Dasatinib overcomes glucocorticoid resistance in B-cell acute lymphoblastic leukemia.

Resistance to glucocorticoids (GC) is associated with an increased risk of relapse in B-cell progenitor acute lymphoblastic leukemia (BCP-ALL). Performing transcriptomic and single-cell proteomic studies in healthy B-cell progenitors, we herein identify coordination between the glucocorticoid receptor pathway with B-cell developmental pathways. Healthy pro-B cells most highly express the glucocorticoid receptor, and this developmental expression is conserved in primary BCP-ALL cells from patients at diagnosis and relapse. In-vitro and in vivo glucocorticoid treatment of primary BCP-ALL cells demonstrate that the interplay between B-cell development and the glucocorticoid pathways is crucial for GC resistance in leukemic cells. Gene set enrichment analysis in BCP-ALL cell lines surviving GC treatment show enrichment of B cell receptor signaling pathways. In addition, primary BCP-ALL cells surviving GC treatment in vitro and in vivo demonstrate a late pre-B cell phenotype with activation of PI3K/mTOR and CREB signaling. Dasatinib, a multi-kinase inhibitor, most effectively targets this active signaling in GC-resistant cells, and when combined with glucocorticoids, results in increased cell death in vitro and decreased leukemic burden and prolonged survival in an in vivo xenograft model. Targeting the active signaling through the addition of dasatinib may represent a therapeutic approach to overcome GC resistance in BCP-ALL.

Subcellular localization of biomolecules and drug distribution by high-definition ion beam imaging.

Simultaneous visualization of the relationship between multiple biomolecules and their ligands or small molecules at the nanometer scale in cells will enable greater understanding of how biological processes operate. We present here high-definition multiplex ion beam imaging (HD-MIBI), a secondary ion mass spectrometry approach capable of high-parameter imaging in 3D of targeted biological entities and exogenously added structurally-unmodified small molecules. With this technology, the atomic constituents of the biomolecules themselves can be used in our system as the "tag" and we demonstrate measurements down to ~30 nm lateral resolution. We correlated the subcellular localization of the chemotherapy drug cisplatin simultaneously with five subnuclear structures. Cisplatin was preferentially enriched in nuclear speckles and excluded from closed-chromatin regions, indicative of a role for cisplatin in active regions of chromatin. Unexpectedly, cells surviving multi-drug treatment with cisplatin and the BET inhibitor JQ1 demonstrated near total cisplatin exclusion from the nucleus, suggesting that selective subcellular drug relocalization may modulate resistance to this important chemotherapeutic treatment. Multiplexed high-resolution imaging techniques, such as HD-MIBI, will enable studies of biomolecules and drug distributions in biologically relevant subcellular microenvironments by visualizing the processes themselves in concert, rather than inferring mechanism through surrogate analyses.

Author Correction: Ultra-high throughput single-cell analysis of proteins and RNAs by split-pool synthesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ultra-high throughput single-cell analysis of proteins and RNAs by split-pool synthesis.

Single-cell omics provide insight into cellular heterogeneity and function. Recent technological advances have accelerated single-cell analyses, but workflows remain expensive and complex. We present a method enabling simultaneous, ultra-high throughput single-cell barcoding of millions of cells for targeted analysis of proteins and RNAs. Quantum barcoding (QBC) avoids isolation of single cells by building cell-specific oligo barcodes dynamically within each cell. With minimal instrumentation (four 96-well plates and a multichannel pipette), cell-specific codes are added to each tagged molecule within cells through sequential rounds of classical split-pool synthesis. Here we show the utility of this technology in mouse and human model systems for as many as 50 antibodies to targeted proteins and, separately, >70 targeted RNA regions. We demonstrate that this method can be applied to multi-modal protein and RNA analyses. It can be scaled by expansion of the split-pool process and effectively renders sequencing instruments as versatile multi-parameter flow cytometers.

Three-dimensional intact-tissue sequencing of single-cell transcriptional states.

Retrieving high-content gene-expression information while retaining three-dimensional (3D) positional anatomy at cellular resolution has been difficult, limiting integrative understanding of structure and function in complex biological tissues. We developed and applied a technology for 3D intact-tissue RNA sequencing, termed STARmap (spatially-resolved transcript amplicon readout mapping), which integrates hydrogel-tissue chemistry, targeted signal amplification, and in situ sequencing. The capabilities of STARmap were tested by mapping 160 to 1020 genes simultaneously in sections of mouse brain at single-cell resolution with high efficiency, accuracy, and reproducibility. Moving to thick tissue blocks, we observed a molecularly defined gradient distribution of excitatory-neuron subtypes across cubic millimeter-scale volumes (>30,000 cells) and a short-range 3D self-clustering in many inhibitory-neuron subtypes that could be identified and described with 3D STARmap.

Highly multiplexed simultaneous detection of RNAs and proteins in single cells.

To enable the detection of expression signatures specific to individual cells, we developed PLAYR (proximity ligation assay for RNA), a method for highly multiplexed transcript quantification by flow and mass cytometry that is compatible with standard antibody staining. When used with mass cytometry, PLAYR allowed for the simultaneous quantification of more than 40 different mRNAs and proteins. In primary cells, we quantified multiple transcripts, with the identity and functional state of each analyzed cell defined on the basis of the expression of a separate set of transcripts or proteins. By expanding high-throughput deep phenotyping of cells beyond protein epitopes to include RNA expression, PLAYR opens a new avenue for the characterization of cellular metabolism.

Sequential Functions of CPEB1 and CPEB4 Regulate Pathologic Expression of Vascular Endothelial Growth Factor and Angiogenesis in Chronic Liver Disease.

BACKGROUND & AIMS: Vascular endothelial growth factor (VEGF) regulates angiogenesis, yet therapeutic strategies to disrupt VEGF signaling can interfere with physiologic angiogenesis. In a search for ways to inhibit pathologic production or activities of VEGF without affecting its normal production or functions, we investigated the post-transcriptional regulation of VEGF by the cytoplasmic polyadenylation element-binding proteins CPEB1 and CPEB4 during development of portal hypertension and liver disease. METHODS: We obtained transjugular liver biopsies from patients with hepatitis C virus-associated cirrhosis or liver tissues removed during transplantation; healthy human liver tissue was obtained from a commercial source (control). We also performed experiments with male Sprague-Dawley rats and CPEB-deficient mice (C57BL6 or mixed C57BL6/129 background) and their wild-type littermates. Secondary biliary cirrhosis was induced in rats by bile duct ligation, and portal hypertension was induced by partial portal vein ligation. Liver and mesenteric tissues were collected and analyzed in angiogenesis, reverse transcription polymerase chain reaction, polyA tail, 3' rapid amplification of complementary DNA ends, Southern blot, immunoblot, histologic, immunohistochemical, immunofluorescence, and confocal microscopy assays. CPEB was knocked down with small interfering RNAs in H5V endothelial cells, and translation of luciferase reporters constructs was assessed. RESULTS: Activation of CPEB1 promoted alternative nuclear processing within noncoding 3'-untranslated regions of VEGF and CPEB4 messenger RNAs in H5V cells, resulting in deletion of translation repressor elements. The subsequent overexpression of CPEB4 promoted cytoplasmic polyadenylation of VEGF messenger RNA, increasing its translation; the high levels of VEGF produced by these cells led to their formation of tubular structures in Matrigel assays. We observed increased levels of CPEB1 and CPEB4 in cirrhotic liver tissues from patients, compared with control tissue, as well as in livers and mesenteries of rats and mice with cirrhosis or/and portal hypertension. Mice with knockdown of CPEB1 or CPEB4 did not overexpress VEGF or have signs of mesenteric neovascularization, and developed less-severe forms of portal hypertension after portal vein ligation. CONCLUSIONS: We identified a mechanism of VEGF overexpression in liver and mesentery that promotes pathologic, but not physiologic, angiogenesis, via sequential and nonredundant functions of CPEB1 and CPEB4. Regulation of CPEB4 by CPEB1 and the CPEB4 autoamplification loop induces pathologic angiogenesis. Strategies to block the activities of CPEBs might be developed to treat chronic liver and other angiogenesis-dependent diseases.

Global Analysis of CPEBs Reveals Sequential and Non-Redundant Functions in Mitotic Cell Cycle.

CPEB (Cytoplasmic Polyadenylation Element Binding) proteins are a family of four RNA-binding proteins that regulate the translation of maternal mRNAs controlling meiotic cell cycle progression. But CPEBs are not limited to the transcriptionally silent germline; they are also expressed, in various combinations, in somatic cells, yet their role in regulation of mitosis-related gene expression is largely unknown. Deregulation of CPEB1 and CPEB4 have been linked to tumor development. However, a systematic analysis addressing their requirements for the temporal regulation of mitotic gene expression has yet to be performed. This study addresses the requirements of each of the four CPEBs for mitotic phase transitions, with a particular focus on cytoplasmic polyadenylation and translational regulation. We demonstrate that CPEB3 is the only member dispensable for mitotic cell division, whereas the other three members, CPEB1, 2, and 4, are essential to successful mitotic cell division. Thus, CPEB1 is required for prophase entry, CPEB2 for metaphase and CPEB4 for cytokinesis. These three CPEBs have sequential non-redundant functions that promote the phase-specific polyadenylation and translational activation of CPE-regulated transcripts in the mitotic cell cycle.

CPEB1 coordinates alternative 3'-UTR formation with translational regulation.

More than half of mammalian genes generate multiple messenger RNA isoforms that differ in their 3' untranslated regions (3' UTRs) and therefore in regulatory sequences, often associated with cell proliferation and cancer; however, the mechanisms coordinating alternative 3'-UTR processing for specific mRNA populations remain poorly defined. Here we report that the cytoplasmic polyadenylation element binding protein 1 (CPEB1), an RNA-binding protein that regulates mRNA translation, also controls alternative 3'-UTR processing. CPEB1 shuttles to the nucleus, where it co-localizes with splicing factors and mediates shortening of hundreds of mRNA 3' UTRs, thereby modulating their translation efficiency in the cytoplasm. CPEB1-mediated 3'-UTR shortening correlates with cell proliferation and tumorigenesis. CPEB1 binding to pre-mRNAs not only directs the use of alternative polyadenylation sites, but also changes alternative splicing by preventing U2AF65 recruitment. Our results reveal a novel function of CPEB1 in mediating alternative 3'-UTR processing, which is coordinated with regulation of mRNA translation, through its dual nuclear and cytoplasmic functions.

Translational control by changes in poly(A) tail length: recycling mRNAs.

Beyond the well-known function of poly(A) tail length in mRNA stability, recent years have witnessed an explosion of information about how changes in tail length and the selection of alternative polyadenylation sites contribute to the translational regulation of a large portion of the genome. The mechanisms and factors mediating nuclear and cytoplasmic changes in poly(A) tail length have been studied in great detail, the targets of these mechanisms have been identified--in some cases by genome-wide screenings--and changes in poly(A) tail length are now implicated in a number of physiological and pathological processes. However, in very few cases have all three levels--mechanisms, targets and functions--been studied together.