RESUMEN
Noncanonical exon usage plays many important roles in cellular phenotypes, but its contribution to human B-cell development remains sketchily understood. To fill this gap, we collected various B-cell fractions from bone marrow (BM) and tonsil donors, performed RNA sequencing, and examined transcript variants. We identified 150 genes that harbor local splicing variations in all pairwise comparisons. One of them encodes FBXW7, an E3 ubiquitin ligase implicated as a driver in several blood cancers. Surprisingly, we discovered that in normal human pro-B cells, the predominant transcript used an alternative first exon to produce the poorly characterized FBXW7ß isoform, previously thought to be restricted to neural tissues. The FBXW7ß transcript was also abundant in cell lines and primary samples of pediatric B-cell acute lymphoblastic leukemia (B-ALL), which originates in the BM. When overexpressed in a heterologous cell system, this transcript yielded the expected protein product, as judged by anti-FLAG immunoblotting and mass spectrometry. Furthermore, in REH B-ALL cells, FBXW7ß mRNA was the only FBXW7 isoform enriched in the polyribosome fraction. To shed light on possible functions of FBXW7ß, we used gain- and loss-of-function approaches and identified an FBXW7-dependent inflammatory gene signature, apparent in a subset of B-ALL with high FBXW7ß expression. This signature contained several members of the tumor necrosis factor superfamily, including those comprising the HLA Class III cluster (LTB, LST1, NCR3, LTA, and NFKBIL1). Our findings suggest that FBXW7ß expression drives proinflammatory responses, which could contribute to normal B-cell development, leukemogenesis, and responses to anticancer therapies.
Asunto(s)
Proteína 7 que Contiene Repeticiones F-Box-WD , Células Precursoras de Linfocitos B , Niño , Humanos , Línea Celular , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Células Precursoras de Linfocitos B/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Activación TranscripcionalRESUMEN
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with poor outcomes with conventional therapy. Nearly 100% of BPDCNs overexpress interleukin 3 receptor subunit alpha (CD123). Given that CD123 is differentially expressed on the surface of BPDCN cells, it has emerged as an attractive therapeutic target. UCART123 is an investigational product consisting of allogeneic T cells expressing an anti-CD123 chimeric antigen receptor (CAR), edited with TALEN® nucleases. In this study, we examine the antitumor activity of UCART123 in preclinical models of BPDCN. We report that UCART123 have selective antitumor activity against CD123-positive primary BPDCN samples (while sparing normal hematopoietic progenitor cells) in the in vitro cytotoxicity and T cell degranulation assays; supported by the increased secretion of IFNγ by UCART123 cells when cultured in the presence of BPDCN cells. UCART123 eradicate BPDCN and result in long-term disease-free survival in a subset of primary patient-derived BPDCN xenograft mouse models. One potential challenge of CD123 targeting therapies is the loss of CD123 antigen through diverse genetic mechanisms, an event observed in one of three BPDCN PDX studied. In summary, these results provide a preclinical proof-of-principle that allogeneic UCART123 cells have potent anti-BPDCN activity.
Asunto(s)
Neoplasias Hematológicas , Trasplante de Células Madre Hematopoyéticas , Trastornos Mieloproliferativos , Neoplasias Cutáneas , Enfermedad Aguda , Animales , Células Dendríticas/metabolismo , Neoplasias Hematológicas/tratamiento farmacológico , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Subunidad alfa del Receptor de Interleucina-3/metabolismo , Ratones , Trastornos Mieloproliferativos/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
Aberrant splicing is a hallmark of leukemias with mutations in splicing factor (SF)-encoding genes. Here we investigated its prevalence in pediatric B-cell acute lymphoblastic leukemias (B-ALL), where SFs are not mutated. By comparing these samples to normal pro-B cells, we found thousands of aberrant local splice variations (LSVs) per sample, with 279 LSVs in 241 genes present in every comparison. These genes were enriched in RNA processing pathways and encoded â¼100 SFs, e.g. hnRNPA1. HNRNPA1 3'UTR was most pervasively mis-spliced, yielding the transcript subject to nonsense-mediated decay. To mimic this event, we knocked it down in B-lymphoblastoid cells and identified 213 hnRNPA1-regulated exon usage events comprising the hnRNPA1 splicing signature in pediatric leukemia. Some of its elements were LSVs in DICER1 and NT5C2, known cancer drivers. We searched for LSVs in other leukemia and lymphoma drivers and discovered 81 LSVs in 41 additional genes. Seventy-seven LSVs out of 81 were confirmed using two large independent B-ALL RNA-seq datasets, and the twenty most common B-ALL drivers, including NT5C2, showed higher prevalence of aberrant splicing than of somatic mutations. Thus, post-transcriptional deregulation of SF can drive widespread changes in B-ALL splicing and likely contributes to disease pathogenesis.
Asunto(s)
Empalme Alternativo , Linfocitos B/metabolismo , Regulación Leucémica de la Expresión Génica , Ribonucleoproteína Nuclear Heterogénea A1/genética , Degradación de ARNm Mediada por Codón sin Sentido , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Regiones no Traducidas 3' , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , Adulto , Linfocitos B/patología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Línea Celular Tumoral , Niño , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Exones , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Humanos , Intrones , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Cultivo Primario de Células , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Factores de Empalme Serina-Arginina/antagonistas & inhibidores , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
We previously described a mechanism of acquired resistance of B-cell acute lymphoblastic leukemia to CD19-directed chimeric antigen receptor T-cell (CART) immunotherapy. It was based on in-frame insertions in or skipping of CD19 exon 2. To distinguish between epitope loss and defects in surface localization, we used retroviral transduction and genome editing to generate cell lines expressing CD19 exon 2 variants (CD19ex2vs) bearing vesicular stomatitis virus G protein (VSVg) tags. These lines were negative by live-cell flow cytometry with an anti-VSVg antibody and resistant to killing by VSVg-directed antibody-drug conjugates (ADCs), suggestive of a defect in surface localization. Indeed, pulse-chase and α-mannosidase inhibitor assays showed that all CD19ex2vs acquired endoplasmic reticulum (ER)-specific high-mannose-type sugars but not complex-type glycans synthesized in the Golgi apparatus. When fused with green fluorescent protein (GFP), CD19ex2vs (including a mutant lacking the relevant disulfide bond) showed colocalization with ER markers, implying protein misfolding. Mass spectrometric profiling of CD19-interacting proteins demonstrated that CD19ex2vs fail to bind to the key tetraspanin CD81 and instead interact with ER-resident chaperones, such as calnexin, and ER transporters involved in antigen presentation. Thus, even the intact domains of CD19ex2vs cannot be easily targeted with ADCs or current CD19 CARTs but could serve as sources of peptides for major histocompatibility complex (MHC)-restricted presentation and T-cell receptor (TCR)-mediated killing.
Asunto(s)
Antígenos CD19/metabolismo , Retículo Endoplásmico/metabolismo , Línea Celular , Edición Génica/métodos , Aparato de Golgi/metabolismo , Células HEK293 , Humanos , Inmunoterapia/métodos , Glicoproteínas de Membrana , Receptores de Antígenos de Linfocitos T/metabolismo , Retroviridae/metabolismo , Proteínas del Envoltorio ViralRESUMEN
We describe a proximity ligation assay (PLA)-based method of assessing association of DNA and RNA in single cells during the cell cycle. Pulse-labeling of DNA with EdU and RNA with BrU and testing their close proximity by PLA demonstrates that RNA synthesis in individual cells resumes about 30-45 min after DNA replication. Consistent with this conclusion, RNA Pol II phosphorylated at Ser2 of its CTD is detected at the same time as RNA transcripts on nascent DNA. Our results also show that RNA is associated with DNA foci during all stages of mitosis.
Asunto(s)
ADN/genética , ADN/metabolismo , Hibridación de Ácido Nucleico , ARN/genética , ARN/metabolismo , Ciclo Celular , Línea Celular , Técnicas Citológicas/métodos , Humanos , Biología Molecular/métodos , Coloración y Etiquetado/métodos , Factores de TiempoRESUMEN
UNLABELLED: The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms. SIGNIFICANCE: CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.
Asunto(s)
Empalme Alternativo , Antígenos CD19/genética , Inmunoterapia , Mutación , Receptores de Antígenos de Linfocitos T/genética , Proteínas Recombinantes de Fusión/genética , Antígenos CD19/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Membrana Celular/metabolismo , Epítopos/inmunología , Exones , Humanos , Inmunoterapia/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Unión Proteica , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Recurrencia , Análisis de Secuencia de ADN , Factores de Empalme Serina-Arginina , Transcripción GenéticaRESUMEN
The mechanism of epigenetic inheritance following DNA replication may involve dissociation of chromosomal proteins from parental DNA and reassembly on daughter strands in a specific order. Here we investigated the behaviour of different types of chromosomal proteins using newly developed methods that allow assessment of the assembly of proteins during DNA replication. Unexpectedly, most chromatin-modifying proteins tested, including methylases, demethylases, acetyltransferases and a deacetylase, are found in close proximity to PCNA or associate with short nascent DNA. Histone modifications occur in a temporal order following DNA replication, mediated by complex activities of different enzymes. In contrast, components of several major nucleosome-remodelling complexes are dissociated from parental DNA, and are later recruited to nascent DNA following replication. Epigenetic inheritance of gene expression patterns may require many aspects of chromatin structure to remain in close proximity to the replication complex followed by reassembly on nascent DNA shortly after replication.
Asunto(s)
Replicación del ADN , ADN/genética , Drosophila/enzimología , Epigénesis Genética , Histonas/metabolismo , Animales , Cromatina/genética , Cromatina/metabolismo , ADN/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Histonas/genética , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismoRESUMEN
Propagation of gene-expression patterns through the cell cycle requires the existence of an epigenetic mark that re-establishes the chromatin architecture of the parental cell in the daughter cells. We devised assays to determine which potential epigenetic marks associate with epigenetic maintenance elements during DNA replication in Drosophila embryos. Histone H3 trimethylated at lysines 4 or 27 is present during transcription but, surprisingly, is replaced by nonmethylated H3 following DNA replication. Methylated H3 is detected on DNA only in nuclei not in S phase. In contrast, the TrxG and PcG proteins Trithorax and Enhancer-of-Zeste, which are H3K4 and H3K27 methylases, and Polycomb continuously associate with their response elements on the newly replicated DNA. We suggest that histone modification enzymes may re-establish the histone code on newly assembled unmethylated histones and thus may act as epigenetic marks.