73 results on '"R. Shaknovich"'
Search Results
2. Mapping and Mutagenesis of the Amino-Terminal Transcriptional Repression Domain of the Drosophila Krüppel Proteint†
- Author
-
Martha J. Grossel, Jonathan D. Licht, M.A. English, Ulla Hansen, R Shaknovich, Wendy Hanna-Rose, M. Ro, and J C Reddy
- Subjects
Protein Folding ,Molecular Sequence Data ,Kruppel-Like Transcription Factors ,Repressor ,Biology ,Polymerase Chain Reaction ,Protein Structure, Secondary ,Animals ,Drosophila Proteins ,Point Mutation ,Amino Acid Sequence ,Psychological repression ,Peptide sequence ,Molecular Biology ,DNA Primers ,chemistry.chemical_classification ,Alanine ,Base Sequence ,Cell Biology ,Amino acid ,N-terminus ,DNA-Binding Proteins ,Repressor Proteins ,Mutagenesis, Insertional ,Biochemistry ,chemistry ,Mutagenesis, Site-Directed ,Protein folding ,Drosophila ,Drosophila Protein ,Plasmids ,Transcription Factors ,Research Article - Abstract
We previously demonstrated that the Drosophila Krüppel protein is a transcriptional repressor with separable DNA-binding and transcriptional repression activities. In this study, the minimal amino (N)-terminal repression region of the Krüppel protein was defined by transferring regions of the Krüppel protein to a heterologous DNA-binding protein, the lacI protein. Fusion of a predicted alpha-helical region from amino acids 62 to 92 in the N terminus of the Krüppel protein was sufficient to transfer repression activity. This putative alpha-helix has several hydrophobic surfaces, as well as a glutamine-rich surface. Mutants containing multiple amino acid substitutions of the glutamine residues demonstrated that this putative alpha-helical region is essential for repression activity of a Krüppel protein containing the entire N-terminal and DNA-binding regions. Furthermore, one point mutant with only a single glutamine on this surface altered to lysine abolished the ability of the Krüppel protein to repress, indicating the importance of the amino acid at residue 86 for repression. The N terminus also contained an adjacent activation region localized between amino acids 86 and 117. Finally, in accordance with predictions from primary amino acid sequence similarity, a repression region from the Drosophila even-skipped protein, which was six times more potent than that of the Krüppel protein in the mammalian cells, was characterized. This segment included a hydrophobic stretch of 11 consecutive alanine residues and a proline-rich region.
- Published
- 1994
3. Reduced and altered DNA-binding and transcriptional properties of the PLZF-retinoic acid receptor-alpha chimera generated in t(11;17)-associated acute promyelocytic leukemia
- Author
-
J D, Licht, R, Shaknovich, M A, English, A, Melnick, J Y, Li, J C, Reddy, S, Dong, S J, Chen, A, Zelent, and S, Waxman
- Subjects
Gene Rearrangement ,Base Sequence ,Transcription, Genetic ,Receptors, Retinoic Acid ,Chromosomes, Human, Pair 11 ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Kruppel-Like Transcription Factors ,DNA ,Translocation, Genetic ,DNA-Binding Proteins ,Leukemia, Promyelocytic, Acute ,Humans ,Promyelocytic Leukemia Zinc Finger Protein ,Chromosomes, Human, Pair 17 ,Transcription Factors - Abstract
Acute promyelocytic leukemia (APL) associated with chromosomal rearrangement t(11;17) is a distinct syndrome which, unlike typical t(15;17) APL, fails to respond to all-trans retinoic acid (ATRA) therapy. In t(11;17) the PLZF gene, encoding a Krüppel-like zinc finger protein, is fused to the retinoic acid receptor-alpha (RAR alpha) gene, yielding two classes of chimeric proteins. PLZF protein was found in the nucleus in a punctate speckled pattern that differed from the nuclear body expression pattern of the PML protein affected in t(15;17) APL. The reciprocal PLZF-RAR alpha and RAR alpha-PLZF fusion proteins were localized to the nucleus both in the presence and absence of ATRA. PLZF-RAR alpha, in combination with the retinoid X receptor (RXR) bound to a retinoic acid-responsive element (RARE) less efficiently than RAR alpha and formed multimeric DNA-protein complexes. PLZF-RAR alpha stimulated ATRA-dependent transcription of RARE-containing reporter genes with diminished activity compared to wild-type RAR alpha. In addition, PLZF-RAR alpha antagonized the function of coexpressed wild-type RAR alpha, an effect relieved by over-expression of RXR. Leukemogenesis in t(11;17) APL may be related to interference with ATRA-mediated differentiation due to sequestration of RXR by the PLZF-RAR alpha chimera. However, disruption of the function of the myeloid-specific PLZF protein may also play an important role.
- Published
- 1996
4. Author Correction: A purine scaffold Hsp90 inhibitor destabilizes BCL-6 and has specific antitumor activity in BCL-6-dependent B cell lymphomas.
- Author
-
Cerchietti LC, Lopes EC, Yang SN, Hatzi K, Bunting KL, Tsikitas LA, Mallik A, Robles AI, Walling J, Varticovski L, Shaknovich R, Bhalla KN, Chiosis G, and Melnick A
- Published
- 2024
- Full Text
- View/download PDF
5. Single-cell spatial analysis of tumor immune architecture in diffuse large B-cell lymphoma.
- Author
-
Colombo AR, Hav M, Singh M, Xu A, Gamboa A, Lemos T, Gerdtsson E, Chen D, Houldsworth J, Shaknovich R, Aoki T, Chong L, Takata K, Chavez EA, Steidl C, Hicks J, Kuhn P, Siddiqi I, and Merchant A
- Subjects
- Hepatitis A Virus Cellular Receptor 2, Humans, Spatial Analysis, Tumor Microenvironment genetics, Hodgkin Disease, Lymphoma, Large B-Cell, Diffuse pathology
- Abstract
Multiplexed immune cell profiling of the tumor microenvironment (TME) in cancer has improved our understanding of cancer immunology, but complex spatial analyses of tumor-immune interactions in lymphoma are lacking. Here, we used imaging mass cytometry (IMC) on 33 cases of diffuse large B-cell lymphoma (DLBCL) to characterize tumor and immune cell architecture and correlate it to clinicopathological features such as cell of origin, gene mutations, and responsiveness to chemotherapy. To understand the poor response of DLBCL to immune checkpoint inhibitors (ICI), we compared our results to IMC data from Hodgkin lymphoma, a cancer highly responsive to ICI, and observed differences in the expression of PD-L1, PD-1, and TIM-3. We created a spatial classification of tumor cells and identified tumor-centric subregions of immune activation, immune suppression, and immune exclusion within the topology of DLBCL. Finally, the spatial analysis allowed us to identify markers such as CXCR3, which are associated with penetration of immune cells into immune desert regions, with important implications for engineered cellular therapies. This is the first study to integrate tumor mutational profiling, cell of origin classification, and multiplexed immuno-phenotyping of the TME into a spatial analysis of DLBCL at the single-cell level. We demonstrate that, far from being histopathologically monotonous, DLBCL has a complex tumor architecture, and that changes in tumor topology can be correlated with clinically relevant features. This analysis identifies candidate biomarkers and therapeutic targets such as TIM-3, CCR4, and CXCR3 that are relevant for combination treatment strategies in immuno-oncology and cellular therapies., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)
- Published
- 2022
- Full Text
- View/download PDF
6. Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology.
- Author
-
Deveson IW, Gong B, Lai K, LoCoco JS, Richmond TA, Schageman J, Zhang Z, Novoradovskaya N, Willey JC, Jones W, Kusko R, Chen G, Madala BS, Blackburn J, Stevanovski I, Bhandari A, Close D, Conroy J, Hubank M, Marella N, Mieczkowski PA, Qiu F, Sebra R, Stetson D, Sun L, Szankasi P, Tan H, Tang LY, Arib H, Best H, Burgher B, Bushel PR, Casey F, Cawley S, Chang CJ, Choi J, Dinis J, Duncan D, Eterovic AK, Feng L, Ghosal A, Giorda K, Glenn S, Happe S, Haseley N, Horvath K, Hung LY, Jarosz M, Kushwaha G, Li D, Li QZ, Li Z, Liu LC, Liu Z, Ma C, Mason CE, Megherbi DB, Morrison T, Pabón-Peña C, Pirooznia M, Proszek PZ, Raymond A, Rindler P, Ringler R, Scherer A, Shaknovich R, Shi T, Smith M, Song P, Strahl M, Thodima VJ, Tom N, Verma S, Wang J, Wu L, Xiao W, Xu C, Yang M, Zhang G, Zhang S, Zhang Y, Shi L, Tong W, Johann DJ Jr, Mercer TR, and Xu J
- Subjects
- High-Throughput Nucleotide Sequencing methods, Humans, Limit of Detection, Practice Guidelines as Topic, Reproducibility of Results, Circulating Tumor DNA genetics, Medical Oncology, Neoplasms genetics, Precision Medicine, Sequence Analysis, DNA standards
- Abstract
Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments and proficiency testing on standardized, cell-line-derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas, below this limit, detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false negatives) were more common than erroneous candidates (false positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best practice guidelines and provides a resource for precision oncology., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)
- Published
- 2021
- Full Text
- View/download PDF
7. Prognostic Significance of Blood-Based Multi-cancer Detection in Plasma Cell-Free DNA.
- Author
-
Chen X, Dong Z, Hubbell E, Kurtzman KN, Oxnard GR, Venn O, Melton C, Clarke CA, Shaknovich R, Ma T, Meixiong G, Seiden MV, Klein EA, Fung ET, and Liu MC
- Subjects
- Aged, Female, Follow-Up Studies, Humans, Longitudinal Studies, Male, Middle Aged, Neoplasms mortality, Prognosis, Survival Rate, Circulating Tumor DNA blood, Early Detection of Cancer methods, Neoplasms blood
- Abstract
Purpose: We recently reported the development of a cell-free DNA (cfDNA) targeted methylation (TM)-based sequencing approach for a multi-cancer early detection (MCED) test that includes cancer signal origin prediction. Here, we evaluated the prognostic significance of cancer detection by the MCED test using longitudinal follow-up data., Experimental Design: As part of a Circulating Cell-free Genome Atlas (CCGA) substudy, plasma cfDNA samples were sequenced using a TM approach, and machine learning classifiers predicted cancer status and cancer signal origin. Overall survival (OS) of cancer participants in the first 3 years of follow-up was evaluated in relation to cancer detection by the MCED test and clinical characteristics., Results: Cancers not detected by the MCED test had significantly better OS ( P < 0.0001) than cancers detected, even after accounting for other covariates, including clinical stage and method of clinical diagnosis (i.e., standard-of-care screening or clinical presentation with signs/symptoms). Additionally, cancers not detected by the MCED test had better OS than was expected when data were adjusted for age, stage, and cancer type from the Surveillance, Epidemiology, and End Results (SEER) program. In cancers with current screening options, the MCED test also differentiated more aggressive cancers from less aggressive cancers ( P < 0.0001)., Conclusions: Cancer detection by the MCED test was prognostic beyond clinical stage and method of diagnosis. Cancers not detected by the MCED test had better prognosis than cancers detected and SEER-based expected survival. Cancer detection and prognosis may be linked by the underlying biological factor of tumor fraction in cfDNA., (©2021 The Authors; Published by the American Association for Cancer Research.)
- Published
- 2021
- Full Text
- View/download PDF
8. The PATHFINDER Study: Assessment of the Implementation of an Investigational Multi-Cancer Early Detection Test into Clinical Practice.
- Author
-
Nadauld LD, McDonnell CH 3rd, Beer TM, Liu MC, Klein EA, Hudnut A, Whittington RA, Taylor B, Oxnard GR, Lipson J, Lopatin M, Shaknovich R, Chung KC, Fung ET, Schrag D, and Marinac CR
- Abstract
To examine the extent of the evaluation required to achieve diagnostic resolution and the test performance characteristics of a targeted methylation cell-free DNA (cfDNA)-based multi-cancer early detection (MCED) test, ~6200 participants ≥50 years with (cohort A) or without (cohort B) ≥1 of 3 additional specific cancer risk factors will be enrolled in PATHFINDER (NCT04241796), a prospective, longitudinal, interventional, multi-center study. Plasma cfDNA from blood samples will be analyzed to detect abnormally methylated DNA associated with cancer (i.e., cancer "signal") and a cancer signal origin (i.e., tissue of origin). Participants with a "signal detected" will undergo further diagnostic evaluation per guiding physician discretion; those with a "signal not detected" will be advised to continue guideline-recommended screening. The primary objective will be to assess the number and types of subsequent diagnostic tests needed for diagnostic resolution. Based on microsimulations (using estimates of cancer incidence and dwell times) of the typical risk profiles of anticipated participants, the median (95% CI) number of participants with a "signal detected" result is expected to be 106 (87-128). Subsequent diagnostic evaluation is expected to detect 52 (39-67) cancers. The positive predictive value of the MCED test is expected to be 49% (39-58%). PATHFINDER will evaluate the integration of a cfDNA-based MCED test into existing clinical cancer diagnostic pathways. The study design of PATHFINDER is described here.
- Published
- 2021
- Full Text
- View/download PDF
9. Cross-oncopanel study reveals high sensitivity and accuracy with overall analytical performance depending on genomic regions.
- Author
-
Gong B, Li D, Kusko R, Novoradovskaya N, Zhang Y, Wang S, Pabón-Peña C, Zhang Z, Lai K, Cai W, LoCoco JS, Lader E, Richmond TA, Mittal VK, Liu LC, Johann DJ Jr, Willey JC, Bushel PR, Yu Y, Xu C, Chen G, Burgess D, Cawley S, Giorda K, Haseley N, Qiu F, Wilkins K, Arib H, Attwooll C, Babson K, Bao L, Bao W, Lucas AB, Best H, Bhandari A, Bisgin H, Blackburn J, Blomquist TM, Boardman L, Burgher B, Butler DJ, Chang CJ, Chaubey A, Chen T, Chierici M, Chin CR, Close D, Conroy J, Cooley Coleman J, Craig DJ, Crawford E, Del Pozo A, Deveson IW, Duncan D, Eterovic AK, Fan X, Foox J, Furlanello C, Ghosal A, Glenn S, Guan M, Haag C, Hang X, Happe S, Hennigan B, Hipp J, Hong H, Horvath K, Hu J, Hung LY, Jarosz M, Kerkhof J, Kipp B, Kreil DP, Łabaj P, Lapunzina P, Li P, Li QZ, Li W, Li Z, Liang Y, Liu S, Liu Z, Ma C, Marella N, Martín-Arenas R, Megherbi DB, Meng Q, Mieczkowski PA, Morrison T, Muzny D, Ning B, Parsons BL, Paweletz CP, Pirooznia M, Qu W, Raymond A, Rindler P, Ringler R, Sadikovic B, Scherer A, Schulze E, Sebra R, Shaknovich R, Shi Q, Shi T, Silla-Castro JC, Smith M, López MS, Song P, Stetson D, Strahl M, Stuart A, Supplee J, Szankasi P, Tan H, Tang LY, Tao Y, Thakkar S, Thierry-Mieg D, Thierry-Mieg J, Thodima VJ, Thomas D, Tichý B, Tom N, Garcia EV, Verma S, Walker K, Wang C, Wang J, Wang Y, Wen Z, Wirta V, Wu L, Xiao C, Xiao W, Xu S, Yang M, Ying J, Yip SH, Zhang G, Zhang S, Zhao M, Zheng Y, Zhou X, Mason CE, Mercer T, Tong W, Shi L, Jones W, and Xu J
- Subjects
- DNA Copy Number Variations, Genetic Testing standards, Genomics standards, Humans, Molecular Diagnostic Techniques methods, Molecular Diagnostic Techniques standards, Mutation, Neoplasms diagnosis, Polymorphism, Single Nucleotide, Reproducibility of Results, Sensitivity and Specificity, Biomarkers, Tumor, Genetic Testing methods, Genomics methods, Neoplasms genetics, Oncogenes
- Abstract
Background: Targeted sequencing using oncopanels requires comprehensive assessments of accuracy and detection sensitivity to ensure analytical validity. By employing reference materials characterized by the U.S. Food and Drug Administration-led SEquence Quality Control project phase2 (SEQC2) effort, we perform a cross-platform multi-lab evaluation of eight Pan-Cancer panels to assess best practices for oncopanel sequencing., Results: All panels demonstrate high sensitivity across targeted high-confidence coding regions and variant types for the variants previously verified to have variant allele frequency (VAF) in the 5-20% range. Sensitivity is reduced by utilizing VAF thresholds due to inherent variability in VAF measurements. Enforcing a VAF threshold for reporting has a positive impact on reducing false positive calls. Importantly, the false positive rate is found to be significantly higher outside the high-confidence coding regions, resulting in lower reproducibility. Thus, region restriction and VAF thresholds lead to low relative technical variability in estimating promising biomarkers and tumor mutational burden., Conclusion: This comprehensive study provides actionable guidelines for oncopanel sequencing and clear evidence that supports a simplified approach to assess the analytical performance of oncopanels. It will facilitate the rapid implementation, validation, and quality control of oncopanels in clinical use.
- Published
- 2021
- Full Text
- View/download PDF
10. Histone demethylase LSD1 is required for germinal center formation and BCL6-driven lymphomagenesis.
- Author
-
Hatzi K, Geng H, Doane AS, Meydan C, LaRiviere R, Cardenas M, Duy C, Shen H, Vidal MNC, Baslan T, Mohammad HP, Kruger RG, Shaknovich R, Haberman AM, Inghirami G, Lowe SW, and Melnick AM
- Subjects
- Animals, CRISPR-Cas Systems, Carcinogenesis, DNA, Intergenic genetics, Germinal Center immunology, Histone Demethylases genetics, Hyperplasia, Immunological Synapses genetics, Introns genetics, Lymphoma genetics, Mice, Mice, Inbred C57BL, Mice, Knockout, Proto-Oncogene Proteins c-bcl-6 genetics, B-Lymphocytes physiology, Germinal Center pathology, Histone Demethylases metabolism, Lymphoma metabolism, Proto-Oncogene Proteins c-bcl-6 metabolism
- Abstract
Germinal center (GC) B cells feature repression of many gene enhancers to establish their characteristic transcriptome. Here we show that conditional deletion of Lsd1 in GCs significantly impaired GC formation, associated with failure to repress immune synapse genes linked to GC exit, which are also direct targets of the transcriptional repressor BCL6. We found that BCL6 directly binds LSD1 and recruits it primarily to intergenic and intronic enhancers. Conditional deletion of Lsd1 suppressed GC hyperplasia caused by constitutive expression of BCL6 and significantly delayed BCL6-driven lymphomagenesis. Administration of catalytic inhibitors of LSD1 had little effect on GC formation or GC-derived lymphoma cells. Using a CRISPR-Cas9 domain screen, we found instead that the LSD1 Tower domain was critical for dependence on LSD1 in GC-derived B cells. These results indicate an essential role for LSD1 in the humoral immune response, where it modulates enhancer function by forming repression complexes with BCL6.
- Published
- 2019
- Full Text
- View/download PDF
11. TET2 Deficiency Causes Germinal Center Hyperplasia, Impairs Plasma Cell Differentiation, and Promotes B-cell Lymphomagenesis.
- Author
-
Dominguez PM, Ghamlouch H, Rosikiewicz W, Kumar P, Béguelin W, Fontán L, Rivas MA, Pawlikowska P, Armand M, Mouly E, Torres-Martin M, Doane AS, Calvo Fernandez MT, Durant M, Della-Valle V, Teater M, Cimmino L, Droin N, Tadros S, Motanagh S, Shih AH, Rubin MA, Tam W, Aifantis I, Levine RL, Elemento O, Inghirami G, Green MR, Figueroa ME, Bernard OA, Aoufouchi S, Li S, Shaknovich R, and Melnick AM
- Subjects
- Animals, CREB-Binding Protein genetics, CREB-Binding Protein metabolism, DNA-Binding Proteins metabolism, Dioxygenases, Epigenesis, Genetic genetics, Gene Expression Profiling methods, Germinal Center pathology, Hematopoietic Stem Cells metabolism, Humans, Hyperplasia, Lymphoma, Large B-Cell, Diffuse metabolism, Lymphoma, Large B-Cell, Diffuse pathology, Mice, Knockout, Mice, Transgenic, Mutation, Plasma Cells pathology, Positive Regulatory Domain I-Binding Factor 1 genetics, Positive Regulatory Domain I-Binding Factor 1 metabolism, Proto-Oncogene Proteins metabolism, Cell Differentiation genetics, DNA-Binding Proteins genetics, Germinal Center metabolism, Lymphoma, Large B-Cell, Diffuse genetics, Plasma Cells metabolism, Proto-Oncogene Proteins genetics
- Abstract
TET2 somatic mutations occur in ∼10% of diffuse large B-cell lymphomas (DLBCL) but are of unknown significance. Herein, we show that TET2 is required for the humoral immune response and is a DLBCL tumor suppressor. TET2 loss of function disrupts transit of B cells through germinal centers (GC), causing GC hyperplasia, impaired class switch recombination, blockade of plasma cell differentiation, and a preneoplastic phenotype. TET2 loss was linked to focal loss of enhancer hydroxymethylation and transcriptional repression of genes that mediate GC exit, such as PRDM1. Notably, these enhancers and genes are also repressed in CREBBP -mutant DLBCLs. Accordingly, TET2 mutation in patients yields a CREBBP -mutant gene-expression signature, CREBBP and TET2 mutations are generally mutually exclusive, and hydroxymethylation loss caused by TET2 deficiency impairs enhancer H3K27 acetylation. Hence, TET2 plays a critical role in the GC reaction, and its loss of function results in lymphomagenesis through failure to activate genes linked to GC exit signals. SIGNIFICANCE: We show that TET2 is required for exit of the GC, B-cell differentiation, and is a tumor suppressor for mature B cells. Loss of TET2 phenocopies CREBBP somatic mutation. These results advocate for sequencing TET2 in patients with lymphoma and for the testing of epigenetic therapies to treat these tumors. See related commentary by Shingleton and Dave, p. 1515 . This article is highlighted in the In This Issue feature, p. 1494 ., (©2018 American Association for Cancer Research.)
- Published
- 2018
- Full Text
- View/download PDF
12. Extracellular vesicles in DLBCL provide abundant clues to aberrant transcriptional programming and genomic alterations.
- Author
-
Rutherford SC, Fachel AA, Li S, Sawh S, Muley A, Ishii J, Saxena A, Dominguez PM, Caldas Lopes E, Agirre X, Chambwe N, Correa F, Jiang Y, Richards KL, Betel D, and Shaknovich R
- Subjects
- Cell Line, Tumor, Extracellular Vesicles genetics, Extracellular Vesicles pathology, Humans, Lymphoma, Large B-Cell, Diffuse genetics, Lymphoma, Large B-Cell, Diffuse pathology, Neoplasm Proteins genetics, RNA, Neoplasm genetics, Extracellular Vesicles metabolism, Lymphoma, Large B-Cell, Diffuse metabolism, Neoplasm Proteins metabolism, RNA, Neoplasm metabolism
- Abstract
The biological role of extracellular vesicles (EVs) in diffuse large B-cell lymphoma (DLBCL) initiation and progression remains largely unknown. We characterized EVs secreted by 5 DLBCL cell lines, a primary DLBCL tumor, and a normal control B-cell sample, optimized their purification, and analyzed their content. We found that DLBCLs secreted large quantities of CD63, Alix, TSG101, and CD81 EVs, which can be extracted using an ultracentrifugation-based method and traced by their cell of origin surface markers. We also showed that tumor-derived EVs can be exchanged between lymphoma cells, normal tonsillar cells, and HK stromal cells. We then examined the content of EVs, focusing on isolation of high-quality total RNA. We sequenced the total RNA and analyzed the nature of RNA species, including coding and noncoding RNAs. We compared whole-cell and EV-derived RNA composition in benign and malignant B cells and discovered that transcripts from EVs were involved in many critical cellular functions. Finally, we performed mutational analysis and found that mutations detected in EVs exquisitely represented mutations in the cell of origin. These results enhance our understanding and enable future studies of the role that EVs may play in the pathogenesis of DLBCL, particularly with regards to the exchange of genomic information. Current findings open a new strategy for liquid biopsy approaches in disease monitoring., (© 2018 by The American Society of Hematology.)
- Published
- 2018
- Full Text
- View/download PDF
13. SOX11 augments BCR signaling to drive MCL-like tumor development.
- Author
-
Kuo PY, Jatiani SS, Rahman AH, Edwards D, Jiang Z, Ahr K, Perumal D, Leshchenko VV, Brody J, Shaknovich R, Ye BH, and Parekh S
- Subjects
- Animals, B-Lymphocytes metabolism, B-Lymphocytes pathology, Biomarkers, Cell Line, Tumor, Clonal Evolution, Gene Expression, Gene Expression Regulation, Neoplastic, High-Throughput Nucleotide Sequencing, Humans, Immunoglobulin Heavy Chains, Lymphoma, Mantle-Cell genetics, Mice, Mice, Transgenic, Phenotype, SOXC Transcription Factors genetics, Lymphoma, Mantle-Cell metabolism, Lymphoma, Mantle-Cell pathology, Receptors, Antigen, B-Cell metabolism, SOXC Transcription Factors metabolism, Signal Transduction, Tumor Microenvironment
- Abstract
Mantle cell lymphoma (MCL) is characterized by increased B-cell receptor (BCR) signaling, and BTK inhibition is an effective therapeutic intervention in MCL patients. The mechanisms leading to increased BCR signaling in MCL are poorly understood, as mutations in upstream regulators of BCR signaling such as CD79A, commonly observed in other lymphomas, are rare in MCL. The transcription factor SOX11 is overexpressed in the majority (78% to 93%) of MCL patients and is considered an MCL-specific oncogene. So far, attempts to understand SOX11 function in vivo have been hampered by the lack of appropriate animal models, because germline deletion of SOX11 is embryonically lethal. We have developed a transgenic mouse model (Eμ-SOX11-EGFP) in the C57BL/6 background expressing murine SOX11 and EGFP under the control of a B-cell-specific IgH-Eμ enhancer. The overexpression of SOX11 exclusively in B cells exhibits oligoclonal B-cell hyperplasia in the spleen, bone marrow, and peripheral blood, with an immunophenotype (CD5
+ CD19+ CD23- ) identical to human MCL. Furthermore, phosphocytometric time-of-flight analysis of the splenocytes from these mice shows hyperactivation of pBTK and other molecules in the BCR signaling pathway, and serial bone marrow transplant from transgenic donors produces lethality with decreasing latency. We report here that overexpression of SOX11 in B cells promotes BCR signaling and a disease phenotype that mimics human MCL., (© 2018 by The American Society of Hematology.)- Published
- 2018
- Full Text
- View/download PDF
14. AICDA drives epigenetic heterogeneity and accelerates germinal center-derived lymphomagenesis.
- Author
-
Teater M, Dominguez PM, Redmond D, Chen Z, Ennishi D, Scott DW, Cimmino L, Ghione P, Chaudhuri J, Gascoyne RD, Aifantis I, Inghirami G, Elemento O, Melnick A, and Shaknovich R
- Subjects
- Animals, B-Lymphocytes metabolism, B-Lymphocytes pathology, Cytidine Deaminase metabolism, Gene Expression Profiling, Gene Expression Regulation, Neoplastic, Humans, Lymphoma, Large B-Cell, Diffuse enzymology, Lymphoma, Large B-Cell, Diffuse metabolism, Mice, Inbred BALB C, Mice, Inbred C57BL, Mice, Knockout, Mice, Transgenic, Mutation, Cytidine Deaminase genetics, Epigenesis, Genetic, Germinal Center metabolism, Lymphoma, Large B-Cell, Diffuse genetics
- Abstract
Epigenetic heterogeneity is emerging as a feature of tumors. In diffuse large B-cell lymphoma (DLBCL), increased cytosine methylation heterogeneity is associated with poor clinical outcome, yet the underlying mechanisms remain unclear. Activation-induced cytidine deaminase (AICDA), an enzyme that mediates affinity maturation and facilitates DNA demethylation in germinal center (GC) B cells, is required for DLBCL pathogenesis and linked to inferior outcome. Here we show that AICDA overexpression causes more aggressive disease in BCL2-driven murine lymphomas. This phenotype is associated with increased cytosine methylation heterogeneity, but not with increased AICDA-mediated somatic mutation burden. Reciprocally, the cytosine methylation heterogeneity characteristic of normal GC B cells is lost upon AICDA depletion. These observations are relevant to human patients, since DLBCLs with high AICDA expression manifest increased methylation heterogeneity vs. AICDA-low DLBCLs. Our results identify AICDA as a driver of epigenetic heterogeneity in B-cell lymphomas with potential significance for other tumors with aberrant expression of cytidine deaminases.
- Published
- 2018
- Full Text
- View/download PDF
15. Precision Medicine for Relapsed Multiple Myeloma on the Basis of an Integrative Multiomics Approach.
- Author
-
Laganà A, Beno I, Melnekoff D, Leshchenko V, Madduri D, Ramdas D, Sanchez L, Niglio S, Perumal D, Kidd BA, Miotto R, Shaknovich R, Chari A, Cho HJ, Barlogie B, Jagannath S, Dudley JT, and Parekh S
- Abstract
Purpose: Multiple myeloma (MM) is a malignancy of plasma cells, with a median survival of 6 years. Despite recent therapeutic advancements, relapse remains mostly inevitable, and the disease is fatal in the majority of patients. A major challenge in the treatment of patients with relapsed MM is the timely identification of treatment options in a personalized manner. Current approaches in precision oncology aim at matching specific DNA mutations to drugs, but incorporation of genome-wide RNA profiles has not yet been clinically assessed., Methods: We have developed a novel computational platform for precision medicine of relapsed and/or refractory MM on the basis of DNA and RNA sequencing. Our approach expands on the traditional DNA-based approaches by integrating somatic mutations and copy number alterations with RNA-based drug repurposing and pathway analysis. We tested our approach in a pilot precision medicine clinical trial with 64 patients with relapsed and/or refractory MM., Results: We generated treatment recommendations in 63 of 64 patients. Twenty-six patients had treatment implemented, and 21 were assessable. Of these, 11 received a drug that was based on RNA findings, eight received a drug that was based on DNA, and two received a drug that was based on both RNA and DNA. Sixteen of the 21 evaluable patients had a clinical response (ie, reduction of disease marker ≥ 25%), giving a clinical benefit rate of 76% and an overall response rate of 66%, with five patients having ongoing responses at the end of the trial. The median duration of response was 131 days., Conclusion: Our results show that a comprehensive sequencing approach can identify viable options in patients with relapsed and/or refractory myeloma, and they represent proof of principle of how RNA sequencing can contribute beyond DNA mutation analysis to the development of a reliable drug recommendation tool., Competing Interests: AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO’s conflictof interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center. Alessandro Laganà No relationship to disclose Itai Beno No relationship to disclose David Melnekoff No relationship to disclose Violetta Leshchenko No relationship to disclose Deepu Madduri Employment: Roivant Sciences (I) Leadership: Roivant Sciences (I) Stock and Other Ownership Interests: Roivant Sciences (I) Honoraria: Abbvie Consulting or Advisory Role: Abbvie Speakers’ Bureau: Baxalta/Shire Patents, Royalties, Other Intellectual Property: Roivant Sciences (I) Dennis Ramdas Employment: Eli Lilly (I) Stock and Other Ownership Interests: Eli Lilly (I) Speakers’ Bureau: Janssen Pharmaceuticals, Sanofi, Takeda Pharmaceuticals (I) Larysa Sanchez No relationship to disclose Scot Niglio No relationship to disclose Deepak Perumal No relationship to disclose Brian A. Kidd No relationship to disclose Riccardo Miotto No relationship to disclose Rita Shaknovich Employment: CGIX Leadership: CGIX Stock and Other Ownership Interests: CGIX Consulting or Advisory Role: AstraZeneca Travel, Accommodations, Expenses: AstraZeneca Ajai Chari Consulting or Advisory Role: Array BioPharma, Celgene, Novartis, Millennium Pharmaceuticals, Amgen, Janssen Oncology, Adaptive Biotechnologies, Bayer AG, Seattle Genetics Research Funding: Array BioPharma, Celgene, Millennium Pharmaceuticals, Novartis, Onyx Pharmaceuticals, Janssen Pharmaceuticals, Pharmacyclics, Acetylon Pharmaceuticals (Inst), Biotest (Inst), Bristol-Myers Squibb (Inst) Travel, Accommodations, Expenses: Takeda Pharmaceuticals, Celgene, Novartis, Amgen, Janssen Oncology, Bristol-Myers Squibb Hearn Jay Cho Honoraria: Genentech/Roche Consulting or Advisory Role: Janssen Research & Development, Genentech, Bristol-Myers Squibb Research Funding: Agenus, Janssen Research & Development, Bristol-Myers Squibb Travel, Accommodations, Expenses: Roche/Genentech, Bristol-Myers Squibb Bart Barlogie No relationship to disclose Sundar Jagannath Honoraria: Celgene, Karyopharm Therapeutics Consulting or Advisory Role: Celgene, Bristol-Myers Squibb, Janssen Pharmaceuticals, Novartis, Karyopharm Therapeutics Research Funding: Celgene (Inst), Karyopharm Therapeutics (Inst), Bristol-Myers Squibb (Inst) Joel T. Dudley Consulting or Advisory Role: Janssen Pharmaceuticals, Allergan Research Funding: AstraZeneca (Inst) Samir Parekh Consulting or Advisory Role: Foundation Medicine
- Published
- 2018
- Full Text
- View/download PDF
16. The new frontier of epigenetic heterogeneity in B-cell neoplasms.
- Author
-
Dominguez PM, Teater M, and Shaknovich R
- Subjects
- Animals, DNA Methylation, Germinal Center metabolism, Humans, Leukemia, B-Cell diagnosis, Leukemia, B-Cell metabolism, Lymphoma, B-Cell diagnosis, Lymphoma, B-Cell metabolism, Signal Transduction, Epigenesis, Genetic, Gene Expression Regulation, Neoplastic, Genetic Heterogeneity, Leukemia, B-Cell genetics, Lymphoma, B-Cell genetics
- Abstract
Purpose of Review: There is mounting evidence that heterogeneity of the epigenome is a feature of many cancers, including B-cell lymphomas, and presents important clinical implications. The purpose of this review is to explain the biological and clinical relevance of this epigenetic phenomenon in B-cell neoplasms., Recent Findings: Here, we summarize new findings demonstrating that B-cell lymphomas display increased DNA methylation heterogeneity compared to their normal counterparts. This plasticity of cytosine methylation manifests both as intertumor and intratumor heterogeneity and is associated with worse prognosis and poor clinical outcome in lymphoma patients. Recent studies of different subtypes of B-cell lymphomas have revealed that epigenetic aberrations and heterogeneous cytosine methylation patterning are common features of all neoplasms derived from B-lymphocytes, irrespective of maturation stage. With regard to mechanisms driving this process, recent reports suggest that cytosine methylation heterogeneity arises through passive and active processes. One factor implicated in active generation of cytosine methylation heterogeneity is activation-induced cytidine deaminase, which mediates DNA methylation changes and introduces epigenetic heterogeneity in normal germinal center B cells, the cells of origin of mature B-cell neoplasms such as diffuse large B-cell lymphoma and follicular lymphoma., Summary: Understanding the scope and mechanism of epigenetic heterogeneity in cancer is of paramount importance to our understanding of clonal plasticity and treatment responses in B-cell lymphomas.
- Published
- 2017
- Full Text
- View/download PDF
17. CREBBP Inactivation Promotes the Development of HDAC3-Dependent Lymphomas.
- Author
-
Jiang Y, Ortega-Molina A, Geng H, Ying HY, Hatzi K, Parsa S, McNally D, Wang L, Doane AS, Agirre X, Teater M, Meydan C, Li Z, Poloway D, Wang S, Ennishi D, Scott DW, Stengel KR, Kranz JE, Holson E, Sharma S, Young JW, Chu CS, Roeder RG, Shaknovich R, Hiebert SW, Gascoyne RD, Tam W, Elemento O, Wendel HG, and Melnick AM
- Subjects
- Acetylation, Animals, CREB-Binding Protein metabolism, Cell Line, Tumor, Enhancer Elements, Genetic, Gene Knockout Techniques, Histone Deacetylases metabolism, Histones metabolism, Humans, Lymphoma, Large B-Cell, Diffuse metabolism, Mice, Neoplasm Transplantation, Nuclear Receptor Co-Repressor 2 genetics, Proto-Oncogene Proteins c-bcl-6 genetics, Transcription, Genetic, CREB-Binding Protein genetics, Germinal Center metabolism, Histone Deacetylases genetics, Lymphoma, Large B-Cell, Diffuse genetics, Mutation
- Abstract
Somatic mutations in CREBBP occur frequently in B-cell lymphoma. Here, we show that loss of CREBBP facilitates the development of germinal center (GC)-derived lymphomas in mice. In both human and murine lymphomas, CREBBP loss-of-function resulted in focal depletion of enhancer H3K27 acetylation and aberrant transcriptional silencing of genes that regulate B-cell signaling and immune responses, including class II MHC. Mechanistically, CREBBP-regulated enhancers are counter-regulated by the BCL6 transcriptional repressor in a complex with SMRT and HDAC3, which we found to bind extensively to MHC class II loci. HDAC3 loss-of-function rescued repression of these enhancers and corresponding genes, including MHC class II, and more profoundly suppressed CREBBP-mutant lymphomas in vitro and in vivo Hence, CREBBP loss-of-function contributes to lymphomagenesis by enabling unopposed suppression of enhancers by BCL6/SMRT/HDAC3 complexes, suggesting HDAC3-targeted therapy as a precision approach for CREBBP-mutant lymphomas., Significance: Our findings establish the tumor suppressor function of CREBBP in GC lymphomas in which CREBBP mutations disable acetylation and result in unopposed deacetylation by BCL6/SMRT/HDAC3 complexes at enhancers of B-cell signaling and immune response genes. Hence, inhibition of HDAC3 can restore the enhancer histone acetylation and may serve as a targeted therapy for CREBBP-mutant lymphomas. Cancer Discov; 7(1); 38-53. ©2016 AACR.See related commentary by Höpken, p. 14This article is highlighted in the In This Issue feature, p. 1., Competing Interests: of Potential Conflicts of Interest: E. Holson is chief scientific officer of KDAc Therapeutics, Inc. No potential conflicts of interest were disclosed by other authors., (©2016 American Association for Cancer Research.)
- Published
- 2017
- Full Text
- View/download PDF
18. Rationally designed BCL6 inhibitors target activated B cell diffuse large B cell lymphoma.
- Author
-
Cardenas MG, Yu W, Beguelin W, Teater MR, Geng H, Goldstein RL, Oswald E, Hatzi K, Yang SN, Cohen J, Shaknovich R, Vanommeslaeghe K, Cheng H, Liang D, Cho HJ, Abbott J, Tam W, Du W, Leonard JP, Elemento O, Cerchietti L, Cierpicki T, Xue F, MacKerell AD Jr, and Melnick AM
- Subjects
- Animals, Cell Line, Tumor, Doxorubicin pharmacology, Drug Screening Assays, Antitumor, HEK293 Cells, Humans, Indoles pharmacology, Ligands, Lymphoma, Large B-Cell, Diffuse pathology, Magnetic Resonance Spectroscopy, Male, Mice, Mice, SCID, Neoplasm Transplantation, Protein Binding, Proto-Oncogene Proteins c-bcl-6 metabolism, Thiazolidinediones pharmacology, Translocation, Genetic, Antineoplastic Agents pharmacology, Drug Design, Gene Expression Regulation, Neoplastic, Lymphoma, Large B-Cell, Diffuse drug therapy, Proto-Oncogene Proteins c-bcl-6 antagonists & inhibitors
- Abstract
Diffuse large B cell lymphomas (DLBCLs) arise from proliferating B cells transiting different stages of the germinal center reaction. In activated B cell DLBCLs (ABC-DLBCLs), a class of DLBCLs that respond poorly to current therapies, chromosomal translocations and amplification lead to constitutive expression of the B cell lymphoma 6 (BCL6) oncogene. The role of BCL6 in maintaining these lymphomas has not been investigated. Here, we designed small-molecule inhibitors that display higher affinity for BCL6 than its endogenous corepressor ligands to evaluate their therapeutic efficacy for targeting ABC-DLBCL. We used an in silico drug design functional-group mapping approach called SILCS to create a specific BCL6 inhibitor called FX1 that has 10-fold greater potency than endogenous corepressors and binds an essential region of the BCL6 lateral groove. FX1 disrupted formation of the BCL6 repression complex, reactivated BCL6 target genes, and mimicked the phenotype of mice engineered to express BCL6 with corepressor binding site mutations. Low doses of FX1 induced regression of established tumors in mice bearing DLBCL xenografts. Furthermore, FX1 suppressed ABC-DLBCL cells in vitro and in vivo, as well as primary human ABC-DLBCL specimens ex vivo. These findings indicate that ABC-DLBCL is a BCL6-dependent disease that can be targeted by rationally designed inhibitors that exceed the binding affinity of natural BCL6 ligands.
- Published
- 2016
- Full Text
- View/download PDF
19. EZH2 and BCL6 Cooperate to Assemble CBX8-BCOR Complex to Repress Bivalent Promoters, Mediate Germinal Center Formation and Lymphomagenesis.
- Author
-
Béguelin W, Teater M, Gearhart MD, Calvo Fernández MT, Goldstein RL, Cárdenas MG, Hatzi K, Rosen M, Shen H, Corcoran CM, Hamline MY, Gascoyne RD, Levine RL, Abdel-Wahab O, Licht JD, Shaknovich R, Elemento O, Bardwell VJ, and Melnick AM
- Subjects
- Animals, Germinal Center pathology, Humans, Lymphoma, Large B-Cell, Diffuse genetics, Lymphoma, Large B-Cell, Diffuse pathology, Mice, Mice, Inbred C57BL, Mice, Transgenic, Mitochondrial Membrane Transport Proteins, Polycomb-Group Proteins metabolism, Promoter Regions, Genetic, Proto-Oncogene Proteins metabolism, Transcription, Genetic, Enhancer of Zeste Homolog 2 Protein metabolism, Germinal Center metabolism, Lymphoma, Large B-Cell, Diffuse metabolism, Polycomb Repressive Complex 1 metabolism, Proto-Oncogene Proteins c-bcl-6 metabolism, Repressor Proteins metabolism
- Abstract
The EZH2 histone methyltransferase mediates the humoral immune response and drives lymphomagenesis through formation of bivalent chromatin domains at critical germinal center (GC) B cell promoters. Herein we show that the actions of EZH2 in driving GC formation and lymphoma precursor lesions require site-specific binding by the BCL6 transcriptional repressor and the presence of a non-canonical PRC1-BCOR-CBX8 complex. The chromodomain protein CBX8 is induced in GC B cells, binds to H3K27me3 at bivalent promoters, and is required for stable association of the complex and the resulting histone modifications. Moreover, oncogenic BCL6 and EZH2 cooperate to accelerate diffuse large B cell lymphoma (DLBCL) development and combinatorial targeting of these repressors results in enhanced anti-lymphoma activity in DLBCLs., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
20. miR-181a negatively regulates NF-κB signaling and affects activated B-cell-like diffuse large B-cell lymphoma pathogenesis.
- Author
-
Kozloski GA, Jiang X, Bhatt S, Ruiz J, Vega F, Shaknovich R, Melnick A, and Lossos IS
- Subjects
- Animals, B-Lymphocytes metabolism, B-Lymphocytes pathology, Cell Line, Tumor, Female, Gene Expression Regulation, Neoplastic, HEK293 Cells, HeLa Cells, Humans, Lymphocyte Activation genetics, Lymphoma, Large B-Cell, Diffuse pathology, Mice, Mice, Inbred NOD, Mice, SCID, NF-kappa B metabolism, Signal Transduction genetics, Xenograft Model Antitumor Assays, Cell Transformation, Neoplastic genetics, Lymphoma, Large B-Cell, Diffuse genetics, MicroRNAs physiology
- Abstract
Distinct subgroups of diffuse large B-cell lymphoma (DLBCL) genetically resemble specific mature B-cell populations that are blocked at different stages of the immune response in germinal centers (GCs). The activated B-cell (ABC)-like subgroup resembles post-GC plasmablasts undergoing constitutive survival signaling, yet knowledge of the mechanisms that negatively regulate this oncogenic signaling remains incomplete. In this study, we report that microRNA (miR)-181a is a negative regulator of nuclear factor κ-light-chain enhancer of activated B-cells (NF-κB) signaling. miR-181a overexpression significantly decreases the expression and activity of key NF-κB signaling components. Moreover, miR-181a decreases DLBCL tumor cell proliferation and survival, and anti-miR-181a abrogates these effects. Remarkably, these effects are augmented in the NF-κB dependent ABC-like subgroup compared with the GC B-cell (GCB)-like DLBCL subgroup. Concordantly, in vivo analyses of miR-181a induction in xenografts results in slower tumor growth rate and prolonged survival in the ABC-like DLBCL xenografts compared with the GCB-like DLBCL. We link these outcomes to relatively lower endogenous miR-181a expression and to NF-κB signaling dependency in the ABC-like DLBCL subgroup. Our findings indicate that miR-181a inhibits NF-κB activity, and that manipulation of miR-181a expression in the ABC-like DLBCL genetic background may result in a significant change in the proliferation and survival phenotype of this malignancy., (© 2016 by The American Society of Hematology.)
- Published
- 2016
- Full Text
- View/download PDF
21. Pharmacoproteomics identifies combinatorial therapy targets for diffuse large B cell lymphoma.
- Author
-
Goldstein RL, Yang SN, Taldone T, Chang B, Gerecitano J, Elenitoba-Johnson K, Shaknovich R, Tam W, Leonard JP, Chiosis G, Cerchietti L, and Melnick A
- Subjects
- Adenine analogs & derivatives, Agammaglobulinaemia Tyrosine Kinase, Benzodioxoles pharmacology, Cell Line, Tumor, HSP90 Heat-Shock Proteins antagonists & inhibitors, HSP90 Heat-Shock Proteins metabolism, Humans, Intracellular Signaling Peptides and Proteins antagonists & inhibitors, Intracellular Signaling Peptides and Proteins metabolism, Lymphoma, Large B-Cell, Diffuse metabolism, Lymphoma, Large B-Cell, Diffuse pathology, Piperidines, Protein-Tyrosine Kinases antagonists & inhibitors, Protein-Tyrosine Kinases metabolism, Proteomics, Purines pharmacology, Pyrazoles pharmacology, Pyrimidines pharmacology, Receptors, Antigen, B-Cell metabolism, Syk Kinase, Antineoplastic Combined Chemotherapy Protocols pharmacology, Lymphoma, Large B-Cell, Diffuse drug therapy, Signal Transduction drug effects
- Abstract
Rationally designed combinations of targeted therapies for refractory cancers, such as activated B cell-like diffuse large B cell lymphoma (ABC DLBCL), are likely required to achieve potent, durable responses. Here, we used a pharmacoproteomics approach to map the interactome of a tumor-enriched isoform of HSP90 (teHSP90). Specifically, we chemically precipitated teHSP90-client complexes from DLBCL cell lines with the small molecule PU-H71 and found that components of the proximal B cell receptor (BCR) signalosome were enriched within teHSP90 complexes. Functional assays revealed that teHSP90 facilitates BCR signaling dynamics by enabling phosphorylation of key BCR signalosome components, including the kinases SYK and BTK. Consequently, treatment of BCR-dependent ABC DLBCL cells with PU-H71 attenuated BCR signaling, calcium flux, and NF-κB signaling, ultimately leading to growth arrest. Combined exposure of ABC DLBCL cell lines to PU-H71 and ibrutinib, a BCR pathway inhibitor, more potently suppressed BCR signaling than either drug alone. Correspondingly, PU-H71 combined with ibrutinib induced synergistic killing of lymphoma cell lines, primary human lymphoma specimens ex vivo, and lymphoma xenografts in vivo, without notable toxicity. Together, our results demonstrate that a pharmacoproteome-driven rational combination therapy has potential to provide more potent BCR-directed therapy for ABC DLCBL patients.
- Published
- 2015
- Full Text
- View/download PDF
22. The histone lysine methyltransferase KMT2D sustains a gene expression program that represses B cell lymphoma development.
- Author
-
Ortega-Molina A, Boss IW, Canela A, Pan H, Jiang Y, Zhao C, Jiang M, Hu D, Agirre X, Niesvizky I, Lee JE, Chen HT, Ennishi D, Scott DW, Mottok A, Hother C, Liu S, Cao XJ, Tam W, Shaknovich R, Garcia BA, Gascoyne RD, Ge K, Shilatifard A, Elemento O, Nussenzweig A, Melnick AM, and Wendel HG
- Subjects
- Animals, B-Lymphocytes pathology, DNA-Binding Proteins genetics, Humans, Mice, Mice, Knockout, Mutation, Neoplasm Proteins genetics, DNA-Binding Proteins physiology, Gene Expression Regulation physiology, Lymphoma, B-Cell etiology, Neoplasm Proteins physiology
- Abstract
The gene encoding the lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma and diffuse large B cell lymphoma; however, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center involution and impedes B cell differentiation and class switch recombination. Integrative genomic analyses indicate that KMT2D affects methylation of lysine 4 on histone H3 (H3K4) and expression of a set of genes, including those in the CD40, JAK-STAT, Toll-like receptor and B cell receptor signaling pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3 and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell-activating pathways.
- Published
- 2015
- Full Text
- View/download PDF
23. DNA Methylation Dynamics of Germinal Center B Cells Are Mediated by AID.
- Author
-
Dominguez PM, Teater M, Chambwe N, Kormaksson M, Redmond D, Ishii J, Vuong B, Chaudhuri J, Melnick A, Vasanthakumar A, Godley LA, Papavasiliou FN, Elemento O, and Shaknovich R
- Subjects
- Animals, B-Lymphocytes cytology, B-Lymphocytes immunology, Cell Differentiation, Cell Movement, Cell Proliferation, Conserved Sequence, Cytidine Deaminase genetics, Cytidine Deaminase immunology, Cytosine metabolism, DNA Methylation, Germinal Center cytology, Germinal Center immunology, Humans, Lymphocyte Activation, Mice, Mice, Inbred BALB C, Mice, Knockout, B-Lymphocytes metabolism, Cytidine Deaminase metabolism, Epigenesis, Genetic, Germinal Center metabolism
- Abstract
Changes in DNA methylation are required for the formation of germinal centers (GCs), but the mechanisms of such changes are poorly understood. Activation-induced cytidine deaminase (AID) has been recently implicated in DNA demethylation through its deaminase activity coupled with DNA repair. We investigated the epigenetic function of AID in vivo in germinal center B cells (GCBs) isolated from wild-type (WT) and AID-deficient (Aicda(-/-)) mice. We determined that the transit of B cells through the GC is associated with marked locus-specific loss of methylation and increased methylation diversity, both of which are lost in Aicda(-/-) animals. Differentially methylated cytosines (DMCs) between GCBs and naive B cells (NBs) are enriched in genes that are targeted for somatic hypermutation (SHM) by AID, and these genes form networks required for B cell development and proliferation. Finally, we observed significant conservation of AID-dependent epigenetic reprogramming between mouse and human B cells., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
24. IL10 receptor is a novel therapeutic target in DLBCLs.
- Author
-
Béguelin W, Sawh S, Chambwe N, Chan FC, Jiang Y, Choo JW, Scott DW, Chalmers A, Geng H, Tsikitas L, Tam W, Bhagat G, Gascoyne RD, and Shaknovich R
- Subjects
- Apoptosis, Biomarkers, Tumor genetics, Blotting, Western, Cell Cycle, Cell Proliferation, High-Throughput Nucleotide Sequencing, Humans, Immunoenzyme Techniques, Interleukin-10 genetics, Lymphoma, Large B-Cell, Diffuse mortality, Neoplasm Staging, Prognosis, RNA, Messenger genetics, Real-Time Polymerase Chain Reaction, Receptors, Interleukin-10 genetics, Reverse Transcriptase Polymerase Chain Reaction, Survival Rate, Tissue Array Analysis, Tumor Cells, Cultured, Biomarkers, Tumor metabolism, Interleukin-10 metabolism, Lymphoma, Large B-Cell, Diffuse metabolism, Lymphoma, Large B-Cell, Diffuse pathology, Receptors, Interleukin-10 metabolism
- Abstract
Diffuse large B-cell lymphoma (DLBCL) is a biologically and clinically heterogeneous disease with marked genomic instability and variable response to conventional R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) chemotherapy. More clinically aggressive cases of DLBCLs have high level of circulating interleukin 10 (IL10) cytokine and evidence of activated intracellular STAT3 (signal transducer and activator of transcription 3) signaling. We investigated the role of IL10 and its surface receptor in supporting the neoplastic phenotype of DLBCLs. We determined that IL10RA gene is amplified in 21% and IL10RB gene in 10% of primary DLBCLs. Gene expression of IL10, IL10RA and IL10RB was markedly elevated in DLBCLs. We hypothesized that DLBCLs depend for their proliferation and survival on IL10-STAT3 signaling and that blocking the IL10 receptor (IL10R) would induce cell death. We used anti-IL10R blocking antibody, which resulted in a dose-dependent cell death in all tested activated B-cell-like subtype of DLBCL cell lines and primary DLBCLs. Response of germinal center B-cell-like subtype of DLBCL cell lines to anti-IL10R antibody varied from sensitive to resistant. Cells underwent cell cycle arrest, followed by induction of apoptosis. Cell death depended on inhibition of STAT3 and, to a lesser extent, STAT1 signaling. Anti-IL10R treatment resulted in interruption of IL10-IL10R autostimulatory loop. We thus propose that IL10R is a novel therapeutic target in DLBCLs.
- Published
- 2015
- Full Text
- View/download PDF
25. Erratum: TET1 is a tumor suppressor of hematopoietic malignancy.
- Author
-
Cimmino L, Dawlaty MM, Ndiaye-Lobry D, Yap YS, Bakogianni S, Yu Y, Bhattacharyya S, Shaknovich R, Geng H, Lobry C, Mullenders J, King B, Trimarchi T, Aranda-Orgilles B, Liu C, Shen S, Verma AK, Jaenisch R, and Aifantis I
- Published
- 2015
- Full Text
- View/download PDF
26. Signatures of accelerated somatic evolution in gene promoters in multiple cancer types.
- Author
-
Smith KS, Yadav VK, Pedersen BS, Shaknovich R, Geraci MW, Pollard KS, and De S
- Subjects
- Adult, Gene Expression, Genomics, Humans, Middle Aged, Neoplasms diagnosis, Software, Mutation, Neoplasms genetics, Promoter Regions, Genetic
- Abstract
Cancer-associated somatic mutations outside protein-coding regions remain largely unexplored. Analyses of the TERT locus have indicated that non-coding regulatory mutations can be more frequent than previously suspected and play important roles in oncogenesis. Using a computational method called SASE-hunter, developed here, we identified a novel signature of accelerated somatic evolution (SASE) marked by a significant excess of somatic mutations localized in a genomic locus, and prioritized those loci that carried the signature in multiple cancer patients. Interestingly, even when an affected locus carried the signature in multiple individuals, the mutations contributing to SASE themselves were rarely recurrent at the base-pair resolution. In a pan-cancer analysis of 906 samples from 12 tumor types, we detected SASE in the promoters of several genes, including known cancer genes such as MYC, BCL2, RBM5 and WWOX. Nucleotide substitution patterns consistent with oxidative DNA damage and local somatic hypermutation appeared to contribute to this signature in selected gene promoters (e.g. MYC). SASEs in selected cancer gene promoters were associated with over-expression, and also correlated with the age of onset of cancer, aggressiveness of the disease and survival. Taken together, our work detects a hitherto under-appreciated and clinically important class of regulatory changes in cancer genomes., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2015
- Full Text
- View/download PDF
27. TET1 is a tumor suppressor of hematopoietic malignancy.
- Author
-
Cimmino L, Dawlaty MM, Ndiaye-Lobry D, Yap YS, Bakogianni S, Yu Y, Bhattacharyya S, Shaknovich R, Geng H, Lobry C, Mullenders J, King B, Trimarchi T, Aranda-Orgilles B, Liu C, Shen S, Verma AK, Jaenisch R, and Aifantis I
- Subjects
- 5-Methylcytosine analogs & derivatives, Animals, Cell Differentiation genetics, Cell Lineage genetics, Chromosomal Instability, Cytosine metabolism, DNA Methylation, DNA Repair, DNA-Binding Proteins genetics, Epigenesis, Genetic, Exome genetics, Gene Expression Profiling, Humans, Mice, Mutation genetics, Proto-Oncogene Proteins genetics, Tumor Suppressor Proteins genetics, B-Lymphocytes physiology, Cytosine analogs & derivatives, DNA-Binding Proteins metabolism, Embryonic Stem Cells physiology, Lymphoma, B-Cell genetics, Proto-Oncogene Proteins metabolism, Tumor Suppressor Proteins metabolism
- Abstract
The methylcytosine dioxygenase TET1 ('ten-eleven translocation 1') is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. The diminished expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we found that deletion of Tet1 promoted the development of B cell lymphoma in mice. TET1 was required for maintenance of the normal abundance and distribution of 5hmC, which prevented hypermethylation of DNA, and for regulation of the B cell lineage and of genes encoding molecules involved in chromosome maintenance and DNA repair. Whole-exome sequencing of TET1-deficient tumors revealed mutations frequently found in non-Hodgkin B cell lymphoma (B-NHL), in which TET1 was hypermethylated and transcriptionally silenced. Our findings provide in vivo evidence of a function for TET1 as a tumor suppressor of hematopoietic malignancy.
- Published
- 2015
- Full Text
- View/download PDF
28. Epigenomic evolution in diffuse large B-cell lymphomas.
- Author
-
Pan H, Jiang Y, Boi M, Tabbò F, Redmond D, Nie K, Ladetto M, Chiappella A, Cerchietti L, Shaknovich R, Melnick AM, Inghirami GG, Tam W, and Elemento O
- Subjects
- DNA Methylation, Gene Expression Regulation, Neoplastic, Gene Regulatory Networks, Humans, Lymphoma, Large B-Cell, Diffuse genetics, Neoplasm Proteins genetics, Neoplasm Proteins metabolism, Recurrence, Transcriptome, Biological Evolution, Epigenomics, Lymphoma, Large B-Cell, Diffuse metabolism
- Abstract
The contribution of epigenomic alterations to tumour progression and relapse is not well characterized. Here we characterize an association between disease progression and DNA methylation in diffuse large B-cell lymphoma (DLBCL). By profiling genome-wide DNA methylation at single-base pair resolution in thirteen DLBCL diagnosis-relapse sample pairs, we show that DLBCL patients exhibit heterogeneous evolution of tumour methylomes during relapse. We identify differentially methylated regulatory elements and determine a relapse-associated methylation signature converging on key pathways such as transforming growth factor-β (TGF-β) receptor activity. We also observe decreased intra-tumour methylation heterogeneity from diagnosis to relapsed tumour samples. Relapse-free patients display lower intra-tumour methylation heterogeneity at diagnosis compared with relapsed patients in an independent validation cohort. Furthermore, intra-tumour methylation heterogeneity is predictive of time to relapse. Therefore, we propose that epigenomic heterogeneity may support or drive the relapse phenotype and can be used to predict DLBCL relapse.
- Published
- 2015
- Full Text
- View/download PDF
29. Epigenetic function of activation-induced cytidine deaminase and its link to lymphomagenesis.
- Author
-
Dominguez PM and Shaknovich R
- Abstract
Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation and class switch recombination of immunoglobulin (Ig) genes during B cell maturation and immune response. Expression of AID is tightly regulated due to its mutagenic and recombinogenic potential, which is known to target not only Ig genes, but also non-Ig genes, contributing to lymphomagenesis. In recent years, a new epigenetic function of AID and its link to DNA demethylation came to light in several developmental systems. In this review, we summarize existing evidence linking deamination of unmodified and modified cytidine by AID to base-excision repair and mismatch repair machinery resulting in passive or active removal of DNA methylation mark, with the focus on B cell biology. We also discuss potential contribution of AID-dependent DNA hypomethylation to lymphomagenesis.
- Published
- 2014
- Full Text
- View/download PDF
30. Epigenetic diversity in hematopoietic neoplasms.
- Author
-
Shaknovich R, De S, and Michor F
- Subjects
- Clonal Evolution, Humans, Lymphoma genetics, DNA Methylation, Epigenesis, Genetic, Hematologic Neoplasms genetics
- Abstract
Tumor cell populations display a remarkable extent of variability in non-genetic characteristics such as DNA methylation, histone modification patterns, and differentiation levels of individual cells. It remains to be elucidated whether non-genetic heterogeneity is simply a byproduct of tumor evolution or instead a manifestation of a higher-order tissue organization that is maintained within the neoplasm to establish a differentiation hierarchy, a favorable microenvironment, or a buffer against changing selection pressures during tumorigenesis. Here, we review recent findings on epigenetic diversity, particularly heterogeneity in DNA methylation patterns in hematologic malignancies. We also address the implications of epigenetic heterogeneity for the clonal evolution of tumors and discuss its effects on gene expression and other genome functions in cancer., (Copyright © 2014. Published by Elsevier B.V.)
- Published
- 2014
- Full Text
- View/download PDF
31. CTCF haploinsufficiency destabilizes DNA methylation and predisposes to cancer.
- Author
-
Kemp CJ, Moore JM, Moser R, Bernard B, Teater M, Smith LE, Rabaia NA, Gurley KE, Guinney J, Busch SE, Shaknovich R, Lobanenkov VV, Liggitt D, Shmulevich I, Melnick A, and Filippova GN
- Subjects
- Animals, CCCTC-Binding Factor, Cell Line, Tumor, Epigenesis, Genetic, Gene Expression Regulation, Neoplastic, Genetic Predisposition to Disease, Haploinsufficiency, Humans, Mice, Mice, Inbred C57BL, Mice, Transgenic, Mutation, Neoplasms metabolism, Protein Binding, Repressor Proteins metabolism, Survival Analysis, DNA Methylation, Genes, Tumor Suppressor, Neoplasms genetics, Repressor Proteins genetics
- Abstract
Epigenetic alterations, particularly in DNA methylation, are ubiquitous in cancer, yet the molecular origins and the consequences of these alterations are poorly understood. CTCF, a DNA-binding protein that regulates higher-order chromatin organization, is frequently altered by hemizygous deletion or mutation in human cancer. To date, a causal role for CTCF in cancer has not been established. Here, we show that Ctcf hemizygous knockout mice are markedly susceptible to spontaneous, radiation-, and chemically induced cancer in a broad range of tissues. Ctcf(+/-) tumors are characterized by increased aggressiveness, including invasion, metastatic dissemination, and mixed epithelial/mesenchymal differentiation. Molecular analysis of Ctcf(+/-) tumors indicates that Ctcf is haploinsufficient for tumor suppression. Tissues with hemizygous loss of CTCF exhibit increased variability in CpG methylation genome wide. These findings establish CTCF as a prominent tumor-suppressor gene and point to CTCF-mediated epigenetic stability as a major barrier to neoplastic progression., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
32. Variability in DNA methylation defines novel epigenetic subgroups of DLBCL associated with different clinical outcomes.
- Author
-
Chambwe N, Kormaksson M, Geng H, De S, Michor F, Johnson NA, Morin RD, Scott DW, Godley LA, Gascoyne RD, Melnick A, Campagne F, and Shaknovich R
- Subjects
- Case-Control Studies, Cells, Cultured, Follow-Up Studies, Humans, Lymphoma, Large B-Cell, Diffuse classification, Prognosis, Survival Rate, DNA Methylation genetics, Epigenesis, Genetic, Gene Expression Regulation, Neoplastic, Genetic Variation genetics, Lymphoma, Large B-Cell, Diffuse genetics, Lymphoma, Large B-Cell, Diffuse mortality, Neoplasm Proteins genetics
- Abstract
Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive form of non-Hodgkin lymphoma with variable biology and clinical behavior. The current classification does not fully explain the biological and clinical heterogeneity of DLBCLs. In this study, we carried out genomewide DNA methylation profiling of 140 DLBCL samples and 10 normal germinal center B cells using the HpaII tiny fragment enrichment by ligation-mediated polymerase chain reaction assay and hybridization to a custom Roche NimbleGen promoter array. We defined methylation disruption as a main epigenetic event in DLBCLs and designed a method for measuring the methylation variability of individual cases. We then used a novel approach for unsupervised hierarchical clustering based on the extent of DNA methylation variability. This approach identified 6 clusters (A-F). The extent of methylation variability was associated with survival outcomes, with significant differences in overall and progression-free survival. The novel clusters are characterized by disruption of specific biological pathways such as cytokine-mediated signaling, ephrin signaling, and pathways associated with apoptosis and cell-cycle regulation. In a subset of patients, we profiled gene expression and genomic variation to investigate their interplay with methylation changes. This study is the first to identify novel epigenetic clusters of DLBCLs and their aberrantly methylated genes, molecular associations, and survival.
- Published
- 2014
- Full Text
- View/download PDF
33. Down-regulation of eIF4GII by miR-520c-3p represses diffuse large B cell lymphoma development.
- Author
-
Mazan-Mamczarz K, Zhao XF, Dai B, Steinhardt JJ, Peroutka RJ, Berk KL, Landon AL, Sadowska M, Zhang Y, Lehrmann E, Becker KG, Shaknovich R, Liu Z, and Gartenhaus RB
- Subjects
- Animals, Cell Line, Tumor, Cellular Senescence genetics, Down-Regulation, Eukaryotic Initiation Factor-4G biosynthesis, Gene Expression Regulation, Neoplastic, Humans, Lymphoma, Large B-Cell, Diffuse pathology, Mice, MicroRNAs biosynthesis, RNA, Messenger genetics, RNA, Small Interfering, Up-Regulation, Xenograft Model Antitumor Assays, Cell Proliferation, Eukaryotic Initiation Factor-4G genetics, Lymphoma, Large B-Cell, Diffuse genetics, MicroRNAs genetics
- Abstract
Deregulation of the translational machinery is emerging as a critical contributor to cancer development. The contribution of microRNAs in translational gene control has been established however; the role of microRNAs in disrupting the cap-dependent translation regulation complex has not been previously described. Here, we established that elevated miR-520c-3p represses global translation, cell proliferation and initiates premature senescence in HeLa and DLBCL cells. Moreover, we demonstrate that miR-520c-3p directly targets translation initiation factor, eIF4GII mRNA and negatively regulates eIF4GII protein synthesis. miR-520c-3p overexpression diminishes cells colony formation and reduces tumor growth in a human xenograft mouse model. Consequently, downregulation of eIF4GII by siRNA decreases translation, cell proliferation and ability to form colonies, as well as induces cellular senescence. In vitro and in vivo findings were further validated in patient samples; DLBCL primary cells demonstrated low miR-520c-3p levels with reciprocally up-regulated eIF4GII protein expression. Our results provide evidence that the tumor suppressor effect of miR-520c-3p is mediated through repression of translation while inducing senescence and that eIF4GII is a key effector of this anti-tumor activity.
- Published
- 2014
- Full Text
- View/download PDF
34. Mechanism-based epigenetic chemosensitization therapy of diffuse large B-cell lymphoma.
- Author
-
Clozel T, Yang S, Elstrom RL, Tam W, Martin P, Kormaksson M, Banerjee S, Vasanthakumar A, Culjkovic B, Scott DW, Wyman S, Leser M, Shaknovich R, Chadburn A, Tabbo F, Godley LA, Gascoyne RD, Borden KL, Inghirami G, Leonard JP, Melnick A, and Cerchietti L
- Subjects
- Adult, Aged, Aged, 80 and over, Antimetabolites, Antineoplastic therapeutic use, Azacitidine adverse effects, Cell Line, Tumor, DNA Damage drug effects, DNA Modification Methylases metabolism, Doxorubicin pharmacology, Drug Resistance, Neoplasm genetics, Epigenesis, Genetic, Humans, Middle Aged, RNA Interference, RNA, Small Interfering, Smad1 Protein genetics, Young Adult, Azacitidine therapeutic use, DNA Methylation genetics, DNA Modification Methylases antagonists & inhibitors, Lymphoma, Large B-Cell, Diffuse drug therapy, Lymphoma, Large B-Cell, Diffuse genetics
- Abstract
Unlabelled: Although aberrant DNA methylation patterning is a hallmark of cancer, the relevance of targeting DNA methyltransferases (DNMT) remains unclear for most tumors. In diffuse large B-cell lymphoma (DLBCL) we observed that chemoresistance is associated with aberrant DNA methylation programming. Prolonged exposure to low-dose DNMT inhibitors (DNMTI) reprogrammed chemoresistant cells to become doxorubicin sensitive without major toxicity in vivo. Nine genes were recurrently hypermethylated in chemoresistant DLBCL. Of these, SMAD1 was a critical contributor, and reactivation was required for chemosensitization. A phase I clinical study was conducted evaluating azacitidine priming followed by standard chemoimmunotherapy in high-risk patients newly diagnosed with DLBCL. The combination was well tolerated and yielded a high rate of complete remission. Pre- and post-azacitidine treatment biopsies confirmed SMAD1 demethylation and chemosensitization, delineating a personalized strategy for the clinical use of DNMTIs., Significance: The problem of chemoresistant DLBCL remains the most urgent challenge in the clinical management of patients with this disease. We describe a mechanism-based approach toward the rational translation of DNMTIs for the treatment of high-risk DLBCL., (©2013 AACR.)
- Published
- 2013
- Full Text
- View/download PDF
35. GobyWeb: simplified management and analysis of gene expression and DNA methylation sequencing data.
- Author
-
Dorff KC, Chambwe N, Zeno Z, Simi M, Shaknovich R, and Campagne F
- Subjects
- Base Sequence, Genomics, Humans, RNA Splicing genetics, User-Computer Interface, DNA Methylation genetics, Database Management Systems, Gene Expression Regulation, High-Throughput Nucleotide Sequencing, Internet, Software
- Abstract
We present GobyWeb, a web-based system that facilitates the management and analysis of high-throughput sequencing (HTS) projects. The software provides integrated support for a broad set of HTS analyses and offers a simple plugin extension mechanism. Analyses currently supported include quantification of gene expression for messenger and small RNA sequencing, estimation of DNA methylation (i.e., reduced bisulfite sequencing and whole genome methyl-seq), or the detection of pathogens in sequenced data. In contrast to previous analysis pipelines developed for analysis of HTS data, GobyWeb requires significantly less storage space, runs analyses efficiently on a parallel grid, scales gracefully to process tens or hundreds of multi-gigabyte samples, yet can be used effectively by researchers who are comfortable using a web browser. We conducted performance evaluations of the software and found it to either outperform or have similar performance to analysis programs developed for specialized analyses of HTS data. We found that most biologists who took a one-hour GobyWeb training session were readily able to analyze RNA-Seq data with state of the art analysis tools. GobyWeb can be obtained at http://gobyweb.campagnelab.org and is freely available for non-commercial use. GobyWeb plugins are distributed in source code and licensed under the open source LGPL3 license to facilitate code inspection, reuse and independent extensions http://github.com/CampagneLaboratory/gobyweb2-plugins.
- Published
- 2013
- Full Text
- View/download PDF
36. Downregulation of FOXP1 is required during germinal center B-cell function.
- Author
-
Sagardoy A, Martinez-Ferrandis JI, Roa S, Bunting KL, Aznar MA, Elemento O, Shaknovich R, Fontán L, Fresquet V, Perez-Roger I, Robles EF, De Smedt L, Sagaert X, Melnick A, and Martinez-Climent JA
- Subjects
- Animals, Cell Differentiation immunology, Cell Line, DNA-Binding Proteins immunology, DNA-Binding Proteins metabolism, Down-Regulation immunology, Forkhead Transcription Factors immunology, Germinal Center immunology, Humans, Lymphoma metabolism, Mice, Mice, Transgenic, Palatine Tonsil cytology, Proto-Oncogene Proteins c-bcl-6, Repressor Proteins immunology, Transcriptional Activation immunology, B-Lymphocytes immunology, B-Lymphocytes metabolism, Forkhead Transcription Factors metabolism, Germinal Center cytology, Lymphoma immunology, Repressor Proteins metabolism
- Abstract
B-cell maturation and germinal center (GC) formation are dependent on the interplay between BCL6 and other transcriptional regulators. FOXP1 is a transcription factor that regulates early B-cell development, but whether it plays a role in mature B cells is unknown. Analysis of human tonsillar B-cell subpopulations revealed that FOXP1 shows the opposite expression pattern to BCL6, suggesting that FOXP1 regulates the transition from resting follicular B cell to activated GC B cell. Chromatin immunoprecipitation-on-chip and gene expression assays on B cells indicated that FOXP1 acts as a transcriptional activator and repressor of genes involved in the GC reaction, half of which are also BCL6 targets. To study FOXP1 function in vivo, we developed transgenic mice expressing human FOXP1 in lymphoid cells. These mice exhibited irregular formation of splenic GCs, showing a modest increase in naïve and marginal-zone B cells and a significant decrease in GC B cells. Furthermore, aberrant expression of FOXP1 impaired transcription of noncoding γ1 germline transcripts and inhibited efficient class switching to the immunoglobulin G1 isotype. These studies show that FOXP1 is physiologically downregulated in GC B cells and that aberrant expression of FOXP1 impairs mechanisms triggered by B-cell activation, potentially contributing to B-cell lymphomagenesis.
- Published
- 2013
- Full Text
- View/download PDF
37. Mechanisms of epigenetic deregulation in lymphoid neoplasms.
- Author
-
Jiang Y, Hatzi K, and Shaknovich R
- Subjects
- Animals, Humans, Epigenesis, Genetic physiology, Gene Expression Regulation, Neoplastic physiology, Leukemia, Lymphocytic, Chronic, B-Cell genetics, Leukemia, Lymphocytic, Chronic, B-Cell physiopathology, Lymphoma, B-Cell genetics, Lymphoma, B-Cell physiopathology
- Published
- 2013
- Full Text
- View/download PDF
38. EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation.
- Author
-
Béguelin W, Popovic R, Teater M, Jiang Y, Bunting KL, Rosen M, Shen H, Yang SN, Wang L, Ezponda T, Martinez-Garcia E, Zhang H, Zheng Y, Verma SK, McCabe MT, Ott HM, Van Aller GS, Kruger RG, Liu Y, McHugh CF, Scott DW, Chung YR, Kelleher N, Shaknovich R, Creasy CL, Gascoyne RD, Wong KK, Cerchietti L, Levine RL, Abdel-Wahab O, Licht JD, Elemento O, and Melnick AM
- Subjects
- Animals, Cell Differentiation, Cell Proliferation, Enhancer of Zeste Homolog 2 Protein, Gene Deletion, Gene Expression Regulation, Neoplastic, Germinal Center drug effects, Histones metabolism, Methylation, Mice, Polycomb Repressive Complex 2 genetics, Polycomb Repressive Complex 2 metabolism, Promoter Regions, Genetic, Proto-Oncogene Proteins c-bcl-2 metabolism, Proto-Oncogene Proteins c-bcl-2 physiology, B-Lymphocytes metabolism, Cell Transformation, Neoplastic genetics, Germinal Center metabolism, Mutation, Polycomb Repressive Complex 2 physiology
- Abstract
The EZH2 histone methyltransferase is highly expressed in germinal center (GC) B cells and targeted by somatic mutations in B cell lymphomas. Here, we find that EZH2 deletion or pharmacologic inhibition suppresses GC formation and functions. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GC B cell (GCB)-type diffuse large B cell lymphomas (DLBCLs) are mostly addicted to EZH2 but not the more differentiated activated B cell (ABC)-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
39. Differential gene body methylation and reduced expression of cell adhesion and neurotransmitter receptor genes in adverse maternal environment.
- Author
-
Oh JE, Chambwe N, Klein S, Gal J, Andrews S, Gleason G, Shaknovich R, Melnick A, Campagne F, and Toth M
- Subjects
- Animals, Cell Adhesion genetics, Disease Models, Animal, Epigenesis, Genetic, Female, Male, Mice, Mice, Transgenic, Pregnancy, Prenatal Exposure Delayed Effects genetics, Vocalization, Animal physiology, Anxiety genetics, CpG Islands genetics, DNA Methylation genetics, Dentate Gyrus metabolism, Receptor, Serotonin, 5-HT1A genetics
- Abstract
Early life adversity, including adverse gestational and postpartum maternal environment, is a contributing factor in the development of autism, attention deficit hyperactivity disorder (ADHD), anxiety and depression but little is known about the underlying molecular mechanism. In a model of gestational maternal adversity that leads to innate anxiety, increased stress reactivity and impaired vocal communication in the offspring, we asked if a specific DNA methylation signature is associated with the emergence of the behavioral phenotype. Genome-wide DNA methylation analyses identified 2.3% of CpGs as differentially methylated (that is, differentially methylated sites, DMSs) by the adverse environment in ventral-hippocampal granule cells, neurons that can be linked to the anxiety phenotype. DMSs were typically clustered and these clusters were preferentially located at gene bodies. Although CpGs are typically either highly methylated or unmethylated, DMSs had an intermediate (20-80%) methylation level that may contribute to their sensitivity to environmental adversity. The adverse maternal environment resulted in either hyper or hypomethylation at DMSs. Clusters of DMSs were enriched in genes that encode cell adhesion molecules and neurotransmitter receptors; some of which were also downregulated, indicating multiple functional deficits at the synapse in adversity. Pharmacological and genetic evidence links many of these genes to anxiety.
- Published
- 2013
- Full Text
- View/download PDF
40. Gene expression and epigenetic deregulation.
- Author
-
Shaknovich R
- Subjects
- Chromatin Assembly and Disassembly, DNA Methylation, Gene Silencing, Humans, Leukemia, Lymphocytic, Chronic, B-Cell etiology, Epigenesis, Genetic, Gene Expression Regulation, Leukemic, Leukemia, Lymphocytic, Chronic, B-Cell genetics
- Abstract
The last decade resulted in many scientific discoveries illuminating epigenetic mechanisms of gene regulation and genome organization. DNA methylation emerged as playing a pivotal role in development and cancer. Genome-wide changes in DNA methylation, including hypermethylation of tumor suppressor genes and genome-wide loss of methylation, are two dominant mechanisms that deregulate gene expression and contribute to chromosomal instability. In this chapter we give an overview of how methylation patterns are established during B-cell development and what machinery is necessary to maintain those patterns. We summarize the current state of knowledge of aberrant changes taking place during and contributing to lymphoid transformation in general and to the development of CLL in particular. We discuss key deregulated biomarkers extensively studied using single-gene approaches and give an overview of a wealth of data that became available from genome-wide approaches, focusing on pathways that are critical for lymphomagenesis. We also highlight epigenetic differences between known prognostic groups of CLL.
- Published
- 2013
- Full Text
- View/download PDF
41. Aberration in DNA methylation in B-cell lymphomas has a complex origin and increases with disease severity.
- Author
-
De S, Shaknovich R, Riester M, Elemento O, Geng H, Kormaksson M, Jiang Y, Woolcock B, Johnson N, Polo JM, Cerchietti L, Gascoyne RD, Melnick A, and Michor F
- Subjects
- Binding Sites, CCCTC-Binding Factor, Cell Line, Tumor, Gene Silencing, Genome, Human, Humans, Insulator Elements genetics, Promoter Regions, Genetic, Repressor Proteins genetics, Repressor Proteins metabolism, B-Lymphocytes metabolism, B-Lymphocytes pathology, DNA Methylation genetics, Epigenesis, Genetic genetics, Lymphoma, Follicular genetics, Lymphoma, Follicular metabolism, Lymphoma, Large B-Cell, Diffuse genetics, Lymphoma, Large B-Cell, Diffuse metabolism, Lymphoma, Large B-Cell, Diffuse pathology
- Abstract
Despite mounting evidence that epigenetic abnormalities play a key role in cancer biology, their contributions to the malignant phenotype remain poorly understood. Here we studied genome-wide DNA methylation in normal B-cell populations and subtypes of B-cell non-Hodgkin lymphoma: follicular lymphoma and diffuse large B-cell lymphomas. These lymphomas display striking and progressive intra-tumor heterogeneity and also inter-patient heterogeneity in their cytosine methylation patterns. Epigenetic heterogeneity is initiated in normal germinal center B-cells, increases markedly with disease aggressiveness, and is associated with unfavorable clinical outcome. Moreover, patterns of abnormal methylation vary depending upon chromosomal regions, gene density and the status of neighboring genes. DNA methylation abnormalities arise via two distinct processes: i) lymphomagenic transcriptional regulators perturb promoter DNA methylation in a target gene-specific manner, and ii) aberrant epigenetic states tend to spread to neighboring promoters in the absence of CTCF insulator binding sites., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2013
- Full Text
- View/download PDF
42. MALT1 small molecule inhibitors specifically suppress ABC-DLBCL in vitro and in vivo.
- Author
-
Fontan L, Yang C, Kabaleeswaran V, Volpon L, Osborne MJ, Beltran E, Garcia M, Cerchietti L, Shaknovich R, Yang SN, Fang F, Gascoyne RD, Martinez-Climent JA, Glickman JF, Borden K, Wu H, and Melnick A
- Subjects
- Animals, B-Lymphocytes metabolism, Caspases metabolism, Catalysis, Cell Line, Tumor, Cell Proliferation drug effects, DNA-Binding Proteins antagonists & inhibitors, DNA-Binding Proteins metabolism, Down-Regulation drug effects, Humans, Lymphoma, Large B-Cell, Diffuse metabolism, Lymphoma, Large B-Cell, Diffuse pathology, Male, Mice, Mice, Inbred NOD, Mice, SCID, Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein, NF-kappa B metabolism, Neoplasm Proteins metabolism, Nuclear Proteins antagonists & inhibitors, Nuclear Proteins metabolism, Proteolysis, Proto-Oncogene Proteins c-rel, Xenograft Model Antitumor Assays, B-Lymphocytes drug effects, Lymphoma, Large B-Cell, Diffuse drug therapy, Neoplasm Proteins antagonists & inhibitors, Protease Inhibitors pharmacology
- Abstract
MALT1 cleavage activity is linked to the pathogenesis of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), a chemoresistant form of DLBCL. We developed a MALT1 activity assay and identified chemically diverse MALT1 inhibitors. A selected lead compound, MI-2, featured direct binding to MALT1 and suppression of its protease function. MI-2 concentrated within human ABC-DLBCL cells and irreversibly inhibited cleavage of MALT1 substrates. This was accompanied by NF-κB reporter activity suppression, c-REL nuclear localization inhibition, and NF-κB target gene downregulation. Most notably, MI-2 was nontoxic to mice, and displayed selective activity against ABC-DLBCL cell lines in vitro and xenotransplanted ABC-DLBCL tumors in vivo. The compound was also effective against primary human non-germinal center B cell-like DLBCLs ex vivo., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
43. Incidence and clinical implications of ATM aberrations in chronic lymphocytic leukemia.
- Author
-
Ouillette P, Li J, Shaknovich R, Li Y, Melnick A, Shedden K, and Malek SN
- Subjects
- Ataxia Telangiectasia Mutated Proteins, Chromosome Deletion, Chromosomes, Human, Pair 11 genetics, Cohort Studies, Humans, Mutation, Prospective Studies, Ataxia Telangiectasia genetics, Cell Cycle Proteins genetics, DNA-Binding Proteins genetics, Leukemia, Lymphocytic, Chronic, B-Cell genetics, Protein Serine-Threonine Kinases genetics, Tumor Suppressor Proteins genetics
- Abstract
A subset of chronic lymphocytic leukemia (CLL) carries mutations in ataxia telangiectasia mutated (ATM). Such ATM mutations may be particularly relevant in the setting of del11q, which invariably results in the deletion of one ATM allele. To improve our understanding of the frequency and type of ATM mutations that exist in CLL, we resequenced all ATM coding exons in 24 CLL with del11q using direct sequencing. We detected two missense mutations, resulting in an ATM mutation frequency of 8%; nonsense and frameshift mutations were not identified. Given the low ATM mutation frequency detected in this cohort, we proceeded with measurements of nonmutational ATM aberrations in CLL through analysis of the activation state of ATM in response to external irradiation. The phosphorylation state of ATM at Ser-1981 was measured using quantitative immunoblotting in purified CLL cells isolated from 251 CLL patients; data were normalized to simultaneous measurements of total ATM protein and actin. Resulting p-ATM/ATM and p-ATM/actin ratios were subsequently analyzed for prognostic significance inclusive and exclusive of TP53 exons 2-10 mutations. From these analyses, conducted in a large prospectively enrolled CLL patient cohort, neither the p-ATM/ATM nor the p-ATM/actin ratios were found to be prognostic for short survival. These data in aggregate demonstrate a low frequency of ATM aberrations in an unselected CLL cohort and do not support a major prognostic role for ATM aberrations in CLL, thus motivating renewed research efforts aimed at understanding the pathobiology of 11q deletions in CLL. © 2012 Wiley Periodicals, Inc., (Copyright © 2012 Wiley Periodicals, Inc.)
- Published
- 2012
- Full Text
- View/download PDF
44. SYK inhibition and response prediction in diffuse large B-cell lymphoma.
- Author
-
Cheng S, Coffey G, Zhang XH, Shaknovich R, Song Z, Lu P, Pandey A, Melnick AM, Sinha U, and Wang YL
- Subjects
- Biomarkers metabolism, Cell Line, Tumor, G1 Phase drug effects, Gene Expression Regulation, Neoplastic, Humans, Intracellular Signaling Peptides and Proteins genetics, Intracellular Signaling Peptides and Proteins metabolism, Lymph Nodes drug effects, Lymph Nodes metabolism, Lymph Nodes pathology, Lymphoma, Large B-Cell, Diffuse metabolism, Lymphoma, Large B-Cell, Diffuse pathology, Molecular Targeted Therapy, Neoplasm Proteins genetics, Neoplasm Proteins metabolism, Phospholipase C gamma antagonists & inhibitors, Phospholipase C gamma genetics, Phospholipase C gamma metabolism, Phosphorylation drug effects, Protein Processing, Post-Translational drug effects, Protein-Tyrosine Kinases genetics, Protein-Tyrosine Kinases metabolism, Proto-Oncogene Proteins c-akt antagonists & inhibitors, Proto-Oncogene Proteins c-akt genetics, Proto-Oncogene Proteins c-akt metabolism, RNA Interference, RNA, Messenger metabolism, RNA, Small Interfering, Signal Transduction drug effects, Syk Kinase, Tumor Cells, Cultured, Antineoplastic Agents pharmacology, Drug Resistance, Neoplasm, Intracellular Signaling Peptides and Proteins antagonists & inhibitors, Lymphoma, Large B-Cell, Diffuse drug therapy, Neoplasm Proteins antagonists & inhibitors, Protein Kinase Inhibitors pharmacology, Protein-Tyrosine Kinases antagonists & inhibitors
- Abstract
Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, and the role of SYK in its pathogenesis is not completely understood. Using tissue microarray, we demonstrated for the first time that SYK protein is activated in 27 of 61 (44%) primary human DLBCL tissues. Among DLBCL cell lines, 7 were sensitive and 3 were resistant to a highly specific SYK inhibitor, PRT060318. In sensitive DLBCL cells, SYK inhibition blocked the G(1)-S transition and caused cell-cycle arrest. This effect was reproduced by genetic reduction of SYK using siRNA. A detailed analysis of the BCR signaling pathways revealed that the consequence of SYK inhibition on PLCγ2 and AKT, as opposed to ERK1/2, was responsible for cell-cycle arrest. Genetic knock-down of these key molecules decelerated the proliferation of lymphoma cells. In addition, BCR signaling can be blocked by PRT060318 in primary lymphoma cells. Together, these findings provide insights into cellular pathways required for lymphoma cell growth and support the rationale for considering SYK inhibition as a potentially useful therapy for DLBCL. The results further suggest the possibility of using PLCγ2 and AKT as biomarkers to predict therapeutic response in prospective clinical trials of specific SYK inhibitors.
- Published
- 2011
- Full Text
- View/download PDF
45. The Eph-receptor A7 is a soluble tumor suppressor for follicular lymphoma.
- Author
-
Oricchio E, Nanjangud G, Wolfe AL, Schatz JH, Mavrakis KJ, Jiang M, Liu X, Bruno J, Heguy A, Olshen AB, Socci ND, Teruya-Feldstein J, Weis-Garcia F, Tam W, Shaknovich R, Melnick A, Himanen JP, Chaganti RS, and Wendel HG
- Subjects
- Animals, Antibodies, Monoclonal, Murine-Derived therapeutic use, Cell Line, Tumor, Chromosomes, Human, Pair 6, Genomics, Humans, Lymphoma, Follicular drug therapy, Lymphoma, Follicular genetics, Male, Mice, Neoplasm Transplantation, RNA Interference, Rituximab, Transplantation, Heterologous, Genes, Tumor Suppressor, Lymphoma, Follicular metabolism, Receptor, EphA7 metabolism
- Abstract
Insights into cancer genetics can lead to therapeutic opportunities. By cross-referencing chromosomal changes with an unbiased genetic screen we identify the ephrin receptor A7 (EPHA7) as a tumor suppressor in follicular lymphoma (FL). EPHA7 is a target of 6q deletions and inactivated in 72% of FLs. Knockdown of EPHA7 drives lymphoma development in a murine FL model. In analogy to its physiological function in brain development, a soluble splice variant of EPHA7 (EPHA7(TR)) interferes with another Eph-receptor and blocks oncogenic signals in lymphoma cells. Consistent with this drug-like activity, administration of the purified EPHA7(TR) protein produces antitumor effects against xenografted human lymphomas. Further, by fusing EPHA7(TR) to the anti-CD20 antibody (rituximab) we can directly target this tumor suppressor to lymphomas in vivo. Our study attests to the power of combining descriptive tumor genomics with functional screens and reveals EPHA7(TR) as tumor suppressor with immediate therapeutic potential., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
46. DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation.
- Author
-
Shaknovich R, Cerchietti L, Tsikitas L, Kormaksson M, De S, Figueroa ME, Ballon G, Yang SN, Weinhold N, Reimers M, Clozel T, Luttrop K, Ekstrom TJ, Frank J, Vasanthakumar A, Godley LA, Michor F, Elemento O, and Melnick A
- Subjects
- Animals, B-Lymphocytes immunology, B-Lymphocytes metabolism, B-Lymphocytes pathology, Cell Differentiation immunology, Cluster Analysis, DNA (Cytosine-5-)-Methyltransferase 1, DNA (Cytosine-5-)-Methyltransferases metabolism, Epigenesis, Genetic physiology, Gene Expression Profiling, Germinal Center metabolism, Humans, Mice, Mice, Inbred C57BL, Mice, Transgenic, Microarray Analysis, Sheep, Validation Studies as Topic, B-Lymphocytes physiology, Cell Differentiation genetics, DNA (Cytosine-5-)-Methyltransferases physiology, DNA Methylation physiology, Germinal Center immunology
- Abstract
The phenotype of germinal center (GC) B cells includes the unique ability to tolerate rapid proliferation and the mutagenic actions of activation induced cytosine deaminase (AICDA). Given the importance of epigenetic patterning in determining cellular phenotypes, we examined DNA methylation and the role of DNA methyltransferases in the formation of GCs. DNA methylation profiling revealed a marked shift in DNA methylation patterning in GC B cells versus resting/naive B cells. This shift included significant differential methylation of 235 genes, with concordant inverse changes in gene expression affecting most notably genes of the NFkB and MAP kinase signaling pathways. GC B cells were predominantly hypomethylated compared with naive B cells and AICDA binding sites were highly overrepresented among hypomethylated loci. GC B cells also exhibited greater DNA methylation heterogeneity than naive B cells. Among DNA methyltransferases (DNMTs), only DNMT1 was significantly up-regulated in GC B cells. Dnmt1 hypomorphic mice displayed deficient GC formation and treatment of mice with the DNA methyltransferase inhibitor decitabine resulted in failure to form GCs after immune stimulation. Notably, the GC B cells of Dnmt1 hypomorphic animals showed evidence of increased DNA damage, suggesting dual roles for DNMT1 in DNA methylation and double strand DNA break repair.
- Published
- 2011
- Full Text
- View/download PDF
47. Epigenetics and B-cell lymphoma.
- Author
-
Shaknovich R and Melnick A
- Subjects
- Animals, DNA Methylation, Epigenesis, Genetic, Humans, Lymphoma, B-Cell pathology, Lymphoma, B-Cell genetics
- Abstract
Purpose of Review: It has only recently become apparent that mutations in epigenetic mechanisms and perturbation of epigenomic patterning are frequent events in B-cell lymphomas. The purpose of this review is to highlight these new findings and provide a conceptual framework for understanding how epigenetic modifications might contribute to lymphomagenesis., Recent Findings: Somatic mutations affecting histone methyltransferases such as enhancer of zeste 2 and mixed lineage leukemia 2, histone demethylases including ubiquitously transcribed X chromosome tetratricopeptide repeat and Jumonji domain-containing 2C, and histone acetyltransferases including CBP and p300 are recurrent and common in lymphomas. These mutations result in disruption of chromatin structure and functions of other proteins, ultimately causing aberrant transcriptional programming affecting multiple gene networks. Widespread perturbation of cytosine methylation patterning now appears to be a hallmark of B-cell lymphomas and occurs in specific patterns that can distinguish disease subtypes. Therapeutic targeting strategies can overcome abnormal epigenetic mechanisms and potently kill lymphoma cells., Summary: Newly discovered epigenetic lesions may provide critical insights into the genesis of B-cell lymphomas, but further studies are required to understand how they affect biological mechanism. Epigenetic lesions offer tremendous opportunities for the development of improved biomarkers and treatments.
- Published
- 2011
- Full Text
- View/download PDF
48. EZH2-mediated epigenetic silencing in germinal center B cells contributes to proliferation and lymphomagenesis.
- Author
-
Velichutina I, Shaknovich R, Geng H, Johnson NA, Gascoyne RD, Melnick AM, and Elemento O
- Subjects
- B-Lymphocytes pathology, Enhancer of Zeste Homolog 2 Protein, Epigenesis, Genetic, Germinal Center pathology, Humans, Lymphoma, Large B-Cell, Diffuse pathology, Polycomb Repressive Complex 2, Promoter Regions, Genetic, Tumor Cells, Cultured, Cell Proliferation, Cell Transformation, Neoplastic, DNA-Binding Proteins physiology, Gene Silencing physiology, Lymphoma, Large B-Cell, Diffuse etiology, Transcription Factors physiology
- Abstract
EZH2 is the catalytic subunit of the PRC2 Polycomb complex and mediates transcriptional repression through its histone methyltransferase activity. EZH2 is up-regulated in normal germinal center (GC) B cells and is implicated in lymphomagenesis. To explore the transcriptional programs controlled by EZH2, we performed chromatin immunoprecipitation (ChIP-on-chip) in GC cells and found that it binds approximately 1800 promoters, often associated with DNA sequences similar to Droso-phila Polycomb response elements. While EZH2 targets overlapped extensively between GC B cells and embryonic stem cells, we also observed a large GC-specific EZH2 regulatory program. These genes are preferentially histone 3 lysine 27-trimethylated and repressed in GC B cells and include several key cell cycle-related tumor suppressor genes. Accordingly, siRNA-mediated down-regulation of EZH2 in diffuse large B-cell lymphoma (DLBCL) cells resulted in acute cell cycle arrest at the G(1)/S transition and up-regulation of its tumor suppressor target genes. At the DNA level, EZH2-bound promoters are hypomethylated in GC B cells, but many of them are aberrantly hypermethylated in DLBCL, suggesting disruption of normal epigenetic processes in these cells. EZH2 is thus involved in regulating a specific epigenetic program in normal GCs, including silencing of antiproliferative genes, which may contribute to the malignant transformation of GC B cells into DLBCLs.
- Published
- 2010
- Full Text
- View/download PDF
49. BCL6 repression of EP300 in human diffuse large B cell lymphoma cells provides a basis for rational combinatorial therapy.
- Author
-
Cerchietti LC, Hatzi K, Caldas-Lopes E, Yang SN, Figueroa ME, Morin RD, Hirst M, Mendez L, Shaknovich R, Cole PA, Bhalla K, Gascoyne RD, Marra M, Chiosis G, and Melnick A
- Subjects
- Animals, Cell Line, Tumor, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, E1A-Associated p300 Protein genetics, E1A-Associated p300 Protein metabolism, Female, Histone Deacetylase Inhibitors pharmacology, Humans, Lymphoma, Large B-Cell, Diffuse metabolism, Lymphoma, Large B-Cell, Diffuse pathology, Male, Mice, Mice, SCID, Molecular Chaperones genetics, Molecular Chaperones metabolism, Proto-Oncogene Proteins c-bcl-6, Xenograft Model Antitumor Assays, Antineoplastic Combined Chemotherapy Protocols pharmacology, DNA-Binding Proteins antagonists & inhibitors, E1A-Associated p300 Protein antagonists & inhibitors, Lymphoma, Large B-Cell, Diffuse drug therapy
- Abstract
B cell lymphoma 6 (BCL6), which encodes a transcriptional repressor, is a critical oncogene in diffuse large B cell lymphomas (DLBCLs). Although a retro-inverted BCL6 peptide inhibitor (RI-BPI) was recently shown to potently kill DLBCL cells, the underlying mechanisms remain unclear. Here, we show that RI-BPI induces a particular gene expression signature in human DLBCL cell lines that included genes associated with the actions of histone deacetylase (HDAC) and Hsp90 inhibitors. BCL6 directly repressed the expression of p300 lysine acetyltransferase (EP300) and its cofactor HLA-B-associated transcript 3 (BAT3). RI-BPI induced expression of p300 and BAT3, resulting in acetylation of p300 targets including p53 and Hsp90. Induction of p300 and BAT3 was required for the antilymphoma effects of RI-BPI, since specific blockade of either protein rescued human DLBCL cell lines from the BCL6 inhibitor. Consistent with this, combination of RI-BPI with either an HDAC inhibitor (HDI) or an Hsp90 inhibitor potently suppressed or even eradicated established human DLBCL xenografts in mice. Furthermore, HDAC and Hsp90 inhibitors independently enhanced RI-BPI killing of primary human DLBCL cells in vitro. We also show that p300-inactivating mutations occur naturally in human DLBCL patients and may confer resistance to BCL6 inhibitors. Thus, BCL6 repression of EP300 provides a basis for rational targeted combinatorial therapy for patients with DLBCL.
- Published
- 2010
- Full Text
- View/download PDF
50. DNA methylation signatures define molecular subtypes of diffuse large B-cell lymphoma.
- Author
-
Shaknovich R, Geng H, Johnson NA, Tsikitas L, Cerchietti L, Greally JM, Gascoyne RD, Elemento O, and Melnick A
- Subjects
- Epigenesis, Genetic, Gene Expression Regulation, Neoplastic, Gene Regulatory Networks genetics, Genes, Neoplasm genetics, Humans, Signal Transduction genetics, Tumor Necrosis Factor-alpha genetics, DNA Methylation genetics, Gene Expression Profiling, Lymphoma, Large B-Cell, Diffuse classification, Lymphoma, Large B-Cell, Diffuse genetics
- Abstract
Expression profiling has shown 2 main and clinically distinct subtypes of diffuse large B-cell lymphomas (DLBCLs): germinal-center B cell-like (GCB) and activated B cell-like (ABC) DLBCLs. Further work has shown that these subtypes are partially characterized by distinct genetic alterations and different survival. Here, we show with the use of an assay that measures DNA methylation levels of 50,000 CpG motifs distributed among more than 14,000 promoters that these 2 DLBCL subtypes are also characterized by distinct epigenetic profiles. DNA methylation and gene expression profiling were performed on a cohort of 69 patients with DLBCL. After assigning ABC or GCB labels with a Bayesian expression classifier trained on an independent dataset, a supervised analysis identified 311 differentially methylated probe sets (263 unique genes) between ABC and GCB DLBCLs. Integrated analysis of methylation and gene expression showed a core tumor necrosis factor-α signaling pathway as the principal differentially perturbed gene network. Sixteen genes overlapped between the core ABC/GCB methylation and expression signatures and encoded important proteins such as IKZF1. This reduced gene set was an accurate predictor of ABC and GCB subtypes. Collectively, the data suggest that epigenetic patterning contributes to the ABC and GCB DLBCL phenotypes and could serve as useful biomarker.
- Published
- 2010
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.