23 results on '"Tiedemann RE"'
Search Results
2. sciCNV: high-throughput paired profiling of transcriptomes and DNA copy number variations at single-cell resolution.
- Author
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Mahdipour-Shirayeh A, Erdmann N, Leung-Hagesteijn C, and Tiedemann RE
- Subjects
- Chromosomes, High-Throughput Nucleotide Sequencing methods, RNA, Messenger, DNA Copy Number Variations, Transcriptome
- Abstract
Chromosome copy number variations (CNVs) are a near-universal feature of cancer; however, their individual effects on cellular function are often incompletely understood. Single-cell ribonucleic acid (RNA) sequencing (scRNA-seq) might be leveraged to reveal the function of intra-clonal CNVs; however, it cannot directly link cellular gene expression to CNVs. Here, we report a high-throughput scRNA-seq analysis pipeline that provides paired CNV profiles and transcriptomes for single cells, enabling exploration of the effects of CNVs on cellular programs. RTAM1 and -2 normalization methods are described, and are shown to improve transcriptome alignment between cells, increasing the sensitivity of scRNA-seq for CNV detection. We also report single-cell inferred chromosomal copy number variation (sciCNV), a tool for inferring single-cell CNVs from scRNA-seq at 19-46 Mb resolution. Comparison of sciCNV with existing RNA-based CNV methods reveals useful advances in sensitivity and specificity. Using sciCNV, we demonstrate that scRNA-seq can be used to examine the cellular effects of cancer CNVs. As an example, sciCNV is used to identify subclonal multiple myeloma (MM) cells with +8q22-24. Studies of the gene expression of intra-clonal MM cells with and without the CNV demonstrate that +8q22-24 upregulates MYC and MYC-target genes, messenger RNA processing and protein synthesis, which is consistent with established models. In conclusion, we provide new tools for scRNA-seq that enable paired profiling of the CNVs and transcriptomes of single cells, facilitating rapid and accurate deconstruction of the effects of cancer CNVs on cellular programming., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
- Published
- 2022
- Full Text
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3. Correction to "Design, Synthesis, and Characterization of 4-Aminoquinazolines as Potent Inhibitors of the G Protein-Coupled Receptor Kinase 6 (GRK6) for the Treatment of Multiple Myeloma".
- Author
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Uehling DE, Joseph B, Chung KC, Zhang AX, Ler S, Prakesch MA, Poda G, Grouleff J, Aman A, Kiyota T, Leung-Hagesteijn C, Konda JD, Marcellus R, Griffin C, Subramaniam R, Abibi A, Strathdee CA, Isaac MB, Al-Awar R, and Tiedemann RE
- Published
- 2022
- Full Text
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4. Combined EZH2 Inhibition and IKAROS Degradation Leads to Enhanced Antitumor Activity in Diffuse Large B-cell Lymphoma.
- Author
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Tong KI, Yoon S, Isaev K, Bakhtiari M, Lackraj T, He MY, Joynt J, Silva A, Xu MC, Privé GG, He HH, Tiedemann RE, Chavez EA, Chong LC, Boyle M, Scott DW, Steidl C, and Kridel R
- Subjects
- Apoptosis genetics, Cell Cycle Checkpoints genetics, Cell Line, Tumor, Humans, Lenalidomide, Enhancer of Zeste Homolog 2 Protein genetics, Lymphoma, Large B-Cell, Diffuse drug therapy, Lymphoma, Large B-Cell, Diffuse genetics, Lymphoma, Large B-Cell, Diffuse pathology
- Abstract
Purpose: The efficacy of EZH2 inhibition has been modest in the initial clinical exploration of diffuse large B-cell lymphoma (DLBCL), yet EZH2 inhibitors are well tolerated. Herein, we aimed to uncover genetic and pharmacologic opportunities to enhance the clinical efficacy of EZH2 inhibitors in DLBCL., Experimental Design: We conducted a genome-wide sensitizing CRISPR/Cas9 screen with tazemetostat, a catalytic inhibitor of EZH2. The sensitizing effect of IKZF1 loss of function was then validated and leveraged for combination treatment with lenalidomide. RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing analyses were performed to elucidate transcriptomic and epigenetic changes underlying synergy., Results: We identified IKZF1 knockout as the top candidate for sensitizing DLBCL cells to tazemetostat. Treating cells with tazemetostat and lenalidomide, an immunomodulatory drug that selectively degrades IKAROS and AIOLOS, phenocopied the effects of the CRISPR/Cas9 screen. The combined drug treatment triggered either cell-cycle arrest or apoptosis in a broad range of DLBCL cell lines, regardless of EZH2 mutational status. Cell-line-based xenografts also showed slower tumor growth and prolonged survival in the combination treatment group. RNA-seq analysis revealed strong upregulation of interferon signaling and antiviral immune response signatures. Gene expression of key immune response factors such as IRF7 and DDX58 were induced in cells treated with lenalidomide and tazemetostat, with a concomitant increase of H3K27 acetylation at their promoters. Furthermore, transcriptome analysis demonstrated derepression of endogenous retroviruses after combination treatment., Conclusions: Our data underscore the synergistic interplay between IKAROS degradation and EZH2 inhibition on modulating epigenetic changes and ultimately enhancing antitumor effects in DLBCL., (©2021 American Association for Cancer Research.)
- Published
- 2021
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5. Design, Synthesis, and Characterization of 4-Aminoquinazolines as Potent Inhibitors of the G Protein-Coupled Receptor Kinase 6 (GRK6) for the Treatment of Multiple Myeloma.
- Author
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Uehling DE, Joseph B, Chung KC, Zhang AX, Ler S, Prakesch MA, Poda G, Grouleff J, Aman A, Kiyota T, Leung-Hagesteijn C, Konda JD, Marcellus R, Griffin C, Subramaniam R, Abibi A, Strathdee CA, Isaac MB, Al-Awar R, and Tiedemann RE
- Subjects
- Animals, Antineoplastic Agents chemical synthesis, Antineoplastic Agents chemistry, Cell Proliferation drug effects, Cell Survival drug effects, Dose-Response Relationship, Drug, Drug Screening Assays, Antitumor, G-Protein-Coupled Receptor Kinases metabolism, Humans, Mice, Models, Molecular, Molecular Structure, Multiple Myeloma metabolism, Multiple Myeloma pathology, Protein Kinase Inhibitors chemical synthesis, Protein Kinase Inhibitors chemistry, Quinazolines chemical synthesis, Quinazolines chemistry, Structure-Activity Relationship, Antineoplastic Agents pharmacology, Drug Design, G-Protein-Coupled Receptor Kinases antagonists & inhibitors, Multiple Myeloma drug therapy, Protein Kinase Inhibitors pharmacology, Quinazolines pharmacology
- Abstract
Both previous and additional genetic knockdown studies reported herein implicate G protein-coupled receptor kinase 6 (GRK6) as a critical kinase required for the survival of multiple myeloma (MM) cells. Therefore, we sought to develop a small molecule GRK6 inhibitor as an MM therapeutic. From a focused library of known kinase inhibitors, we identified two hits with moderate biochemical potencies against GRK6. From these hits, we developed potent (IC
50 < 10 nM) analogues with selectivity against off-target kinases. Further optimization led to the discovery of an analogue ( 18 ) with an IC50 value of 6 nM against GRK6 and selectivity against a panel of 85 kinases. Compound 18 has potent cellular target engagement and antiproliferative activity against MM cells and is synergistic with bortezomib. In summary, we demonstrate that targeting GRK6 with small molecule inhibitors represents a promising approach for MM and identify 18 as a novel, potent, and selective GRK6 inhibitor.- Published
- 2021
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6. Listeria Susceptibility in Patients With Multiple Myeloma Receiving Daratumumab-Based Therapy.
- Author
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Khan S, Vaisman A, Hota SS, Bennett S, Trudel S, Reece D, and Tiedemann RE
- Subjects
- Case-Control Studies, Disease Susceptibility, Humans, Listeria monocytogenes, Risk Factors, Antibodies, Monoclonal therapeutic use, Antineoplastic Agents therapeutic use, Listeriosis epidemiology, Multiple Myeloma drug therapy
- Published
- 2020
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7. Chromosomal Instability and mTORC1 Activation through PTEN Loss Contribute to Proteotoxic Stress in Ovarian Carcinoma.
- Author
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Chui MH, Doodnauth SA, Erdmann N, Tiedemann RE, Sircoulomb F, Drapkin R, Shaw P, and Rottapel R
- Subjects
- Animals, Bortezomib pharmacology, Carcinoma, Ovarian Epithelial drug therapy, Carcinoma, Ovarian Epithelial genetics, Carcinoma, Ovarian Epithelial pathology, Cell Line, Tumor, Cell Transformation, Neoplastic drug effects, Cell Transformation, Neoplastic genetics, Cell Transformation, Neoplastic pathology, Chromosomal Instability drug effects, Cystadenocarcinoma, Serous drug therapy, Cystadenocarcinoma, Serous genetics, Cystadenocarcinoma, Serous pathology, Epithelium drug effects, Epithelium pathology, Fallopian Tube Neoplasms genetics, Fallopian Tube Neoplasms pathology, Female, Gene Expression Regulation, Neoplastic drug effects, Gene Expression Regulation, Neoplastic genetics, Humans, Mice, Mice, Inbred NOD, Mice, SCID, Ovarian Neoplasms drug therapy, Proteasome Endopeptidase Complex metabolism, Proteasome Inhibitors pharmacology, Signal Transduction drug effects, Signal Transduction genetics, Unfolded Protein Response drug effects, Unfolded Protein Response genetics, Chromosomal Instability genetics, Mechanistic Target of Rapamycin Complex 1 genetics, Ovarian Neoplasms genetics, PTEN Phosphohydrolase genetics
- Abstract
High-grade serous ovarian carcinoma commonly arises from fallopian tube secretory epithelium and is characterized by a high level of chromosomal instability. To model the acquisition of aneuploidy during early carcinogenesis, chromosome missegregation was induced in immortalized tubal epithelial cells, which proved acutely detrimental to cellular fitness. The phenotype was characterized by accumulation of misfolded proteins, activation of the unfolded protein response (UPR), decreased protein synthesis, and enhanced vulnerability to proteasome inhibition. However, chromosome missegregation also resulted in heightened transformation potential, assessed by colony formation in soft agar. Ovarian cancer cells retained intrinsic sensitivity to proteasome inhibitors under adherent culture conditions, but acquired resistance as spheroids (recapitulating their native configuration in ascites) by downregulating protein synthesis via mTORC1 suppression. Loss of PTEN drove constitutive mTORC1 activity, enhanced proteotoxic stress, as evidenced by UPR induction, and resensitized tumor spheroids to proteasome inhibition both in vitro and in vivo . In cohorts of primary ovarian carcinomas, mTORC1 and UPR signaling pathways were closely associated. These results implicate attenuation of protein synthesis as a protective mechanism in tumor spheroids, which may explain the overall poor response to bortezomib in clinical trials of patients with advanced ovarian cancer. However, patients with PTEN-deficient tumors may represent a subpopulation potentially amenable to treatment with proteasome inhibitors or other therapeutic agents that disrupt protein homeostasis. SIGNIFICANCE: Chromosome instability and protein synthesis are important factors that determine the efficacy of proteotoxic stress-inducing agents, such as proteasome inhibitors, in the treatment of ovarian cancer. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/21/5536/F1.large.jpg., (©2019 American Association for Cancer Research.)
- Published
- 2019
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8. Xbp1s-Negative Tumor B Cells and Pre-Plasmablasts Mediate Therapeutic Proteasome Inhibitor Resistance in Multiple Myeloma.
- Author
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Leung-Hagesteijn C, Erdmann N, Cheung G, Keats JJ, Stewart AK, Reece DE, Chung KC, and Tiedemann RE
- Published
- 2015
- Full Text
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9. Clinical characteristics and early treatment outcomes of follicular lymphoma in young adults.
- Author
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Gangatharan SA, Maganti M, Kuruvilla JG, Kukreti V, Tiedemann RE, Gospodarowicz MK, Hodgson DC, Sun A, Tsang RW, Pintilie M, and Crump M
- Subjects
- Adult, Age Factors, Aged, Disease-Free Survival, Female, Follow-Up Studies, Humans, Male, Middle Aged, Neoplasm Staging, Retrospective Studies, Survival Rate, Young Adult, Databases, Factual, Lymphoma, Follicular mortality, Lymphoma, Follicular therapy
- Abstract
Follicular lymphoma (FL) in young adults (YA, <40 years old) is uncommon, and the clinical characteristics and outcomes of this group are not well defined. We conducted a retrospective database review of 427 patients with newly diagnosed FL aged 65 years or less registered at Princess Margaret Cancer Centre between 1995 and 2010. YA (n = 61) and those 40-65 (n = 366) were compared with regards to clinical stage at diagnosis, FL International Prognostic Index (FLIPI) score, and the following clinical outcomes: time to second treatment, cause-specific survival (CSS) and overall survival (OS). At diagnosis, stage and FLIPI score were similar, as were the proportion of patients requiring therapy (YA 75% versus older adults 71%). Median follow-up was 8.1 years. Time to second therapy was similar in both age groups (5-year probability 23% YA versus 27% older adults; Gray's P-value = 0.76). Ten-year OS was significantly higher for YA (87% versus older adults 72%; P = 0.029). On multivariate analysis, age <40 years, low FLIPI score and observation as initial management were favourable prognostic factors for OS and CSS. We conclude that YA with FL have a favourable prognosis compared to older patients; whether this reflects competing mortality risks or age-related differences in lymphoma biology warrants further investigation., (© 2015 John Wiley & Sons Ltd.)
- Published
- 2015
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10. BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies.
- Author
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Bogenberger JM, Kornblau SM, Pierceall WE, Lena R, Chow D, Shi CX, Mantei J, Ahmann G, Gonzales IM, Choudhary A, Valdez R, Camoriano J, Fauble V, Tiedemann RE, Qiu YH, Coombes KR, Cardone M, Braggio E, Yin H, Azorsa DO, Mesa RA, Stewart AK, and Tibes R
- Subjects
- Biphenyl Compounds pharmacology, Bridged Bicyclo Compounds, Heterocyclic pharmacology, Cell Line, Tumor, Humans, Myeloid Cell Leukemia Sequence 1 Protein antagonists & inhibitors, Myeloid Cell Leukemia Sequence 1 Protein physiology, Myeloproliferative Disorders drug therapy, Nitrophenols pharmacology, Piperazines pharmacology, Proto-Oncogene Proteins c-bcl-2 antagonists & inhibitors, RNA Interference, Sulfonamides pharmacology, bcl-X Protein antagonists & inhibitors, bcl-X Protein physiology, Antimetabolites, Antineoplastic pharmacology, Azacitidine pharmacology, Leukemia, Myeloid, Acute drug therapy, Myelodysplastic Syndromes drug therapy, Proto-Oncogene Proteins c-bcl-2 physiology
- Abstract
Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response.
- Published
- 2014
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11. Getting to the root of the problem: the causes of relapse in multiple myeloma.
- Author
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Chan Chung KC and Tiedemann RE
- Subjects
- Disease Progression, Drug Resistance, Neoplasm, Humans, Multiple Myeloma drug therapy, Multiple Myeloma genetics, Neoplastic Stem Cells metabolism, Recurrence, Antineoplastic Agents pharmacology, Multiple Myeloma pathology, Proteasome Inhibitors pharmacology
- Abstract
Multiple myeloma (MM) remains incurable, and ultimately, patients exhibit disease progression under current treatment regimens. Proteasome inhibitors have emerged as frontline treatment of relapsed and refractory MM however, resistance to these drugs occur through poorly defined mechanisms. Numerous studies have identified different acquired resistance models such as β5 proteasome subunit mutations and stabilization of tumor suppressors and apoptotic proteins. In addition, recent findings have identified a progenitor organization in MM whereby early progenitor tumor cells show resistance to proteasome inhibitor therapy and cause progressive disease with maturation arrest. This editorial highlights the potential causes of MM relapse in the context of these tumor progenitor cells and the role these cells play in treatment failure.
- Published
- 2014
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12. Genome-wide studies in multiple myeloma identify XPO1/CRM1 as a critical target validated using the selective nuclear export inhibitor KPT-276.
- Author
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Schmidt J, Braggio E, Kortuem KM, Egan JB, Zhu YX, Xin CS, Tiedemann RE, Palmer SE, Garbitt VM, McCauley D, Kauffman M, Shacham S, Chesi M, Bergsagel PL, and Stewart AK
- Subjects
- Animals, Cell Line, Tumor, Cell Nucleus metabolism, Gene Expression Profiling, Humans, Karyopherins drug effects, Mice, RNA Interference, Receptors, Cytoplasmic and Nuclear drug effects, Reverse Transcriptase Polymerase Chain Reaction, Xenograft Model Antitumor Assays, Exportin 1 Protein, Acrylamides pharmacology, Biological Transport drug effects, Cell Nucleus drug effects, Genome-Wide Association Study, Karyopherins genetics, Multiple Myeloma genetics, Receptors, Cytoplasmic and Nuclear genetics, Thiazoles pharmacology
- Abstract
RNA interference screening identified XPO1 (exportin 1) among the 55 most vulnerable targets in multiple myeloma (MM). XPO1 encodes CRM1, a nuclear export protein. XPO1 expression increases with MM disease progression. Patients with MM have a higher expression of XPO1 compared with normal plasma cells (P<0.04) and to patients with monoclonal gammopathy of undetermined significance/smoldering MM (P<0.0001). The highest XPO1 level was found in human MM cell lines (HMCLs). A selective inhibitor of nuclear export compound KPT-276 specifically and irreversibly inhibits the nuclear export function of XPO1. The viability of 12 HMCLs treated with KTP-276 was significantly reduced. KPT-276 also actively induced apoptosis in primary MM patient samples. In gene expression analyses, two genes of probable relevance were dysregulated by KPT-276: cell division cycle 25 homolog A (CDC25A) and bromodomain-containing protein 4 (BRD4), both of which are associated with c-MYC pathway. Western blotting and reverse transcription-PCR confirm that c-MYC, CDC25A and BRD4 are all downregulated after treatment with KPT-276. KPT-276 reduced monoclonal spikes in the Vk*MYC transgenic MM mouse model, and inhibited tumor growth in a xenograft MM mouse model. A phase I clinical trial of an analog of KPT-276 is ongoing in hematological malignancies including MM.
- Published
- 2013
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13. Xbp1s-negative tumor B cells and pre-plasmablasts mediate therapeutic proteasome inhibitor resistance in multiple myeloma.
- Author
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Leung-Hagesteijn C, Erdmann N, Cheung G, Keats JJ, Stewart AK, Reece DE, Chung KC, and Tiedemann RE
- Subjects
- Activating Transcription Factor 6 metabolism, Boronic Acids pharmacology, Boronic Acids therapeutic use, Bortezomib, Cell Survival drug effects, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Endoplasmic Reticulum Stress, Endoribonucleases metabolism, Humans, Immunophenotyping, Membrane Proteins metabolism, Multiple Myeloma metabolism, Mutation, Plasma Cells metabolism, Plasma Cells pathology, Precursor Cells, B-Lymphoid metabolism, Precursor Cells, B-Lymphoid pathology, Proteasome Inhibitors pharmacology, Protein Serine-Threonine Kinases metabolism, Pyrazines pharmacology, Pyrazines therapeutic use, Regulatory Factor X Transcription Factors, Signal Transduction, Transcription Factors genetics, Transcription Factors metabolism, X-Box Binding Protein 1, eIF-2 Kinase metabolism, DNA-Binding Proteins deficiency, Drug Resistance, Neoplasm genetics, Multiple Myeloma drug therapy, Multiple Myeloma genetics, Proteasome Inhibitors therapeutic use, Transcription Factors deficiency
- Abstract
Proteasome inhibitor (PI) resistance mechanisms in multiple myeloma (MM) remain controversial. We report the existence of a progenitor organization in primary MM that recapitulates maturation stages between B cells and plasma cells and that contributes to clinical PI resistance. Xbp1s(-) tumor B cells and pre-plasmablasts survive therapeutic PI, preventing cure, while maturation arrest of MM before the plasmablast stage enables progressive disease on PI treatment. Mechanistically, suppression of Xbp1s in MM is shown to induce bortezomib resistance via de-commitment to plasma cell maturation and immunoglobulin production, diminishing endoplasmic reticulum (ER) front-loading and cytotoxic susceptibility to PI-induced inhibition of ER-associated degradation. These results reveal the tumor progenitor structure in MM and highlight its role in therapeutic failure., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
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14. Identification of molecular vulnerabilities in human multiple myeloma cells by RNA interference lethality screening of the druggable genome.
- Author
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Tiedemann RE, Zhu YX, Schmidt J, Shi CX, Sereduk C, Yin H, Mousses S, and Stewart AK
- Subjects
- Antineoplastic Agents pharmacology, Biomarkers, Tumor genetics, Cell Line, Tumor, Cell Survival drug effects, Cell Survival genetics, Cyclin-Dependent Kinases genetics, Gene Expression Profiling, Gene Expression Regulation, Neoplastic, Genetic Predisposition to Disease genetics, HEK293 Cells, Humans, Methyltransferases genetics, Multiple Myeloma drug therapy, Multiple Myeloma pathology, Myeloid Cell Leukemia Sequence 1 Protein, Protein-Tyrosine Kinases genetics, Proto-Oncogene Proteins c-bcl-2 genetics, Genes, Lethal genetics, Genome, Human genetics, Multiple Myeloma genetics, RNA Interference
- Abstract
Despite recent advances in targeted treatments for multiple myeloma, optimal molecular therapeutic targets have yet to be identified. To functionally identify critical molecular targets, we conducted a genome-scale lethality study in multiple myeloma cells using siRNAs. We validated the top 160 lethal hits with four siRNAs per gene in three multiple myeloma cell lines and two non-myeloma cell lines, cataloging a total of 57 potent multiple myeloma survival genes. We identified the Bcl2 family member MCL1 and several 26S proteasome subunits among the most important and selective multiple myeloma survival genes. These results provided biologic validation of our screening strategy. Other essential targets included genes involved in RNA splicing, ubiquitination, transcription, translation, and mitosis. Several of the multiple myeloma survival genes, especially MCL1, TNK2, CDK11, and WBSCR22, exhibited differential expression in primary plasma cells compared with other human primary somatic tissues. Overall, the most striking differential functional vulnerabilities between multiple myeloma and non-multiple myeloma cells were found to occur within the 20S proteasome subunits, MCL1, RRM1, USP8, and CKAP5. We propose that these genes should be investigated further as potential therapeutic targets in multiple myeloma., (©2011 AACR.)
- Published
- 2012
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15. Kinome-wide RNAi studies in human multiple myeloma identify vulnerable kinase targets, including a lymphoid-restricted kinase, GRK6.
- Author
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Tiedemann RE, Zhu YX, Schmidt J, Yin H, Shi CX, Que Q, Basu G, Azorsa D, Perkins LM, Braggio E, Fonseca R, Bergsagel PL, Mousses S, and Stewart AK
- Subjects
- Animals, Cell Line, Tumor, Chromosomes, Human genetics, Chromosomes, Human metabolism, G-Protein-Coupled Receptor Kinases antagonists & inhibitors, G-Protein-Coupled Receptor Kinases genetics, Gene Silencing drug effects, HSP90 Heat-Shock Proteins genetics, HSP90 Heat-Shock Proteins metabolism, Humans, Mice, Multiple Myeloma drug therapy, Multiple Myeloma genetics, Phosphorylation drug effects, Phosphorylation genetics, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors therapeutic use, Protein Kinases genetics, Protein Kinases metabolism, STAT3 Transcription Factor genetics, STAT3 Transcription Factor metabolism, Translocation, Genetic drug effects, Translocation, Genetic genetics, G-Protein-Coupled Receptor Kinases metabolism, Multiple Myeloma enzymology, RNA, Small Interfering
- Abstract
A paucity of validated kinase targets in human multiple myeloma has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in myeloma tumor lines bearing common t(4;14), t(14;16), and t(11;14) translocations to identify critically vulnerable kinases in myeloma tumor cells without regard to preconceived mechanistic notions. Fifteen kinases were repeatedly vulnerable in myeloma cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. Whereas several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly G protein-coupled receptor kinase, GRK6, appeared selectively vulnerable in myeloma. GRK6 inhibition was lethal to 6 of 7 myeloma tumor lines but was tolerated in 7 of 7 human cell lines. GRK6 exhibits lymphoid-restricted expression, and from coimmunoprecipitation studies we demonstrate that expression in myeloma cells is regulated via direct association with the heat shock protein 90 (HSP90) chaperone. GRK6 silencing causes suppression of signal transducer and activator of transcription 3 (STAT3) phosphorylation associated with reduction in MCL1 levels and phosphorylation, illustrating a potent mechanism for the cytotoxicity of GRK6 inhibition in multiple myeloma (MM) tumor cells. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma.
- Published
- 2010
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16. Identification of a potent natural triterpenoid inhibitor of proteosome chymotrypsin-like activity and NF-kappaB with antimyeloma activity in vitro and in vivo.
- Author
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Tiedemann RE, Schmidt J, Keats JJ, Shi CX, Zhu YX, Palmer SE, Mao X, Schimmer AD, and Stewart AK
- Subjects
- Animals, Antineoplastic Agents chemistry, Apoptosis drug effects, Biological Products chemistry, Bone Marrow Cells cytology, Bone Marrow Cells drug effects, Cell Lineage, Cells, Cultured, Chymases metabolism, Coculture Techniques, Combinatorial Chemistry Techniques, Cyclin D, Cyclins genetics, Cyclins metabolism, Drug Screening Assays, Antitumor, Enzyme Activation drug effects, Gene Expression Regulation, Neoplastic drug effects, Humans, Mice, Molecular Structure, Multiple Myeloma pathology, NF-kappa B metabolism, Pentacyclic Triterpenes, Proteasome Endopeptidase Complex metabolism, Transcriptional Activation drug effects, Triterpenes chemistry, Xenograft Model Antitumor Assays, Antineoplastic Agents pharmacology, Biological Products pharmacology, Chymases antagonists & inhibitors, Multiple Myeloma metabolism, NF-kappa B antagonists & inhibitors, Proteasome Inhibitors, Triterpenes pharmacology
- Abstract
As multiple myeloma tumors universally dysregulate cyclin D genes we conducted high-throughput chemical library screens for compounds that induce suppression of cyclin D2 promoter transcription. The top-ranked compound was a natural triterpenoid, pristimerin. Strikingly, the early transcriptional response of cells treated with pristimerin closely resembles cellular responses elicited by proteosome inhibitors, with rapid induction of heat shock proteins, activating transcription factor 3 (ATF3), and CHOP. Enzymatic assays and immunoblotting confirm that pristimerin rapidly (< 90 minutes) and specifically inhibits chymotrypsin-like proteosome activity at low concentrations (< 100 nM) and causes accumulation of cellular ubiquitinated proteins. Notably, cytotoxic triterpenoids including pristimerin inhibit NF-kappaB activation via inhibition of IKK alpha or IKK beta, whereas proteosome inhibitors instead suppress NF-kappaB function by impairing degradation of ubiquitinated I kappaB. By inhibiting both IKK and the proteosome, pristimerin causes overt suppression of constitutive NF-kappaB activity in myeloma cells that may mediate its suppression of cyclin D. Multiple myeloma is exquisitely sensitive to proteosome or NF-kappaB pathway inhibition. Consistent with this, pristimerin is potently and selectively lethal to primary myeloma cells (IC(50) < 100 nM), inhibits xenografted plasmacytoma tumors in mice, and is synergistically cytotoxic with bortezomib--providing the rationale for pharmaceutical development of triterpenoid dual-function proteosome/NF-kappaB inhibitors as therapeutics for human multiple myeloma and related malignancies.
- Published
- 2009
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17. Identification of kinetin riboside as a repressor of CCND1 and CCND2 with preclinical antimyeloma activity.
- Author
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Tiedemann RE, Mao X, Shi CX, Zhu YX, Palmer SE, Sebag M, Marler R, Chesi M, Fonseca R, Bergsagel PL, Schimmer AD, and Stewart AK
- Subjects
- Animals, Cell Cycle physiology, Cell Line, Tumor drug effects, Cyclin D, Cyclin D2, Female, Gene Expression Regulation, Humans, Kinetin metabolism, Lentivirus genetics, Lentivirus metabolism, Mice, Mice, Nude, Molecular Structure, NIH 3T3 Cells, Promoter Regions, Genetic, RNA Interference, Transplantation, Heterologous, Antineoplastic Agents metabolism, Cyclins genetics, Cyclins metabolism, Kinetin genetics, Multiple Myeloma drug therapy, Multiple Myeloma genetics, Multiple Myeloma metabolism, Nucleosides genetics, Nucleosides metabolism, Nucleosides pharmacology, Nucleosides therapeutic use
- Abstract
Knockout and transgenic studies in mice demonstrate that normal somatic tissues redundantly express 3 cyclin D proteins, whereas tumor cells seem dependent on a single overexpressed cyclin D. Thus, selective suppression of the individual cyclin D deregulated in a tumor represents a biologically valid approach to targeted cancer therapy. In multiple myeloma, overexpression of 1 of the cyclin D proteins is a ubiquitous feature, unifying at least 7 different initiating genetic events. We demonstrate here that RNAi of genes encoding cyclin D1 and cyclin D2 (CCND1 and CCND2, respectively) inhibits proliferation and is progressively cytotoxic in human myeloma cells. By screening a chemical library using a cell-based assay for inhibition of CCND2 trans-activation, we identified the plant cytokinin kinetin riboside as an inhibitor of CCND2 trans-activation. Kinetin riboside induced marked suppression of CCND2 transcription and rapidly suppressed cyclin D1 and D2 protein expression in primary myeloma cells and tumor lines, causing cell-cycle arrest, tumor cell-selective apoptosis, and inhibition of myeloma growth in xenografted mice. Mechanistically, kinetin riboside upregulated expression of transcription repressor isoforms of cAMP-response element modulator (CREM) and blocked both trans-activation of CCND2 by various myeloma oncogenes and cis-activation of translocated CCND1, suggesting induction of an overriding repressor activity that blocks multiple oncogenic pathways targeting cyclin D genes. These data support targeted repression of cyclin D genes as a therapeutic strategy for human malignancies.
- Published
- 2008
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18. Genetic aberrations and survival in plasma cell leukemia.
- Author
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Tiedemann RE, Gonzalez-Paz N, Kyle RA, Santana-Davila R, Price-Troska T, Van Wier SA, Chng WJ, Ketterling RP, Gertz MA, Henderson K, Greipp PR, Dispenzieri A, Lacy MQ, Rajkumar SV, Bergsagel PL, Stewart AK, and Fonseca R
- Subjects
- Adult, Aged, Aged, 80 and over, Epigenesis, Genetic, Female, Humans, Leukemia, Plasma Cell diagnosis, Leukemia, Plasma Cell mortality, Male, Middle Aged, Molecular Epidemiology, Neoplasms, Second Primary diagnosis, Neoplasms, Second Primary mortality, Survival Rate, Translocation, Genetic, Leukemia, Plasma Cell genetics, Mutation, Neoplasms, Second Primary genetics
- Abstract
Plasma cell leukemia (PCL) is an aggressive and rare hematological malignancy that originates either as primary disease (pPCL) or as a secondary leukemic transformation (sPCL) of multiple myeloma (MM). We report here the genetic aberrations and survival of 80 patients with pPCL or sPCL and make comparisons with 439 cases of MM. pPCL presents a decade earlier than sPCL (54.7 vs 65.3 years) and is associated with longer median overall survival (11.1 vs 1.3 months; P<0.001). 14q32 (IgH) translocations are highly prevalent in both sPCL and pPCL (82-87%); in pPCL IgH translocations almost exclusively involve 11q13 (CCND1), supporting a central etiological role, while in sPCL multiple partner oncogenes are involved, including 11q13, 4p16 (FGFR3/MMSET) and 16q23 (MAF), recapitulating MM. Both show ubiquitous inactivation of TP53 (pPCL 56%; sPCL 83%) by coding mutation or 17p13 deletion; complemented by p14ARF epigenetic silencing in sPCL (29%). Both show frequent N-RAS or K-RAS mutation. Poor survival in pPCL was predicted by MYC translocation (P=0.006). Survival in sPCL was consistently short. Overall pPCL and sPCL are different disorders with distinct natural histories, genetics and survival.
- Published
- 2008
- Full Text
- View/download PDF
19. Preclinical studies of the pan-Bcl inhibitor obatoclax (GX015-070) in multiple myeloma.
- Author
-
Trudel S, Li ZH, Rauw J, Tiedemann RE, Wen XY, and Stewart AK
- Subjects
- Apoptosis Regulatory Proteins drug effects, Apoptosis Regulatory Proteins genetics, Bcl-2-Like Protein 11, Caspase 3 drug effects, Caspase 3 metabolism, Cell Line, Tumor, Cytochromes c drug effects, Cytochromes c metabolism, Drug Screening Assays, Antitumor, Humans, Indoles, Membrane Proteins drug effects, Membrane Proteins genetics, Myeloid Cell Leukemia Sequence 1 Protein, Neoplasm Proteins metabolism, Protein Binding drug effects, Proto-Oncogene Proteins drug effects, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins c-bcl-2 metabolism, Pyrroles therapeutic use, Up-Regulation drug effects, bcl-2 Homologous Antagonist-Killer Protein metabolism, Multiple Myeloma drug therapy, Pyrroles pharmacology
- Abstract
Bcl family members Bcl-2, Bcl-x(L), and Mcl-1, are frequently expressed and implicated in the survival of myeloma cells. Obatoclax (GX015-070) is a novel, small-molecule antagonist of the BH3-binding groove of the Bcl family of proteins. We show that GX015-070 inhibits the binding of Bak to Mcl-1, up-regulates Bim, induces cytochrome c release, and activates capase-3 in human myeloma cell lines (HMCLs), confirming the predicted mechanism of action. Consequently, GX015-070 potently inhibited the viability of 15 of 16 HMCLs (mean IC(50) of 246 nM), including those resistant to melphalan and dexamethasone. In combination studies, GX015-070 enhanced the antimyeloma activity induced by melphalan, dexamethasone, or bortezomib. Sensitivity to GX015-070 correlated with the absence or near absence of Bcl-x(L). Coculture with interleukin-6 or adherence to bone marrow stroma conferred modest resistance; however, it did not overcome GX015-070-induced cytotoxicity. Of importance, GX015-070 as a single agent induced potent cytotoxic responses against patient-derived tumor cells. GX015-070 inhibited normal bone marrow-derived colony formation; however, cytotoxicity to human blood lymphocytes was not observed. Taken together, these studies describe a novel BH3 mimic with selectivity for Mcl-1, and support the therapeutic application of GX015-070 for diverse neoplasias including multiple myeloma.
- Published
- 2007
- Full Text
- View/download PDF
20. The superantigen streptococcal pyrogenic exotoxin C (SPE-C) exhibits a novel mode of action.
- Author
-
Li PL, Tiedemann RE, Moffat SL, and Fraser JD
- Subjects
- Cell Line, Crystallography, X-Ray, Dimerization, Exotoxins chemistry, Exotoxins genetics, Histocompatibility Antigens Class II metabolism, Humans, Lymphocyte Activation drug effects, Protein Binding immunology, Pyrogens chemistry, Pyrogens genetics, Recombinant Proteins metabolism, Structure-Activity Relationship, Superantigens chemistry, Superantigens genetics, Zinc pharmacology, Bacterial Proteins, Exotoxins pharmacology, Membrane Proteins, Pyrogens pharmacology, Streptococcus pyogenes immunology, Superantigens pharmacology
- Abstract
Recombinant streptococcal pyrogenic exotoxin C (SPE-C) is a potent superantigen that stimulates Vbeta2-bearing human T cells, but is inactive in mice. SPE-C binds with high affinity to both human HLA-DR and murine I-E molecules, but not to murine I-A molecules in a zinc-dependent fashion. Competition binding studies with other recombinant toxins revealed that SPE-C lacks the generic low affinity major histocompatibility complex (MHC) class II alpha-chain binding site common to all other bacterial superantigens. Despite this, SPE-C cross-links MHC class II to induce homotypic aggregation of class II-bearing B cells. Nondenaturing sodium dodecyl sulfate electrophoresis and size exclusion chromatography revealed that both wild-type and recombinant SPE-C exist in a stable dimer at neutral or alkaline pH. These data support a recent crystal structure of SPE-C and reveal yet another mechanism by which bacterial superantigens ligate and cross-link MHC class II.
- Published
- 1997
- Full Text
- View/download PDF
21. Cross-linking of MHC class II molecules by staphylococcal enterotoxin A is essential for antigen-presenting cell and T cell activation.
- Author
-
Tiedemann RE and Fraser JD
- Subjects
- Antigens, Bacterial genetics, Antigens, Bacterial metabolism, Binding Sites, Enterotoxins genetics, Enterotoxins metabolism, Gene Expression Regulation, Histidine chemistry, Humans, Interleukin-1 biosynthesis, Interleukin-1 genetics, Leukemia, Monocytic, Acute pathology, Mutagenesis, Site-Directed, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins immunology, Staphylococcus aureus genetics, Staphylococcus aureus immunology, Superantigens genetics, Superantigens metabolism, Tumor Cells, Cultured, Tumor Necrosis Factor-alpha biosynthesis, Tumor Necrosis Factor-alpha genetics, Antigen Presentation, Antigens, Bacterial immunology, Enterotoxins immunology, HLA-DR1 Antigen immunology, Immunologic Capping, Lymphocyte Activation, Superantigens immunology, T-Lymphocyte Subsets immunology
- Abstract
Two binding sites for MHC class II have previously been identified on opposite sides of the superantigen, staphylococcal enterotoxin A (SEA). The sites mediate separate binding reactions with nonoverlapping regions of class II, and in solution cause SEA to complex with purified HLA-DR1 to form DR1.SEA2 trimers. Here, a set of complementary SEA class II-binding mutants was used to study the interaction of SEA with cell surface MHC class II. The results indicate that both class II binding sites are required on the same toxin molecule for maximal activity, demonstrating that simultaneous ligation of two MHC class II molecules on APCs by a single SEA is essential for effective superantigen function. Coalescence of MHC class II by SEA results in protein tyrosine kinase activation and contributes to the induction of cell:cell adhesion, pro-inflammatory cytokine gene transcription, and T cell proliferation.
- Published
- 1996
22. Isolation of HLA-DR1.(staphylococcal enterotoxin A)2 trimers in solution.
- Author
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Tiedemann RE, Urban RJ, Strominger JL, and Fraser JD
- Subjects
- B-Lymphocytes, Binding Sites, Cell Line, Enterotoxins isolation & purification, HLA-DR1 Antigen isolation & purification, Humans, Isoelectric Focusing, Macromolecular Substances, Models, Structural, Recombinant Proteins chemistry, Recombinant Proteins immunology, Recombinant Proteins isolation & purification, Solutions, Staphylococcus aureus immunology, Superantigens chemistry, Superantigens immunology, Superantigens isolation & purification, Zinc Fingers, Enterotoxins chemistry, Enterotoxins immunology, HLA-DR1 Antigen chemistry, Protein Structure, Secondary
- Abstract
Mutational studies indicate that the superantigen staphylococcal enterotoxin A (SEA) has two separate binding sites for major histocompatibility complex (MHC) class II molecules. Direct evidence is provided here for the formation of SEA-MHC class II trimers in solution. Isoelectric focusing separated SEA-HLA-DR1 complexes into both dimers and HLA-DR1.SEA2 trimers. The molar ratio of components was determined by dual isotope labeling. The SEA mutant SEA-F47S, L48S, Y92A, which is deficient in MHC class II alpha-chain binding, formed only dimers with HLA-DR1, whereas a second SEA mutant, SEA-H225A, which lacks high-affinity MHC class II beta-chain binding was incapable of forming any complexes. Thus SEA binding to its MHC receptor is a two-step process involving initial beta-chain binding followed by cooperative binding of a second SEA molecule to the class II alpha chain.
- Published
- 1995
- Full Text
- View/download PDF
23. Staphylococcal enterotoxin A has two cooperative binding sites on major histocompatibility complex class II.
- Author
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Hudson KR, Tiedemann RE, Urban RG, Lowe SC, Strominger JL, and Fraser JD
- Subjects
- Amino Acid Sequence, Binding Sites, Enterotoxins chemistry, Enterotoxins immunology, Humans, Molecular Sequence Data, Peptide Fragments chemistry, Peptide Fragments immunology, Protein Binding, Recombinant Proteins chemistry, Recombinant Proteins immunology, Recombinant Proteins metabolism, Superantigens chemistry, Zinc physiology, Enterotoxins metabolism, HLA-DR1 Antigen metabolism, Models, Molecular, Peptide Fragments metabolism, Protein Conformation, Superantigens metabolism
- Abstract
The superantigen staphylococcal enterotoxin A (SEA) binds to major histocompatibility complex (MHC) class II molecules at two sites on either side of the peptide groove. Two separate but cooperative interactions to the human class II molecule HLA-DR1 were detected. The first high affinity interaction to the DR1 beta chain is mediated by a zinc atom coordinated by H187, H225, and D227 in SEA and H81 in the polymorphic DR1 beta chain. The second low affinity site is to the DR1 alpha chain analogous to SEB binding and is mediated by residue F47 in SEA. Binding of one SEA to the DR1 beta chain enhances the binding of a second SEA molecule to the DR1 alpha chain. The zinc site is on the opposite side of the SEA molecule from residue F47 so that one SEA molecule can readily bind two class II molecules. Both binding sites on SEA are required for maximal activity. Thus, unlike, SEB, SEA requires two separate binding sites for optimal activity, which may allow it to stabilize SEA interaction with T cell receptors, as well as to activate the antigen-presenting cell by cross-linking MHC class II.
- Published
- 1995
- Full Text
- View/download PDF
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