Single-cell mass cytometry reveals intracellular survival/proliferative signaling in FLT3-ITD-mutated AML stem/progenitor cells

Cytometry A. 2015 Apr;87(4):346-56. doi: 10.1002/cyto.a.22628. Epub 2015 Jan 16.

Abstract

Understanding the unique phenotypes and complex signaling pathways of leukemia stem cells (LSCs) will provide insights and druggable targets that can be used to eradicate acute myeloid leukemia (AML). Current work on AML LSCs is limited by the number of parameters that conventional flow cytometry (FCM) can analyze because of cell autofluorescence and fluorescent dye spectral overlap. Single-cell mass cytometry (CyTOF) substitutes rare earth elements for fluorophores to label antibodies, which allows measurements of up to 120 parameters in single cells without correction for spectral overlap. The aim of this study was the evaluation of intracellular signaling in antigen-defined stem/progenitor cell subsets in primary AML. CyTOF and conventional FCM yielded comparable results on LSC phenotypes defined by CD45, CD34, CD38, CD123, and CD99. Intracellular phosphoprotein responses to ex vivo cell signaling inhibitors and cytokine stimulation were assessed in myeloid leukemia cell lines and one primary AML sample. CyTOF and conventional FCM results were confirmed by western blotting. In the primary AML sample, we investigated the cell responses to ex vivo stimulation with stem cell factor and BEZ235-induced inhibition of PI3K and identified activation patterns in multiple PI3K downstream signaling pathways including p-4EBP1, p-AKT, and p-S6, particularly in CD34(+) subsets. We evaluated multiple signaling pathways in antigen-defined subpopulations in primary AML cells with FLT3-ITD mutations. The data demonstrated the heterogeneity of cell phenotype distribution and distinct patterns of signaling activation across AML samples and between AML and normal samples. The mTOR targets p-4EBP1 and p-S6 were exclusively found in FLT3-ITD stem/progenitor cells, but not in their normal counterparts, suggesting both as novel targets in FLT3 mutated AML. Our data suggest that CyTOF can identify functional signaling pathways in antigen-defined subpopulations in primary AML, which may provide a rationale for designing therapeutics targeting LSC-enriched cell populations.

Keywords: Key terms: acute myeloid leukemia; flow cytometry; leukemia stem cells; mass cytometry; western blotting.

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism
  • Antigens, CD / genetics
  • Antigens, CD / immunology
  • Cell Cycle Proteins
  • Cell Line, Tumor
  • Cell Proliferation / genetics
  • Cell Survival / genetics
  • Cytokines / metabolism
  • Flow Cytometry / methods*
  • Humans
  • Imidazoles / pharmacology
  • Leukemia, Myeloid, Acute / genetics*
  • Mass Spectrometry / methods
  • Neoplastic Stem Cells / cytology*
  • Phosphatidylinositol 3-Kinases / metabolism
  • Phosphoinositide-3 Kinase Inhibitors
  • Phosphoproteins / metabolism
  • Proto-Oncogene Proteins c-akt / metabolism
  • Quinolines / pharmacology
  • Ribosomal Protein S6 Kinases / metabolism
  • Signal Transduction / genetics*
  • Staining and Labeling
  • Stem Cell Factor / pharmacology
  • TOR Serine-Threonine Kinases / metabolism
  • fms-Like Tyrosine Kinase 3 / genetics*

Substances

  • Adaptor Proteins, Signal Transducing
  • Antigens, CD
  • Cell Cycle Proteins
  • Cytokines
  • EIF4EBP1 protein, human
  • Imidazoles
  • Phosphoinositide-3 Kinase Inhibitors
  • Phosphoproteins
  • Quinolines
  • Stem Cell Factor
  • MTOR protein, human
  • TOR Serine-Threonine Kinases
  • FLT3 protein, human
  • fms-Like Tyrosine Kinase 3
  • Proto-Oncogene Proteins c-akt
  • Ribosomal Protein S6 Kinases
  • dactolisib