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.
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.
|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|
|Abell, Amy N; Johnson, Gary L (2014) Implications of Mesenchymal Cells in Cancer Stem Cell Populations: Relevance to EMT. Curr Pathobiol Rep 2:21-26|
|Stinchcombe, Thomas E; Johnson, Gary L (2014) MEK inhibition in non-small cell lung cancer. Lung Cancer 86:121-5|
|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|
|Stuhlmiller, T J; Earp, H S; Johnson, G L (2014) Adaptive reprogramming of the breast cancer kinome. Clin Pharmacol Ther 95:413-5|
|Cooper, Matthew J; Cox, Nathan J; Zimmerman, Eric I et al. (2013) Application of multiplexed kinase inhibitor beads to study kinome adaptations in drug-resistant leukemia. PLoS One 8:e66755|
|Jordan, Nicole Vincent; Prat, Aleix; Abell, Amy N et al. (2013) SWI/SNF chromatin-remodeling factor Smarcd3/Baf60c controls epithelial-mesenchymal transition by inducing Wnt5a signaling. Mol Cell Biol 33:3011-25|
|Dillon, Patrick J; Gregory, Sean M; Tamburro, Kristen et al. (2013) Tousled-like kinases modulate reactivation of gammaherpesviruses from latency. Cell Host Microbe 13:204-14|
|Zawistowski, Jon S; Nakamura, Kazuhiro; Parker, Joel S et al. (2013) MicroRNA 9-3p targets ýý1 integrin to sensitize claudin-low breast cancer cells to MEK inhibition. Mol Cell Biol 33:2260-74|