RESUMEN
Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two most common non-Hodgkin lymphomas (NHLs). Here we sequenced tumour and matched normal DNA from 13 DLBCL cases and one FL case to identify genes with mutations in B-cell NHL. We analysed RNA-seq data from these and another 113 NHLs to identify genes with candidate mutations, and then re-sequenced tumour and matched normal DNA from these cases to confirm 109 genes with multiple somatic mutations. Genes with roles in histone modification were frequent targets of somatic mutation. For example, 32% of DLBCL and 89% of FL cases had somatic mutations in MLL2, which encodes a histone methyltransferase, and 11.4% and 13.4% of DLBCL and FL cases, respectively, had mutations in MEF2B, a calcium-regulated gene that cooperates with CREBBP and EP300 in acetylating histones. Our analysis suggests a previously unappreciated disruption of chromatin biology in lymphomagenesis.
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Histonas/metabolismo , Linfoma no Hodgkin/genética , Mutación/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genoma Humano/genética , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Pérdida de Heterocigocidad/genética , Linfoma Folicular/enzimología , Linfoma Folicular/genética , Linfoma de Células B Grandes Difuso/enzimología , Linfoma de Células B Grandes Difuso/genética , Linfoma no Hodgkin/enzimología , Proteínas de Dominio MADS/genética , Proteínas de Dominio MADS/metabolismo , Factores de Transcripción MEF2 , Factores Reguladores Miogénicos/genética , Factores Reguladores Miogénicos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMEN
Diffuse large B-cell lymphoma (DLBCL) accounts for 30% to 40% of newly diagnosed lymphomas and has an overall cure rate of approximately 60%. Previously, we observed FOXO1 mutations in non-Hodgkin lymphoma patient samples. To explore the effects of FOXO1 mutations, we assessed FOXO1 status in 279 DLBCL patient samples and 22 DLBCL-derived cell lines. FOXO1 mutations were found in 8.6% (24/279) of DLBCL cases: 92.3% (24/26) of mutations were in the first exon, 46.2% (12/26) were recurrent mutations affecting the N-terminal region, and another 38.5% (10/26) affected the Forkhead DNA binding domain. Recurrent mutations in the N-terminal region resulted in diminished T24 phosphorylation, loss of interaction with 14-3-3, and nuclear retention. FOXO1 mutation was associated with decreased overall survival in patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (P = .037), independent of cell of origin (COO) and the revised International Prognostic Index (R-IPI). This association was particularly evident (P = .003) in patients in the low-risk R-IPI categories. The independent relationship of mutations in FOXO1 to survival, transcending the prognostic influence of the R-IPI and COO, indicates that FOXO1 mutation is a novel prognostic factor that plays an important role in DLBCL pathogenesis.
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Factores de Transcripción Forkhead/genética , Linfoma de Células B Grandes Difuso/genética , Mutación , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Análisis Mutacional de ADN , Femenino , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/fisiología , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Linfoma de Células B Grandes Difuso/diagnóstico , Linfoma de Células B Grandes Difuso/patología , Masculino , Persona de Mediana Edad , Modelos Moleculares , Mutación/fisiología , Pronóstico , Adulto JovenRESUMEN
Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous cancer composed of at least 2 molecular subtypes that differ in gene expression and distribution of mutations. Recently, application of genome/exome sequencing and RNA-seq to DLBCL has revealed numerous genes that are recurrent targets of somatic point mutation in this disease. Here we provide a whole-genome-sequencing-based perspective of DLBCL mutational complexity by characterizing 40 de novo DLBCL cases and 13 DLBCL cell lines and combining these data with DNA copy number analysis and RNA-seq from an extended cohort of 96 cases. Our analysis identified widespread genomic rearrangements including evidence for chromothripsis as well as the presence of known and novel fusion transcripts. We uncovered new gene targets of recurrent somatic point mutations and genes that are targeted by focal somatic deletions in this disease. We highlight the recurrence of germinal center B-cell-restricted mutations affecting genes that encode the S1P receptor and 2 small GTPases (GNA13 and GNAI2) that together converge on regulation of B-cell homing. We further analyzed our data to approximate the relative temporal order in which some recurrent mutations were acquired and demonstrate that ongoing acquisition of mutations and intratumoral clonal heterogeneity are common features of DLBCL. This study further improves our understanding of the processes and pathways involved in lymphomagenesis, and some of the pathways mutated here may indicate new avenues for therapeutic intervention.
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Biomarcadores de Tumor/química , Biomarcadores de Tumor/genética , Variaciones en el Número de Copia de ADN/genética , Genoma Humano , Linfoma de Células B Grandes Difuso/genética , Mutación/genética , Subunidades alfa de la Proteína de Unión al GTP G12-G13/química , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Pediatric acute myeloid leukemia (pAML) is characterized by heterogeneous cellular composition, driver alterations and prognosis. Characterization of this heterogeneity and how it affects treatment response remains understudied in pediatric patients. We used single-cell RNA sequencing and single-cell ATAC sequencing to profile 28 patients representing different pAML subtypes at diagnosis, remission and relapse. At diagnosis, cellular composition differed between genetic subgroups. Upon relapse, cellular hierarchies transitioned toward a more primitive state regardless of subtype. Primitive cells in the relapsed tumor were distinct compared to cells at diagnosis, with under-representation of myeloid transcriptional programs and over-representation of other lineage programs. In some patients, this was accompanied by the appearance of a B-lymphoid-like hierarchy. Our data thus reveal the emergence of apparent subtype-specific plasticity upon treatment and inform on potentially targetable processes.
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Leucemia Mieloide Aguda , Humanos , Niño , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Pronóstico , RecurrenciaRESUMEN
CIC encodes a transcriptional repressor and MAPK signalling effector that is inactivated by loss-of-function mutations in several cancer types, consistent with a role as a tumour suppressor. Here, we used bioinformatic, genomic, and proteomic approaches to investigate CIC's interaction networks. We observed both previously identified and novel candidate interactions between CIC and SWI/SNF complex members, as well as novel interactions between CIC and cell cycle regulators and RNA processing factors. We found that CIC loss is associated with an increased frequency of mitotic defects in human cell lines and an in vivo mouse model and with dysregulated expression of mitotic regulators. We also observed aberrant splicing in CIC-deficient cell lines, predominantly at 3' and 5' untranslated regions of genes, including genes involved in MAPK signalling, DNA repair, and cell cycle regulation. Our study thus characterises the complexity of CIC's functional network and describes the effect of its loss on cell cycle regulation, mitotic integrity, and transcriptional splicing, thereby expanding our understanding of CIC's potential roles in cancer. In addition, our work exemplifies how multi-omic, network-based analyses can be used to uncover novel insights into the interconnected functions of pleiotropic genes/proteins across cellular contexts.
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Glioblastomas (GBMs) are aggressive brain tumors characterized by extensive inter- and intratumor heterogeneity. Patient-derived models, such as organoids and explants, have recently emerged as useful models to study such heterogeneity, although the extent to which they can recapitulate GBM genomic features remains unclear. Here, we analyze bulk exome and single-cell genome and transcriptome profiles of 12 IDH wild-type GBMs, including two recurrent tumors, and of patient-derived explants (PDEs) and gliomasphere (GS) lines derived from these tumors. We find that PDEs are genetically similar to, and variably retain gene expression characteristics of, their parent tumors. Notably, PDEs appear to exhibit similar levels of transcriptional heterogeneity compared with their parent tumors, whereas GS lines tend to be enriched for cells in a more uniform transcriptional state. The approaches and datasets introduced here will provide a valuable resource to help guide experiments using GBM-derived models, especially in the context of studying cellular heterogeneity.
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Neoplasias Encefálicas , Glioblastoma , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular , Genómica , Glioblastoma/genética , Glioblastoma/patología , Humanos , Recurrencia Local de NeoplasiaRESUMEN
RNA sequencing (RNAseq) has been widely used to generate bulk gene expression measurements collected from pools of cells. Only relatively recently have single-cell RNAseq (scRNAseq) methods provided opportunities for gene expression analyses at the single-cell level, allowing researchers to study heterogeneous mixtures of cells at unprecedented resolution. Tumors tend to be composed of heterogeneous cellular mixtures and are frequently the subjects of such analyses. Extensive method developments have led to several protocols for scRNAseq but, owing to the small amounts of RNA in single cells, technical constraints have required compromises. For example, the majority of scRNAseq methods are limited to sequencing only the 3' or 5' termini of transcripts. Other protocols that facilitate full-length transcript profiling tend to capture only polyadenylated mRNAs and are generally limited to processing only 96 cells at a time. Here, we address these limitations and present a novel protocol that allows for the high-throughput sequencing of full-length, total RNA at single-cell resolution. We demonstrate that our method produced strand-specific sequencing data for both polyadenylated and non-polyadenylated transcripts, enabled the profiling of transcript regions beyond only transcript termini, and yielded data rich enough to allow identification of cell types from heterogeneous biological samples.
RESUMEN
Purpose Children with acute myeloid leukemia (AML) whose disease is refractory to standard induction chemotherapy therapy or who experience relapse after initial response have dismal outcomes. We sought to comprehensively profile pediatric AML microRNA (miRNA) samples to identify dysregulated genes and assess the utility of miRNAs for improved outcome prediction. Patients and Methods To identify miRNA biomarkers that are associated with treatment failure, we performed a comprehensive sequence-based characterization of the pediatric AML miRNA landscape. miRNA sequencing was performed on 1,362 samples-1,303 primary, 22 refractory, and 37 relapse samples. One hundred sixty-four matched samples-127 primary and 37 relapse samples-were analyzed by using RNA sequencing. Results By using penalized lasso Cox proportional hazards regression, we identified 36 miRNAs the expression levels at diagnosis of which were highly associated with event-free survival. Combined expression of the 36 miRNAs was used to create a novel miRNA-based risk classification scheme (AMLmiR36). This new miRNA-based risk classifier identifies those patients who are at high risk (hazard ratio, 2.830; P ≤ .001) or low risk (hazard ratio, 0.323; P ≤ .001) of experiencing treatment failure, independent of conventional karyotype or mutation status. The performance of AMLmiR36 was independently assessed by using 878 patients from two different clinical trials (AAML0531 and AAML1031). Our analysis also revealed that miR-106a-363 was abundantly expressed in relapse and refractory samples, and several candidate targets of miR-106a-5p were involved in oxidative phosphorylation, a process that is suppressed in treatment-resistant leukemic cells. Conclusion To assess the utility of miRNAs for outcome prediction in patients with pediatric AML, we designed and validated a miRNA-based risk classification scheme. We also hypothesized that the abundant expression of miR-106a could increase treatment resistance via modulation of genes that are involved in oxidative phosphorylation.
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Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/genética , MicroARNs/genética , Biomarcadores de Tumor/genética , Niño , Supervivencia sin Enfermedad , Femenino , Perfilación de la Expresión Génica , Pruebas Genéticas , Humanos , Masculino , MicroARNs/biosíntesis , ARN Mensajero/genéticaRESUMEN
BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is an aggressive disease, with 30% to 40% of patients failing to be cured with available primary therapy. microRNAs (miRNAs) are RNA molecules that attenuate expression of their mRNA targets. To characterize the DLBCL miRNome, we sequenced miRNAs from 92 DLBCL and 15 benign centroblast fresh frozen samples and from 140 DLBCL formalin-fixed, paraffin-embedded tissue samples for validation. RESULTS: We identify known and candidate novel miRNAs, 25 of which are associated with survival independently of cell-of-origin and International Prognostic Index scores, which are established indicators of outcome. Of these 25 miRNAs, six miRNAs are significantly associated with survival in our validation cohort. Abundant expression of miR-28-5p, miR-214-5p, miR-339-3p, and miR-5586-5p is associated with superior outcome, while abundant expression of miR-324-5p and NOVELM00203M is associated with inferior outcome. Comparison of DLBCL miRNA-seq expression profiles with those from other cancer types identifies miRNAs that were more abundant in B-cell contexts. Unsupervised clustering of miRNAs identifies two clusters of patients that have distinct differences in their outcomes. Our integrative miRNA and mRNA expression analyses reveal that miRNAs increased in abundance in DLBCL appear to regulate the expression of genes involved in metabolism, cell cycle, and protein modification. Additionally, these miRNAs, including one candidate novel miRNA, miR-10393-3p, appear to target chromatin modification genes that are frequent targets of somatic mutation in non-Hodgkin lymphomas. CONCLUSIONS: Our comprehensive sequence analysis of the DLBCL miRNome identifies candidate novel miRNAs and miRNAs associated with survival, reinforces results from previous mutational analyses, and reveals regulatory networks of significance for lymphomagenesis.
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Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/mortalidad , MicroARNs/genética , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Análisis por Conglomerados , Ciclofosfamida/uso terapéutico , Doxorrubicina/uso terapéutico , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , MicroARNs/química , Prednisona/uso terapéutico , Pronóstico , Interferencia de ARN , ARN Mensajero/genética , Rituximab , Transcriptoma , Vincristina/uso terapéuticoRESUMEN
The p53 tumor suppressor induces apoptosis in response to genotoxic and environmental stresses. Separately from its functions as a transcription factor, it is also capable to be translocated to the mitochondria and plays a critical role in transcription-independent mitochondrial apoptosis. We previously demonstrated that Tid1 interacts with p53, resulting in mitochondrial translocation of the complex and induction of intrinsic apoptosis [1]; however, the mechanism how they interact has been unknown. In this study, far western analyses demonstrated that Tid1 directly interacted with p53. Using domain deletion mutant constructs, we determined that DnaJ domain of Tid1 was necessary for the interaction, while either N- or C-terminal domains of p53 were sufficient for the interaction. In breast cancer cells, depletion of Tid1 by short hairpin RNA (shRNA) led to absence of p53 accumulation at mitochondria and resistance to apoptosis under hypoxic or genotoxic stresses. Our studies imply that Tid1 could be important in the potential combination chemotherapies of p53-related cancers.
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Apoptosis , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas del Choque Térmico HSP40/metabolismo , Mitocondrias/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Western Blotting , Neoplasias de la Mama/genética , Femenino , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/genética , Humanos , Inmunoprecipitación , Transporte de Proteínas , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Fracciones Subcelulares , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Tamoxifen resistance is one of the overarching challenges in the treatment of patients with estrogen receptor (ER)-positive breast cancer. Through a genome-wide RNA interference screen to discover genes responsible for tamoxifen resistance in vitro, we identified insulin-like growth factor binding protein 5 (IGFBP5) as a determinant of drug sensitivity. Specific knockdown of IGFBP5 by retroviral infection with short hairpin RNA-expressing cassette in MCF7 human breast cancer cells (pRS-shIGFBP5) conferred tamoxifen resistance in vitro due to concomitant loss of ERalpha expression and signaling. IGFBP5 expression was also reduced in MCF7 cells selected for tamoxifen resistance in culture (TAMR). Both tamoxifen-resistant MCF7-TAMR and MCF7-pRS-shIGFBP5 cells could be resensitized to drug by treatment with exogenous recombinant IGFBP5 (rIGFBP5) protein. Treatment with rIGFBP5 protein in mouse tumor xenografts reversed the in vivo tamoxifen resistance of MCF7-pRS-shIGFBP5 cell-derived tumors by reducing tumor cell proliferation. IGFBP5 immunohistochemical staining in a cohort of 153 breast cancer patients showed that low IGFBP5 expression was associated with shorter overall survival after tamoxifen therapy. Thus, IGFBP5 warrants investigation as an agent to reverse tamoxifen resistance.