Protein microarray technology for profiling signaling pathways: Insights into pro-oncogenic Notch signaling in T cell acute lymphoblastic leukemia

We have developed a multiplexed reverse phase protein (RPP) microarray platform for simultaneous monitoring of site-specific phosphorylation of numerous signaling proteins using nanogram amounts of lysates derived from stimulated living cells. We first show the application of RPP microarrays to the study of signaling kinetics and pathway delineation in Jurkat T lymphocytes. RPP microarrays were used to profile the phosphorylation state of 62 signaling components in Jurkat T cells stimulated through their membrane CD3 and CD28 receptors, identifying a previously unrecognized link between CD3 crosslinking and dephosphorylation of Raf-1 at Ser259. Finally, the potential of this technology to analyze rare primary cell populations is shown in a study of differential STAT protein phosphorylation in interleukin (IL)-2-stimulated CD4+ CD25+ regulatory T cells. RPP microarrays, prepared using simple procedures and standard microarray equipment, represent a powerful new tool for the study of signal transduction in both health and disease.; Protein microarrays have the potential to expedite the discovery of unexpected connections between signaling pathways. We applied RPP microarrays to study Notch signaling in cancer cells. Members of the conserved Notch family of transmembrane receptors are critically involved in the control of differentiation, proliferation, and apoptosis for numerous cell types. Furthermore, constitutive Notch activation is required for the proliferation of a subgroup of T cell acute lymphoblastic leukemias (T-ALLs). Downstream pathways that transmit prooncogenic signals are not well characterized. To identify these pathways, RPP microarrays were employed to profile the phosphorylation state of 108 epitopes on 82 signaling proteins in a panel of 13 T cell leukemia cell lines. Notch inhibition resulted in suppression of both phosphorylation and activity of effectors downstream of mTOR in a phosphatidylinositol-3 kinase (PI3K) independent manner. Simultaneous inhibition of both mTOR and Notch signals suppressed T-ALL growth in a highly synergistic manner, pointing to a new therapeutic strategy for Notch-dependent cancers. This study represents a novel approach to the study of signaling pathways and also a paradigm for drug target and drug combination identification in a variety of disease settings.