Proteomic analysis defines kinase taxonomies specific for subtypes of breast cancer

Abstract

Multiplexed small molecule inhibitors covalently bound to Sepharose beads (MIBs) were used to capture functional kinases in luminal, HER2-enriched and triple negative (basal-like and claudin-low) breast cancer cell lines and tumors. Kinase MIB-binding profiles at baseline without perturbation proteomically distinguished the four breast cancer subtypes. Understudied kinases, whose disease associations and pharmacology are generally unexplored, were highly represented in MIB-binding taxonomies and are integrated into signaling subnetworks with kinases that have been previously well characterized in breast cancer. Computationally it was possible to define subtypes using profiles of less than 50 of the more than 300 kinases bound to MIBs that included understudied as well as metabolic and lipid kinases. Furthermore, analysis of MIB-binding profiles established potential functional annotations for these understudied kinases. Thus, comprehensive MIBs-based capture of kinases provides a unique proteomics-based method for integration of poorly characterized kinases of the understudied kinome into functional subnetworks in breast cancer cells and tumors that is not possible using genomic strategies. The MIB-binding profiles readily defined subtype-selective differential adaptive kinome reprogramming in response to targeted kinase inhibition, demonstrating how MIB profiles can be used in determining dynamic kinome changes that result in subtype selective phenotypic state changes.

Competitive Kinase Enrichment Proteomics Reveals that Abemaciclib Inhibits GSK3β and Activates WNT Signaling

“…Lysates were incubated with MIBs and nutated at 4˚C for 15 min (8). The MIB mix contained VI16832 (22% V/V), CTx-0294885 (22% V/V), Purvalanol B (14% V/V), PP58 (14% V/V), UNC21474 (14% V/V), and Shokat (14% V/V) inhibitors conjugated to sepharose beads (8,9,19–22). Kinase-bound MIBs were washed once each with MIB lysis buffer, MIB low salt buffer (0.5% Triton X-100, 50 mM Hepes-NaOH [pH 8.0], 150 mM NaCl, 1 mM EDTA, and 1 mM EGTA), and MIB high salt buffer (0.5% Triton X-100, 50 mM Hepes-NaOH [pH 8.0], 1 M NaCl, 1 mM EDTA, and 1 mM EGTA).…”

High-Throughput Assessment of Kinome-wide Activation States

“…In current proteomics workflows, detection of these doubly phosphorylated peptides can be hampered because of poor ionization efficiency and inefficient elution after phosphopeptide enrichment. Our final list of targeted kinases comprise clinically relevant kinases with inhibitors approved by the Food and Drug Administration (FDA) such as Met, Abl, Src, BTK, Jak3, and Kit (Fabbro et al., 2015) (Table S4) as well as numerous kinases classified as understudied (Collins et al., 2018) (Figure S1).…”

“…Using a starting amount of merely 300 μg of protein, we were able to detect and quantify 46 phosphorylation sites in the T-loop region of 43 kinase groups, spanning a dynamic range of more than 3 orders of magnitude (Figure 3E; Table S5). The detected kinases include, besides a substantial number of understudied kinases such as CDK11A, CRK7, DYRK1A, DYRK2, DYRK4, HIPK3, NEK6, and PKN2 (Collins et al., 2018), various crucial players in control of growth and proliferation (Figure 3F), a variety of which are targets for potent novel inhibitors currently in clinical trials. This includes detection of cdc2/CDK1 and CDK2, which can be inhibited by Dinaciclib and ERK1 and ERK2, the phosphorylation of which can be inhibited by various MEK inhibitors such as Selumetinib.…”

Synthesis and Evaluation of Novel 1,2,6-Thiadiazinone Kinase Inhibitors as Potent Inhibitors of Solid Tumors

“…Diluted lysates were passed over a mixture of 25 mL of settled beads of each of the following inhibitors conjugated to ECH Sepharose beads: Purvalanol B, PP58, VI-16832, UNC21474A, UNC8088A, and 37.5 mL of settled beads conjugated to CTx-0294885 and VI-16832 [ 69 ]. The kinase inhibitor–bead conjugates were previously equilibrated in high-salt buffer (50 mM HEPES pH 7.5, 1M NaCl, 0.5% Triton X-100, 1 mM EDTA, and 1 mM EGTA).…”