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 claudinlow breast cancer cell lines and tumors. Kinase MIB-binding profiles at baseline without perturbation proteomically distinguished the four breast cancer subtypes. Kinases lacking defined functions in breast cancer were highly represented in the MIB-binding taxonomies. We show that these understudied kinases, whose disease associations and pharmacology are generally unexplored, are integrated in kinase 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 . CC-BY 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Defining the KRAS-regulated kinome in KRAS-mutant pancreatic cancer

“…It is likely that even this is a substantial underestimate: the human kinome encodes over 500 protein kinases. Some kinases were not detected here because they are not recognized by any one of the six broadspectrum kinase inhibitors used in the affinity purification step; others were not detected due to low or lack of expression in pancreatic cancer (58).…”

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 mg 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.…”

Kinome state is predictive of cell viability in pancreatic cancer tumor and stroma cell lines

“…Linked with their role in disease, the druggability of kinases has led to increased interest in the development of kinase inhibitors, with over 70 now having achieved FDA approvalCohen et al (2021). Recent proteomics techniques including kinobeads and multiplexed inhibitor beads linked with mass spectroscopy (MIB/MS) are a relatively new mechanism which affords the ability to assess the state of the protein kinome en masse (Duncan et al, 2012;Collins et al, 2018). When combined with perturbation with targeted kinase inhibitors, the quantification of the dynamic response of the kinome provides a novel platform for the study of cell signaling and potential design of drug therapies.…”