Kinase-Substrate Enrichment Analysis Provides Insights into the Heterogeneity of Signaling Pathway Activation in Leukemia Cells

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.

Empirical inference of circuitry and plasticity in a kinase signaling network

“…S2. Motif analysis (14) further revealed that the inhibitor pairs exhibited strongly correlated impacts on specific phosphorylation motifs while mirroring the relationships seen in Fig. 1 C and D (Fig.…”

“…Phosphorylated peptides were enriched through the use of TiO 2 beads (GL Sciences) in a similar manner to that previously described (14,19) with some modifications, and analyzed by LC-MS/MS using a LTQ-Orbitrap mass spectrometer. Peptides were identified by means of Mascot searches against the SwissProt human protein database.…”

“…Following quantile normalization of the data (39), the magnitude and statistical significance of differences between conditions were computed by means of empirical Bayes shrinkage of SDs (40) using the limma package within the R computing environment (41,42). The abundance of CTAMs was monitored systematically by using KSEA (14,18,19,30).…”

“…Peptides were identified by means of Mascot searches against the SwissProt human protein database. Peptide quantification was achieved by using Pescal as previously described (14).…”

“…The maturation of phosphoproteomics techniques based on mass spectrometry (MS) is now allowing the simultaneous quantification of several thousands of phosphorylation sites per experiment, and approaches to derive kinase activity from these large-scale phosphoproteomics datasets have been reported (11)(12)(13)(14). One such approach, named kinase substrate enrichment analysis (KSEA), is based on the premise that, because each phosphorylation site is the result of a kinase's catalytic activity, phosphoproteomic profiling provides a means by which to capture and measure the activities of all kinases expressed in the system under investigation (14).…”