RESUMO
Tumors may contain billions of cells, including distinct malignant clones and nonmalignant cell types. Clarifying the evolutionary histories, prevalence, and defining molecular features of these cells is essential for improving clinical outcomes, since intratumoral heterogeneity provides fuel for acquired resistance to targeted therapies. Here we present a statistically motivated strategy for deconstructing intratumoral heterogeneity through multiomic and multiscale analysis of serial tumor sections (MOMA). By combining deep sampling of IDH-mutant astrocytomas with integrative analysis of single-nucleotide variants, copy-number variants, and gene expression, we reconstruct and validate the phylogenies, spatial distributions, and transcriptional profiles of distinct malignant clones. By genotyping nuclei analyzed by single-nucleus RNA-seq for truncal mutations, we further show that commonly used algorithms for identifying cancer cells from single-cell transcriptomes may be inaccurate. We also demonstrate that correlating gene expression with tumor purity in bulk samples can reveal optimal markers of malignant cells and use this approach to identify a core set of genes that are consistently expressed by astrocytoma truncal clones, including AKR1C3, whose expression is associated with poor outcomes in several types of cancer. In summary, MOMA provides a robust and flexible strategy for precisely deconstructing intratumoral heterogeneity and clarifying the core molecular properties of distinct cellular populations in solid tumors.
RESUMO
Treatment failure for the lethal brain tumor glioblastoma (GBM) is attributed to intratumoral heterogeneity and tumor evolution. We utilized 3D neuronavigation during surgical resection to acquire samples representing the whole tumor mapped by 3D spatial coordinates. Integrative tissue and single-cell analysis revealed sources of genomic, epigenomic, and microenvironmental intratumoral heterogeneity and their spatial patterning. By distinguishing tumor-wide molecular features from those with regional specificity, we inferred GBM evolutionary trajectories from neurodevelopmental lineage origins and initiating events such as chromothripsis to emergence of genetic subclones and spatially restricted activation of differential tumor and microenvironmental programs in the core, periphery, and contrast-enhancing regions. Our work depicts GBM evolution and heterogeneity from a 3D whole-tumor perspective, highlights potential therapeutic targets that might circumvent heterogeneity-related failures, and establishes an interactive platform enabling 360° visualization and analysis of 3D spatial patterns for user-selected genes, programs, and other features across whole GBM tumors.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Modelos Biológicos , Humanos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Epigenômica , Genômica , Glioblastoma/genética , Glioblastoma/patologia , Análise de Célula Única , Microambiente Tumoral , Heterogeneidade GenéticaRESUMO
Tumors may contain billions of cells including distinct malignant clones and nonmalignant cell types. Clarifying the evolutionary histories, prevalence, and defining molecular features of these cells is essential for improving clinical outcomes, since intratumoral heterogeneity provides fuel for acquired resistance to targeted therapies. Here we present a statistically motivated strategy for deconstructing intratumoral heterogeneity through multiomic and multiscale analysis of serial tumor sections (MOMA). By combining deep sampling of IDH-mutant astrocytomas with integrative analysis of single-nucleotide variants, copy-number variants, and gene expression, we reconstruct and validate the phylogenies, spatial distributions, and transcriptional profiles of distinct malignant clones. By genotyping nuclei analyzed by single-nucleus RNA-seq for truncal mutations, we further show that commonly used algorithms for identifying cancer cells from single-cell transcriptomes may be inaccurate. We also demonstrate that correlating gene expression with tumor purity in bulk samples can reveal optimal markers of malignant cells and use this approach to identify a core set of genes that is consistently expressed by astrocytoma truncal clones, including AKR1C3, whose expression is associated with poor outcomes in several types of cancer. In summary, MOMA provides a robust and flexible strategy for precisely deconstructing intratumoral heterogeneity and clarifying the core molecular properties of distinct cellular populations in solid tumors.
RESUMO
Diagnosis of spinal cord injury (SCI) severity at the ultra-acute stage is of great importance for emergency clinical care of patients as well as for potential enrollment into clinical trials. The lack of a diagnostic biomarker for SCI has played a major role in the poor results of clinical trials. We analyzed global gene expression in peripheral white blood cells during the acute injury phase and identified 197 genes whose expression changed after SCI compared with healthy and trauma controls and in direct relation to SCI severity. Unsupervised coexpression network analysis identified several gene modules that predicted injury severity (AIS grades) with an overall accuracy of 72.7% and included signatures of immune cell subtypes. Specifically, for complete SCIs (AIS A), ROC analysis showed impressive specificity and sensitivity (AUC: 0.865). Similar precision was also shown for AIS D SCIs (AUC: 0.938). Our findings indicate that global transcriptomic changes in peripheral blood cells have diagnostic and potentially prognostic value for SCI severity.
Assuntos
RNA/sangue , Traumatismos da Medula Espinal/sangue , Traumatismos da Medula Espinal/diagnóstico , Estudos de Casos e Controles , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Leucócitos/metabolismo , Modelos Logísticos , RNA/genética , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologia , Transcriptoma/genéticaRESUMO
Developmental silencing of fetal globins serves as both a paradigm of spatiotemporal gene regulation and an opportunity for therapeutic intervention of ß-hemoglobinopathy. The nucleosome remodeling and deacetylase (NuRD) chromatin complex participates in γ-globin repression. We used pooled CRISPR screening to disrupt NuRD protein coding sequences comprehensively in human adult erythroid precursors. Essential for fetal hemoglobin (HbF) control is a non-redundant subcomplex of NuRD protein family paralogs, whose composition we corroborated by affinity chromatography and proximity labeling mass spectrometry proteomics. Mapping top functional guide RNAs identified key protein interfaces where in-frame alleles resulted in loss-of-function due to destabilization or altered function of subunits. We ascertained mutations of CHD4 that dissociate its requirement for cell fitness from HbF repression in both primary human erythroid precursors and transgenic mice. Finally we demonstrated that sequestering CHD4 from NuRD phenocopied these mutations. These results indicate a generalizable approach to discover protein complex features amenable to rational biochemical targeting.
Assuntos
Cromatina/genética , Células Eritroides/metabolismo , Hemoglobina Fetal/metabolismo , Regulação da Expressão Gênica , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Mutagênese , Animais , Cromatina/metabolismo , Células Eritroides/citologia , Hemoglobina Fetal/genética , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Camundongos , Camundongos Transgênicos , Domínios e Motivos de Interação entre ProteínasRESUMO
CRISPR/Cas9 pooled screening permits parallel evaluation of comprehensive guide RNA libraries to systematically perturb protein coding sequences in situ and correlate with functional readouts. For the analysis and visualization of the resulting datasets, we develop CRISPRO, a computational pipeline that maps functional scores associated with guide RNAs to genomes, transcripts, and protein coordinates and structures. No currently available tool has similar functionality. The ensuing genotype-phenotype linear and three-dimensional maps raise hypotheses about structure-function relationships at discrete protein regions. Machine learning based on CRISPRO features improves prediction of guide RNA efficacy. The CRISPRO tool is freely available at gitlab.com/bauerlab/crispro .
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Genoma , Mutagênese/genética , Fases de Leitura Aberta/genética , Linhagem Celular , Humanos , Anotação de Sequência Molecular , Estrutura Secundária de Proteína , RNA Guia de Cinetoplastídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The lack of mechanistic explanations for many genotype-phenotype associations identified by GWAS precludes thorough assessment of their impact on human health. Here, we conducted an expression quantitative trait locus (eQTL) mapping analysis in erythroblasts and found erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of red blood cells (rbc). The same SNPs were previously associated with mean corpuscular hemoglobin concentration (MCHC) and susceptibility to severe malaria infection. We showed that Atp2b4-/- mice demonstrate increased MCHC, confirming ATP2B4 as the causal gene at this GWAS locus. Using CRISPR-Cas9, we fine mapped the genetic signal to an erythroid-specific enhancer of ATP2B4. Erythroid cells with a deletion of the ATP2B4 enhancer had abnormally high intracellular calcium levels. These results illustrate the power of combined transcriptomic, epigenomic, and genome-editing approaches in characterizing noncoding regulatory elements in phenotype-relevant cells. Our study supports ATP2B4 as a potential target for modulating rbc hydration in erythroid disorders and malaria infection.
Assuntos
ATPases Transportadoras de Cálcio/genética , Eritrócitos/citologia , Predisposição Genética para Doença , Malária/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Animais , Sistemas CRISPR-Cas , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Mapeamento Cromossômico , Elementos Facilitadores Genéticos , Epigenômica , Eritroblastos/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Células HEK293 , Humanos , Malária/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Polimorfismo de Nucleotídeo Único , Locos de Características QuantitativasRESUMO
Gene editing technologies offer new options for developing novel biomedical research models and for gene and stem cell based therapies. However, applications in many species demand high efficiencies, specificity, and a thorough understanding of likely editing outcomes. To date, overall efficiencies, rates of off-targeting and degree of genetic mosaicism have not been well-characterized for most species, limiting our ability to optimize methods. As a model gene for measuring these parameters of the CRISPR/Cas9 application in a primate species (rhesus monkey), we selected the ß-hemoglobin gene (HBB), which also has high relevance to the potential application of gene editing and stem-cell technologies for treating human disease. Our data demonstrate an ability to achieve a high efficiency of gene editing in rhesus monkey zygotes, with no detected off-target effects at selected off-target loci. Considerable genetic mosaicism and variation in the fraction of embryonic cells bearing targeted alleles are observed, and the timing of editing events is revealed using a new model. The uses of Cas9-WT protein combined with optimized concentrations of sgRNAs are two likely areas for further refinement to enhance efficiency while limiting unfavorable outcomes that can be exceedingly costly for application of gene editing in primate species.
Assuntos
Hemoglobina Fetal/genética , Globinas beta/genética , Alelos , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Caspase 9/administração & dosagem , Caspase 9/genética , Feminino , Edição de Genes/métodos , Macaca mulatta , Microinjeções , Mosaicismo/embriologia , Gravidez , RNA Mensageiro/administração & dosagemRESUMO
Benzoxaboroles are effective against bacterial, fungal and protozoan pathogens. We report potent activity of the benzoxaborole AN3661 against Plasmodium falciparum laboratory-adapted strains (mean IC50 32 nM), Ugandan field isolates (mean ex vivo IC50 64 nM), and murine P. berghei and P. falciparum infections (day 4 ED90 0.34 and 0.57 mg kg-1, respectively). Multiple P. falciparum lines selected in vitro for resistance to AN3661 harboured point mutations in pfcpsf3, which encodes a homologue of mammalian cleavage and polyadenylation specificity factor subunit 3 (CPSF-73 or CPSF3). CRISPR-Cas9-mediated introduction of pfcpsf3 mutations into parental lines recapitulated AN3661 resistance. PfCPSF3 homology models placed these mutations in the active site, where AN3661 is predicted to bind. Transcripts for three trophozoite-expressed genes were lost in AN3661-treated trophozoites, which was not observed in parasites selected or engineered for AN3661 resistance. Our results identify the pre-mRNA processing factor PfCPSF3 as a promising antimalarial drug target.
Assuntos
Antimaláricos/farmacologia , Compostos de Boro/farmacologia , Fator de Especificidade de Clivagem e Poliadenilação/química , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/química , RNA Mensageiro/genética , Sequência de Aminoácidos , Animais , Antimaláricos/síntese química , Compostos de Boro/síntese química , Sistemas CRISPR-Cas , Domínio Catalítico , Fator de Especificidade de Clivagem e Poliadenilação/antagonistas & inibidores , Fator de Especificidade de Clivagem e Poliadenilação/genética , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Resistência a Medicamentos/genética , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Edição de Genes/métodos , Humanos , Malária/tratamento farmacológico , Malária/parasitologia , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Camundongos , Simulação de Acoplamento Molecular , Mutação , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/genética , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Trofozoítos/efeitos dos fármacos , Trofozoítos/genética , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/metabolismoRESUMO
Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.