RESUMEN
Glioblastoma (GBM) is a deadly cancer in which cancer stem cells (CSCs) sustain tumor growth and contribute to therapeutic resistance. Protein arginine methyltransferase 5 (PRMT5) has recently emerged as a promising target in GBM. Using two orthogonal-acting inhibitors of PRMT5 (GSK591 or LLY-283), we show that pharmacological inhibition of PRMT5 suppresses the growth of a cohort of 46 patient-derived GBM stem cell cultures, with the proneural subtype showing greater sensitivity. We show that PRMT5 inhibition causes widespread disruption of splicing across the transcriptome, particularly affecting cell cycle gene products. We identify a GBM splicing signature that correlates with the degree of response to PRMT5 inhibition. Importantly, we demonstrate that LLY-283 is brain-penetrant and significantly prolongs the survival of mice with orthotopic patient-derived xenografts. Collectively, our findings provide a rationale for the clinical development of brain penetrant PRMT5 inhibitors as treatment for GBM.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Animales , Apoptosis , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Descubrimiento de Drogas , Epigenómica , Femenino , Regulación Neoplásica de la Expresión Génica , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/patología , Humanos , Ratones , Células Madre Neoplásicas/metabolismo , Proteína-Arginina N-Metiltransferasas/efectos de los fármacos , Proteína-Arginina N-Metiltransferasas/genética , Empalme del ARN , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chromatin accessibility discriminates stem from mature cell populations, enabling the identification of primitive stem-like cells in primary tumors, such as glioblastoma (GBM) where self-renewing cells driving cancer progression and recurrence are prime targets for therapeutic intervention. We show, using single-cell chromatin accessibility, that primary human GBMs harbor a heterogeneous self-renewing population whose diversity is captured in patient-derived glioblastoma stem cells (GSCs). In-depth characterization of chromatin accessibility in GSCs identifies three GSC states: Reactive, Constructive, and Invasive, each governed by uniquely essential transcription factors and present within GBMs in varying proportions. Orthotopic xenografts reveal that GSC states associate with survival, and identify an invasive GSC signature predictive of low patient survival, in line with the higher invasive properties of Invasive state GSCs compared to Reactive and Constructive GSCs as shown by in vitro and in vivo assays. Our chromatin-driven characterization of GSC states improves prognostic precision and identifies dependencies to guide combination therapies.
Asunto(s)
Autorrenovación de las Células , Cromatina/metabolismo , Glioblastoma/secundario , Células Madre Neoplásicas/fisiología , Línea Celular Tumoral , Femenino , Humanos , Masculino , Análisis de la Célula IndividualRESUMEN
We investigated the role of 3D genome architecture in instructing functional properties of glioblastoma stem cells (GSCs) by generating sub-5-kb resolution 3D genome maps by in situ Hi-C. Contact maps at sub-5-kb resolution allow identification of individual DNA loops, domain organization, and large-scale genome compartmentalization. We observed differences in looping architectures among GSCs from different patients, suggesting that 3D genome architecture is a further layer of inter-patient heterogeneity for glioblastoma. Integration of DNA contact maps with chromatin and transcriptional profiles identified specific mechanisms of gene regulation, including the convergence of multiple super enhancers to individual stemness genes within individual cells. We show that the number of loops contacting a gene correlates with elevated transcription. These results indicate that stemness genes are hubs of interaction between multiple regulatory regions, likely to ensure their sustained expression. Regions of open chromatin common among the GSCs tested were poised for expression of immune-related genes, including CD276 We demonstrate that this gene is co-expressed with stemness genes in GSCs and that CD276 can be targeted with an antibody-drug conjugate to eliminate self-renewing cells. Our results demonstrate that integrated structural genomics data sets can be employed to rationally identify therapeutic vulnerabilities in self-renewing cells.
Asunto(s)
Neoplasias Encefálicas/genética , Cromatina/ultraestructura , Mapeo Cromosómico/métodos , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Proteínas de Neoplasias/genética , Antígenos B7/antagonistas & inhibidores , Antígenos B7/genética , Antígenos B7/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Proliferación Celular , Cromatina/química , Elementos de Facilitación Genéticos , Perfilación de la Expresión Génica , Heterogeneidad Genética , Genoma Humano , Genómica/métodos , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Terapia Molecular Dirigida , Proteínas de Neoplasias/clasificación , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transcripción GenéticaRESUMEN
Glioblastoma therapies have remained elusive due to limitations in understanding mechanisms of growth and survival of the tumorigenic population. Using CRISPR-Cas9 approaches in patient-derived GBM stem cells (GSCs) to interrogate function of the coding genome, we identify actionable pathways responsible for growth, which reveal the gene-essential circuitry of GBM stemness and proliferation. In particular, we characterize members of the SOX transcription factor family, SOCS3, USP8, and DOT1L, and protein ufmylation as important for GSC growth. Additionally, we reveal mechanisms of temozolomide resistance that could lead to combination strategies. By reaching beyond static genome analysis of bulk tumors, with a genome-wide functional approach, we reveal genetic dependencies within a broad range of biological processes to provide increased understanding of GBM growth and treatment resistance.
Asunto(s)
Neoplasias Encefálicas/patología , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Glioblastoma/patología , Células Madre Neoplásicas/metabolismo , Temozolomida/farmacología , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/mortalidad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Endopeptidasas/genética , Endopeptidasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Biblioteca de Genes , Glioblastoma/tratamiento farmacológico , Glioblastoma/mortalidad , Histona Metiltransferasas/metabolismo , Humanos , Ratones , Ratones SCID , Células Madre Neoplásicas/efectos de los fármacos , Proteína 3 Supresora de la Señalización de Citocinas/genética , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Análisis de Supervivencia , Temozolomida/uso terapéutico , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismoRESUMEN
Pediatric glioblastoma (pGBM) is a lethal cancer with no effective therapies. To understand the mechanisms of tumor evolution in this cancer, we performed whole-genome sequencing with linked reads on longitudinally resected pGBM samples. Our analyses showed that all diagnostic and recurrent samples were collections of genetically diverse subclones. Clonal composition rapidly evolved at recurrence, with less than 8% of nonsynonymous single-nucleotide variants being shared in diagnostic-recurrent pairs. To track the origins of the mutational events observed in pGBM, we generated whole-genome datasets for two patients and their parents. These trios showed that genetic variants could be (i) somatic, (ii) inherited from a healthy parent, or (iii) de novo in the germlines of pGBM patients. Analysis of variant allele frequencies supported a model of tumor growth involving slow-cycling cancer stem cells that give rise to fast-proliferating progenitor-like cells and to nondividing cells. Interestingly, radiation and antimitotic chemotherapeutics did not increase overall tumor burden upon recurrence. These findings support an important role for slow-cycling stem cell populations in contributing to recurrences, because slow-cycling cell populations are expected to be less prone to genotoxic stress induced by these treatments and therefore would accumulate few mutations. Our results highlight the need for new targeted treatments that account for the complex functional hierarchies and genomic heterogeneity of pGBM. SIGNIFICANCE: This work challenges several assumptions regarding the genetic organization of pediatric GBM and highlights mutagenic programs that start during early prenatal development.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/9/2111/F1.large.jpg.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Glioblastoma/genética , Mutación , Recurrencia Local de Neoplasia/genética , Células Madre Neoplásicas/metabolismo , Animales , Neoplasias Encefálicas/patología , Niño , Perfilación de la Expresión Génica , Glioblastoma/patología , Humanos , Estudios Longitudinales , Ratones , Recurrencia Local de Neoplasia/patología , Células Madre Neoplásicas/patología , Células Tumorales Cultivadas , Secuenciación Completa del Genoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Alteration of tissue mechanical properties is a physical hallmark of solid tumors including gliomas. How tumor cells sense and regulate tissue mechanics is largely unknown. Here, we show that mechanosensitive ion channel Piezo regulates mitosis and tissue stiffness of Drosophila gliomas, but not non-transformed brains. PIEZO1 is overexpressed in aggressive human gliomas and its expression inversely correlates with patient survival. Deleting PIEZO1 suppresses the growth of glioblastoma stem cells, inhibits tumor development, and prolongs mouse survival. Focal mechanical force activates prominent PIEZO1-dependent currents from glioma cell processes, but not soma. PIEZO1 localizes at focal adhesions to activate integrin-FAK signaling, regulate extracellular matrix, and reinforce tissue stiffening. In turn, a stiffer mechanical microenvironment elevates PIEZO1 expression to promote glioma aggression. Therefore, glioma cells are mechanosensory in a PIEZO1-dependent manner, and targeting PIEZO1 represents a strategy to break the reciprocal, disease-aggravating feedforward circuit between tumor cell mechanotransduction and the aberrant tissue mechanics. VIDEO ABSTRACT.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Canales Iónicos/biosíntesis , Mecanotransducción Celular/fisiología , Adulto , Anciano , Animales , Animales Modificados Genéticamente , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Drosophila melanogaster , Femenino , Glioma/genética , Glioma/patología , Humanos , Canales Iónicos/genética , Masculino , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Microambiente Tumoral/fisiología , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare 'outlier' clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Rastreo Celular , Glioblastoma/patología , Células Madre Neoplásicas/patología , Animales , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Proliferación Celular , Células Clonales/efectos de los fármacos , Células Clonales/patología , Epigénesis Genética , Femenino , Glioblastoma/tratamiento farmacológico , Xenoinjertos , Humanos , Ratones , Invasividad Neoplásica , Trasplante de Neoplasias , Células Madre Neoplásicas/efectos de los fármacos , Fenotipo , Procesos EstocásticosRESUMEN
Mutations in the histone 3 variant H3.3 have been identified in one-third of pediatric glioblastomas (GBMs), but not in adult tumors. Here we show that H3.3 is a dynamic determinant of functional properties in adult GBM. H3.3 is repressed by mixed lineage leukemia 5 (MLL5) in self-renewing GBM cells. MLL5 is a global epigenetic repressor that orchestrates reorganization of chromatin structure by punctuating chromosomes with foci of compacted chromatin, favoring tumorigenic and self-renewing properties. Conversely, H3.3 antagonizes self-renewal and promotes differentiation. We exploited these epigenetic states to rationally identify two small molecules that effectively curb cancer stem cell properties in a preclinical model. Our work uncovers a role for MLL5 and H3.3 in maintaining self-renewal hierarchies in adult GBM.