RESUMEN
The sphingosine-1-phosphate receptor S1PR2 and its downstream signaling pathway are commonly silenced in diffuse large B-cell lymphoma (DLBCL), either by mutational inactivation or through negative regulation by the oncogenic transcription factor FOXP1. In this study, we examined the upstream regulators of S1PR2 expression and have newly identified the transforming growth factor-ß (TGF-ß)/TGF-ßR2/SMAD1 axis as critically involved in S1PR2 transcriptional activation. Phosphorylated SMAD1 directly binds to regulatory elements in the S1PR2 locus as assessed by chromatin immunoprecipitation, and the CRISPR-mediated genomic editing of S1PR2, SMAD1, or TGFBR2 in DLBCL cell lines renders cells unresponsive to TGF-ß-induced apoptosis. DLBCL clones lacking any 1 of the 3 factors have a clear growth advantage in vitro, as well as in subcutaneous xenotransplantation models, and in a novel model of orthotopic growth of DLBCL cells in the spleens and bone marrow of MISTRG mice expressing various human cytokines. The loss of S1pr2 induces hyperproliferation of the germinal center (GC) B-cell compartment of immunized mice and accelerates MYC-driven lymphomagenesis in spontaneous and serial transplantation models. The specific loss of Tgfbr2 in murine GC B-cell phenocopies the effects of S1pr2 loss on GC B-cell hyperproliferation. Finally, we show that SMAD1 expression is aberrantly downregulated in >85% of analyzed DLBCL patients. The combined results uncover an important novel tumor suppressive function of the TGF-ß/TGF-ßR2/SMAD1/S1PR2 axis in DLBCL, and show that DLBCL cells have evolved to inactivate the pathway at the level of SMAD1 expression.
Asunto(s)
Linfoma de Células B Grandes Difuso/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal , Proteína Smad1/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Apoptosis , Sistemas CRISPR-Cas , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Marcación de Gen , Centro Germinal/patología , Humanos , Ratones , Ratones Noqueados , Modelos Biológicos , Receptores de Esfingosina-1-FosfatoRESUMEN
The genes encoding the histone acetyl-transferases (HATs) CREB binding protein (CREBBP) and EP300 are recurrently mutated in the activated B cell-like and germinal center (GC) B cell-like subtypes of diffuse large B cell lymphoma (DLBCL). Here, we introduced a patient mutation into a human DLBCL cell line using CRISPR and deleted Crebbp and Ep300 in the GC B cell compartment of mice. CREBBP-mutant DLBCL clones exhibited reduced histone H3 acetylation, expressed significantly less MHCII, and grew faster than wild-type clones in s.c. and orthotopic xenograft models. Mice lacking Crebbp in GC B cells exhibited hyperproliferation of their GC compartment upon immunization, had reduced MHCII surface expression on GC cells, and developed accelerated MYC-driven lymphomas. Ep300 inactivation reproduced some, but not all, consequences of Crebbp inactivation. MHCII deficiency phenocopied the effects of CREBBP loss in spontaneous and serial transplantation models of MYC-driven lymphomagenesis, supporting the idea that the mutational inactivation of CREBBP promotes immune evasion. Indeed, the depletion of CD4+ T cells greatly facilitated the engraftment of lymphoma cells in serial transplantation models. In summary, we provide evidence that both HATs are bona fide tumor suppressors that control MHCII expression and promote tumor immune control; mutational inactivation of CREBBP, but not of EP300, has additional cell-intrinsic engraftment and growth-promoting effects.
Asunto(s)
Proteína de Unión a CREB/antagonistas & inhibidores , Proteína de Unión a CREB/genética , Linfoma de Células B Grandes Difuso/genética , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Proteína de Unión a CREB/deficiencia , Proteína de Unión a CREB/inmunología , Sistemas CRISPR-Cas , Línea Celular Tumoral , Proliferación Celular/genética , Regulación hacia Abajo , Proteína p300 Asociada a E1A/antagonistas & inhibidores , Proteína p300 Asociada a E1A/deficiencia , Proteína p300 Asociada a E1A/genética , Proteína p300 Asociada a E1A/inmunología , Eliminación de Gen , Genes MHC Clase II , Centro Germinal/inmunología , Centro Germinal/patología , Antígenos HLA/genética , Xenoinjertos , Código de Histonas/genética , Humanos , Linfoma de Células B Grandes Difuso/inmunología , Linfoma de Células B Grandes Difuso/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/inmunologíaRESUMEN
The pro-apoptotic multidomain Bcl-2 proteins Bax and Bak (also known as BAK1) are considered the gatekeepers of the intrinsic pathway of apoptosis by triggering the mitochondrial release of cytochrome c The role of the third Bax- and Bak-homologous multidomain protein Bok, however, is still unresolved. As cells doubly deficient for Bax and Bak are largely resistant to various apoptotic stimuli, it has been proposed that Bok is either dispensable for apoptosis or that its role is dependent on Bax and Bak. Here, we demonstrate, in several cell systems, that Bok efficiently induces cytochrome c release and apoptosis even in the complete absence of both Bak and Bax. Moreover, modulation of endogenous Bok levels affects the apoptosis response. By RNA interference and targeted deletion of the Bok gene, we demonstrate that Bok can significantly influence the apoptotic response to chemotherapeutic drugs in ovarian carcinoma cells. Hence, our results not only establish Bok as a Bak- and Bax-independent apoptosis inducer, but also suggest a potential impact of Bok expression in ovarian cancer therapy.
Asunto(s)
Apoptosis , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Apoptosis/efectos de los fármacos , Citocromos c/metabolismo , Citostáticos/farmacología , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/metabolismo , Células HCT116 , Humanos , Células MCF-7 , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismoRESUMEN
Numerous targeted therapies have been developed for diffuse large B-cell lymphoma, but the results of late-stage clinical trials were mostly disappointing and have led to very few new regulatory approvals. Here, we use single and combinatorial drug response profiling to show that the combined inhibition of the anti-apoptotic protein Bcl-2 and of the tyrosine kinase BTK with the small molecules venetoclax and ibrutinib efficiently kills DLBCL cells in vitro. High Bcl-2 expression due to either BCL2 amplifications or translocations, in conjunction with chronic active BCR signaling accurately predict responses to dual Bcl-2/BTK inhibition. Orthotopic xenotransplantation and patient-derived xenograft models confirm that the combinatorial is superior to single-agent treatment in reducing the lymphoma burden. Combinatorial treatment further efficiently overcomes both primary and acquired resistance to venetoclax, which we could link to reduced expression of the Bcl-2 family members Bcl-XL and Bcl-2A1 under ibrutinib. We found in a Swiss DLBCL cohort that ~15% of patients are projected to respond to the venetoclax/ibrutinib combination based on their high Bcl-2 expression and nuclear NF-κB localization. Our data show that drug sensitivities exposed by drug response profiling can be attributed to specific mutational signatures and immunohistochemical biomarkers, and point to combined Bcl-2/BTK inhibition as a promising therapeutic strategy in DLBCL.
Asunto(s)
Apoptosis , Linfoma de Células B Grandes Difuso , Agammaglobulinemia Tirosina Quinasa , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/uso terapéutico , Trasplante HeterólogoRESUMEN
Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy arising from germinal center or post-germinal center B-cells that retain many of the properties of normal B-cells. Here we show that a subset of DLBCL express the cytokine IL-10 and its receptor. The genetic ablation of IL-10 receptor signaling abrogates the autocrine STAT3 phosphorylation triggered by tumor cell-intrinsic IL-10 expression and impairs growth of DLBCL cell lines in subcutaneous and orthotopic xenotransplantation models. Furthermore, we demonstrate using an immunocompetent Myc-driven model of DLBCL that neutralization of IL-10 signaling reduces tumor growth, which can be attributed to reduced Treg infiltration, stronger intratumoral effector T-cell responses, and restored tumor-specific MHCII expression. The effects of IL-10R neutralization were phenocopied by the genetic ablation of IL-10 signaling in the Treg compartment and could be reversed by MHCII blockade. The BTK inhibitor ibrutinib effectively blocked tumor cell-intrinsic IL-10 expression and tumor growth in this Myc-driven model. Tumors from patients with high IL-10RA expression are infiltrated by higher numbers of Tregs than IL-10RAlow patients. Finally, we show in 16 cases of DLBCL derived from transplant patients on immunosuppressive therapy that IL-10RA expression is less common in this cohort, and Treg infiltration is not observed.
Asunto(s)
Interleucina-10 , Linfoma de Células B Grandes Difuso , Línea Celular Tumoral , Proliferación Celular , Centro Germinal , Humanos , Interleucina-10/genética , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genéticaRESUMEN
Currently, a major focus of biomedical research and clinical application are the so-called advanced therapy medicinal products (ATMPs), which are highly complex medicines that enable the targeted and personalised treatment of patients. The potential of ATMPs in future cancer treatment is invaluable. However, this novel class of treatments is often extremely expensive. Consequently, these therapies push established reimbursement models to their limits. Because of the high costs, as well as the lack of appropriate reimbursement models, access to these potentially lifesaving therapies is currently not guaranteed to all patients. This paper analyses the current legal framework in Switzerland and critically evaluates existing reimbursement models, particularly with respect to their adaptation for ATMPs. As a promising reimbursement arrangement, this paper proposes a model combining outcome-based instalment payments with aspects of the pay for performance and the annuity payment model. According to this performance-based shared risk model, instalment payments are due when defined treatment goals are achieved.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Reembolso de Incentivo , Humanos , SuizaRESUMEN
The depletion of eosinophils represents an efficient strategy to alleviate allergic asthma, but the consequences of prolonged eosinophil deficiency for human health remain poorly understood. We show here that the ablation of eosinophils severely compromises antitumor immunity in syngeneic and genetic models of colorectal cancer (CRC), which can be attributed to defective Th1 and CD8+ T cell responses. The specific loss of GM-CSF signaling or IRF5 expression in the eosinophil compartment phenocopies the loss of the entire lineage. GM-CSF activates IRF5 in vitro and in vivo and can be administered recombinantly to improve tumor immunity. IL-10 counterregulates IRF5 activation by GM-CSF. CRC patients whose tumors are infiltrated by large numbers of eosinophils also exhibit robust CD8 T cell infiltrates and have a better prognosis than patients with eosinophillow tumors. The combined results demonstrate a critical role of eosinophils in tumor control in CRC and introduce the GM-CSF-IRF5 axis as a critical driver of the antitumor activities of this versatile cell type.
Asunto(s)
Eosinófilos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Inmunidad , Factores Reguladores del Interferón/metabolismo , Neoplasias/inmunología , Transducción de Señal , Células TH1/inmunología , Adenoma/tratamiento farmacológico , Adenoma/inmunología , Adenoma/patología , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Eosinófilos/efectos de los fármacos , Eosinófilos/patología , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunidad/efectos de los fármacos , Interleucina-10/metabolismo , Interleucina-5/metabolismo , Intestinos/patología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/patología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Masculino , Ratones Endogámicos C57BL , Neoplasias/metabolismo , Neoplasias/patología , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Análisis de Supervivencia , Células TH1/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Transgenes , Microambiente Tumoral/efectos de los fármacosRESUMEN
The sphingosine-1-phosphate (S1P) receptor S1PR2 and its downstream adaptor Gα13 are recurrently mutationally inactivated in the germinal center B-cell subtype of diffuse large B-cell lymphoma (DLBCL) and are silenced by the S1PR2 repressor FOXP1 in the activated B-cell like subtype of the disease. Loss of S1PR2 signaling relieves the germinal center confinement that is maintained by an S1P gradient and allows cells to resist S1P-induced apoptosis. We have shown previously that S1PR2 expression is induced in normal B cells through a newly described transforming growth factor-ß (TGF-ß)/TGF-ßRII/SMAD1 signaling axis that is inactivated in >85% of DLBCL patients. DLBCL cell lines lacking S1PR2, TGFBRII, or SMAD1 as the result of genomic editing all have a strong growth advantage in vitro, as well as in subcutaneous and orthotopic xenotransplantation models. Here, we show that the TGF-ß signaling pathway in DLBCL is blocked at the level of SMAD1 in DLBCL cell lines and patient samples by hypermethylation of CpG-rich regions surrounding the SMAD1 transcription start site. The pharmacologic restoration of SMAD1 expression by the demethylating agent decitabine (DAC) sensitizes cells to TGF-ß-induced apoptosis and reverses the growth of initially SMAD1- cell lines in ectopic and orthotopic models. This effect of DAC is reduced in a SMAD1-knockout cell line. We further show that DAC restores SMAD1 expression and reduces the tumor burden in a novel patient-derived orthotopic xenograft model. The combined data lend further support to the concept of an altered epigenome as a major driver of DLBCL pathogenesis.
Asunto(s)
Antineoplásicos/farmacología , Desmetilación del ADN/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Smad1/genética , Proteína Smad1/metabolismo , Animales , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Silenciador del Gen , Humanos , Inmunohistoquímica , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/patología , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Interleukin-6 (IL-6) is a growth factor for normal B cells and plasma cell-derived malignancies. Here, we show that the IL-6 signaling pathway is also active in a subset of diffuse large B-cell lymphoma (DLBCL) patients with particularly poor prognosis. Primary DLBCL cells and DLBCL cell lines expressing IL-6R engraft and form orthotopic lymphomas in humanized mice that ectopically produce human IL-6, and in mice reconstituted with a human immune system. We show that a subset of DLBCL cases have evolved mechanisms that ensure constitutive activation of the IL-6 signaling pathway, i.e., the expression of both chains of the IL-6R, the expression of the cytokine itself, and the mutational inactivation of a negative regulator of IL-6 signaling, SOCS1. IL-6 signaling promotes MYC-driven lymphomagenesis in a genetically engineered model, and treatment with the IL-6R-specific antibody tocilizumab reduces growth of primary DLBCL cells and of DLBCL cell lines in various therapeutic settings. The combined results uncover the IL-6 signaling pathway as a driver and negative prognosticator in aggressive DLBCL that can be targeted with a safe and well-tolerated biologic.