Identification of Novel Signalling Pathways in Myeloblasts from Patients with Acute Myeloid Leukemia Using a Phosphoproteomic Strategy.

Acquired somatic mutations which result in constitutive activation of signal transduction pathways are increasingly recognised in acute myeloid leukemia (AML). Reasoning that since protein tyrosine phosphorylation is an important mechanism mediating the transduction of proliferative and survival signals we have utilised a proteomic strategy to identify dysregulated phosphoproteins in myeloblasts from patients with AML. Using an anti-phosphotyrosine antibody we have immunoprecipitated proteins from AML blasts, separated proteins by SDS PAGE and identified proteins within distinct bands by mass spectrometry. Results in primary AML blasts have been compared with CD34+ hemopoietic progenitors harvested from GCSF mobilised normal donors. The methodology was validated using vanadate-stimulated HL60 cells. 10 patients with a median age of 51 (range 16–90 yrs) were studied. One patient had favorable risk cytogenetics, three adverse risk cytogenetics and six a normal karyotype. Mutations in the flt-3 tyrosine kinase were present in four patients. Blasts from every patient demonstrated phosphorylation of MAP kinase (MAPK) implying activation of the ras-MAPK cascade. In contrast CD34+ cells from three normal donors demonstrated weak or no MAPK phosphorylation. Since each AML sample demonstrated MAPK phosphorylation irrespective of flt-3 status we next examined their phosphotyrosine-proteome. This identified a number of phosphorylated and non-phosphorylated proteins in signalling complexes in anti-phosphotyrosine immunoprecipitates. Notable examples amongst these were receptors (Ephrin type-A3, interleukin-13, GPCR43), signalling intermediates (TAPP1, RASA1, GDI-2, LARG, SGEF, cortactin, Cd-2 associated protein) and transcription factors (ELK-1, HFK-1). These proteins identify three functionally distinct groups in AML blasts which were not detected in normal CD34+ progenitors: (i) intermediates of a tyrosine kinase mediated ras-MAPK signalling cascade (ii) intermediates in PtdIns-3-kinase mediated signalling presumably suppressing apoptosis and (iii) regulators of the actin cytoskeleton and thus cell movement. Surprisingly the strategy also identified tyrosine phosphorylation of the 5-HT3A receptor and the MRCP1-ABC transporter in a number of patients suggesting a role for these proteins in the pathogenesis of certain subtypes of AML. The phosphorylation of a number of these identified proteins was confirmed immunochemically. Thus adoption of a phosphoproteomic methodology has identified novel phosphoproteins in AML which require further validation. Our data also demonstrate a common intracellular signalling pathway (ras-MAPK) in distinct AML patients regulated by differing primary events. These observations provide information of value in the rational development of targeted therapies in AML.