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

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.

Blockade of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase and murine double minute synergistically induces Apoptosis in acute myeloid leukemia via BH3-only proteins Puma and Bim.

Molecular aberrations of the Ras/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK and/or Murine double minute (MDM2)/p53 signaling pathways have been reported in 80% and 50% of primary acute myeloid leukemia (AML) samples and confer poor outcome. In this study, antileukemic effects of combined MEK inhibition by AZD6244 and nongenotoxic p53 activation by MDM2 antagonist Nutlin-3a were investigated. Simultaneous blockade of MEK and MDM2 signaling by AZD6244 and Nutlin-3a triggered synergistic proapoptotic responses in AML cell lines [combination index (CI) = 0.06 +/- 0.03 and 0.43 +/- 0.03 in OCI/AML3 and MOLM13 cells, respectively] and in primary AML cells (CI = 0.52 +/- 0.01). Mechanistically, the combination upregulated levels of BH3-only proteins Puma and Bim, in part via transcriptional upregulation of the FOXO3a transcription factor. Suppression of Puma and Bim by short interfering RNA rescued OCI/AML3 cells from AZD/Nutlin-induced apoptosis. These results strongly indicate the therapeutic potential of combined MEK/MDM2 blockade in AML and implicate Puma and Bim as major regulators of AML cell survival.

Peroxisome proliferator-activated receptor gamma and retinoid X receptor ligands are potent inducers of differentiation and apoptosis in leukemias.

The peroxisome proliferator-activated receptor gamma (PPAR gamma) is a member of the nuclear receptor family that forms heterodimers with retinoid X receptor. These heterodimers bind to DNA and activate the transcription of target genes. Here, we report that the PPAR gamma receptor protein is expressed in primary myeloid and lymphoid leukemias and in lymphoma and myeloma cell lines. In this study, we compared the activity of several PPAR gamma ligands including BRL49653 (rosiglitazone), 15-deoxy-Delta 12,14-prostaglandin J(2), and the novel triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid on leukemia cells. Exposure to these PPAR gamma ligands induced apoptosis in myeloid (U937 and HL-60) and lymphoid (Su-DHL, Sup-M2, Ramos, Raji, Hodgkin's cell lines, and primary chronic lymphocytic leukemia) cells. A similar exposure to these PPAR gamma ligands induced the differentiation of myeloid leukemic cells. A combination of PPAR gamma ligands with a retinoid X receptor agonist (i.e., LG100268) or a retinoic acid receptor agonist (i.e., all trans-retinoic acid) enhanced differentiating and growth-inhibitory effects. 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid induced differentiation and apoptosis with much greater potency than the other PPAR gamma ligands in established cell lines and primary chronic lymphocytic leukemia samples. Exposure to 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid induced mitochondrial depolarization and caspase activation, which was associated with apoptosis induction. In Bcl-2-overexpressing chronic lymphocytic leukemia cells, the small-molecule Bcl-2 inhibitor HA14-1 sensitized these cells to 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-induced apoptosis. These results suggest that PPAR gamma ligation alone and in combination with retinoids holds promise as novel therapy for leukemias by activating the transcriptional activity of target genes that control apoptosis and differentiation in leukemias.