Assuntos
Proteínas F-Box , Leucemia Mieloide Aguda , Células Mieloides , Proteólise , Humanos , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/genética , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Células Mieloides/metabolismo , Células Mieloides/patologiaRESUMO
Biomarkers that predict response to lenalidomide maintenance therapy in patients with multiple myeloma (MM) have remained elusive. We have shown that immunomodulatory drugs (IMiDs) exert anti-MM activity via destabilization of MCT1 and CD147. In this study, cell samples of 654 patients with MM who received lenalidomide (n = 455), thalidomide (n = 98), or bortezomib (n = 101) maintenance were assessed by gene expression profiling and RNA sequencing, followed by correlation of MCT1 and CD147 expression with data for progression-free survival (PFS) and overall survival (OS). Patients with high expression levels of MCT1 showed significantly reduced PFS (31.9 months vs 48.2 months in MCT1high vs MCT1low; P = .03) and OS (75.9 months vs not reached [NR] in MCT1high vs MCT1low; P = .001) in cases with lenalidomide maintenance, whereas MCT1 expression had no significant impact on PFS or OS in cases with bortezomib maintenance. We validated the predictive role of MCT1 for IMiD-based maintenance in an independent cohort of patients who received thalidomide (OS, 83.6 months vs NR in MCT1high vs MCT1low; P = .03). Functional validation showed that MCT1 overexpression in human MM cell lines significantly reduced the efficacy of lenalidomide, whereas no change was observed with bortezomib treatment, either in vitro or in a MM xenograft model. Our findings have established MCT1 expression as a predictive marker for response to lenalidomide-based maintenance in patients with MM.
Assuntos
Mieloma Múltiplo , Biomarcadores , Bortezomib/farmacologia , Bortezomib/uso terapêutico , Humanos , Lenalidomida/uso terapêutico , Mieloma Múltiplo/terapia , Talidomida/farmacologia , Talidomida/uso terapêuticoRESUMO
Personalized medicine aims to match the right drug with the right patient by using specific features of the individual patient's tumor. However, current strategies of personalized therapy matching provide treatment opportunities for less than 10% of patients with cancer. A promising method may be drug profiling of patient biopsy specimens with single-cell resolution to directly quantify drug effects. We prospectively tested an image-based single-cell functional precision medicine (scFPM) approach to guide treatments in 143 patients with advanced aggressive hematologic cancers. Fifty-six patients (39%) were treated according to scFPM results. At a median follow-up of 23.9 months, 30 patients (54%) demonstrated a clinical benefit of more than 1.3-fold enhanced progression-free survival compared with their previous therapy. Twelve patients (40% of responders) experienced exceptional responses lasting three times longer than expected for their respective disease. We conclude that therapy matching by scFPM is clinically feasible and effective in advanced aggressive hematologic cancers. SIGNIFICANCE: This is the first precision medicine trial using a functional assay to instruct n-of-one therapies in oncology. It illustrates that for patients lacking standard therapies, high-content assay-based scFPM can have a significant value in clinical therapy guidance based on functional dependencies of each patient's cancer.See related commentary by Letai, p. 290.This article is highlighted in the In This Issue feature, p. 275.
Assuntos
Neoplasias Hematológicas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Áustria , Estudos de Coortes , Feminino , Neoplasias Hematológicas/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Medicina de Precisão , Intervalo Livre de Progressão , Adulto JovemRESUMO
The complex architecture of transmembrane proteins requires quality control (QC) of folding, membrane positioning, and trafficking as prerequisites for cellular homeostasis and intercellular communication. However, it has remained unclear whether transmembrane protein-specific QC hubs exist. Here we identify cereblon (CRBN), the target of immunomodulatory drugs (IMiDs), as a co-chaperone that specifically determines chaperone activity of HSP90 toward transmembrane proteins by means of counteracting AHA1. This function is abrogated by IMiDs, which disrupt the interaction of CRBN with HSP90. Among the multiple transmembrane protein clients of CRBN-AHA1-HSP90 revealed by cell surface proteomics, we identify the amino acid transporter LAT1/CD98hc as a determinant of IMiD activity in multiple myeloma (MM) and present an Anticalin-based CD98hc radiopharmaceutical for MM radio-theranostics. These data establish the CRBN-AHA1-HSP90 axis in the biogenesis of transmembrane proteins, link IMiD activity to tumor metabolism, and nominate CD98hc and LAT1 as attractive diagnostic and therapeutic targets in MM.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Fatores Imunológicos/farmacologia , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Chaperonas Moleculares/metabolismo , Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Células Tumorais CultivadasRESUMO
Multiple myeloma (MM) is the second most common hematological malignancy and results from the clonal amplification of plasma cells. Despite recent advances in treatment, MM remains incurable with a median survival time of only 5-6years, thus necessitating further insights into MM biology and exploitation of novel therapeutic approaches. Both the ubiquitin proteasome system (UPS) and the PI3K/Akt/mTOR signaling pathways have been implicated in the pathogenesis, and treatment of MM and different lines of evidence suggest a close cross talk between these central cell-regulatory signaling networks. In this review, we outline the interplay between the UPS and mTOR pathways and discuss their implications for the pathophysiology and therapy of MM.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Ubiquitina/antagonistas & inibidores , Animais , Antibióticos Antineoplásicos/química , Inibidores Enzimáticos/química , Humanos , Mieloma Múltiplo/metabolismo , Sirolimo/química , Ubiquitina/metabolismoRESUMO
Solute carriers (SLCs) are transmembrane proteins that transport various nutrients, metabolites, and drugs across cellular membranes. Despite the relevance of SLCs to cell homeostasis, metabolism, and disease states, for the majority of SLCs we lack experimental evidence regarding the nature of the cognate ligands, whether endobiotic or xenobiotic. Moreover, even for the roughly 20 SLCs for which inhibitors have been characterized, engagement assays in cells are limited to the accessibility of radiolabeled or fluorescent probes. The cellular thermal shift assay (CETSA) has been introduced as a powerful method to assess target engagement by monitoring ligand-induced changes in the thermal stability of cellular proteins. We addressed the question of whether CETSA could be modified to become routinely applicable to membrane transporters such as SLCs. We used SLC16A1 (MCT1) and SLC1A2 (EAAT2) as targets to establish robust conditions by which chemical engagement of SLCs can be detected. Using immunoblotting, we demonstrate that treatment with the SLC16A1 inhibitors AZD3965 and AR-C155858 stabilized endogenous SLC16A1 in HEK293 cell lysates as well as intact cells. In addition, the high-affinity ligand of SLC16A1, l-lactate, and the low-affinity ligand, formate, resulted in strong and weak stabilization of SLC16A1, respectively. Moreover, we observed stabilization of SLC1A2 upon treatment with the selective inhibitor WAY-213613. We propose that the experimental approach presented here should be generally and easily applicable for monitoring the engagement of chemical ligands by SLCs in cellular settings and thus assisting in their deorphanization.
Assuntos
Bioensaio/métodos , Proteínas de Transporte de Glutamato da Membrana Plasmática/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportador 2 de Aminoácido Excitatório , Formiatos/metabolismo , Proteínas de Transporte de Glutamato da Membrana Plasmática/antagonistas & inibidores , Células HEK293 , Calefação , Humanos , Ácido Láctico/metabolismo , Ligantes , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Ligação Proteica , Estabilidade Proteica , Pirimidinonas/metabolismo , Tiofenos/metabolismo , Uracila/análogos & derivados , Uracila/metabolismoRESUMO
Macrophages represent the first line of immune defense against pathogens, and phagosome acidification is a necessary step in pathogen clearance. Here, we identified the bicarbonate transporter SLC4A7, which is strongly induced upon macrophage differentiation, as critical for phagosome acidification. Loss of SLC4A7 reduced acidification of phagocytosed beads or bacteria and impaired the intracellular microbicidal capacity in human macrophage cell lines. The phenotype was rescued by wild-type SLC4A7, but not by SLC4A7 mutants, affecting transport capacity or cell surface localization. Loss of SLC4A7 resulted in increased cytoplasmic acidification during phagocytosis, suggesting that SLC4A7-mediated, bicarbonate-driven maintenance of cytoplasmic pH is necessary for phagosome acidification. Altogether, we identify SLC4A7 and bicarbonate-driven cytoplasmic pH homeostasis as an important element of phagocytosis and the associated microbicidal functions in macrophages.
Assuntos
Bicarbonatos/metabolismo , Macrófagos/metabolismo , Fagossomos/metabolismo , Simportadores de Sódio-Bicarbonato/fisiologia , Sistemas CRISPR-Cas , Proteínas de Transporte de Cátions/metabolismo , Citoplasma/metabolismo , Técnicas de Inativação de Genes , Homeostase , Humanos , Concentração de Íons de Hidrogênio , Fagocitose , Simportadores de Sódio-Bicarbonato/genética , Células THP-1 , Transcriptoma , Células U937RESUMO
Immunomodulatory drugs (IMiDs), such as thalidomide and its derivatives lenalidomide and pomalidomide, are key treatment modalities for hematologic malignancies, particularly multiple myeloma (MM) and del(5q) myelodysplastic syndrome (MDS). Cereblon (CRBN), a substrate receptor of the CRL4 ubiquitin ligase complex, is the primary target by which IMiDs mediate anticancer and teratogenic effects. Here we identify a ubiquitin-independent physiological chaperone-like function of CRBN that promotes maturation of the basigin (BSG; also known as CD147) and solute carrier family 16 member 1 (SLC16A1; also known as MCT1) proteins. This process allows for the formation and activation of the CD147-MCT1 transmembrane complex, which promotes various biological functions, including angiogenesis, proliferation, invasion and lactate export. We found that IMiDs outcompete CRBN for binding to CD147 and MCT1, leading to destabilization of the CD147-MCT1 complex. Accordingly, IMiD-sensitive MM cells lose CD147 and MCT1 expression after being exposed to IMiDs, whereas IMiD-resistant cells retain their expression. Furthermore, del(5q) MDS cells have elevated CD147 expression, which is attenuated after IMiD treatment. Finally, we show that BSG (CD147) knockdown phenocopies the teratogenic effects of thalidomide exposure in zebrafish. These findings provide a common mechanistic framework to explain both the teratogenic and pleiotropic antitumor effects of IMiDs.
Assuntos
Basigina/efeitos dos fármacos , Proteínas de Ciclo Celular/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Imunossupressores/farmacologia , Proteínas Oncogênicas/efeitos dos fármacos , Peptídeo Hidrolases/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Teratogênese/efeitos dos fármacos , Talidomida/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Basigina/genética , Basigina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Humanos , Lenalidomida , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Teratogênese/genética , Talidomida/análogos & derivados , Ubiquitina-Proteína LigasesAssuntos
Medula Óssea/diagnóstico por imagem , Vértebras Lombares/diagnóstico por imagem , Fraturas por Osteoporose/etiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico por imagem , Sacro/diagnóstico por imagem , Fraturas da Coluna Vertebral/etiologia , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Medula Óssea/patologia , Exame de Medula Óssea , Fluordesoxiglucose F18 , Humanos , Dor Lombar/etiologia , Dor Lombar/prevenção & controle , Vértebras Lombares/lesões , Vértebras Lombares/cirurgia , Imageamento por Ressonância Magnética , Masculino , Imagem Multimodal , Fraturas por Osteoporose/fisiopatologia , Fraturas por Osteoporose/cirurgia , Tomografia por Emissão de Pósitrons , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/fisiopatologia , Compostos Radiofarmacêuticos , Indução de Remissão , Sacro/lesões , Sacro/patologia , Fraturas da Coluna Vertebral/fisiopatologia , Fraturas da Coluna Vertebral/cirurgia , Fusão Vertebral , Imagem Corporal TotalRESUMO
Two families of E3 ubiquitin ligases are prominent in cell cycle regulation and mediate the timely and precise ubiquitin-proteasome-dependent degradation of key cell cycle proteins: the SCF (Skp1/Cul1/F-box protein) complex and the APC/C (anaphase promoting complex or cyclosome). While certain SCF ligases drive cell cycle progression throughout the cell cycle, APC/C (in complex with either of two substrate recruiting proteins: Cdc20 and Cdh1) orchestrates exit from mitosis (APC/C(Cdc20)) and establishes a stable G1 phase (APC/C(Cdh1)). Upon DNA damage or perturbation of the normal cell cycle, both ligases are involved in checkpoint activation. Mechanistic insight into these processes has significantly improved over the last ten years, largely due to a better understanding of APC/C and the functional characterization of multiple F-box proteins, the variable substrate recruiting components of SCF ligases. Here, we review the role of SCF- and APC/C-mediated ubiquitylation in the normal and perturbed cell cycle and discuss potential clinical implications of SCF and APC/C functions. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.
Assuntos
Ciclo Celular/fisiologia , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/fisiologia , Ubiquitina/fisiologia , Animais , Humanos , Mitose/fisiologiaAssuntos
Medula Óssea/patologia , Linfoma de Células B/diagnóstico , Imageamento por Ressonância Magnética , Imagem Corporal Total/métodos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Artralgia/etiologia , Biópsia , Medula Óssea/diagnóstico por imagem , Exame de Medula Óssea , Reações Falso-Negativas , Humanos , Linfoma de Células B/diagnóstico por imagem , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/patologia , Masculino , Imagem Multimodal , Tomografia por Emissão de Pósitrons , Indução de Remissão , Tomografia Computadorizada por Raios X , Adulto JovemRESUMO
The Tel2 (also known as Telo2) and Tti1 proteins control the cellular abundance of mammalian PIKKs and are integral components of mTORC1 and mTORC2. Here we report that Tel2 and Tti1 are targeted for degradation within mTORC1 by the SCFFbxo9 ubiquitin ligase to adjust mTOR signalling to growth factor availability. This process is primed by CK2, which translocates to the cytoplasm to mediate mTORC1-specific phosphorylation of Tel2/Tti1, subsequent to growth factor deprivation. As a consequence, mTORC1 is inactivated to restrain cell growth and protein translation whereas relief of feedback inhibition activates the PI(3)K/TORC2/Akt pathway to sustain survival. Significantly, primary human multiple myelomas exhibit high levels of Fbxo9. In this setting, PI(3)K/TORC2/Akt signalling and survival of multiple myeloma cells is dependent on Fbxo9 expression. Thus, mTORC1-specific degradation of the Tel2 and Tti1 proteins represents a central mTOR regulatory mechanism with implications in multiple myeloma, both in promoting survival and in providing targets for the specific treatment of multiple myeloma with high levels of Fbxo9 expression.
Assuntos
Proteínas de Transporte/metabolismo , Caseína Quinase II/fisiologia , Sobrevivência Celular , Proteínas F-Box/fisiologia , Mieloma Múltiplo/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-ets/metabolismo , Sequência de Aminoácidos , Animais , Estudos de Casos e Controles , Linhagem Celular Tumoral , Meios de Cultura Livres de Soro , Intervalo Livre de Doença , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Estimativa de Kaplan-Meier , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mieloma Múltiplo/patologia , Complexos Multiproteicos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosforilação , Plasmócitos/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
PAX5 is the main target of somatic mutations in acute B lymphoblastic leukemia (B-ALL). We analyzed 153 adult and child B-ALL harboring karyotypic abnormalities at chromosome 9p, to determine the frequency and the nature of PAX5 alterations. We found PAX5 internal rearrangements in 21% of the cases. To isolate fusion partners, we used classic and innovative techniques (rolling circle amplification-rapid amplification of cDNA ends) and single nucleotide polymorphism-comparative genomic hybridization arrays. Recurrent and novel fusion partners were identified, including NCoR1, DACH2, GOLGA6, and TAOK1 genes showing the high variability of the partners. We noted that half the fusion genes can give rise to truncated PAX5 proteins. Furthermore, malignant cells carrying PAX5 fusion genes displayed a simple karyotype. These data strongly suggest that PAX5 fusion genes are early players in leukemogenesis. In addition, PAX5 deletion was observed in 60% of B-ALL with 9p alterations. Contrary to cases with PAX5 fusions, deletions were associated with complex karyotypes and common recurrent translocations. This supports the hypothesis of the secondary nature of the deletion. Our data shed more light on the high variability of PAX5 alterations in B-ALL. Therefore, it is probable that gene fusions occur early, whereas deletions should be regarded as a late/secondary event.