Astraea Jager

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. Using mass cytometry, we profiled surface and intracellular signaling proteins simultaneously in millions of healthy and leukemic cells. We developed PhenoGraph, which algorithmically defines phenotypes in high-dimensional single-cell data. PhenoGraph revealed that the surface phenotypes of leukemic blasts do not necessarily reflect their intracellular state. Using hematopoietic progenitors, we defined a signaling-based measure of cellular phenotype, which led to isolation of a gene expression signature that was predictive of survival in independent cohorts. This study presents new methods for large-scale analysis of single-cell heterogeneity and demonstrates their utility, yielding insights into AML pathophysiology.

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. Here, samples were mixed with polystyrene beads embedded with metal lanthanides, allowing monitoring of mass cytometry instrument performance over multiple days of data acquisition. The protocol described here includes simultaneous measurements of beads and cells on the mass cytometer, subsequent extraction of the bead‐based signature, and the application of an algorithm enabling correction of both short‐ and long‐term signal fluctuations. The variation in the intensity of the beads that remains after normalization may also be used to determine data quality. Application of the algorithm to a one‐month longitudinal analysis of a human peripheral blood sample reduced the range of median signal fluctuation from 4.9‐fold to 1.3‐fold.

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. Notably, conjugation to the capsid did not significantly perturb the binding of the antibodies, as indicated by binding affinities similar to those obtained for the parent antibodies. An array of conjugates was synthesized with various reporters on the interior surface of the capsids to be used in cell studies, including fluorescence-based flow cytometry, confocal microscopy, and mass cytometry. The results of these studies lay the foundation for further exploration of these constructs in the context of clinically relevant applications, including drug delivery and in vivo diagnostics.

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.

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 CRLF2 gene (hiCRLF2) 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 hiCRLF2 cells. To identify additional activated networks, we used single-cell mass cytometry to examine 15 BCP-ALL primary patient samples. We uncovered a coordinated signaling network downstream of CRLF2 characterized by co-activation of JAK/STAT, PI3K, and CREB pathways. This CRLF2-driven network could be more effectively disrupted by SRC/ABL inhibition than single-agent JAK or PI3K inhibition, and this could be demonstrated even in primary minimal residual disease (MRD) cells. Our study suggests SCR/ABL inhibition as effective in disrupting the cooperative functional networks present in hiCRLF2 BCP-ALL patients, supporting further investigation of this strategy in pre-clinical studies.

Glucocorticoids Exert a Dual Role in B-Cell Acute Lymphoblastic Leukemia: Apoptosis and Differentiation of Early B-Cell Populations

Differentiation of Early B-Cell Populations DTU Orbit (02/12/2018) Glucocorticoids Exert a Dual Role in B-Cell Acute Lymphoblastic Leukemia: Apoptosis and Differentiation of Early B-Cell Populations Glucocorticoids (GCs) are routinely administered for B-cell acute lymphoblastic leukemia (BCP-ALL) treatment and the response to glucocorticoids is considered a strong prognostic marker of relapse risk. Despite this, their mechanism of action and resistance are still poorly understood.

Abstract 2693: Relapse in BCP-ALL predicted by activated signaling in pro-B cell subsets

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common type of childhood cancer and is characterized by the malignant expansion of B-lymphocyte progenitors in the bone marrow (BM). Current therapy improves the relapse-free survival in children to over 80%. However, the ∼20% of patients who relapse have a poor prognosis and there are no reliable tests that predict relapse using diagnostic samples. We reasoned that aligning BCP-ALL cells according to a formalized context of normal B-lymphocyte development would reveal hidden cell states associated with relapse, and potentially expose targets to augment therapy for patients at risk. Until recently, our ability to pinpoint the identities of B-cell progenitors had been hindered by the vast cellular diversity within the BM and by the scarcity of the primary BM samples. We applied a single-cell proteomics platform termed mass cytometry by time-of-flight (CyTOF). In CyTOF, elemental mass reporter tagged antibodies probe proteins defining cellular identity and signaling within those cells. CyTOF simultaneously quantifies > 40 proteins per cell in millions of individual cells. We defined a cell-state signature for 15 developmental populations of B lymphocytes within the normal human BM. Using this signature we assigned each leukemia cell from 52 primary diagnostic samples to its closest match in B lymphopoiesis using a classifier based on Mahalanobis distance. When applied to BM samples from 4 healthy donors our classifier correctly assigned cells to the true developmental population (accuracy = 0.92, F-measure = 0.92). Using this classifier it was determined that each BCP-ALL sample contains a mix of developmental populations - with 97% of samples enriched in populations that span the pre-pro-B to pre-B transition. We identified 20 predictors (using a machine learning approach) in diagnostic samples that perfectly separate patients who will relapse from those who will not (lasso; predictive AUC = 0.83). This is superior to the NCI risk that is currently employed at clinical diagnosis. These predictors are informative and suggest that high basal activation of IL-7 signaling nodes (pSTAT5, pAKT) in pre-pro-B to pro-BII cells and poor response following pre-B-cell receptor engagement in pre-BI cells portend relapse. As such, these pathways might eventually be targeted via drug repurposing to improve outcomes and to guide therapy in the high-risk childhood BCP-ALL patients identified with our predictor signature. Such an approach to cancer cell developmental classification could be generally applicable across various investigations on understanding and preventing relapse. Citation Format: Zinaida Good, Jolanda Sarno, Astraea Jager, Nikolay Samusik, Wendy Fantl, Nima Aghaeepour, Robert Tibshirani, Sean C. Bendall, Giuseppe Gaipa, Andrea Biondi, Garry P. Nolan, Kara L. Davis. Relapse in BCP-ALL predicted by activated signaling in pro-B cell subsets. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2693.

Abstract 3307: Improved survival in AML by in vivo drug targeting of conserved regulatory pathways in phenotypically distinct tumor-initiating compartments

Increasing evidence suggests tumors are maintained by cancer stem cells, however their nature remains controversial. In a HoxA9-Meis1 (H9M) driven 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 + ). Each compartment clonally recapitulated the original range of tumor cell immunophenotypes in vivo, including cells with a less-differentiated immunophenotype. These distinct populations largely shared signaling networks, and in vivo pharmacologic targeting of shared pathways (DNA methyltransferase and MEK phosphorylation) significantly increased survival. Collectively, these data show that H9M AML is organized as an atypical hierarchy that defies the strict lineage marker boundaries and unidirectional differentiation of normal hematopoiesis. Moreover, in some malignancies, tumor-initiation ability (or “cancer-stemness”) can represent a targetable, cellular state that can exithat exists independently of distinct immunophenotypic definition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3307. doi:1538-7445.AM2012-3307