Genome editing-induced t(4;11) chromosomal translocations model B cell precursor acute lymphoblastic leukemias with KMT2A-AFF1 fusion.

A t(4;11) leukemia model established from CRISPR-engineered chromosomal translocations between the KMT2A and AFF1 genes recapitulate proteomic, epigenomic, and transcriptomic features of primary patient leukemias.

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.

CytofIn enables integrated analysis of public mass cytometry datasets using generalized anchors.

The increasing use of mass cytometry for analyzing clinical samples offers the possibility to perform comparative analyses across public datasets. However, challenges in batch normalization and data integration limit the comparison of datasets not intended to be analyzed together. Here, we present a data integration strategy, CytofIn, using generalized anchors to integrate mass cytometry datasets from the public domain.

Mass Cytometry of Hematopoietic Cells.

Mass cytometry is now a well-established method that enables the measurement of 40-50 markers (generally proteins but transcripts are also possible) in single cells. Analytes are detected via antibodies tagged with heavy metal and detected by using a time-of-flight mass spectrometer. Over the past decade, mass cytometry has proven to be a valuable method for immunophenotyping hematopoietic cells with remarkable precision in both healthy and malignant scenarios.

Progressive B Cell Loss in Revertant X-SCID.

We report the case of a patient with X-linked severe combined immunodeficiency (X-SCID) who survived for over 20 years without hematopoietic stem cell transplantation (HSCT) because of a somatic reversion mutation. An important feature of this rare case included the strategy to validate the pathogenicity of a variant of the IL2RG gene when the T and B cell lineages comprised only revertant cells. We studied the X-inactivation of sorted T cells from the mother to show that the pathogenic variant was indeed the cause of his SCID.

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.

High-efficiency CRISPR induction of t(9;11) chromosomal translocations and acute leukemias in human blood stem cells.

Chromosomal rearrangements involving the mixed lineage leukemia () gene, also known as , are often observed in human leukemias and are generally associated with a poor prognosis. To model these leukemias, we applied clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce chromosomal rearrangements in human hematopoietic stem and progenitor cells purified from umbilical cord blood. Electroporation of ribonucleoprotein complexes containing chemically modified synthetic single guide RNAs and purified Cas9 protein induced translocations between chromosomes 9 and 11 [t(9;11)] at an efficiency >1%.

SRC/ABL inhibition disrupts CRLF2-driven signaling to induce cell death in B-cell acute lymphoblastic leukemia.

Children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) overexpressing the gene () have poor prognosis. CRLF2 protein overexpression leads to activated JAK/STAT signaling and trials are underway using JAK inhibitors to overcome treatment failure. Pre-clinical studies indicated limited efficacy of single JAK inhibitors, thus additional pathways must be targeted in cells.

Publisher Correction: High-resolution myogenic lineage mapping by single-cell mass cytometry.

In the version of this Article originally published, the name of author Andrew Tri Van Ho was coded wrongly, resulting in it being incorrect when exported to citation databases. This has been corrected, though no visible changes will be apparent.

Single-cell developmental classification of B cell precursor acute lymphoblastic leukemia at diagnosis reveals predictors of relapse.

Insight into the cancer cell populations that are responsible for relapsed disease is needed to improve outcomes. Here we report a single-cell-based study of B cell precursor acute lymphoblastic leukemia at diagnosis that reveals hidden developmentally dependent cell signaling states that are uniquely associated with relapse. By using mass cytometry we simultaneously quantified 35 proteins involved in B cell development in 60 primary diagnostic samples.

High-resolution myogenic lineage mapping by single-cell mass cytometry.

Muscle regeneration is a dynamic process during which cell state and identity change over time. A major roadblock has been a lack of tools to resolve a myogenic progression in vivo. Here we capitalize on a transformative technology, single-cell mass cytometry (CyTOF), to identify in vivo skeletal muscle stem cell and previously unrecognized progenitor populations that precede differentiation.

Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells that Correlate with Prognosis.

Acute myeloid leukemia (AML) manifests as phenotypically and functionally diverse cells, often within the same patient. Intratumor phenotypic and functional heterogeneity have been linked primarily by physical sorting experiments, which assume that functionally distinct subpopulations can be prospectively isolated by surface phenotypes. This assumption has proven problematic, and we therefore developed a data-driven approach.

Synthetically Modified Viral Capsids as Versatile Carriers for Use in Antibody-Based Cell Targeting.

The present study describes an efficient and reliable method for the preparation of MS2 viral capsids that are synthetically modified with antibodies using a rapid oxidative coupling strategy. The overall protocol delivers conjugates in high yields and recoveries, requires a minimal excess of antibody to achieve modification of more than 95% of capsids, and can be completed in a short period of time. Antibody-capsid conjugates targeting extracellular receptors on human breast cancer cell lines were prepared and characterized.

Normalization of mass cytometry data with bead standards.

Mass cytometry uses atomic mass spectrometry combined with isotopically pure reporter elements to currently measure as many as 40 parameters per single cell. As with any quantitative technology, there is a fundamental need for quality assurance and normalization protocols. In the case of mass cytometry, the signal variation over time due to changes in instrument performance combined with intervals between scheduled maintenance must be accounted for and then normalized.

Decoupling of tumor-initiating activity from stable immunophenotype in HoxA9-Meis1-driven AML.

Increasing evidence suggests tumors are maintained by cancer stem cells; however, their nature remains controversial. In a HoxA9-Meis1 (H9M) model of acute myeloid leukemia (AML), we found that tumor-initiating activity existed in three, immunophenotypically distinct compartments, corresponding to disparate lineages on the normal hematopoietic hierarchy--stem/progenitor cells (Lin(-)kit(+)) and committed progenitors of the myeloid (Gr1(+)kit(+)) and lymphoid lineages (Lym(+)kit(+)). These distinct tumor-initiating cells (TICs) clonally recapitulated the immunophenotypic spectrum of the original tumor in vivo (including cells with a less-differentiated immunophenotype) and shared signaling networks, such that in vivo pharmacologic targeting of conserved TIC survival pathways (DNA methyltransferase and MEK phosphorylation) significantly increased survival.