The Graves Lab is investigating cellular mechanisms of drug resistance in leukemia. This is a major problem that limits the efficacy of many commonly used therapeutics. 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. To accomplish this 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. 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.
Kinase-dependent regulation of microRNAs. Micro RNAs (miRs) are small non-coding RNAs that regulate protein expression in cells. MiR expression is commonly dysregulated in cancer and has important consequences for cell function and survival. We are interested in Lyn, a Src family tyrosine kinase involved in promoting cell survival and anti-apoptotic responses in both cell models and leukemia patients. We recently showed that Lyn down-regulates a tumor suppressor microRNA (miR181) in drug-resistant CML leukemia cells. This miR in turn regulates the expression of a key cell survival protein, Mcl-1. Thus, Lyn-dependent repression of miR181 favors the survival these cells (Zimmerman et al, Mol Pharm 2010). We are investigating the cellular mechanisms by which Lyn represses miR181 expression and the signals involved in Lyn activation in these cells. We are also investigating the ability of Flt3 receptor tyrosine kinase and Flt3 mutants to regulate miRs in drug resistant AML cells. (This is a collaborative project with Dr. Scott Hammond and Dr. Kristy Richard at UNC).
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 mixed pan kinase inhibitor beads (MIBs), 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. Using a cell model of drug (imatinib-resistant) leukemia, we have identified unique changes in the kinome that may contribute to imatinib resistance. Targeting this kinome response is currently being tested as an approach to prevent drug resistance in leukemia. In addition, we are profiling the kinome in AML and drug-resistant AML patient samples. One goal of this research is to identify the kinome changes and to develop novel kinase assays for these enzymes (Wang et al. ACS Chem Biol, 2010). This is expected both increase the ability to diagnose as well as target unique kinase changes in leukemia. (This is a collaborative project with Dr. Gary Johnson, Dr. David Lawrence and others at UNC).