A novel approach has been developed to study the reprogramming of protein kinases """"""""en masse"""""""" allowing ~40-60% of the expressed kinome assayed in a single mass spectroscopy run. Our methods utilize Multiplexed Inhibitor Beads (MIBs), consisting of Sepharose beads with covalently immobilized, linker adapted, kinase inhibitors. The technique allows interrogation of kinases known by sequence but which have been understudied due to lack of biologic or phenotypic knowledge or the availability of reagents. MIB/MS identified a kinome response signature to a select MEK1/2 inhibitor AZD6244 that is currently in clinical testing for triple negative breast cancer. The only defined substrate for MEK1/2 are ERK1 and 2, yet we observed changes in activity of kinases in every subfamily of the kinome in response to MEK inhibition. Kinome assessment showed a time-dependent reprogramming that involved an early loss of ERK feedback regulation of RAF and MEK that allowed upstream reactivation of the MEK-ERK pathway. The time dependent change in MIB binding of specific receptor tyrosine kinases (RTKs) such as PDGFR? and DDR1 was readily detected and provided the critical experimental observation that MEK inhibition was driving the expression and activation of multiple RTKs. c-Myc degradation was a key mechanism mediating kinome reprogramming. The fact that multiple RTKs are activated in response to MEK inhibition demonstrates the difficulty in using single kinase inhibitors to arrest tumor progression.
The aims of this proposal include: 1. Define kinome activation state in the human basal/claudin-low like SUM159 cell line and the pre-clinical C3Tag GEMM for basal/claudin-low breast cancer. Kinase signatures will be defined in response to MEK1/2 and PI3K inhibitors alone and in combination, which are currently in clinical testing. 2. Define mechanisms of kinome reprogramming in MEK and PI3K inhibitor resistant tumors and cell lines. 3. Develop rational predictions of kinase inhibitor combinations based on kinome signatures of sensitive and resistant tumors for testing in the C3Tag GEMM for basal/claudin-low breast cancer. The goal is to define combination therapies that overcome resistance and cause apoptosis and tumor regression.

Public Health Relevance

Rationally devising novel kinase inhibitor combination therapies requires detailed knowledge of kinome activity. Currently, there is no discovery mechanism to completely define the dynamic activity of the kinome in response to inhibitors. The MIB/MS technology will be used to assess global kinome behavior and its response to small molecule inhibitors leading to new and effective combination therapies to treat disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM101141-03
Application #
8607578
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Dunsmore, Sarah
Project Start
2012-04-15
Project End
2016-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Pharmacology
Type
Schools of Medicine
DUNS #
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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Miller, Samantha M; Goulet, Daniel R; Johnson, Gary L (2017) Targeting the Breast Cancer Kinome. J Cell Physiol 232:53-60
Mobley, Robert J; Raghu, Deepthi; Duke, Lauren D et al. (2017) MAP3K4 Controls the Chromatin Modifier HDAC6 during Trophoblast Stem Cell Epithelial-to-Mesenchymal Transition. Cell Rep 18:2387-2400
Troutman, Scott; Moleirinho, Susana; Kota, Smitha et al. (2016) Crizotinib inhibits NF2-associated schwannoma through inhibition of focal adhesion kinase 1. Oncotarget 7:54515-54525
Stuhlmiller, Timothy J; Miller, Samantha M; Zawistowski, Jon S et al. (2015) Inhibition of Lapatinib-Induced Kinome Reprogramming in ERBB2-Positive Breast Cancer by Targeting BET Family Bromodomains. Cell Rep 11:390-404
Tsai, Yihsuan S; Dominguez, Daniel; Gomez, Shawn M et al. (2015) Transcriptome-wide identification and study of cancer-specific splicing events across multiple tumors. Oncotarget 6:6825-39
Zawistowski, Jon S; Graves, Lee M; Johnson, Gary L (2014) Assessing adaptation of the cancer kinome in response to targeted therapies. Biochem Soc Trans 42:765-9
Johnson, Gary L; Stuhlmiller, Timothy J; Angus, Steven P et al. (2014) Molecular pathways: adaptive kinome reprogramming in response to targeted inhibition of the BRAF-MEK-ERK pathway in cancer. Clin Cancer Res 20:2516-22
Turowec, Jacob P; Zukowski, Stephanie A; Knight, James D R et al. (2014) An unbiased proteomic screen reveals caspase cleavage is positively and negatively regulated by substrate phosphorylation. Mol Cell Proteomics 13:1184-97
Stuhlmiller, T J; Earp, H S; Johnson, G L (2014) Adaptive reprogramming of the breast cancer kinome. Clin Pharmacol Ther 95:413-5

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