RESUMEN
Research over the past decade has suggested important roles for pseudogenes in physiology and disease. In vitro experiments demonstrated that pseudogenes contribute to cell transformation through several mechanisms. However, in vivo evidence for a causal role of pseudogenes in cancer development is lacking. Here, we report that mice engineered to overexpress either the full-length murine B-Raf pseudogene Braf-rs1 or its pseudo "CDS" or "3' UTR" develop an aggressive malignancy resembling human diffuse large B cell lymphoma. We show that Braf-rs1 and its human ortholog, BRAFP1, elicit their oncogenic activity, at least in part, as competitive endogenous RNAs (ceRNAs) that elevate BRAF expression and MAPK activation in vitro and in vivo. Notably, we find that transcriptional or genomic aberrations of BRAFP1 occur frequently in multiple human cancers, including B cell lymphomas. Our engineered mouse models demonstrate the oncogenic potential of pseudogenes and indicate that ceRNA-mediated microRNA sequestration may contribute to the development of cancer.
Asunto(s)
Linfoma de Células B Grandes Difuso/genética , Proteínas Proto-Oncogénicas B-raf/genética , Seudogenes , ARN/metabolismo , Animales , Secuencia de Bases , Humanos , Linfoma de Células B Grandes Difuso/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas B-raf/metabolismoRESUMEN
T-cell/histiocyte-rich large B-cell lymphoma (TCRLBCL) is an aggressive variant of diffuse large B-cell lymphoma (DLBCL) characterized by rare malignant B cells within a robust but ineffective immune cell infiltrate. The mechanistic basis of immune escape in TCRLBCL is poorly defined and not targeted therapeutically. We performed a genetic and quantitative spatial analysis of the PD-1/PD-L1 pathway in a multi-institutional cohort of TCRLBCLs and found that malignant B cells harbored PD-L1/PD-L2 copy gain or amplification in 64% of cases, which was associated with increased PD-L1 expression (P = .0111). By directed and unsupervised spatial analyses of multiparametric cell phenotypic data within the tumor microenvironment, we found that TCRLBCL is characterized by tumor-immune "neighborhoods" in which malignant B cells are surrounded by exceptionally high numbers of PD-L1-expressing TAMs and PD-1+ T cells. Furthermore, unbiased clustering of spatially resolved immune signatures distinguished TCRLBCL from related subtypes of B-cell lymphoma, including classic Hodgkin lymphoma (cHL) and DLBCL-NOS. Finally, we observed clinical responses to PD-1 blockade in 3 of 5 patients with relapsed/refractory TCRLBCL who were enrolled in clinical trials for refractory hematologic malignancies (NCT03316573; NCT01953692), including 2 complete responses and 1 partial response. Taken together, these data implicate PD-1 signaling as an immune escape pathway in TCRLBCL and also support the potential utility of spatially resolved immune signatures to aid the diagnostic classification and immunotherapeutic prioritization of diverse tumor types.
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Histiocitos/inmunología , Linfoma de Células B Grandes Difuso/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Linfocitos T/inmunología , Escape del Tumor , Antígeno B7-H1/análisis , Antígeno B7-H1/inmunología , Histiocitos/patología , Humanos , Linfoma de Células B Grandes Difuso/patología , Receptor de Muerte Celular Programada 1/análisis , Linfocitos T/patologíaRESUMEN
Inhibition of the B-cell receptor (BCR) signaling pathway is a promising treatment strategy in multiple B-cell malignancies. However, the role of BCR blockade in diffuse large B-cell lymphoma (DLBCL) remains undefined. We recently characterized primary DLBCL subsets with distinct genetic bases for perturbed BCR/phosphoinositide 3-kinase (PI3K) signaling and dysregulated B-cell lymphoma 2 (BCL-2) expression. Herein, we explore the activity of PI3K inhibitors and BCL-2 blockade in a panel of functionally and genetically characterized DLBCL cell line models. A PI3K inhibitor with predominant α/δ activity, copanlisib, exhibited the highest cytotoxicity in all BCR-dependent DLBCLs. The proapoptotic effect of copanlisib was associated with DLBCL subtype-specific dysregulated expression of BCL-2 family members including harakiri (HRK) and its antiapoptotic partner BCL extra large (BCL-xL), BCL2 related protein A1, myeloid cell leukemia 1 (MCL-1), and BCL2 interacting mediator of cell death. Using functional BH3 profiling, we found that the cytotoxic activity of copanlisib was primarily mediated through BCL-xL and MCL-1-dependent mechanisms that might complement BCL-2 blockade. For these reasons, we evaluated single-agent activity of venetoclax in the DLBCLs and identified a subset with limited sensitivity to BCL-2 blockade despite having genetic bases of BCL-2 dysregulation. As these were largely BCR-dependent DLBCLs, we hypothesized that combined inhibition of PI3Kα/δ and BCL-2 would perturb BCR-dependent and BCL-2-mediated survival pathways. Indeed, we observed synergistic activity of copanlisib/venetoclax in BCR-dependent DLBCLs with genetic bases for BCL-2 dysregulation in vitro and confirmed these findings in a xenograft model. These results provide preclinical evidence for the rational combination of PI3Kα/δ and BCL-2 blockade in genetically defined DLBCLs.
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Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Sinergismo Farmacológico , Linfoma de Células B Grandes Difuso/patología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Pirimidinas/farmacología , Quinazolinas/farmacología , Sulfonamidas/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis , Proliferación Celular , Femenino , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Primary mediastinal large B-cell lymphomas (PMBLs) are aggressive tumors that typically present as large mediastinal masses in young women. PMBLs share clinical, transcriptional, and molecular features with classical Hodgkin lymphoma (cHL), including constitutive activation of nuclear factor κB (NF-κB), JAK/STAT signaling, and programmed cell death protein 1 (PD-1)-mediated immune evasion. The demonstrated efficacy of PD-1 blockade in relapsed/refractory PMBLs led to recent approval by the US Food and Drug Administration and underscored the importance of characterizing targetable genetic vulnerabilities in this disease. Here, we report a comprehensive analysis of recurrent genetic alterations -somatic mutations, somatic copy number alterations, and structural variants-in a cohort of 37 newly diagnosed PMBLs. We identified a median of 9 genetic drivers per PMBL, including known and newly identified components of the JAK/STAT and NF-κB signaling pathways and frequent B2M alterations that limit major histocompatibility complex class I expression, as in cHL. PMBL also exhibited frequent, newly identified driver mutations in ZNF217 and an additional epigenetic modifier, EZH2. The majority of these alterations were clonal, which supports their role as early drivers. In PMBL, we identified several previously uncharacterized molecular features that may increase sensitivity to PD-1 blockade, including high tumor mutational burden, microsatellite instability, and an apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) mutational signature. The shared genetic features between PMBL and cHL provide a framework for analyzing the mechanism of action of PD-1 blockade in these related lymphoid malignancies.
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Antineoplásicos Inmunológicos/uso terapéutico , Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica , Linfoma de Células B Grandes Difuso/patología , Neoplasias del Mediastino/patología , Mutación , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Adulto , Estudios de Cohortes , Variaciones en el Número de Copia de ADN , Femenino , Genómica , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genética , Masculino , Neoplasias del Mediastino/tratamiento farmacológico , Neoplasias del Mediastino/genética , Pronóstico , Transactivadores/genéticaRESUMEN
In classical Hodgkin lymphoma (cHL), the host antitumor immune response is ineffective. Hodgkin Reed-Sternberg (HRS) cells have multifaceted mechanisms to evade the immune system, including 9p24.1/CD274(PD-L1)/PDCD1LG2(PD-L2) genetic alterations, overexpression of PD-1 ligands, and associated T-cell exhaustion and additional structural bases of aberrant antigen presentation. The clinical success of PD-1 blockade in cHL suggests that the tumor microenvironment (TME) contains reversibly exhausted T effector cells (Teffs). However, durable responses are observed in patients with ß2-microglobulin/major histocompatibility complex (MHC) class I loss on HRS cells, raising the possibility of non-CD8+ T cell-mediated mechanisms of efficacy of PD-1 blockade. These observations highlight the need for a detailed analysis of the cHL TME. Using a customized time-of-flight mass cytometry panel, we simultaneously assessed cell suspensions from diagnostic cHL biopsies and control reactive lymph node/tonsil (RLNT) samples. Precise phenotyping of immune cell subsets revealed salient differences between cHLs and RLNTs. The TME in cHL is CD4+ T-cell rich, with frequent loss of MHC class I expression on HRS cells. In cHLs, we found concomitant expansion of T helper 1 (Th1)-polarized Teffs and regulatory T cells (Tregs). The cHL Th1 Tregs expressed little or no PD-1, whereas the Th1 Teffs were PD-1+ The differential PD-1 expression and likely functional Th1-polarized CD4+ Tregs and exhausted Teffs may represent complementary mechanisms of immunosuppression in cHL.
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Biomarcadores de Tumor/inmunología , Citofotometría , Enfermedad de Hodgkin/inmunología , Células de Reed-Sternberg/inmunología , Linfocitos T Reguladores/inmunología , Microambiente Tumoral/inmunología , Enfermedad de Hodgkin/patología , Humanos , Células de Reed-Sternberg/patología , Linfocitos T Reguladores/patologíaRESUMEN
B-cell receptor (BCR) signaling pathway components represent promising treatment targets in multiple B-cell malignancies including diffuse large B-cell lymphoma (DLBCL). In in vitro and in vivo model systems, a subset of DLBCLs depend upon BCR survival signals and respond to proximal BCR/phosphoinositide 3 kinase (PI3K) blockade. However, single-agent BCR pathway inhibitors have had more limited activity in patients with DLBCL, underscoring the need for indicators of sensitivity to BCR blockade and insights into potential resistance mechanisms. Here, we report highly significant transcriptional upregulation of C-X-C chemokine receptor 4 (CXCR4) in BCR-dependent DLBCL cell lines and primary tumors following chemical spleen tyrosine kinase (SYK) inhibition, molecular SYK depletion or chemical PI3K blockade. SYK or PI3K inhibition also selectively upregulated cell surface CXCR4 protein expression in BCR-dependent DLBCLs. CXCR4 expression was directly modulated by fork-head box O1 via the PI3K/protein kinase B/forkhead box O1 signaling axis. Following chemical SYK inhibition, all BCR-dependent DLBCLs exhibited significantly increased stromal cell-derived factor-1α (SDF-1α) induced chemotaxis, consistent with the role of CXCR4 signaling in B-cell migration. Select PI3K isoform inhibitors also augmented SDF-1α induced chemotaxis. These data define CXCR4 upregulation as an indicator of sensitivity to BCR/PI3K blockade and identify CXCR4 signaling as a potential resistance mechanism in BCR-dependent DLBCLs.
Asunto(s)
Linfoma de Células B Grandes Difuso , Fosfatidilinositol 3-Quinasas , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genética , Fosfatidilinositol 3-Quinasa , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Regulación hacia ArribaRESUMEN
Primary central nervous system (CNS) lymphoma (PCNSL) and primary testicular lymphoma (PTL) are rare extranodal large B-cell lymphomas with similar genetic signatures. There are no standard-of-care treatment options for patients with relapsed and refractory PCNSL and PTL, and the overall prognosis is poor. PCNSLs and PTLs exhibit frequent 9p24.1 copy-number alterations and infrequent translocations of 9p24.1 and associated increased expression of the programmed cell death protein 1 (PD-1) ligands, PD-L1 and PD-L2. The activity of PD-1 blockade in other lymphomas with 9p24.1 alterations prompted us to test the efficacy of the anti-PD1 antibody, nivolumab, in 4 patients with relapsed/refractory PCNSL and 1 patient with CNS relapse of PTL. All 5 patients had clinical and radiographic responses to PD-1 blockade, and 3 patients remain progression-free at 13+ to 17+ months. Our data suggest that nivolumab is active in relapsed/refractory PCNSL and PTL and support further investigation of PD-1 blockade in these diseases.
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Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias del Sistema Nervioso Central/terapia , Linfoma de Células B/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Neoplasias Testiculares/terapia , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/efectos adversos , Antineoplásicos/efectos adversos , Neoplasias del Sistema Nervioso Central/diagnóstico por imagen , Humanos , Linfoma de Células B/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/diagnóstico por imagen , Recurrencia Local de Neoplasia/terapia , Nivolumab , Receptor de Muerte Celular Programada 1/inmunología , Neoplasias Testiculares/diagnóstico por imagen , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
Cancer cells develop under immune surveillance, thus necessitating immune escape for successful growth. Loss of MHC class I expression provides a key immune evasion strategy in many cancers, although the molecular mechanisms remain elusive. MHC class I transactivator (CITA), known as "NLRC5" [NOD-like receptor (NLR) family, caspase recruitment (CARD) domain containing 5], has recently been identified as a critical transcriptional coactivator of MHC class I gene expression. Here we show that the MHC class I transactivation pathway mediated by CITA/NLRC5 constitutes a target for cancer immune evasion. In all the 21 tumor types we examined, NLRC5 expression was highly correlated with the expression of MHC class I, with cytotoxic T-cell markers, and with genes in the MHC class I antigen-presentation pathway, including LMP2/LMP7, TAP1, and ß2-microglobulin. Epigenetic and genetic alterations in cancers, including promoter methylation, copy number loss, and somatic mutations, were most prevalent in NLRC5 among all MHC class I-related genes and were associated with the impaired expression of components of the MHC class I pathway. Strikingly, NLRC5 expression was significantly associated with the activation of CD8(+) cytotoxic T cells and patient survival in multiple cancer types. Thus, NLRC5 constitutes a novel prognostic biomarker and potential therapeutic target of cancers.
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Presentación de Antígeno , Biomarcadores de Tumor/inmunología , Regulación Neoplásica de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Proteínas de Neoplasias/inmunología , Neoplasias/inmunología , Transactivadores/inmunología , Activación Transcripcional/inmunología , Escape del Tumor , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2/genética , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2/inmunología , Biomarcadores de Tumor/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/inmunología , Femenino , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/mortalidad , Neoplasias/patología , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/inmunología , Transactivadores/genética , Microglobulina beta-2/genética , Microglobulina beta-2/inmunologíaRESUMEN
BACKGROUND: Preclinical studies suggest that Reed-Sternberg cells exploit the programmed death 1 (PD-1) pathway to evade immune detection. In classic Hodgkin's lymphoma, alterations in chromosome 9p24.1 increase the abundance of the PD-1 ligands, PD-L1 and PD-L2, and promote their induction through Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling. We hypothesized that nivolumab, a PD-1-blocking antibody, could inhibit tumor immune evasion in patients with relapsed or refractory Hodgkin's lymphoma. METHODS: In this ongoing study, 23 patients with relapsed or refractory Hodgkin's lymphoma that had already been heavily treated received nivolumab (at a dose of 3 mg per kilogram of body weight) every 2 weeks until they had a complete response, tumor progression, or excessive toxic effects. Study objectives were measurement of safety and efficacy and assessment of the PDL1 and PDL2 (also called CD274 and PDCD1LG2, respectively) loci and PD-L1 and PD-L2 protein expression. RESULTS: Of the 23 study patients, 78% were enrolled in the study after a relapse following autologous stem-cell transplantation and 78% after a relapse following the receipt of brentuximab vedotin. Drug-related adverse events of any grade and of grade 3 occurred in 78% and 22% of patients, respectively. An objective response was reported in 20 patients (87%), including 17% with a complete response and 70% with a partial response; the remaining 3 patients (13%) had stable disease. The rate of progression-free survival at 24 weeks was 86%; 11 patients were continuing to participate in the study. Reasons for discontinuation included stem-cell transplantation (in 6 patients), disease progression (in 4 patients), and drug toxicity (in 2 patients). Analyses of pretreatment tumor specimens from 10 patients revealed copy-number gains in PDL1 and PDL2 and increased expression of these ligands. Reed-Sternberg cells showed nuclear positivity of phosphorylated STAT3, indicative of active JAK-STAT signaling. CONCLUSIONS: Nivolumab had substantial therapeutic activity and an acceptable safety profile in patients with previously heavily treated relapsed or refractory Hodgkin's lymphoma. (Funded by Bristol-Myers Squibb and others; ClinicalTrials.gov number, NCT01592370.).
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos/administración & dosificación , Enfermedad de Hodgkin/tratamiento farmacológico , Inmunoterapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Adulto , Anticuerpos Monoclonales/efectos adversos , Antineoplásicos/efectos adversos , Brentuximab Vedotina , Femenino , Enfermedad de Hodgkin/metabolismo , Enfermedad de Hodgkin/terapia , Humanos , Inmunoconjugados/uso terapéutico , Quinasas Janus/metabolismo , Masculino , Persona de Mediana Edad , Nivolumab , Proteína 2 Ligando de Muerte Celular Programada 1/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Recurrencia , Células de Reed-Sternberg/efectos de los fármacos , Factores de Transcripción STAT/metabolismo , Trasplante de Células MadreRESUMEN
Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease defined by transcriptional classifications, specific signaling and survival pathways, and multiple low-frequency genetic alterations. Preclinical model systems that capture the genetic and functional heterogeneity of DLBCL are urgently needed. Here, we generated and characterized a panel of large B-cell lymphoma (LBCL) patient-derived xenograft (PDX) models, including 8 that reflect the immunophenotypic, transcriptional, genetic, and functional heterogeneity of primary DLBCL and 1 that is a plasmablastic lymphoma. All LBCL PDX models were subjected to whole-transcriptome sequencing to classify cell of origin and consensus clustering classification (CCC) subtypes. Mutations and chromosomal rearrangements were evaluated by whole-exome sequencing with an extended bait set. Six of the 8 DLBCL models were activated B-cell (ABC)-type tumors that exhibited ABC-associated mutations such as MYD88, CD79B, CARD11, and PIM1. The remaining 2 DLBCL models were germinal B-cell type, with characteristic alterations of GNA13, CREBBP, and EZH2, and chromosomal translocations involving IgH and either BCL2 or MYC Only 25% of the DLBCL PDX models harbored inactivating TP53 mutations, whereas 75% exhibited copy number alterations of TP53 or its upstream modifier, CDKN2A, consistent with the reported incidence and type of p53 pathway alterations in primary DLBCL. By CCC criteria, 6 of 8 DLBCL PDX models were B-cell receptor (BCR)-type tumors that exhibited selective surface immunoglobulin expression and sensitivity to entospletinib, a recently developed spleen tyrosine kinase inhibitor. In summary, we have established and characterized faithful PDX models of DLBCL and demonstrated their usefulness in functional analyses of proximal BCR pathway inhibition.
Asunto(s)
Linfoma de Células B Grandes Difuso/genética , Animales , Linaje de la Célula , Aberraciones Cromosómicas , Regulación Neoplásica de la Expresión Génica , Genes Relacionados con las Neoplasias , Heterogeneidad Genética , Xenoinjertos , Humanos , Inmunofenotipificación , Linfoma de Células B Grandes Difuso/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mutación , Análisis de Secuencia de ADN , Ensayo de Capsula Subrrenal , TranscriptomaRESUMEN
Primary central nervous system lymphomas (PCNSLs) and primary testicular lymphomas (PTLs) are extranodal large B-cell lymphomas (LBCLs) with inferior responses to current empiric treatment regimens. To identify targetable genetic features of PCNSL and PTL, we characterized their recurrent somatic mutations, chromosomal rearrangements, copy number alterations (CNAs), and associated driver genes, and compared these comprehensive genetic signatures to those of diffuse LBCL and primary mediastinal large B-cell lymphoma (PMBL). These studies identify unique combinations of genetic alterations in discrete LBCL subtypes and subtype-selective bases for targeted therapy. PCNSLs and PTLs frequently exhibit genomic instability, and near-uniform, often biallelic, CDKN2A loss with rare TP53 mutations. PCNSLs and PTLs also use multiple genetic mechanisms to target key genes and pathways and exhibit near-uniform oncogenic Toll-like receptor signaling as a result of MYD88 mutation and/or NFKBIZ amplification, frequent concurrent B-cell receptor pathway activation, and deregulation of BCL6. Of great interest, PCNSLs and PTLs also have frequent 9p24.1/PD-L1/PD-L2 CNAs and additional translocations of these loci, structural bases of immune evasion that are shared with PMBL.
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Neoplasias del Sistema Nervioso Central/genética , Sitios Genéticos , Linfoma de Células B Grandes Difuso/genética , Proteínas de Neoplasias/genética , Neoplasias Testiculares/genética , Translocación Genética , Neoplasias del Sistema Nervioso Central/metabolismo , Neoplasias del Sistema Nervioso Central/patología , Femenino , Humanos , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Masculino , Neoplasias del Mediastino/genética , Neoplasias del Mediastino/metabolismo , Neoplasias del Mediastino/patología , Proteínas de Neoplasias/metabolismo , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologíaRESUMEN
BCL6 is the product of a proto-oncogene implicated in the pathogenesis of human B-cell lymphomas. By binding specific DNA sequences, BCL6 controls the transcription of a variety of genes involved in B-cell development, differentiation and activation. BCL6 is overexpressed in the majority of patients with aggressive diffuse large B-cell lymphoma (DLBCL), the most common lymphoma in adulthood, and transgenic mice constitutively expressing BCL6 in B cells develop DLBCLs similar to the human disease. In many DLBCL patients, BCL6 overexpression is achieved through translocation (~40%) or hypermutation of its promoter (~15%). However, many other DLBCLs overexpress BCL6 through an unknown mechanism. Here we show that BCL6 is targeted for ubiquitylation and proteasomal degradation by a SKP1CUL1F-box protein (SCF) ubiquitin ligase complex that contains the orphan F-box protein FBXO11 (refs 5, 6). The gene encoding FBXO11 was found to be deleted or mutated in multiple DLBCL cell lines, and this inactivation of FBXO11 correlated with increased levels and stability of BCL6. Similarly, FBXO11 was either deleted or mutated in primary DLBCLs. Notably, tumour-derived FBXO11 mutants displayed an impaired ability to induce BCL6 degradation. Reconstitution of FBXO11 expression in FBXO11-deleted DLBCL cells promoted BCL6 ubiquitylation and degradation, inhibited cell proliferation, and induced cell death. FBXO11-deleted DLBCL cells generated tumours in immunodeficient mice, and the tumorigenicity was suppressed by FBXO11 reconstitution. We reveal a molecular mechanism controlling BCL6 stability and propose that mutations and deletions in FBXO11 contribute to lymphomagenesis through BCL6 stabilization. The deletions/mutations found in DLBCLs are largely monoallelic, indicating that FBXO11 is a haplo-insufficient tumour suppressor gene.
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Proteínas de Unión al ADN/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Mutación/genética , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteolisis , Alelos , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al ADN/genética , Eliminación de Gen , Genes Supresores de Tumor , Células HEK293 , Humanos , Linfoma de Células B Grandes Difuso/enzimología , Linfoma de Células B Grandes Difuso/patología , Ratones , Trasplante de Neoplasias , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Proteína-Arginina N-Metiltransferasas/deficiencia , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-bcl-6 , Proteínas Ligasas SKP Cullina F-box/metabolismo , UbiquitinaciónRESUMEN
Tumors are composed of phenotypically heterogeneous cell populations. The nongenomic mechanisms underlying transitions and interactions between cell populations are largely unknown. Here, we show that diffuse large B-cell lymphomas possess a self-organized infrastructure comprising side population (SP) and non-SP cells, where transitions between clonogenic states are modulated by exosome-mediated Wnt signaling. DNA methylation modulated SP-non-SP transitions and was correlated with the reciprocal expressions of Wnt signaling pathway agonist Wnt3a in SP cells and the antagonist secreted frizzled-related protein 4 in non-SP cells. Lymphoma SP cells exhibited autonomous clonogenicity and exported Wnt3a via exosomes to neighboring cells, thus modulating population equilibrium in the tumor.
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Proliferación Celular , Células Clonales/patología , Exosomas/fisiología , Linfoma de Células B Grandes Difuso/patología , Células Madre Neoplásicas/patología , Vía de Señalización Wnt/fisiología , Recuento de Células , Progresión de la Enfermedad , Células HEK293 , Homeostasis/fisiología , Humanos , Linfoma de Células B Grandes Difuso/metabolismo , Transporte de Proteínas , Células Tumorales CultivadasRESUMEN
MYC is one of the most frequently deregulated oncogenes in human malignancies. It encodes a leucine zipper transcription factor that modulates a broad spectrum of cellular genes responsible for enhancing cell proliferation, cellular metabolism, growth, angiogenesis, metastasis, genomic instability, stem cell self-renewal and reduced differentiation. MYC functions predominantly as an amplifier of expression of already active genes, potentiating the pre-existing transcriptional program, although it can also repress certain transcriptional targets. In mouse models, MYC induces lymphomas, but requires cooperation with other lesions, including inactivation of the p53 pathway, structural alterations of BCL2 family members, or increased PI3K activity. In human B-cell tumors, MYC rearrangements involving the 8q24 region and immunoglobulin heavy or light genes are a hallmark of Burkitt lymphoma (BL), but can also occur in other lymphoid malignancies, that include diffuse large B-cell lymphoma (DLBCL), B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma (BCLU), plasma cell myeloma (PCM), mantle cell lymphoma (MCL) and plasmablastic lymphoma. For non-BL lymphoid malignancies, MYC fusions represent secondary genetic events and exist in the context of complex karyotypes. Regardless of the mechanism deregulating MYC, lymphomas over-expressing MYC are addicted to this oncogene, highlighting the potential clinical utility of MYC targeting strategies. Several promising approaches for pharmaceutical intervention have been suggested which are now in preclinical or clinical development. Herein, we therefore review the molecular pathogenetic mechanisms associated with MYC deregulation in human B-cell lymphomas and their implications for therapies targeting MYC.
Asunto(s)
Linfoma de Células B/genética , Proteínas Proto-Oncogénicas c-myc/fisiología , Animales , Regulación Neoplásica de la Expresión Génica , Genes myc , Humanos , Linfoma de Células B/etiología , Linfoma de Células B/terapia , RatonesRESUMEN
BACKGROUND: Daratumumab (DARA), a promising novel therapy for multiple myeloma, is an IgG1κ monoclonal antibody that recognizes CD38 on myeloma cells. During routine compatibility testing, we observed that the plasma of five of five DARA-treated patients demonstrated a positive antibody screen and panreactivity on red blood cell (RBC) panel testing. We hypothesized that the observed panreactivity reflected DARA binding to CD38 on reagent RBCs, and we investigated methods to prevent this binding. STUDY DESIGN AND METHODS: DARA binding to CD38+ or CD38- HL60 cells was assessed by flow cytometry. To remove cell surface CD38, cells were incubated with dithiothreitol (DTT) or trypsin. Soluble CD38 or anti-DARA was used to neutralize DARA in solution. Routine blood bank serologic methods were used to test samples from DARA-treated patients and normal plasma samples spiked with DARA and/or alloantibodies. RESULTS: Normal plasma samples spiked with DARA (0.1-10 µg/mL) and incubated with reagent RBCs recapitulated the interference observed with samples from DARA-treated patients. Flow cytometry experiments confirmed DARA binding to CD38+ HL60 cells, but not to CD38- controls. DTT treatment of CD38+ HL60 cells reduced DARA binding by 92% by denaturing cell surface CD38. Treating DARA-containing plasma with soluble CD38 or anti-DARA idiotype also inhibited DARA binding. CONCLUSION: DARA causes panreactivity in vitro by binding to CD38 on reagent RBCs. Treating reagent RBCs with DTT is a robust method to negate the DARA interference, enabling the safe provision of blood to DARA-treated patients. Because DTT denatures Kell antigens, K- units are provided to these patients.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Tipificación y Pruebas Cruzadas Sanguíneas , Eritrocitos/inmunología , ADP-Ribosil Ciclasa 1/genética , ADP-Ribosil Ciclasa 1/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Reacciones Cruzadas/efectos de los fármacos , Ditiotreitol/farmacología , Relación Dosis-Respuesta a Droga , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Citometría de Flujo , Células HL-60 , Humanos , Mieloma Múltiple/inmunología , Mieloma Múltiple/terapia , Pruebas Serológicas , TransfecciónRESUMEN
To gain insight into the genomic basis of diffuse large B-cell lymphoma (DLBCL), we performed massively parallel whole-exome sequencing of 55 primary tumor samples from patients with DLBCL and matched normal tissue. We identified recurrent mutations in genes that are well known to be functionally relevant in DLBCL, including MYD88, CARD11, EZH2, and CREBBP. We also identified somatic mutations in genes for which a functional role in DLBCL has not been previously suspected. These genes include MEF2B, MLL2, BTG1, GNA13, ACTB, P2RY8, PCLO, and TNFRSF14. Further, we show that BCL2 mutations commonly occur in patients with BCL2/IgH rearrangements as a result of somatic hypermutation normally occurring at the IgH locus. The BCL2 point mutations are primarily synonymous, and likely caused by activation-induced cytidine deaminase-mediated somatic hypermutation, as shown by comprehensive analysis of enrichment of mutations in WRCY target motifs. Those nonsynonymous mutations that are observed tend to be found outside of the functionally important BH domains of the protein, suggesting that strong negative selection against BCL2 loss-of-function mutations is at play. Last, by using an algorithm designed to identify likely functionally relevant but infrequent mutations, we identify KRAS, BRAF, and NOTCH1 as likely drivers of DLBCL pathogenesis in some patients. Our data provide an unbiased view of the landscape of mutations in DLBCL, and this in turn may point toward new therapeutic strategies for the disease.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Linfoma de Células B Grandes Difuso/genética , Mutación , Secuencias de Aminoácidos , Análisis por Conglomerados , Análisis Mutacional de ADN , Exoma , Exones , Humanos , Modelos Genéticos , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN , Translocación GenéticaRESUMEN
Targeting the surface of malignant cells has evolved into a cornerstone in cancer therapy, paradigmatically introduced by the success of humoral immunotherapy against CD20 in malignant lymphoma. However, tumor cell susceptibility to immunochemotherapy varies, with mostly a fatal outcome in cases of resistant disease. Here, we show that lymphoma exosomes shield target cells from antibody attack and that exosome biogenesis is modulated by the lysosome-related organelle-associated ATP-binding cassette (ABC) transporter A3 (ABCA3). B-cell lymphoma cells released exosomes that carried CD20, bound therapeutic anti-CD20 antibodies, consumed complement, and protected target cells from antibody attack. ABCA3, previously shown to mediate resistance to chemotherapy, was critical for the amounts of exosomes released, and both pharmacological blockade and the silencing of ABCA3 enhanced susceptibility of target cells to antibody-mediated lysis. Mechanisms of cancer cell resistance to drugs and antibodies are linked in an ABCA3-dependent pathway of exosome secretion.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/inmunología , Exosomas/inmunología , Evasión Inmune/inmunología , Inmunidad Humoral/inmunología , Inmunoterapia , Linfoma de Células B/inmunología , Linfoma de Células B/terapia , Absorción , Anticuerpos Monoclonales de Origen Murino/farmacología , Anticuerpos Antineoplásicos/inmunología , Antígenos CD20/inmunología , Línea Celular , Citotoxicidad Inmunológica/efectos de los fármacos , Exosomas/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Humanos , Evasión Inmune/efectos de los fármacos , Inmunidad Humoral/efectos de los fármacos , Linfoma de Células B/patología , RituximabRESUMEN
In patients with chronic lymphocytic leukemia, Richter transformation (RT) reflects the development of an aggressive lymphoma that is associated with poor response to chemotherapy and short survival. We initiated an international, investigator-initiated, prospective, open-label phase 2 study in which patients with RT received a combination of the PD-1 inhibitor tislelizumab plus the BTK inhibitor zanubrutinib for 12 cycles. Patients responding to treatment underwent maintenance treatment with both agents. The primary end point was overall response rate after six cycles. Of 59 enrolled patients, 48 patients received at least two cycles of treatment and comprised the analysis population according to the study protocol. The median observation time was 13.9 months, the median age was 67 (range 45-82) years. Ten patients (20.8%) had received previous RT-directed therapy. In total, 28 out of 48 patients responded to induction therapy with an overall response rate of 58.3% (95% confidence interval (CI) 43.2-72.4), including 9 (18.8%) complete reponse and 19 (39.6%) partial response, meeting the study's primary end point by rejecting the predefined null hypothesis of 40% (P = 0.008). Secondary end points included duration of response, progression-free survival and overall survival. The median duration of response was not reached, the median progression-free survival was 10.0 months (95% CI 3.8-16.3). Median overall survival was not reached with a 12-month overall survival rate of 74.7% (95% CI 58.4-91.0). The most common adverse events were infections (18.0%), gastrointestinal disorders (13.0%) and hematological toxicities (11.4%). These data suggest that combined checkpoint and BTK inhibition by tislelizumab plus zanubrutinib is an effective and well-tolerated treatment strategy for patients with RT. ClinicalTrials.gov Identifier: NCT04271956 .
Asunto(s)
Anticuerpos Monoclonales Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica , Leucemia Linfocítica Crónica de Células B , Piperidinas , Pirazoles , Pirimidinas , Humanos , Persona de Mediana Edad , Anciano , Anciano de 80 o más Años , Estudios Prospectivos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológicoRESUMEN
Although BCR-ABL1 tyrosine kinase inhibitors reliably induce disease remission for patients with chronic myeloid leukaemia (CML), unlimited extension of therapy is necessary to prevent relapse from persistent leukaemic cells. Here, we analysed model cell lines and primary CML cells for the expression and functions of the ABC transporter A3 (ABCA3) as well as the embryonic stem cell-associated transcription factor SALL4. ABCA3 protected leukaemic cells from the cytotoxic effects of the tyrosine kinase inhibitors imatinib, dasatinib, and nilotinib. In the surviving cells, exposure to tyrosine kinase inhibitors significantly enhanced ABCA3 expression in vivo and in vitro, and was associated with increased expression of SALL4, which binds the ABCA3 promoter. Inhibition of ABCA3 or SALL4 by genetic silencing or indomethacin, but not interferon gamma, interrupted SALL4-dependent regulation of ABCA3 and restored susceptibility of leukaemic cells to tyrosine kinase inhibition. Tyrosine kinase inhibitor exposure facilitates a protective loop of SALL4 and ABCA3 cooperation in persistent leukaemic cells.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/biosíntesis , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Proteínas de Neoplasias/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Factores de Transcripción/metabolismo , Animales , Benzamidas/farmacología , Dasatinib , Femenino , Células HL-60 , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Ratones , Piperazinas/farmacología , Pirimidinas/farmacología , Tiazoles/farmacologíaRESUMEN
Patients with advanced-stage bronchial cancer benefit from systemic cytostatic therapy, in particular from regimens integrating cisplatin and taxanes. However, eventual disease progression leads to a fatal outcome in most cases, originating from tumor cells resisting chemotherapy. We here show that the intracellular ATP-binding cassette transporter A3 (ABCA3), previously recognized as critical for the secretion of surfactant components from type 2 pneumocytes, is expressed in non-small-cell lung cancer (NSCLC) cells. With some heterogeneity in a given specimen, expression levels detected immunohistochemically in primary cancer tissue were highest in adenocarcinomas and lowest in small cell lung cancers. Genetic silencing of ABCA3 in the NSCLC cell line models A549, NCI-H1650 and NCI-H1975 significantly increased tumor cell susceptibility to the cytostatic effects of both cisplatin (in all cell lines) and paclitaxel (in two of three cell lines). Taken together, ABCA3 emerges as a modulator of NSCLC cell susceptibility to cytostatic therapy.