RESUMEN
Aberrant splicing is typically attributed to splice-factor (SF) mutation and contributes to malignancies including acute myeloid leukemia (AML). Here, we discovered a mutation-independent means to extensively reprogram alternative splicing (AS). We showed that the dysregulated expression of eukaryotic translation initiation factor eIF4E elevated selective splice-factor production, thereby impacting multiple spliceosome complexes, including factors mutated in AML such as SF3B1 and U2AF1. These changes generated a splicing landscape that predominantly supported altered splice-site selection for ~800 transcripts in cell lines and ~4,600 transcripts in specimens from high-eIF4E AML patients otherwise harboring no known SF mutations. Nuclear RNA immunoprecipitations, export assays, polysome analyses, and mutational studies together revealed that eIF4E primarily increased SF production via its nuclear RNA export activity. By contrast, eIF4E dysregulation did not induce known SF mutations or alter spliceosome number. eIF4E interacted with the spliceosome and some pre-mRNAs, suggesting its direct involvement in specific splicing events. eIF4E induced simultaneous effects on numerous SF proteins, resulting in a much larger range of splicing alterations than in the case of mutation or dysregulation of individual SFs and providing a novel paradigm for splicing control and dysregulation.
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Empalme Alternativo , Leucemia Mieloide Aguda , Humanos , Factores de Empalme de ARN/metabolismo , Factor 4E Eucariótico de Iniciación/metabolismo , Empalme del ARN , Factores Eucarióticos de Iniciación/genética , Leucemia Mieloide Aguda/genética , MutaciónRESUMEN
ABSTRACT: Cells in the tumor microenvironment (TME) of diffuse large B-cell lymphoma (DLBCL) show enormous diversity and plasticity, with functions that can range from tumor inhibitory to tumor supportive. The patient's age, immune status, and DLBCL treatments are factors that contribute to the shaping of this TME, but evidence suggests that genetic factors, arising principally in lymphoma cells themselves, are among the most important. Here, we review the current understanding of the role of these genetic drivers of DLBCL in establishing and modulating the lymphoma microenvironment. A better comprehension of the relationship between lymphoma genetic factors and TME biology should lead to better therapeutic interventions, especially immunotherapies.
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Linfoma de Células B Grandes Difuso , Microambiente Tumoral , Humanos , Microambiente Tumoral/genética , Linfoma de Células B Grandes Difuso/patología , PronósticoRESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and often incurable disease. To uncover therapeutic vulnerabilities, we first developed T-ALL patient-derived tumor xenografts (PDXs) and exposed PDX cells to a library of 433 clinical-stage compounds in vitro. We identified 39 broadly active drugs with antileukemia activity. Because endothelial cells (ECs) can alter drug responses in T-ALL, we developed an EC/T-ALL coculture system. We found that ECs provide protumorigenic signals and mitigate drug responses in T-ALL PDXs. Whereas ECs broadly rescued several compounds in most models, for some drugs the rescue was restricted to individual PDXs, suggesting unique crosstalk interactions and/or intrinsic tumor features. Mechanistically, cocultured T-ALL cells and ECs underwent bidirectional transcriptomic changes at the single-cell level, highlighting distinct "education signatures." These changes were linked to bidirectional regulation of multiple pathways in T-ALL cells as well as in ECs. Remarkably, in vitro EC-educated T-ALL cells transcriptionally mirrored ex vivo splenic T-ALL at single-cell resolution. Last, 5 effective drugs from the 2 drug screenings were tested in vivo and shown to effectively delay tumor growth and dissemination thus prolonging overall survival. In sum, we developed a T-ALL/EC platform that elucidated leukemia-microenvironment interactions and identified effective compounds and therapeutic vulnerabilities.
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Células Endoteliales , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Humanos , Células Endoteliales/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Comunicación Celular , Técnicas de Cocultivo , Microambiente TumoralRESUMEN
Peripheral T-cell lymphomas (PTCL) with T-follicular helper phenotype (PTCL-TFH) has recurrent mutations affecting epigenetic regulators, which may contribute to aberrant DNA methylation and chemoresistance. This phase 2 study evaluated oral azacitidine (CC-486) plus cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) as initial treatment for PTCL. CC-486 at 300 mg daily was administered for 7 days before C1 of CHOP, and for 14 days before CHOP C2-6. The primary end point was end-of-treatment complete response (CR). Secondary end points included safety and survival. Correlative studies assessed mutations, gene expression, and methylation in tumor samples. Grade 3 to 4 hematologic toxicities were mostly neutropenia (71%), with febrile neutropenia uncommon (14%). Nonhematologic toxicities included fatigue (14%) and gastrointestinal symptoms (5%). In 20 evaluable patients, CR was 75%, including 88.2% for PTCL-TFH (n = 17). The 2-year progression-free survival (PFS) was 65.8% for all and 69.2% for PTCL-TFH, whereas 2-year overall survival (OS) was 68.4% for all and 76.1% for PTCL-TFH. The frequencies of the TET2, RHOA, DNMT3A, and IDH2 mutations were 76.5%, 41.1%, 23.5%, and 23.5%, respectively, with TET2 mutations significantly associated with CR (P = .007), favorable PFS (P = .004) and OS (P = .015), and DNMT3A mutations associated with adverse PFS (P = .016). CC-486 priming contributed to the reprograming of the tumor microenvironment by upregulation of genes related to apoptosis (P < .01) and inflammation (P < .01). DNA methylation did not show significant shift. This safe and active regimen is being further evaluated in the ALLIANCE randomized study A051902 in CD30-negative PTCL. This trial was registered at www.clinicaltrials.gov as #NCT03542266.
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Linfoma de Células T Periférico , Humanos , Linfoma de Células T Periférico/patología , Azacitidina/efectos adversos , Doxorrubicina , Prednisona/efectos adversos , Vincristina , Ciclofosfamida/efectos adversos , Factores Inmunológicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Microambiente TumoralRESUMEN
Resistance to standard immunochemotherapy remains an unmet challenge in diffuse large B-cell lymphoma (DLBCL), and aberrant DNA methylation may contribute to chemoresistance. Promising early-phase results were reported with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) plus subcutaneous azacitidine, a hypomethylating agent. In this phase 1 study, we evaluated CC-486 (oral azacitidine) plus 6 cycles of R-CHOP in patients with previously untreated intermediate- to high-risk DLBCL or grade 3B/transformed follicular lymphoma. CC-486 doses of 100, 150, 200, or 300 mg given 7 days before cycle 1 and on days 8-21 of cycles 1-5 were evaluated; additional patients were enrolled in the expansion phase to examine preliminary efficacy. The primary objectives were to determine the safety and the maximum tolerated dose (MTD) of CC-486 in combination with R-CHOP. The most common grade 3/4 toxicities were hematologic, including neutropenia (62.7%) and febrile neutropenia (25.4%); grade 3/4 nonhematologic toxicities were uncommon (<7%). The MTD was not established; 2 patients had dose-limiting toxicities (1 with grade 4 febrile neutropenia; 1 with grade 4 prolonged neutropenia). The recommended phase 2 dose was established as 300 mg. The overall response rate was 94.9%, with 52 patients (88.1%) achieving complete responses. With a median follow-up of 28.9 months, estimated 1- and 2-year progression-free survival rates were 84.1% and 78.6%, respectively. Overall, epigenetic priming with CC-486 before R-CHOP can be delivered with acceptable safety to patients with previously untreated intermediate- to high-risk DLBCL or grade 3B/transformed follicular lymphoma. ClinicalTrials.gov: NCT02343536.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Azacitidina/administración & dosificación , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/mortalidad , Administración Oral , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Azacitidina/efectos adversos , Ciclofosfamida/administración & dosificación , Ciclofosfamida/efectos adversos , Supervivencia sin Enfermedad , Doxorrubicina/administración & dosificación , Doxorrubicina/efectos adversos , Femenino , Estudios de Seguimiento , Humanos , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Prednisona/administración & dosificación , Prednisona/efectos adversos , Factores de Riesgo , Rituximab/administración & dosificación , Rituximab/efectos adversos , Tasa de Supervivencia , Vincristina/administración & dosificación , Vincristina/efectos adversosRESUMEN
Methyl-7-guanosine (m7G) "capping" of coding and some noncoding RNAs is critical for their maturation and subsequent activity. Here, we discovered that eukaryotic translation initiation factor 4E (eIF4E), itself a cap-binding protein, drives the expression of the capping machinery and increased capping efficiency of â¼100 coding and noncoding RNAs. To quantify this, we developed enzymatic (cap quantification; CapQ) and quantitative cap immunoprecipitation (CapIP) methods. The CapQ method has the further advantage that it captures information about capping status independent of the type of 5' cap, i.e., it is not restricted to informing on m7G caps. These methodological advances led to unanticipated revelations: 1) Many RNA populations are inefficiently capped at steady state (â¼30 to 50%), and eIF4E overexpression increased this to â¼60 to 100%, depending on the RNA; 2) eIF4E physically associates with noncoding RNAs in the nucleus; and 3) approximately half of eIF4E-capping targets identified are noncoding RNAs. eIF4E's association with noncoding RNAs strongly positions it to act beyond translation. Coding and noncoding capping targets have activities that influence survival, cell morphology, and cell-to-cell interaction. Given that RNA export and translation machineries typically utilize capped RNA substrates, capping regulation provides means to titrate the protein-coding capacity of the transcriptome and, for noncoding RNAs, to regulate their activities. We also discovered a cap sensitivity element (CapSE) which conferred eIF4E-dependent capping sensitivity. Finally, we observed elevated capping for specific RNAs in high-eIF4E leukemia specimens, supporting a role for cap dysregulation in malignancy. In all, levels of capping RNAs can be regulated by eIF4E.
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Factor 4E Eucariótico de Iniciación/metabolismo , Guanosina/análogos & derivados , Caperuzas de ARN/metabolismo , ARN Mensajero/metabolismo , Línea Celular Tumoral , Factor 4E Eucariótico de Iniciación/química , Factor 4E Eucariótico de Iniciación/genética , Guanosina/química , Guanosina/genética , Guanosina/metabolismo , Humanos , Polirribosomas/metabolismo , Caperuzas de ARN/química , Caperuzas de ARN/genética , ARN Mensajero/química , ARN Mensajero/genética , Transcriptoma/genéticaRESUMEN
Enhancers are regulatory regions of DNA, which play a key role in cell-type specific differentiation and development. Most active enhancers are transcribed into enhancer RNA (eRNA) that can regulate transcription of target genes by means of in cis as well as in trans action. eRNA stabilize contacts between distal genomic regions and mediate the interaction of DNA with master transcription factors. Here, we characterized an enhancer eRNA, GECPAR (germinal center proliferative adapter RNA), which is specifically transcribed in normal and neoplastic germinal center B cells from the super-enhancer of POU2AF1, a key regulatory gene of the germinal center reaction. Using diffuse large B-cell lymphoma cell line models, we demonstrated the tumor suppressor activity of GECPAR, which is mediated via its transcriptional regulation of proliferation and differentiation genes, particularly MYC and the Wnt pathway.
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Elementos de Facilitación Genéticos , Linfoma de Células B Grandes Difuso , Humanos , Linfoma de Células B Grandes Difuso/genética , ARN/genética , ARN no Traducido , Transcripción GenéticaRESUMEN
Transient, multi-protein complexes are important facilitators of cellular functions. This includes the chaperome, an abundant protein family comprising chaperones, co-chaperones, adaptors, and folding enzymes-dynamic complexes of which regulate cellular homeostasis together with the protein degradation machinery. Numerous studies have addressed the role of chaperome members in isolation, yet little is known about their relationships regarding how they interact and function together in malignancy. As function is probably highly dependent on endogenous conditions found in native tumours, chaperomes have resisted investigation, mainly due to the limitations of methods needed to disrupt or engineer the cellular environment to facilitate analysis. Such limitations have led to a bottleneck in our understanding of chaperome-related disease biology and in the development of chaperome-targeted cancer treatment. Here we examined the chaperome complexes in a large set of tumour specimens. The methods used maintained the endogenous native state of tumours and we exploited this to investigate the molecular characteristics and composition of the chaperome in cancer, the molecular factors that drive chaperome networks to crosstalk in tumours, the distinguishing factors of the chaperome in tumours sensitive to pharmacologic inhibition, and the characteristics of tumours that may benefit from chaperome therapy. We find that under conditions of stress, such as malignant transformation fuelled by MYC, the chaperome becomes biochemically 'rewired' to form a network of stable, survival-facilitating, high-molecular-weight complexes. The chaperones heat shock protein 90 (HSP90) and heat shock cognate protein 70 (HSC70) are nucleating sites for these physically and functionally integrated complexes. The results indicate that these tightly integrated chaperome units, here termed the epichaperome, can function as a network to enhance cellular survival, irrespective of tissue of origin or genetic background. The epichaperome, present in over half of all cancers tested, has implications for diagnostics and also provides potential vulnerability as a target for drug intervention.
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Chaperonas Moleculares/metabolismo , Complejos Multiproteicos/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Descubrimiento de Drogas , Femenino , Genes myc/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ratones , Chaperonas Moleculares/antagonistas & inhibidores , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/química , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Especificidad de ÓrganosRESUMEN
The discovery of the human histamine H4 receptor (H4R) has contributed to our understanding of the role of histamine in numerous physiological and pathological conditions, including tumor development and progression. The lymph nodes of patients with malignant lymphomas have shown to contain high levels of histamine, however, less is known regarding the expression and function of the H4R in T-cell lymphoma (TCL). In this work we demonstrate the expression of H4R isoforms (mRNA and protein) in three human aggressive TCL (OCI-Ly12, Karpas 299, and HuT78). Histamine and specific H4R agonists (VUF8430 and JNJ28610244) significantly reduced cell viability in a dose-dependent manner (p < 0.05). The combined treatment with the H4R antagonist (JNJ7777120, 10 µM) reversed the effects of the H4R ligands. Importantly, we screened a drug repurposing library of 433 FDA-approved compounds (1 µM) in combination with histamine (10 µM) in Hut78 cells. Histamine produced a favorable antitumor effect with 18 of these compounds, including the histone deacetylase inhibitor panobinostat. Apoptosis, proliferation, and oxidative stress studies confirmed the antitumoral effects of the combination. We conclude that the H4R is expressed in TCL, and it is involved in histamine-mediated responses.
Asunto(s)
Antineoplásicos/farmacología , Agonistas de los Receptores Histamínicos/farmacología , Linfoma de Células T/tratamiento farmacológico , Receptores Histamínicos H4/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células HEK293 , Histamina/metabolismo , Antagonistas de los Receptores Histamínicos/farmacología , Humanos , Linfoma de Células T/metabolismo , Estrés Oxidativo/efectos de los fármacosRESUMEN
We report 5-year follow-up of a multicenter phase 2 study of lenalidomide plus rituximab (LR) as initial treatment of mantle cell lymphoma (MCL). The regimen includes induction and maintenance with the LR doublet. Treatment was continuous until progression, with optional discontinuation after 3 years. The median age of the 38 participants was 65 years, with MCL international prognostic index scores balanced among low, intermediate, and high risk (34%, 34%, and 32%, respectively). Twenty-seven (75%) of the 36 evaluable patients completed ≥3 years of study treatment. At a median follow-up of 64 months (range, 21-78), the 3-year progression-free survival (PFS) and overall survival (OS) were 80% and 90%, respectively, with 5-year estimated PFS and OS of 64% and 77%, respectively. During maintenance, hematologic adverse events (AEs) included asymptomatic grade 3 or 4 cytopenias (42% neutropenia, 5% thrombocytopenia, 3% anemia) and mostly grade 1 or 2 infections managed in the outpatient setting (45% upper respiratory infection, 21% urinary tract infection, 13% sinusitis, 11% cellulitis, 8% pneumonia). Nonhematologic AEs, such as constitutional and inflammatory symptoms, occurred at reduced frequency and intensity compared with induction. A peripheral blood minimal residual disease (MRD) assay (clonoSEQ) showed MRD-negative complete remission in 8 of 10 subjects who had completed ≥3 years of treatment and with available samples for analysis. With longer follow-up, LR continues to demonstrate durable responses and manageable safety as initial induction and maintenance therapy for MCL (ClinicalTrials.gov NCT01472562).
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Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Lenalidomida/uso terapéutico , Linfoma de Células del Manto/tratamiento farmacológico , Rituximab/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Inhibidores de la Angiogénesis/efectos adversos , Antineoplásicos Inmunológicos/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Femenino , Estudios de Seguimiento , Humanos , Lenalidomida/efectos adversos , Masculino , Persona de Mediana Edad , Neutropenia/inducido químicamente , Rituximab/efectos adversos , Análisis de Supervivencia , Trombocitopenia/inducido químicamente , Resultado del TratamientoRESUMEN
Drug resistance is a major hurdle in oncology. Responses of acute myeloid leukaemia (AML) patients to cytarabine (Ara-C)-based therapies are often short lived with a median overall survival of months. Therapies are under development to improve outcomes and include targeting the eukaryotic translation initiation factor (eIF4E) with its inhibitor ribavirin. In a Phase II clinical trial in poor prognosis AML, ribavirin monotherapy yielded promising responses including remissions; however, all patients relapsed. Here we identify a novel form of drug resistance to ribavirin and Ara-C. We observe that the sonic hedgehog transcription factor glioma-associated protein 1 (GLI1) and the UDP glucuronosyltransferase (UGT1A) family of enzymes are elevated in resistant cells. UGT1As add glucuronic acid to many drugs, modifying their activity in diverse tissues. GLI1 alone is sufficient to drive UGT1A-dependent glucuronidation of ribavirin and Ara-C, and thus drug resistance. Resistance is overcome by genetic or pharmacological inhibition of GLI1, revealing a potential strategy to overcome drug resistance in some patients.
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Resistencia a Antineoplásicos , Ácido Glucurónico/metabolismo , Glucuronosiltransferasa/metabolismo , Proteínas Hedgehog/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Factores de Transcripción/metabolismo , Línea Celular Tumoral , Citarabina/metabolismo , Citarabina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Eliminación de Gen , Glucuronosiltransferasa/biosíntesis , Humanos , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/patología , Ribavirina/metabolismo , Ribavirina/farmacología , Transducción de Señal , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Proteína con Dedos de Zinc GLI1RESUMEN
Aggressive double- and triple-hit (DH/TH) diffuse large B-cell lymphomas (DLBCLs) feature activation of Hsp90 stress pathways. Herein, we show that Hsp90 controls posttranscriptional dynamics of key messenger RNA (mRNA) species including those encoding BCL6, MYC, and BCL2. Using a proteomics approach, we found that Hsp90 binds to and maintains activity of eIF4E. eIF4E drives nuclear export and translation of BCL6, MYC, and BCL2 mRNA. eIF4E RNA-immunoprecipitation sequencing in DLBCL suggests that nuclear eIF4E controls an extended program that includes B-cell receptor signaling, cellular metabolism, and epigenetic regulation. Accordingly, eIF4E was required for survival of DLBCL including the most aggressive subtypes, DH/TH lymphomas. Indeed, eIF4E inhibition induces tumor regression in cell line and patient-derived tumorgrafts of TH-DLBCL, even in the presence of elevated Hsp90 activity. Targeting Hsp90 is typically limited by counterregulatory elevation of Hsp70B, which induces resistance to Hsp90 inhibitors. Surprisingly, we identify Hsp70 mRNA as an eIF4E target. In this way, eIF4E inhibition can overcome drug resistance to Hsp90 inhibitors. Accordingly, rational combinatorial inhibition of eIF4E and Hsp90 inhibitors resulted in cooperative antilymphoma activity in DH/TH DLBCL in vitro and in vivo.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Núcleo Celular/metabolismo , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Línea Celular Tumoral , Núcleo Celular/patología , Humanos , Linfoma de Células B/patología , Proteínas de Neoplasias/metabolismoRESUMEN
The interaction of lymphoid tumor cells with components of the extracellular matrix via integrin αvß3 allows tumor survival and growth. This integrin was demonstrated to be the membrane receptor for thyroid hormones (THs) in several tissues. We found that THs, acting as soluble integrin αvß3 ligands, activated growth-related signaling pathways in T-cell lymphomas (TCLs). Specifically, TH-activated αvß3 integrin signaling promoted TCL proliferation and angiogenesis, in part, via the upregulation of vascular endothelial growth factor (VEGF). Consequently, genetic or pharmacologic inhibition of integrin αvß3 decreased VEGF production and induced TCL cell death in vitro and in human xenograft models. In sum, we show that integrin αvß3 transduces prosurvival signals into TCL nuclei, suggesting a novel mechanism for the endocrine modulation of TCL pathophysiology. Targeting this mechanism could constitute an effective and potentially low-toxicity chemotherapy-free treatment of TCL patients.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Integrina alfaVbeta3/genética , Linfoma de Células T/genética , Linfocitos T/inmunología , Hormonas Tiroideas/genética , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Integrina alfaVbeta3/antagonistas & inhibidores , Integrina alfaVbeta3/inmunología , Células Jurkat , Linfoma de Células T/inmunología , Linfoma de Células T/patología , Masculino , Ratones , Ratones SCID , Trasplante de Neoplasias , Neovascularización Patológica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Venenos de Serpiente/farmacología , Linfocitos T/patología , Hormonas Tiroideas/inmunología , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
Tyrosine kinase inhibitors (TKIs) are widely used to treat patients with leukaemia driven by BCR-ABL1 (ref. 1) and other oncogenic tyrosine kinases. Recent efforts have focused on developing more potent TKIs that also inhibit mutant tyrosine kinases. However, even effective TKIs typically fail to eradicate leukaemia-initiating cells (LICs), which often cause recurrence of leukaemia after initially successful treatment. Here we report the discovery of a novel mechanism of drug resistance, which is based on protective feedback signalling of leukaemia cells in response to treatment with TKI. We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones.
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Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Inhibidores de Proteínas Quinasas/farmacología , Factor 1 de Ribosilacion-ADP/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-6 , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
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.
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Linfocitos B , Metilación de ADN/genética , Epigénesis Genética/genética , Linfoma Folicular , Linfoma de Células B Grandes Difuso , Linfocitos B/metabolismo , Linfocitos B/patología , Sitios de Unión , Factor de Unión a CCCTC , Línea Celular Tumoral , Silenciador del Gen , Genoma Humano , Humanos , Elementos Aisladores/genética , Linfoma Folicular/genética , Linfoma Folicular/metabolismo , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
The neurotrophin receptor p75(NTR) has been implicated in mediating neuronal apoptosis after injury to the CNS. Despite its frequent induction in pathologic states, there is limited understanding of the mechanisms that regulate p75(NTR) expression after injury. Here, we show that after focal cerebral ischemia in vivo or oxygen-glucose deprivation in organotypic hippocampal slices or neurons, p75(NTR) is rapidly induced. A concomitant induction of proNGF, a ligand for p75(NTR), is also observed. Induction of this ligand/receptor system is pathologically relevant, as a decrease in apoptosis, after oxygen-glucose deprivation, is observed in hippocampal neurons or slices after delivery of function-blocking antibodies to p75(NTR) or proNGF and in p75(NTR) and ngf haploinsufficient slices. Furthermore, a significant decrease in infarct volume was noted in p75(NTR)-/- mice compared with the wild type. We also investigated the regulatory mechanisms that lead to post-ischemic induction of p75(NTR). We demonstrate that induction of p75(NTR) after ischemic injury is independent of transcription but requires active translation. Basal levels of p75(NTR) in neurons are maintained in part by the expression of microRNA miR-592, and an inverse correlation is seen between miR-592 and p75(NTR) levels in the adult brain. After cerebral ischemia, miR-592 levels fall, with a corresponding increase in p75(NTR) levels. Importantly, overexpression of miR-592 in neurons decreases the level of ischemic injury-induced p75(NTR) and attenuates activation of pro-apoptotic signaling and cell death. These results identify miR-592 as a key regulator of p75(NTR) expression and point to a potential therapeutic candidate to limit neuronal apoptosis after ischemic injury.
Asunto(s)
Apoptosis/fisiología , Regulación de la Expresión Génica/fisiología , Infarto de la Arteria Cerebral Media/patología , MicroARNs/metabolismo , Neuronas/fisiología , Receptores de Factor de Crecimiento Nervioso/metabolismo , Factores de Edad , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Glucosa/deficiencia , Hipocampo/patología , Humanos , Hipoxia , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/genética , Factor de Crecimiento Nervioso/metabolismo , Precursores de Proteínas/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores de Factor de Crecimiento Nervioso/genéticaRESUMEN
Pericytes and vascular smooth muscle cells (VSMCs), which are recruited to developing blood vessels by platelet-derived growth factor BB, support endothelial cell survival and vascular stability. Here, we report that imatinib, a tyrosine kinase inhibitor of platelet-derived growth factor receptor ß (PDGFRß), impaired growth of lymphoma in both human xenograft and murine allograft models. Lymphoma cells themselves neither expressed PDGFRß nor were growth inhibited by imatinib. Tumor growth inhibition was associated with decreased microvascular density and increased vascular leakage. In vivo, imatinib induced apoptosis of tumor-associated PDGFRß(+) pericytes and loss of perivascular integrity. In vitro, imatinib inhibited PDGFRß(+) VSMC proliferation and PDGF-BB signaling, whereas small interfering RNA knockdown of PDGFRß in pericytes protected them against imatinib-mediated growth inhibition. Fluorescence-activated cell sorter analysis of tumor tissue revealed depletion of pericytes, endothelial cells, and their progenitors following imatinib treatment. Compared with imatinib, treatment with an anti-PDGFRß monoclonal antibody partially inhibited lymphoma growth. Last, microarray analysis (Gene Expression Omnibus database accession number GSE30752) of PDGFRß(+) VSMCs following imatinib treatment showed down-regulation of genes implicated in vascular cell proliferation, survival, and assembly, including those representing multiple pathways downstream of PDGFRß. Taken together, these data indicate that PDGFRß(+) pericytes may represent a novel, nonendothelial, antiangiogenic target for lymphoma therapy.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Anticuerpos Monoclonales/farmacología , Benzamidas/farmacología , Linfoma de Células B Grandes Difuso/prevención & control , Linfoma de Células T/prevención & control , Neovascularización Patológica/prevención & control , Pericitos/efectos de los fármacos , Piperazinas/farmacología , Pirimidinas/farmacología , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Diferenciación Celular , Proliferación Celular , Perfilación de la Expresión Génica , Humanos , Mesilato de Imatinib , Técnicas para Inmunoenzimas , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Linfoma de Células T/metabolismo , Linfoma de Células T/patología , Ratones , Ratones SCID , Análisis de Secuencia por Matrices de Oligonucleótidos , Pericitos/inmunología , Pericitos/patología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Células Tumorales CultivadasRESUMEN
PU-H71 is a purine-scaffold Hsp90 inhibitor that, in contrast to other Hsp90 inhibitors, displays unique selectivity for binding the fraction of Hsp90 that is preferentially associated with oncogenic client proteins and enriched in tumor cells (teHsp90). This property allows PU-H71 to potently suppress teHsp90 without inducing toxicity in normal cells. We found that lymphoma cells infected by Epstein-Barr virus or Kaposi sarcoma-associated herpes virus (KSHV) are exquisitely sensitive to this compound. Using PU-H71 affinity capture and proteomics, an unbiased approach to reveal oncogenic networks, we identified the teHsp90 interactome in KSHV(+) primary effusion lymphoma cells. Viral and cellular proteins were identified, including many involved in nuclear factor (NF)-κB signaling, apoptosis, and autophagy. KSHV vFLIP is a viral oncoprotein homologous to cFLIPs, with NF-κB-activating and antiapoptotic activities. We show that teHsp90 binds vFLIP but not cFLIPs. Treatment with PU-H71 induced degradation of vFLIP and IKKγ, NF-κB downregulation, apoptosis and autophagy in vitro, and more importantly, tumor responses in mice. Analysis of the interactome revealed apoptosis as a central pathway; therefore, we tested a BCL2 family inhibitor in primary effusion lymphoma cells. We found strong activity and synergy with PU-H71. Our findings demonstrate PU-H71 affinity capture identifies actionable networks that may help design rational combinations of effective therapies.
Asunto(s)
Benzodioxoles/química , Proteínas HSP90 de Choque Térmico/metabolismo , Infecciones por Herpesviridae/metabolismo , Neoplasias/metabolismo , Neoplasias/virología , Purinas/química , Proteínas Virales/metabolismo , Animales , Apoptosis , Autofagia , Línea Celular Tumoral , Proliferación Celular , Gammaherpesvirinae , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Ratones , FN-kappa B/metabolismo , Trasplante de Neoplasias , Proteoma , Proteómica/métodos , Transducción de SeñalRESUMEN
INTRODUCTION: The transcription factor GATA3 is involved in mammary gland development and is crucial for the maintenance of the differentiated status of luminal epithelial cells. The role of GATA3 in breast cancer as a tumor suppressor has been established, although insights into the mechanism of GATA3 expression loss are still required. METHODS: Chromatin immunoprecipitation assays were conducted to study progestin modulation of recruitment of transcription factors to GATA3 promoter. We performed western blot and reverse RT-qPCR experiments to explore progestin regulation of GATA3 protein and mRNA expression respectively. Confocal microscopy and in vitro phosphorylation studies were conducted to examine progestin capacity to induce GATA3 serine phosphorylation in its 308 residue. GATA3 participation in progestin-induced breast cancer growth was addressed in in vitro proliferation and in vivo tumor growth experiments. RESULTS: In this study, we demonstrate that progestin-activated progesterone receptor (PR) reduces GATA3 expression through regulation at the transcriptional and post-translational levels in breast cancer cells. In the former mechanism, the histone methyltransferase enhancer of zeste homolog 2 is co-recruited with activated PR to a putative progesterone response element in the GATA3 proximal promoter, increasing H3K27me3 levels and inducing chromatin compaction, resulting in decreased GATA3 mRNA levels. This transcriptional regulation is coupled with increased GATA3 protein turnover through progestin-induced GATA3 phosphorylation at serine 308 followed by 26S proteasome-mediated degradation. Both molecular mechanisms converge to accomplish decreased GATA3 expression levels in breast cancer cells upon PR activation. In addition, we demonstrated that decreased GATA3 levels are required for progestin-induced upregulation of cyclin A2, which mediates the G1 to S phase transition of the cell cycle and was reported to be associated with poor prognosis in breast cancer. Finally, we showed that downregulation of GATA3 is required for progestin stimulation of both in vitro cell proliferation and in vivo tumor growth. CONCLUSIONS: In the present study, we reveal that progestin-induced PR activation leads to loss of GATA3 expression in breast cancer cells through transcriptional and post-translational regulation. Importantly, we demonstrate that GATA3 downregulation is required for progestin-induced upregulation of cyclin A2 and for progestin-induced in vitro and in vivo breast cancer cell growth.
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Neoplasias de la Mama/genética , Ciclina A2/genética , Factor de Transcripción GATA3/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Animales/genética , Progestinas/metabolismo , Receptores de Progesterona/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Ciclina A2/metabolismo , Regulación hacia Abajo , Femenino , Factor de Transcripción GATA3/metabolismo , Humanos , Neoplasias Mamarias Animales/metabolismo , Ratones , Fosforilación , Receptores de EstrógenosRESUMEN
PURPOSE OF REVIEW: Cellular to animal to human studies are shedding light on metabolic pathways that contribute to sustaining lymphomagenesis. Old players with new metabolic tricks and new metabolic players come into the scene. The purpose of this review is to discuss the recent advances made in the field of lymphoma metabolism with special focus on the metabolic modulation of tumor promoting and suppressing pathways and, conversely, on the effect of these pathways on metabolite addiction. RECENT FINDINGS: The basis for the high glucose uptake and glycolytic activity in lymphoma cells is now beginning to be understood. Recent findings suggest a greater role of nucleotide biosynthesis as a major driving force for glycolysis, especially during proliferation and cellular stress conditions. There is new evidence for an increasing contribution of glycine-folate metabolism deregulation in nucleotide biosynthesis, genome integrity and epigenetic maintenance. Expanding roles for MYC, PI3K and TP53 in regulating reactive oxygen production, glycolysis and glutaminolysis in lymphoma cells have been described. The identification of novel pathways has allowed the emergence of new 'antimetabolite' strategies to increase the therapeutic efficacy of current approaches. SUMMARY: Metabolism in lymphomas must fulfill the general demands from cell proliferation and those specific to lymphomagenesis. Data emerging from preclinical studies are elucidating the metabolic pathways that contribute to maintaining the malignant phenotype in lymphomas. This has resulted in identification of novel pathways, some of which may have a clinical impact in the diagnosis, characterization and treatment of lymphoma subtypes.