RESUMO
A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild-type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.
Assuntos
Enzimas Desubiquitinantes/genética , Endopeptidases/genética , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Proteases Específicas de Ubiquitina/genética , Alanina/genética , Substituição de Aminoácidos/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Catálise , Cisteína/genética , Enzimas Desubiquitinantes/química , Endopeptidases/química , Humanos , Mutação/genética , Conformação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Transativadores/química , Ubiquitina/química , Ubiquitina/genética , Proteases Específicas de Ubiquitina/química , Ubiquitinação/genéticaRESUMO
Waldenström's macroglobulinemia (WM) is a complex disease characterized by apparent morphological heterogeneity within the malignant clonal cells representing a continuum of small lymphocytes, plasmacytoid lymphocytes, and plasma cells. At the molecular level, the neoplastic B cell-derived clone has undergone somatic hypermutation, but not isotype switching, and retains the capability of plasmacytic differentiation. Although by classical definition, WM is formed by monoclonal expansion, long-lived clonal B lymphocytes are of heterogeneous origin. Even more, according to current opinion, plasma cells also conform certain population with pathogenic and clinical significance. In this article, we review the recent advances in the WM clonal architecture, briefly describe B-cell development during which the molecular changes lead to the malignant transformation and mainly focus on differences between two principal B-lineage clones, including analysis of their genome and transcriptome profiles, as well as immunophenotype features. We assume that the correct identification of a number of specific immunophenotypic molecular and expression alterations leading to proper aberrant clone detection can help to guide patient monitoring throughout treatment and successfully implement therapy strategies directed against both B- and plasma cell tumor WM clones.
Assuntos
Macroglobulinemia de Waldenstrom/diagnóstico , Macroglobulinemia de Waldenstrom/etiologia , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Evolução Clonal/genética , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Variação Genética , Humanos , Imunofenotipagem , Fenótipo , Plasmócitos/metabolismo , Plasmócitos/patologia , Transdução de Sinais , Carga TumoralAssuntos
Amiloidose , Amiloidose de Cadeia Leve de Imunoglobulina , Amiloidose/diagnóstico , Amiloidose/genética , Humanos , Cadeias Leves de Imunoglobulina/genética , Amiloidose de Cadeia Leve de Imunoglobulina/diagnóstico , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Mutação , Patologia Molecular , PlasmócitosAssuntos
Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Leucemia Plasmocitária/tratamento farmacológico , Terapia de Alvo Molecular , Sulfonamidas/uso terapêutico , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Bortezomib/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 14/genética , Terapia Combinada , Ciclofosfamida/administração & dosagem , Dexametasona/administração & dosagem , Doxorrubicina/administração & dosagem , Transplante de Células-Tronco Hematopoéticas , Humanos , Leucemia Plasmocitária/complicações , Leucemia Plasmocitária/genética , Leucemia Plasmocitária/terapia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasia Residual , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Recidiva , Sulfonamidas/farmacologia , Translocação Genética , Vincristina/administração & dosagemRESUMO
The identification of the essential role of cyclin-dependent kinases (CDKs) in the control of cell division has prompted the development of small-molecule CDK inhibitors as anticancer drugs. For many of these compounds, the precise mechanism of action in individual tumor types remains unclear as they simultaneously target different classes of CDKs - enzymes controlling the cell cycle progression as well as CDKs involved in the regulation of transcription. CDK inhibitors are also capable of activating p53 tumor suppressor in tumor cells retaining wild-type p53 gene by modulating MDM2 levels and activity. In the current study, we link, for the first time, CDK activity to the overexpression of the MDM4 (MDMX) oncogene in cancer cells. Small-molecule drugs targeting the CDK9 kinase, dinaciclib, flavopiridol, roscovitine, AT-7519, SNS-032, and DRB, diminished MDM4 levels and activated p53 in A375 melanoma and MCF7 breast carcinoma cells with only a limited effect on MDM2. These results suggest that MDM4, rather than MDM2, could be the primary transcriptional target of pharmacological CDK inhibitors in the p53 pathway. CDK9 inhibitor atuveciclib downregulated MDM4 and enhanced p53 activity induced by nutlin-3a, an inhibitor of p53-MDM2 interaction, and synergized with nutlin-3a in killing A375 melanoma cells. Furthermore, we found that human pluripotent stem cell lines express significant levels of MDM4, which are also maintained by CDK9 activity. In summary, we show that CDK9 activity is essential for the maintenance of high levels of MDM4 in human cells, and drugs targeting CDK9 might restore p53 tumor suppressor function in malignancies overexpressing MDM4.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Melanoma/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Sinergismo Farmacológico , Humanos , Imidazóis/farmacologia , Células MCF-7 , Melanoma/genética , Melanoma/patologia , Camundongos , Piperazinas/farmacologia , Células-Tronco Pluripotentes/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2/biossíntese , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Roscovitina/farmacologia , Sulfonamidas/farmacologia , Transcrição Gênica , Transfecção , Triazinas/farmacologiaRESUMO
The reason why a few myeloma cells egress from the bone marrow (BM) into peripheral blood (PB) remains unknown. Here, we investigated molecular hallmarks of circulating tumor cells (CTCs) to identify the events leading to myeloma trafficking into the bloodstream. After using next-generation flow to isolate matched CTCs and BM tumor cells from 32 patients, we found high correlation in gene expression at single-cell and bulk levels (r ≥ 0.94, P = 10-16), with only 55 genes differentially expressed between CTCs and BM tumor cells. CTCs overexpressed genes involved in inflammation, hypoxia, or epithelial-mesenchymal transition, whereas genes related with proliferation were downregulated in CTCs. The cancer stem cell marker CD44 was overexpressed in CTCs, and its knockdown significantly reduced migration of MM cells towards SDF1-α and their adhesion to fibronectin. Approximately half (29/55) of genes differentially expressed in CTCs were prognostic in patients with newly-diagnosed myeloma (n = 553; CoMMpass). In a multivariate analysis including the R-ISS, overexpression of CENPF and LGALS1 was significantly associated with inferior survival. Altogether, these results help understanding the presence of CTCs in PB and suggest that hypoxic BM niches together with a pro-inflammatory microenvironment induce an arrest in proliferation, forcing tumor cells to circulate in PB and seek other BM niches to continue growing.
Assuntos
Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Células Neoplásicas Circulantes/patologia , Transcrição Gênica/genética , Medula Óssea/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Expressão Gênica/genética , Humanos , Hipóxia/genética , Hipóxia/patologia , Inflamação/genética , Inflamação/patologia , Células-Tronco Neoplásicas/patologia , Prognóstico , Microambiente Tumoral/genéticaRESUMO
Inhibitors of antiapoptotic proteins of the BCL2 family can successfully restart the deregulated process of apoptosis in malignant cells. Whereas nonselective agents have been limited by their affinity to different BCL2 members, thus inducing excessive toxicity, the highly selective BCL2 inhibitor venetoclax (ABT-199, Venclexta™) has an acceptable safety profile. To date, it has been approved in monotherapy for the treatment of relapsed or refractory chronic lymphocytic leukemia (CLL) with 17p deletion. Extension of indications can be expected in monotherapy and in combination regimens. Sensitivity to venetoclax is not common in lymphomas, but promising outcomes have been achieved in the mantle cell lymphoma group. Venetoclax is also active in multiple myeloma patients, especially in those with translocation t(11;14), even if high-risk features such as del17p are also present. Surprisingly, positive results are being obtained in elderly acute myeloid leukemia patients, in whom inhibition of BCL2 is able to substantially increase the efficacy of low-dose cytarabine or hypomethylating agents. Here, we provide a summary of available results from clinical trials and describe a specific mechanism of action that stands behind the efficacy of venetoclax in hematological malignancies.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Sulfonamidas/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Protocolos Clínicos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologiaRESUMO
AIMS: Amyloidosis is caused by deposition of abnormal protein fibrils, leading to damage of organ function. Hereditary amyloidosis represents a monogenic disease caused by germline mutations in 11 amyloidogenic precursor protein genes. One of the important but non-specific symptoms of amyloidosis is hypertrophic cardiomyopathy. Diagnostics of hereditary amyloidosis is complicated and the real cause can remain overlooked. We aimed to design hereditary amyloidosis gene panel and to introduce new next-generation sequencing (NGS) approach to investigate hereditary amyloidosis in a cohort of patients with hypertrophic cardiomyopathy of unknown significance. METHODS: Design of target enrichment DNA library preparation using Haloplex Custom Kit containing 11 amyloidogenic genes was followed by MiSeq Illumina sequencing and bioinformatics identification of germline variants using tool VarScan in a cohort of 40 patients. RESULTS: We present design of NGS panel for 11 genes (TTR, FGA, APOA1, APOA2, LYZ, GSN, CST3, PRNP, APP, B2M, ITM2B) connected to various forms of amyloidosis. We detected one mutation, which is responsible for hereditary amyloidosis. Some other single nucleotide variants are so far undescribed or rare variants or represent common polymorphisms in European population. CONCLUSIONS: We report one positive case of hereditary amyloidosis in a cohort of patients with hypertrophic cardiomyopathy of unknown significance and set up first panel for NGS in hereditary amyloidosis. This work may facilitate successful implementation of the NGS method by other researchers or clinicians and may improve the diagnostic process after validation.
Assuntos
Amiloidose Familiar/genética , Cardiomiopatia Hipertrófica/genética , Análise Mutacional de DNA/métodos , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Polimorfismo de Nucleotídeo Único , Transcriptoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Amiloidose Familiar/diagnóstico , Cardiomiopatia Hipertrófica/diagnóstico , Biologia Computacional , República Tcheca , Feminino , Frequência do Gene , Marcadores Genéticos , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Projetos Piloto , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Fatores de RiscoRESUMO
MdmX overexpression contributes to the development of cancer by inhibiting tumor suppressor p53. A switch in the alternative splicing of MdmX transcript, leading to the inclusion of exon 6, has been identified as the primary mechanism responsible for increased MdmX protein levels in human cancers, including melanoma. However, there are no approved drugs, which could translate these new findings into clinical applications. We analyzed the anti-melanoma activity of enoxacin, a fluoroquinolone antibiotic inhibiting the growth of some human cancers in vitro and in vivo by promoting miRNA maturation. We found that enoxacin inhibited the growth and viability of human melanoma cell lines much stronger than a structurally related fluoroquinolone ofloxacin, which only weakly modulates miRNA processing. A microarray analysis identified a set of miRNAs significantly dysregulated in enoxacin-treated A375 melanoma cells. They had the potential to target multiple signaling pathways required for cancer cell growth, among them the RNA splicing. Recent studies showed that interfering with cellular splicing machinery can result in MdmX downregulation in cancer cells. We, therefore, hypothesized that enoxacin could, by modulating miRNAs targeting splicing machinery, activate p53 in melanoma cells overexpressing MdmX. We found that enoxacin and ciprofloxacin, a related fluoroquinolone capable of promoting microRNA processing, but not ofloxacin, strongly activated wild type p53-dependent transcription in A375 melanoma without causing significant DNA damage. On the molecular level, the drugs promoted MdmX exon 6 skipping, leading to a dose-dependent downregulation of MdmX. Not only in melanoma, but also in MCF7 breast carcinoma and A2780 ovarian carcinoma cells overexpressing MdmX. Together, our results suggest that some clinically approved fluoroquinolones could potentially be repurposed as activators of p53 tumor suppressor in cancers overexpressing MdmX oncoprotein and that p53 activation might contribute to the previously reported activity of enoxacin towards human cancer cells.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Enoxacino/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Ofloxacino/farmacologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismoAssuntos
Mieloma Múltiplo , Humanos , Mieloma Múltiplo/genética , Mutação , Neoplasia Residual , Medicina de PrecisãoRESUMO
Immunoglobulin light chain amyloidosis (ALA) is a plasma cell dyscrasia characterized by deposition of amyloid fibrils in various organs and tissues. The current paper is devoted to clarify if ALA has a unique gene expression profile and to its pathogenetic argumentation. The meta-analysis of ALA patients vs. healthy donors, monoclonal gammopathy of undetermined significance, smoldering and multiple myeloma patients' cohorts have revealed molecular signature of ALA consists of 256 genes representing mostly ribosomal proteins and immunoglobulin regions. This signature appears pathogenetically supported and elucidates for the first time the role of ribosome dysfunction in ALA. In summary of our findings with literature overview, we hypothesize that ALA development is associated not only with changes in genes, coding amyloidogenic protein itself, but with post-transcriptional disbalance as well. Based on our data analysis in ALA, ribosome machinery is impaired and the affected link mainly involves translational initiation, elongation and co-translational protein folding.