Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human patients with acute myeloid leukemia (AML) were stimulated stimulated with MPL receptor agonist (SB559457) or recombinant human Tpo (rhTpo) for 6 hours

ABSTRACT: Biologic characterization of SB-559457 (SB), a non-peptidyl hydrazone class of thrombopoietin receptor (Mpl) agonist, revealed toxicity towards human leukemia cells. Anti-proliferative effects followed by significant, non-apoptotic, cell death within 72 hours occurred in 24/26 AML, 0/6 ALL, and 3/6 CML patient samples exposed to SB, but not recombinant human thrombopoietin (rhTpo), in liquid suspension culture. Further investigation revealed increased phosphorylation of p70S6/S6 kinases in SB, but not in rhTpo, treated cells. Expression profiling of cells exposed to SB vs rhTpo revealed statistically significant, ~2-fold changes in GAPDH and REDD1 gene expression, confirmed by QRT-PCR. These genes, induced in energy or hypoxia stressed cells, have been implicated in cell death pathways, and may provide important clues to the mechanism of SB induced, leukemic cell death. These results suggest that nonpeptidyl, hydrazone class Mpl agonists may be clinically useful anti-leukemic agents by virtue of their combined thrombopoietic and anti-leukemic effects. Experiment Overall Design: Primary cells collected from 5 patients with acute myeloid leukemia (AML) were stimulated stimulated with MPL receptor agonist (SB559457) or recombinant human Tpo (rhTpo) for 6 hours. Next, RNA was isolated from these cells. RNA was used for gene profile analyis to compare genes regulated in AML cells by SB559457 verus rhTpo. For each sample half of the cells were stimulated with Mpl agonist and half with rhTpo.

ORGANISM(S): Homo sapiens

SUBMITTER: Anna Kalota

PROVIDER: E-GEOD-18673 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Acute myeloid leukemia (AML) is a malignancy of transformed hematopoietic progenitors, which is curable in only 25-50% of patients thus the risk of relapse remains the major challenge. Considerable efforts in improving outcome through the understanding of AML biology elucidate that AML is not autonomous but rather supported by a complex multicellular-bone marrow microenvironment for leukemogenesis, leukemia expansion, and chemoresistance. Within the microenvironment, bone marrow mesenchymal stroma cells (BMSC) safeguard leukemia allowing rapid growth and enable resistance to therapy. Herein, we modeled leukemia-stroma interactions to unravel the reciprocal signaling processes regulating leukemic cell growth and survival. First, we conducted a proteomic analysis of primary AML samples (n=13) cocultured with non-malignant stroma cells by utilizing liquid chromatography mass spectrometry (LC-MS/MS) to understand how the stroma contributes to AML cellular signaling. In the analysis, 84 proteins were significantly affected including upregulation of rate-limiting metabolic enzymatic proteins NAMPT, FASN, and HK1 in AML-stroma cocultures relative to AML monoculture. Through an epigenetic drug screen, we modeled stroma mediated protection of leukemia and uncovered prominent drug resistance by histone deacytlase inhibitor (HDACi) treatment in cocultured leukemic cell lines and cocultured primary AML. We performed a quantitative phosphoproteomics analysis of AML-stroma interactions of leukemic KG1a cells in monoculture and in coculture with stroma (Hs5) treated with HDACi. We uncovered phosphorylation networks of stroma mediated protection enriched in pathways involved in AMP-activated signaling, glucose catabolic processes and EGF/EGFR signaling with the most potent changes in hyper-phosphorylation of acetyl-coenzyme A synthetase (ACSS2, S30) and of hypo-phosphorylation acetyl-coenzyme A carboxylase alpha, (ACACA, S80) both of which are involved in acetyl-CoA (acetyl-CoA) utilization for fatty acid synthesis pathway. Validating these findings, we found that ACSS2 substrate, acetate, contributed to leukemic proliferative growth and ACSS2 loss impacted leukemia metabolic fitness. In addition, we uncovered a novel subgroup of AML patients based on high expression of ACSS1 or ACSS2, which significantly correlated with inferior outcome. The differentially upregulated genes of this ACSS1/2-high subgroup revealed a metabolic gene signature related to mitochondrial processes suggesting the metabolic state of AML may aid in predicting outcome. Our findings elucidate phosphoproteome network of AML-stroma interactions whereby overriding stroma mediated protection, we conclude, requires intervening the metabolic support by ACSS2 in AML.

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Transcription profiling of human patients with acute myeloid leukemia (AML) were stimulated stimulated with MPL receptor agonist (SB559457) or recombinant human Tpo (rhTpo) for 6 hours

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