Label-free phosphoproteomics method to investigate signaling pathways downstream of novel PI3K/mTOR inhibitors in cancer cells

摘要

Mass spectrometry-based phosphoproteomics is a powerful tool for the analysis of signaling in an unbiased fashion. Here we used phosphoproteomics to investigate how PI3K/mTOR inhibitors affect phosphorylation in Acute Myeloid Leukemia (AML), a blood malignancy that presents aberrant activation of the PI3K/AKT/mTOR axis in a high percentage of patients. Several PI3K/AKT/mTOR inhibitors are now in clinical trials but the mechanisms that make cells sensitive or resistant to these compounds is unknown. In this work, AML cell lines sensitive (Kasumi-1) or resistant (P31/Fuj) to PI3K/mTOR inhibitors were treated with two kinase inhibitors (AZ123: pan-PI3K/mTOR inhibitor; AZ125: mTORC1/2 inhibitor). Label-free phosphoproteomics revealed hundreds of phosphopeptides regulated by these compounds in a reproducible manner, corresponding to specific patterns of protein kinase phosphorylation motifs.