[show abstract][hide abstract] ABSTRACT: Kinases determine the phenotypes of many cancer cells, but the frequency with which individual kinases are activated in primary tumors remains largely unknown. We used a computational approach, termed kinase-substrate enrichment analysis (KSEA), to systematically infer the activation of given kinase pathways from mass spectrometry-based phosphoproteomic analysis of acute myeloid leukemia (AML) cells. Experiments conducted in cell lines validated the approach and, furthermore, revealed that DNA-dependent protein kinase (DNA-PK) was activated as a result of inhibiting the phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) signaling pathway. Application of KSEA to primary AML cells identified PI3K, casein kinases (CKs), cyclin-dependent kinases (CDKs), and p21-activated kinases (PAKs) as the kinase substrate groups most frequently enriched in this cancer type. Substrates phosphorylated by extracellular signal-regulated kinase (ERK) and cell division cycle 7 (CDC7) were enriched in primary AML cells that were resistant to inhibition of PI3K-mTOR signaling, whereas substrates of the kinases Abl, Lck, Src, and CDK1 were increased in abundance in inhibitor-sensitive cells. Modeling based on the abundances of these substrate groups accurately predicted sensitivity to a dual PI3K and mTOR inhibitor in two independent sets of primary AML cells isolated from patients. Thus, our study demonstrates KSEA as an untargeted method for the systematic profiling of kinase pathway activities and for increasing our understanding of diseases caused by the dysregulation of signaling pathways.
[show abstract][hide abstract] ABSTRACT: Lipopolysaccharide activates plasma-membrane signaling and endosomal signaling by Toll-like receptor 4 (TLR4) through the TIRAP-MyD88 and TRAM-TRIF adaptor complexes, respectively, but it is unclear how the signaling switch between these cell compartments is coordinated. In dendritic cells, we found that the p110δ isoform of phosphatidylinositol-3-OH kinase (PI(3)K) induced internalization of TLR4 and dissociation of TIRAP from the plasma membrane, followed by calpain-mediated degradation of TIRAP. Accordingly, inactivation of p110δ prolonged TIRAP-mediated signaling from the plasma membrane, which augmented proinflammatory cytokine production while decreasing TRAM-dependent endosomal signaling that generated anti-inflammatory cytokines (interleukin 10 and interferon-β). In line with that altered signaling output, p110δ-deficient mice showed enhanced endotoxin-induced death. Thus, by controlling the 'topology' of TLR4 signaling complexes, p110δ balances overall homeostasis in the TLR4 pathway.
[show abstract][hide abstract] ABSTRACT: BACKGROUND: Tumor classification based on their predicted responses to kinase inhibitors is a major goal for advancing targeted personalized therapies. Here, we used a phosphoproteomic approach to investigate biological heterogeneity across hematological cancer cell lines including acute myeloid leukemia, lymphoma and multiple myeloma. RESULTS: Mass spectrometry was used to quantify 2,000 phosphorylation sites across three acute myeloid leukemia, three lymphoma and three multiple myeloma cell lines in six biological replicates. The intensities of the phosphorylation sites grouped these cancer cell lines according to their tumor type. In addition, a phosphoproteomic analysis of seven acute myeloid leukemia cell lines revealed a battery of phosphorylation sites whose combined intensities correlated with the growth-inhibitory responses to three kinase inhibitors with remarkable correlation coefficients and fold changes (>100 between the most resistant and sensitive cells). Modeling based on regression analysis indicated that a subset of phosphorylation sites could be used to predict response to the tested drugs. Quantitative analysis of phosphorylation motifs indicated that resistant and sensitive cells differed in their patterns of kinase activities, but, interestingly, phosphorylations correlating with responses were not on members of the pathway being targeted; instead, these mainly were on parallel kinase pathways. CONCLUSION: This study reveals that the information on kinase activation encoded in phosphoproteomics data correlates remarkably well with the phenotypic responses of cancer cells to compounds that target kinase signaling and could be useful for the identification of novel markers of resistance or sensitivity to drugs that target the signaling network.