RESUMEN
Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoid malignancy affecting adults. The NF-κB transcription factor family is activated by 2 main pathways, the canonical and the alternative NF-κB activation pathway, with different functions. The alternative NF-κB pathway leads to activation of the transcriptionally active RelB NF-κB subunit. Alternative NF-κB activation status and its role in DLBCL pathogenesis remain undefined. Here, we reveal a frequent activation of RelB in a large cohort of DLBCL patients and cell lines, independently of their activated B-cell-like or germinal center B-cell-like subtype. RelB activity defines a new subset of patients with DLBCL and a peculiar gene expression profile and mutational pattern. Importantly, RelB activation does not correlate with the MCD genetic subtype, enriched for activated B-cell-like tumors carrying MYD88L265P and CD79B mutations that cooperatively activate canonical NF-κB, thus indicating that current genetic tools to evaluate NF-κB activity in DLBCL do not provide information on the alternative NF-κB activation. Furthermore, the newly defined RelB-positive subgroup of patients with DLBCL exhibits a dismal outcome after immunochemotherapy. Functional studies revealed that RelB confers DLBCL cell resistance to DNA damage-induced apoptosis in response to doxorubicin, a genotoxic agent used in the front-line treatment of DLBCL. We also show that RelB positivity is associated with high expression of cellular inhibitor of apoptosis protein 2 (cIAP2). Altogether, RelB activation can be used to refine the prognostic stratification of DLBCL and may contribute to subvert the therapeutic DNA damage response in a segment of patients with DLBCL.
Asunto(s)
Linfoma de Células B Grandes Difuso/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción ReIB/metabolismo , Apoptosis , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Linfoma de Células B Grandes Difuso/genética , FN-kappa B/genética , Factor de Transcripción ReIB/genética , Activación TranscripcionalRESUMEN
Patients with multiple myeloma (MM) carrying standard- or high-risk cytogenetic abnormalities (CAs) achieve similar complete response (CR) rates, but the later have inferior progression-free survival (PFS). This questions the legitimacy of CR as a treatment endpoint and represents a biological conundrum regarding the nature of tumor reservoirs that persist after therapy in high-risk MM. We used next-generation flow (NGF) cytometry to evaluate measurable residual disease (MRD) in MM patients with standard- vs high-risk CAs (n = 300 and 90, respectively) enrolled in the PETHEMA/GEM2012MENOS65 trial, and to identify mechanisms that determine MRD resistance in both patient subgroups (n = 40). The 36-month PFS rates were higher than 90% in patients with standard- or high-risk CAs achieving undetectable MRD. Persistent MRD resulted in a median PFS of â¼3 and 2 years in patients with standard- and high-risk CAs, respectively. Further use of NGF to isolate MRD, followed by whole-exome sequencing of paired diagnostic and MRD tumor cells, revealed greater clonal selection in patients with standard-risk CAs, higher genomic instability with acquisition of new mutations in high-risk MM, and no unifying genetic event driving MRD resistance. Conversely, RNA sequencing of diagnostic and MRD tumor cells uncovered the selection of MRD clones with singular transcriptional programs and reactive oxygen species-mediated MRD resistance in high-risk MM. Our study supports undetectable MRD as a treatment endpoint for patients with MM who have high-risk CAs and proposes characterizing MRD clones to understand and overcome MRD resistance. This trial is registered at www.clinicaltrials.gov as #NCT01916252.
Asunto(s)
Resistencia a Antineoplásicos/genética , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Neoplasia Residual/patología , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Compuestos de Boro/uso terapéutico , Bortezomib/uso terapéutico , Aberraciones Cromosómicas , Dexametasona/uso terapéutico , Femenino , Citometría de Flujo , Glicina/análogos & derivados , Glicina/uso terapéutico , Humanos , Lenalidomida/uso terapéutico , Masculino , Persona de Mediana Edad , Supervivencia sin Progresión , Resultado del TratamientoRESUMEN
Refractory or relapsed diffuse large B-cell lymphoma (DLBCL) often associates with the activated B-cell-like (ABC) subtype and genetic alterations that drive constitutive NF-κB activation and impair B-cell terminal differentiation. Here, we show that DNA damage response by p53 is a central mechanism suppressing the pathogenic cooperation of IKK2ca-enforced canonical NF-κB and impaired differentiation resulting from Blimp1 loss in ABC-DLBCL lymphomagenesis. We provide evidences that the interplay between these genetic alterations and the tumor microenvironment select for additional molecular addictions that promote lymphoma progression, including aberrant coexpression of FOXP1 and the B-cell mutagenic enzyme activation-induced deaminase, and immune evasion through major histocompatibility complex class II downregulation, PD-L1 upregulation, and T-cell exhaustion. Consistently, PD-1 blockade cooperated with anti-CD20-mediated B-cell cytotoxicity, promoting extended T-cell reactivation and antitumor specificity that improved long-term overall survival in mice. Our data support a pathogenic cooperation among NF-κB-driven prosurvival, genetic instability, and immune evasion mechanisms in DLBCL and provide preclinical proof of concept for including PD-1/PD-L1 blockade in combinatorial immunotherapy for ABC-DLBCL.
Asunto(s)
Linfocitos B/inmunología , Antígeno B7-H1/inmunología , Regulación Neoplásica de la Expresión Génica , Activación de Linfocitos , Linfoma de Células B Grandes Difuso/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Escape del Tumor , Proteína p53 Supresora de Tumor/inmunología , Animales , Linfocitos B/patología , Antígeno B7-H1/genética , Femenino , Humanos , Inmunoterapia , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Linfoma de Células B Grandes Difuso/terapia , Masculino , Ratones , Ratones Transgénicos , Receptor de Muerte Celular Programada 1/genética , Linfocitos T/inmunología , Linfocitos T/patología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Regulatory T (Treg) cells can weaken antitumor immune responses, and inhibition of their function appears to be a promising therapeutic approach in cancer patients. Mice with targeted deletion of the gene encoding the Cl-/HCO3- anion exchanger AE2 (also termed SLC4A2), a membrane-bound carrier involved in intracellular pH regulation, showed a progressive decrease in the number of Treg cells. We therefore challenged AE2 as a potential target for tumor therapy, and generated linear peptides designed to bind the third extracellular loop of AE2, which is crucial for its exchange activity. Peptide p17AE2 exhibited optimal interaction ability and indeed promoted apoptosis in mouse and human Treg cells, while activating effector T-cell function. Interestingly, this linear peptide also induced apoptosis in different types of human leukemia, lymphoma and multiple myeloma cell lines and primary malignant samples, while it showed only moderate effects on normal B lymphocytes. Finally, a macrocyclic AE2 targeting peptide exhibiting increased stability in vivo was effective in mice xenografted with B-cell lymphoma. These data suggest that targeting the anion exchanger AE2 with specific peptides may represent an effective therapeutic approach in B-cell malignancies.
Asunto(s)
Antineoplásicos/farmacología , Antiportadores de Cloruro-Bicarbonato/antagonistas & inhibidores , Leucemia de Células B/metabolismo , Linfoma de Células B/metabolismo , Péptidos/farmacología , Animales , Aniones/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Leucemia de Células B/tratamiento farmacológico , Leucemia de Células B/patología , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/patología , Ratones , Ratones Noqueados , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
SUMMARY: While the current approach to precursor hematologic conditions is to "watch and wait," this may change with the development of therapies that are safe and extend survival or delay the onset of symptomatic disease. The goal of future therapies in precursor hematologic conditions is to improve survival and prevent or delay the development of symptomatic disease while maximizing safety. Clinical trial considerations in this field include identifying an appropriate at-risk population, safety assessments, dose selection, primary and secondary trial endpoints including surrogate endpoints, control arms, and quality-of-life metrics, all of which may enable more precise benefit-risk assessment.
Asunto(s)
Ensayos Clínicos como Asunto , Mieloma Múltiple , Mieloma Múltiple/terapia , Mieloma Múltiple/tratamiento farmacológico , Humanos , Ensayos Clínicos como Asunto/métodos , Proyectos de Investigación , Calidad de VidaRESUMEN
Tumor recognition by T cells is essential for antitumor immunity. A comprehensive characterization of T cell diversity may be key to understanding the success of immunomodulatory drugs and failure of PD-1 blockade in tumors such as multiple myeloma (MM). Here, we use single-cell RNA and T cell receptor sequencing to characterize bone marrow T cells from healthy adults (n = 4) and patients with precursor (n = 8) and full-blown MM (n = 10). Large T cell clones from patients with MM expressed multiple immune checkpoints, suggesting a potentially dysfunctional phenotype. Dual targeting of PD-1 + LAG3 or PD-1 + TIGIT partially restored their function in mice with MM. We identify phenotypic hallmarks of large intratumoral T cell clones, and demonstrate that the CD27- and CD27+ T cell ratio, measured by flow cytometry, may serve as a surrogate of clonal T cell expansions and an independent prognostic factor in 543 patients with MM treated with lenalidomide-based treatment combinations.
Asunto(s)
Mieloma Múltiple , Adulto , Humanos , Animales , Ratones , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Linfocitos T , Receptor de Muerte Celular Programada 1/genética , Lenalidomida , Células ClonalesRESUMEN
Using high-resolution genomic microarray analysis, a distinct genomic profile was defined in 114 samples from patients with splenic marginal zone lymphoma (SMZL). Deletion or uniparental disomy of chromosome 7q were detected in 42 of 114 (37%) SMZLs but in only nine of 170 (5%) mature B-cell lymphomas (P < 0·00001). The presence of unmutated IGHV, genomic complexity, 17p13-TP53 deletion and 8q-MYC gain, but not 7q deletion, correlated with shorter overall survival of SMZL patients. Mapping studies narrowed down a commonly deleted region of 2·7 Mb in 7q32.1-q32.2 spanning a region between the SND1 and COPG2 genes. High-throughput sequencing analysis of the 7q32-deleted segment did not identify biallelic deletions/insertions or clear pathogenic gene mutations, but detected six nucleotide changes in IRF5 (n = 2), TMEM209 (n = 2), CALU (n = 1) and ZC3HC1 (n = 1) not found in healthy individuals. Comparative expression analysis found a fourfold down-regulation of IRF5 gene in lymphomas with 7q32 deletion versus non-deleted tumours (P = 0·032). Ectopic expression of IRF5 in marginal-zone lymphoma cells decreased proliferation and increased apoptosis in vitro, and impaired lymphoma development in vivo. These results show that cryptic deletions, insertions and/or point mutations inactivating genes within 7q32 are not common in SMZL, and suggest that IRF5 may be a haploinsufficient tumour suppressor in this lymphoma entity.
Asunto(s)
Cromosomas Humanos Par 7/genética , Genes Supresores de Tumor , Estudios de Asociación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Factores Reguladores del Interferón/genética , Linfoma de Células B de la Zona Marginal/genética , Proteínas de Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Eliminación de Secuencia , Neoplasias del Bazo/genética , Animales , Apoptosis/genética , División Celular/efectos de los fármacos , Línea Celular Tumoral/trasplante , Cromosomas Humanos Par 7/ultraestructura , Hibridación Genómica Comparativa , Regulación Neoplásica de la Expresión Génica , Genes de Inmunoglobulinas , Humanos , Factores Reguladores del Interferón/biosíntesis , Factores Reguladores del Interferón/deficiencia , Factores Reguladores del Interferón/fisiología , Estimación de Kaplan-Meier , Linfoma de Células B de la Zona Marginal/mortalidad , Linfoma de Células B de la Zona Marginal/patología , Ratones , Ratones Noqueados , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/fisiología , Mutación Puntual , Reacción en Cadena en Tiempo Real de la Polimerasa , Neoplasias del Bazo/mortalidad , Neoplasias del Bazo/patología , Translocación GenéticaRESUMEN
Normal cell counterparts of solid and myeloid tumors accumulate mutations years before disease onset; whether this occurs in B lymphocytes before lymphoma remains uncertain. We sequenced multiple stages of the B lineage in elderly individuals and patients with lymphoplasmacytic lymphoma, a singular disease for studying lymphomagenesis because of the high prevalence of mutated MYD88. We observed similar accumulation of random mutations in B lineages from both cohorts and unexpectedly found MYD88L265P in normal precursor and mature B lymphocytes from patients with lymphoma. We uncovered genetic and transcriptional pathways driving malignant transformation and leveraged these to model lymphoplasmacytic lymphoma in mice, based on mutated MYD88 in B cell precursors and BCL2 overexpression. Thus, MYD88L265P is a preneoplastic event, which challenges the current understanding of lymphomagenesis and may have implications for early detection of B cell lymphomas.
Asunto(s)
Linfoma de Células B , Linfoma , Macroglobulinemia de Waldenström , Anciano , Animales , Humanos , Linfoma de Células B/metabolismo , Ratones , Mutación , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Macroglobulinemia de Waldenström/diagnóstico , Macroglobulinemia de Waldenström/genética , Macroglobulinemia de Waldenström/patologíaRESUMEN
Intraclonal subpopulations of circulating chronic lymphocytic leukemia (CLL) cells with different proliferative histories and reciprocal surface expression of CXCR4 and CD5 have been observed in the peripheral blood of CLL patients and named proliferative (PF), intermediate (IF), and resting (RF) cellular fractions. Here, we found that these intraclonal circulating fractions share persistent DNA methylation signatures largely associated with the mutation status of the immunoglobulin heavy chain locus (IGHV) and their origins from distinct stages of differentiation of antigen-experienced B cells. Increased leukemic birth rate, however, showed a very limited impact on DNA methylation of circulating CLL fractions independent of IGHV mutation status. Additionally, DNA methylation heterogeneity increased as leukemic cells advanced from PF to RF in the peripheral blood. This frequently co-occurred with heterochromatin hypomethylation and hypermethylation of Polycomb-repressed regions in the PF, suggesting accumulation of longevity-associated epigenetic features in recently born cells. On the other hand, transcriptional differences between paired intraclonal fractions confirmed their proliferative experience and further supported a linear advancement from PF to RF in the peripheral blood. Several of these differentially expressed genes showed unique associations with clinical outcome not evident in the bulk clone, supporting the pathological and therapeutic relevance of studying intraclonal CLL fractions. We conclude that independent methylation and transcriptional landscapes reflect both preexisting cell-of-origin fingerprints and more recently acquired hallmarks associated with the life cycle of circulating CLL cells.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Linfocitos B , Metilación de ADN , Humanos , Leucemia Linfocítica Crónica de Células B/genéticaRESUMEN
Epigenetic regulators that exhibit aberrant enzymatic activities or expression profiles are potential therapeutic targets for cancers. Specifically, enzymes responsible for methylation at histone-3 lysine-9 (like G9a) and aberrant DNA hypermethylation (DNMTs) have been implicated in a number of cancers. Recently, molecules bearing a 4-aminoquinoline scaffold were reported as dual inhibitors of these targets and showed a significant in vivo efficacy in animal models of hematological malignancies. Here, we report a detailed exploration around three growing vectors born by this chemotype. Exploring this chemical space led to the identification of features to navigate G9a and DNMT1 biological spaces: not only their corresponding exclusive areas, selective compounds, but also common spaces. Thus, we identified from selective G9a and first-in-class DNMT1 inhibitors, >1 log unit between their IC50 values, with IC50 < 25 nM (e.g., 43 and 26, respectively) to equipotent inhibitors with IC50 < 50 nM for both targets (e.g., 13). Their ADME/Tox profiling and antiproliferative efficacies, versus some cancer cell lines, are also reported.
Asunto(s)
Aminoquinolinas/química , Aminoquinolinas/farmacología , Metilasas de Modificación del ADN/antagonistas & inhibidores , Diseño de Fármacos , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Aminoquinolinas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Metilasas de Modificación del ADN/química , Metilasas de Modificación del ADN/metabolismo , Antígenos de Histocompatibilidad/química , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Conformación ProteicaRESUMEN
Using knowledge- and structure-based approaches, we designed and synthesized reversible chemical probes that simultaneously inhibit the activity of two epigenetic targets, histone 3 lysine 9 methyltransferase (G9a) and DNA methyltransferases (DNMT), at nanomolar ranges. Enzymatic competition assays confirmed our design strategy: substrate competitive inhibitors. Next, an initial exploration around our hit 11 was pursued to identify an adequate tool compound for in vivo testing. In vitro treatment of different hematological neoplasia cell lines led to the identification of molecules with clear antiproliferative efficacies (GI50 values in the nanomolar range). On the basis of epigenetic functional cellular responses (levels of lysine 9 methylation and 5-methylcytosine), an acceptable therapeutic window (around 1 log unit) and a suitable pharmacokinetic profile, 12 was selected for in vivo proof-of-concept ( Nat. Commun. 2017 , 8 , 15424 ). Herein, 12 achieved a significant in vivo efficacy: 70% overall tumor growth inhibition of a human acute myeloid leukemia (AML) xenograft in a mouse model.
Asunto(s)
Antineoplásicos/farmacología , Metilasas de Modificación del ADN/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Metilasas de Modificación del ADN/química , Metilasas de Modificación del ADN/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacocinética , Antígenos de Histocompatibilidad/química , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Ratones , Simulación del Acoplamiento Molecular , Conformación Proteica , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chronic myelogenous leukemia (CML) is characterized by the expression of the BCR-ABL tyrosine kinase, which results in increased cell proliferation and inhibition of apoptosis. In this study, we show in both BCR-ABL cells (Mo7e-p210 and BaF/3-p210) and primary CML CD34+ cells that STI571 inhibition of BCR-ABL tyrosine kinase activity results in a G(1) cell cycle arrest mediated by the PI3K pathway. This arrest is associated with a nuclear accumulation of p27(Kip1) and down-regulation of cyclins D and E. As a result, there is a reduction of the cyclin E/Cdk2 kinase activity and of the retinoblastoma protein phosphorylation. By quantitative reverse transcription-PCR we show that BCR-ABL/PI3K regulates the expression of p27(Kip1) at the level of transcription. We further show that BCR-ABL also regulates p27(Kip1) protein levels by increasing its degradation by the proteasome. This degradation depends on the ubiquitinylation of p27(Kip1) by Skp2-containing SFC complexes: silencing the expression of Skp2 with a small interfering RNA results in the accumulation of p27(Kip1). We also demonstrate that BCR-ABL cells show transcriptional up-regulation of Skp2. Finally, expression of a p27(Kip1) mutant unable of being recognized by Skp2 results in inhibition of proliferation of BCR-ABL cells, indicating that the degradation of p27(Kip1) contributes to the pathogenesis of CML. In conclusion, these results suggest that BCR-ABL regulates cell cycle in CML cells at least in part by inducing proteasome-mediated degradation of the cell cycle inhibitor p27(Kip1) and provide a rationale for the use of inhibitors of the proteasome in patients with BCR-ABL leukemias.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Fusión bcr-abl/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas Asociadas a Fase-S/biosíntesis , Benzamidas , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Ciclo Celular/efectos de los fármacos , Procesos de Crecimiento Celular/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Péptidos y Proteínas de Señalización Intracelular/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Fosforilación , Piperazinas , Pirimidinas/farmacología , Proteína de Retinoblastoma/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Transcripción GenéticaRESUMEN
The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.
Asunto(s)
Antineoplásicos/farmacología , Metilasas de Modificación del ADN/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Neoplasias Hematológicas/tratamiento farmacológico , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Cristalografía por Rayos X , Metilasas de Modificación del ADN/química , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Epigénesis Genética/efectos de los fármacos , Femenino , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/mortalidad , Antígenos de Histocompatibilidad/química , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Interferones/inmunología , Interferones/metabolismo , Ratones , Ratones Endogámicos BALB C , Microsomas Hepáticos , Simulación del Acoplamiento Molecular , Análisis de Supervivencia , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Although conventional therapies for acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL) are effective in inducing remission, many patients relapse upon treatment. Hence, there is an urgent need for novel therapies. PIM kinases are often overexpressed in AML and DLBCL and are therefore an attractive therapeutic target. However, in vitro experiments have demonstrated that intrinsic resistance to PIM inhibition is common. It is therefore likely that only a minority of patients will benefit from single agent PIM inhibitor treatment. In this study, we performed an shRNA-based genetic screen to identify kinases whose suppression is synergistic with PIM inhibition. Here, we report that suppression of p38α (MAPK14) is synthetic lethal with the PIM kinase inhibitor AZD1208. PIM inhibition elevates reactive oxygen species (ROS) levels, which subsequently activates p38α and downstream AKT/mTOR signaling. We found that p38α inhibitors sensitize hematological tumor cell lines to AZD1208 treatment in vitro and in vivo. These results were validated in ex vivo patient-derived AML cells. Our findings provide mechanistic and translational evidence supporting the rationale to test a combination of p38α and PIM inhibitors in clinical trials for AML and DLBCL.
Asunto(s)
Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Células K562 , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Ratones Transgénicos , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In hematopoietic malignancies, oncogenic alterations interfere with cellular differentiation and lead to tumoral development. Identification of the proteins regulating differentiation is essential to understand how they are altered in malignancies. Chronic myelogenous leukemia (CML) is a biphasic disease initiated by an alteration taking place in hematopoietic stem cells. CML progresses to a blast crisis (BC) due to a secondary differentiation block in any of the hematopoietic lineages. However, the molecular mechanisms of CML evolution to T-cell BC remain unclear. Here, we have profiled the changes in DNA methylation patterns in human samples from BC-CML, in order to identify genes whose expression is epigenetically silenced during progression to T-cell lineage-specific BC. We have found that the CpG-island of the ENGRAILED-2 (EN2) gene becomes methylated in this progression. Afterwards, we demonstrate that En2 is expressed during T-cell development in mice and humans. Finally, we further show that genetic deletion of En2 in a CML transgenic mouse model induces a T-cell lineage BC that recapitulates human disease. These results identify En2 as a new regulator of T-cell differentiation whose disruption induces a malignant T-cell fate in CML progression, and validate the strategy used to identify new developmental regulators of hematopoiesis.
Asunto(s)
Crisis Blástica/metabolismo , Diferenciación Celular/inmunología , Regulación Neoplásica de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Proteínas del Tejido Nervioso/metabolismo , Linfocitos T/inmunología , Animales , Islas de CpG/genética , Metilación de ADN/genética , Cartilla de ADN/genética , Progresión de la Enfermedad , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Ratones , Ratones Transgénicos , Análisis por Micromatrices , Proteínas del Tejido Nervioso/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma and can be separated into two subtypes based upon molecular features with similarities to germinal centre B-cells (GCB-like) or activated B-cells (ABC-like). Here we identify gain of 3q27.2 as being significantly associated with adverse outcome in DLBCL and linked with the ABC-like subtype. This lesion includes the BCL6 oncogene, but does not alter BCL6 transcript levels or target-gene repression. Separately, we identify expression of BCL6 in a subset of human haematopoietic stem/progenitor cells (HSPCs). We therefore hypothesize that BCL6 may act by 'hit-and-run' oncogenesis. We model this hit-and-run mechanism by transiently expressing Bcl6 within murine HSPCs, and find that it causes mature B-cell lymphomas that lack Bcl6 expression and target-gene repression, are transcriptionally similar to post-GCB cells, and show epigenetic changes that are conserved from HSPCs to mature B-cells. Together, these results suggest that BCL6 may function in a 'hit-and-run' role in lymphomagenesis.
Asunto(s)
Linfocitos B/metabolismo , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Linfoma de Células B Grandes Difuso/genética , Animales , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ciclofosfamida/uso terapéutico , Variaciones en el Número de Copia de ADN , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Doxorrubicina/uso terapéutico , Epigénesis Genética , Femenino , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/metabolismo , Masculino , Ratones , Ratones Transgénicos , Fenotipo , Prednisona/uso terapéutico , Pronóstico , Proteínas Proto-Oncogénicas c-bcl-6 , Rituximab , Vincristina/uso terapéuticoRESUMEN
MALT1 cleavage activity is linked to the pathogenesis of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), a chemoresistant form of DLBCL. We developed a MALT1 activity assay and identified chemically diverse MALT1 inhibitors. A selected lead compound, MI-2, featured direct binding to MALT1 and suppression of its protease function. MI-2 concentrated within human ABC-DLBCL cells and irreversibly inhibited cleavage of MALT1 substrates. This was accompanied by NF-κB reporter activity suppression, c-REL nuclear localization inhibition, and NF-κB target gene downregulation. Most notably, MI-2 was nontoxic to mice, and displayed selective activity against ABC-DLBCL cell lines in vitro and xenotransplanted ABC-DLBCL tumors in vivo. The compound was also effective against primary human non-germinal center B cell-like DLBCLs ex vivo.
Asunto(s)
Linfocitos B/efectos de los fármacos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Animales , Linfocitos B/metabolismo , Caspasas/metabolismo , Catálisis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Humanos , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas c-rel , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Splenic marginal zone lymphomas (SMZL) are an uncommon type of B-cell non-Hodgkin's lymphoma (NHL-B) in which no specific chromosomal translocations have been described. In contrast, the most frequent cytogenetic abnormality is the loss of the long arm of chromosome 7 (7q). Previous reports have located this loss in the 7q32 region. In order to better characterize the genomic imbalances in SMZL, molecular studies were carried out in 73 patients with SMZL. To gain insight into the mapping at 7q a tiling array was also used. The results confirmed the loss of 7q as the most frequent change. In addition, several abnormalities, including 4q22.1, 1q21.3-q22, 6q25.3, 20q13.33, 3q28, 2q23.3-q24.1 and 17p13, were also present. A loss of 7q22.1 at 99925039-101348479 bp was observed in half of the cases. The region of 7q22.1 has not previously been characterised in SMZL. Our results confirmed the presence of a new region of loss on chromosome 7 in these NHL.
Asunto(s)
Cromosomas Humanos Par 7/genética , Linfoma de Células B de la Zona Marginal/genética , Neoplasias del Bazo/genética , Adulto , Anciano , Anciano de 80 o más Años , Deleción Cromosómica , Cromosomas Artificiales Bacterianos/genética , Hibridación Genómica Comparativa , Femenino , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Reproducibilidad de los ResultadosRESUMEN
Genetic immunization (GI), which is primarily used for vaccine purposes, is a method for producing antibodies to a protein by delivering the gene encoding the protein as a eukaryotic expression vector instead of the protein itself. The mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) is one of the most likely candidates for involvement in pathogenesis of MALT lymphoma and probably of multiple myelomas. In the present work we describe the production and characterization of a mouse monoclonal antibody (mAb) directed against MALT1 and the study of MALT1 protein expression in a large series of lymphomas and myeloma cell lines. The full-length coding sequence of human MALT1 was inserted into pcDNA3 vector and delivered into mouse skin using a helium gene gun. Six new mAbs against the MALT1 molecule were produced. In order to characterize and confirm the specificity of these mAbs, Western blot (WB) and immunoprecipitation (IP) analyses were performed. A new anti-MALT1 mAb was selected and tested in a large series of cell lines. These results confirm that GI is a reliable and effective alternative method for production of mAbs, allowing accurate and sensitive detection and screening of proteins by WB.