Proteome and phospho-proteome study of Molm-13 cell line in Acute Myeloid Leukemia (AML)

Abstract

The main goal of this thesis was to study the changes in the proteome of Molm-13 cells from Acute Myeloid Leukemia when treated with a proliferation and cell differentiation inductor (FLT3 ligand) and with an apoptosis inductor (Okadaic Acid, OA, FLT3 ligand binds to FLT3 receptor which auto phosphorylates inducing cell proliferation and differentiation. FLT3 receptor is commonly mutated in AML, this mutation is named FLT3-ITD. Signaling pathways like RAS/MAP kinase, AKT/PI-3 kinase and JAK/ STAT are activated depending on ITD localization. Some other cells were treated with Okadaic Acid, a toxin and a potent inhibitor of the Serine/ Threonine (Ser/Thr) protein phosphatases 1 and 2, which blocks the activation of ERK5 a protein present in the MAP kinase pathway. To perform proteomics analysis, protein content of each sample was digested by Filter Aided Sample Preparation Double Digestion method and a peptide aliquot was enriched by Titanium Dioxide to get phosphorylated peptides. Samples were analysed by mass spectrometry with the Orbitrap Elite. More than 3000 proteins were identified in the different samples analysed. Bcl-6, Bcl-2, STAT and TNFAIP8 are important proteins which contribute to tumor cell progression and inhibit the apoptosis process by inhibiting caspases function. These mentioned proteins were found in Wild type (untreated cells) and FLT3- ligand induced cells. Whereas in Okadaic Acid treated cells different proteins were expressed as apoptotic inducer, these proteins are p53 and TNF along more proteins. FLT3- ITD mutation is probably localized in the Endoplasmatic Reticulum (ER).