RESUMEN
Aberrant B-cell receptor/NF-κB signaling is a hallmark feature of B-cell non-Hodgkin lymphomas, especially in diffuse large B-cell lymphoma (DLBCL). Recurrent mutations in this cascade, for example, in CD79B, CARD11, or NFKBIZ, and also in the Toll-like receptor pathway transducer MyD88, all deregulate NF-κB, but their differential impact on lymphoma development and biology remains to be determined. Here, we functionally investigate primary mouse lymphomas that formed in recipient mice of Eµ-myc transgenic hematopoietic stem cells stably transduced with naturally occurring NF-κB mutants. Although most mutants supported Myc-driven lymphoma formation through repressed apoptosis, CARD11- or MyD88-mutant lymphoma cells selectively presented with a macrophage-activating secretion profile, which, in turn, strongly enforced transforming growth factor ß (TGF-ß)-mediated senescence in the lymphoma cell compartment. However, MyD88- or CARD11-mutant Eµ-myc lymphomas exhibited high-level expression of the immune-checkpoint mediator programmed cell death ligand 1 (PD-L1), thus preventing their efficient clearance by adaptive host immunity. Conversely, these mutant-specific dependencies were therapeutically exploitable by anti-programmed cell death 1 checkpoint blockade, leading to direct T-cell-mediated lysis of predominantly but not exclusively senescent lymphoma cells. Importantly, mouse-based mutant MyD88- and CARD11-derived signatures marked DLBCL subgroups exhibiting mirroring phenotypes with respect to the triad of senescence induction, macrophage attraction, and evasion of cytotoxic T-cell immunity. Complementing genomic subclassification approaches, our functional, cross-species investigation unveils pathogenic principles and therapeutic vulnerabilities applicable to and testable in human DLBCL subsets that may inform future personalized treatment strategies.
Asunto(s)
Inmunidad Adaptativa , Proteínas Adaptadoras de Señalización CARD/genética , Senescencia Celular/fisiología , Guanilato Ciclasa/genética , Linfoma de Células B Grandes Difuso/inmunología , Factor 88 de Diferenciación Mieloide/genética , Proteínas de Neoplasias/genética , Linfocitos T Citotóxicos/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antígeno B7-H1/antagonistas & inhibidores , Antígenos CD79/genética , Línea Celular Tumoral , Quimiotaxis , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Genes Reporteros , Genes myc , Humanos , Inhibidores de Puntos de Control Inmunológico , Linfoma de Células B Grandes Difuso/patología , Linfoma de Células B Grandes Difuso/terapia , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense , FN-kappa B/genética , FN-kappa B/metabolismo , Mutación Puntual , Proteína 2 Ligando de Muerte Celular Programada 1/antagonistas & inhibidores , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , TranscriptomaRESUMEN
Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). We have developed the Phenol Toluol extraction (PTex) protocol that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a global RNA-bound proteome of human HEK293 cells and the bacterium Salmonella Typhimurium.