- Protein phosphorylation and protein kinase-mediated signaling pathways; application of proteomics
- Protein kinase inhibitors as therapeutics
- Regulation of metabolic enzymes by phosphorylation and interacting proteins
The Graves Lab is investigating cellular mechanisms of drug resistance in cancer. Protein kinases are key mediators of extracellular and intracellular signaling and are frequently mutated or dysregulated in cancer. We are focusing on global changes in protein kinases or the “kinome” in response to highly targeted therapeutics. In this regard we are collaborating with Dr. Gary Johnson, Dr. Ben Major and others here at UNC. To accomplish our goals we are using new methods of affinity capture and proteomics mass spectrometry to gain insight into changes in kinase expression or activity. We are specifically interested in identifying these kinome adaptations and connecting them to changes in intracellular signaling and metabolic pathways that are disrupted in cancers. We are particularly interested in studying unique kinases that are part of the “untargeted” kinome. These studies have the potential to identify new and unexpected cellular responses to drugs and to provide an experimental rationale for alternative treatments for drug resistant cancers.
Kinome profiling in drug resistant leukemias. The kinome is composed of 518 kinases, many of them known to be oncogenes when dysregulated. How the kinome responds to drug exposure or contributes to cancer drug resistance is poorly understood. Using multiplexed inhibitor beads (MIBs) developed here at UNC, we are performing high affinity capture of a large portion of the kinome from leukemia cell models and patient samples. Analysis of the peptides derived from these kinases by mass spectrometry (MALDI TOF/TOF or OrbiTRAP LC/MS), allows the identification and assessment of the phosphorylation status of individual kinases. We recently completed the kinome analysis of a model of drug (imatinib-resistant) leukemia (Cooper et al., 2013). This study identified unique changes in the kinome, in particular activation of Lyn kinase that we showed contributes to imatinib resistance. In parallel with the kinome analysis, we are performing phosphopeptide analysis with the goal of establishing kinase-substrate relationships. We are currently studying one phosphorylated protein (BIRC6) that we identified by this approach and have shown contributes to imatinib resistance in these cells. Studies are underway to determine how Lyn regulates BIRC6 and cell survival.
Kinome profiling in pancreatic cancer. This is a large project that involves multiple collaborators from UNC (Drs. Johnson, Der, Baldwin, Yeh, Major, Gomez) and other institutions NCCU (Dr. Baines) and Duke (Dr. Haystead). The focus of these studies is to establish the basal kinome of pancreatic cancer and to establish the kinome adaptations to perturbations. Again, this project is using the MIBs technology described in project #1. As Director of the Michael Hooker Proteomics Facility, I am working with my Associate Director Dr. Herring to develop and apply methods of proteomics to accomplish these aims. Much of our recent focus has been to study the effects of established therapeutics (gemcitabine) and compare these treatments to targeted inhibition of Mek, PIM, IKK and other kinases. We are developing approaches to study the effects of drugs in 3D pancreatic cancer spheroids. Because of the large amount of data generated by these studies, we are working with Computational Biologists (Gomez) to evaluate quantitative changes in the kinome.
Developing novel small inhibitors of “untargeted” kinases. Our kinome analysis has identified multiple kinases that show promise as novel targets for select kinase inhibitors. We are have selected a small number of these kinases to develop and test small molecules as inhibitors. This is a collaboration with Drs. Frye, Kireev and Wang (UNC-School of Pharmacy). Our recent studies have focused on MELK, a key kinase in cancer progression whose function is poorly understood. Working with the Frye group, we have identified a new class of small molecule inhibitors of MELK. We are currently investigating the specificity and potency of the compounds for inhibition of MELK and inhibition of pancreatic cancer growth.