Molecularly targeted therapies against kinases are changing the face of cancer treatment;however, there are no assays currently in clinical use that directly monitor the inhibition of these drugs in patient cells. Moreover, cellular heterogeneity both at the genetic and biochemical level underlies resistance to these therapies, while bulk cellular assays do not accurately predict or monitor therapeutic efficacy. A biochemical assay for monitoring BCR-Abl kinase activity in single cells has been developed. The assay quantitatively measures the enzymatic activity of BCR-Abl kinase in patients with Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). The goal of this grant is to optimize the assay to test the hypothesis that the enzymatic activity of BCR-Abl exhibits a great degree of variability amongst individual cells of Ph+ ALL patients allowing prediction of response or resistance to targeted therapies. The proposed research takes into consideration the design, synthesis and evaluation of fluorescently labeled peptide substrates as a novel method of quantitatively estimate BCR-Abl kinase activity in patient samples.
Aim 1 is focused on reporter synthesis by standard FMOC solid phase peptide protocol and characterization by MALDI and HPLC. Two design strategies are selected to generate reporters with increased intracellular resistance to degradation: 1) a beta-hairpin peptide attached through a linker to N-terminus of the substrate, and 2) strategic incorporation of un-natural amino acids into the substrate.
In Aim 2, the substrates are evaluated in vitro for determination of kinetic parameters KM and Vmax, and also in cell lysates and intact BCR-Abl+ cells for quantification of phosphorylation and determination of substrate half-life. Capillary electrophoreses is used for quantification with an automated system for cell lysate studies and a custom-built instrument for single cell analyses. Ultimately, the substrates will be selected based on phosphorylation and resistance to degradation.
Aim 3 will quantify BCR-Abl kinase activity in Ph+ ALL patient samples at the single-cell level using the lead substrates developed in Aim 2. Microinjection is utilized to deliver the substrates into cells but other methods ranging from electroporation to myristoylation will be tested. These data will be used to gain a greater understanding on kinase phosphorylation in tumor cells, to predict the efficacy of drug-targeted therapy for ALL patients and to facilitate development of individualized therapies.
The goal of this project is to optimize an assay to test whether a specific protein activity differs amongst individual cells of patients with acute lymphoblastic leukemia (ALL). Reporter molecules of protein activity will be designed and used to evaluate the bone marrow cells obtained from patients with ALL and analyzed using single-cell capillary electrophoresis. These findings will help predict whether a drug will work for ALL patients and it will help in developing more effective therapies for those patients with this form of cancer.