median follow-up time was 4.4 y. Survival analyses were performed using the Kaplan-Meier method. None of the patients had a known history of human immunodeficiency virus infection. MYD88 L265P mutation was both assessed by Q-probe system that can detect low levels of mutant DNA and allele-specific TaqMan polymerase chain reaction assay. We performed the direct sequence method using 3130 Applied Biosystem Genetic Analyzer as antithesis. The cell-of-origin was determined based on immunohistochemical (IHC) stains for CD10, BCL-6 and MUM-1 by Hans' algorithm. MYD88 L265Pmutation was detected in 25.8% (23/89) in various tissues of DLBCL. MYD88 mutations occurred more frequently in males (P<0.05), cases without B symptoms (P<0.05). MYD88 mutation was infrequent in DLBCL arising in lymph nodes (10.6%), but more frequently found in extranodal sites such as testes (83%, 5/6), nasal (75%,9/12), central nervous system (50%,2/4), and leg (100%,1/1). In agreement with recent studies, we found no mutated cases among gastric cases. As somatic mutations in MYD88 was reported to be the most frequent alterations found in non-GCB type, we further analyzed GCB or non-GCB type by IHC. MYD88 mutations were predominantly observed in the non-GCB type (74%, 17/23), compared with 26%, 6/23 in GCB type. Overall survival (OS) for 3 years were 84.2% and 70.2% in patients with wild-type MYD88 and in MYD88 mutation group (P=0.366), respectively. Progression-free survival (PFS) for 3 years, 76.9% and 64.3% in patients with wild type and in mutated group (P=0.156), respectively. However, all four cases with CNS relapse had this mutation, 2 originated from testis, and remained 2 from lymph nodes. Our results confirm the remarkable site-specific occurrence of MYD88 mutation. In addition, Q-probe system for detection of MYD88 mutation was very useful because of its sensitivity and in the case who obtained only a small amount of biopsy specimen. MYD88 L265Ppromotes survival of malignant lymphoid cells through several mechanisms. Further large scale study should be necessary for more understanding of biological and clinical significance of DLBCL patients with MYD88 mutation. Disclosures No relevant conflicts of interest to declare.

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Recurrent DNA Mutations In Non-Hodgkin Lymphomas Reveal Candidate Therapeutic Targets

Blood ◽

10.1182/blood.v116.21.632.632 ◽

2010 ◽

Vol 116 (21) ◽

pp. 632-632

Author(s):

Andrew J Mungall ◽

Ryan D Morin ◽

Jianghong An ◽

Oleksandr Yakovenko ◽

Merrill Boyle ◽

...

Keyword(s):

Cell Proliferation ◽

B Cell ◽

Cell Lines ◽

Small Molecule ◽

Cell Lymphoma ◽

Three Dimensional ◽

Therapeutic Targets ◽

B Cell Lymphoma ◽

Wild Type ◽

Proliferation Assays

Abstract Abstract 632 Introduction: Non-Hodgkin lymphomas (NHL) are the most common type of lymphoma and can be broadly classified as indolent (slow-growing) diseases, progressing over many years; and aggressive (fast-growing) diseases, which progress rapidly. The latter class includes diffuse large B-cell lymphoma (DLBCL), which accounts for approximately 30% of all NHL diagnoses. Three DLBCL subtypes have been identified based on gene expression profiling, namely: germinal center B-cell (GCB), activated B-cell (ABC) and primary mediastinal B-cell lymphoma (PMBCL). These subtypes show substantial differences in response to treatment and ultimate disease outcome, suggesting that molecular subtyping is an important prognostic indicator and that each subtype may benefit from a distinct treatment regimen. Despite recent advances in cancer genomics revealing molecular and mutational differences between these subtypes, further studies focused on the common NHL subtypes are required to identify critical players in the pathogenesis of DLBCL that may be targeted by pharmacological intervention to improve patient outcome. Methods: Using ultra-high throughput whole genome shotgun sequencing (WGSS) and whole transcriptome shotgun sequencing (WTSS/RNA-seq) we have discovered protein-coding mutations in NHL genomes. With a focus on recurrent and likely gain-of-function mutations we have established procedures to model the three-dimensional structures of mutant proteins and using a computational “molecular docking” pipeline have identified candidate molecules with specificity for the mutant protein. These small molecule compounds are acquired and tested in cell proliferation assays against a suite of DLBCL cell lines characterized for target mutations. Results: Mutations affecting a single key tyrosine in the catalytic site of enhancer of zeste, homolog 2 (EZH2), a member of the Polycomb-group family involved in transcriptional repression were identified (Morin, R. et al. 2010 Nature Genetics 42(2):181-5). This mutation, in a gene previously unknown to be mutated in cancer, is restricted to the GCB subtype of lymphomas and is highly prevalent in patient samples and DLBCL cell lines. Mutations have also been observed in other proteins involved in epigenetic regulation and thus afford potentially novel therapeutic targets. In proof-of-principle experiments small molecule inhibitors were identified using molecular docking approaches to target the effect of EZH2 mutations in both mutant and wild-type DLBCL cell lines. We identified and imported 96 compounds from the Developmental Therapeutic Program NCI/NIH repository. These compounds were tested in alamarBlue cell proliferation assays revealing three with activity at 10uM concentration in EZH2 mutant but not wild-type cells. Computational optimization of these compounds is underway to identify related compounds with improved activities at reduced concentrations. Conclusions: High-throughput sequencing platforms have enabled the identification of recurrent, non-synonymous protein mutations in tumor genomes and transcriptomes. Such a catalogue of mutations provides new avenues of exploration for targeted therapy including small molecule inhibitors. Despite intensive efforts launched in recent years to determine the crystal structure for every human protein, many (including EZH2) do not currently have three dimensional structures. This poses a challenge to novel drug discovery but can be overcome using homology modeling and/or targeting other members of a pathway. Our observations also demonstrate the importance of epigenetic regulation in NHL tumorigenesis and thus provide potential new therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

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