Use of peptide biosensors for monitoring activity of kinases in chronic myelogenous leukemia

Tzu-Yi Yang, Purdue University

Abstract

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder that is characterized by massive expansion of myeloid progenitors and the reciprocal translocation of chromosomes 9 and 22. This leads to the production of the BCRABL gene and the expression of the oncogenic Bcr-Abl protein. Abl kinase is an essential kinase that plays important roles in governing survival, proliferation, and differentiation of hematopoietic cells. The catalytic activity of Abl kinase is normally under tight control in order to maintain tissue homeostasis. Bcr-Abl oncogenic protein is constitutively activated and interrupts normal intracellular signal transduction, leading to aberrant cell proliferation and ransformation. As a hallmark of CML, Bcr-Abl is an attractive therapeutic target. Indeed, the first tyrosine kinase inhibitor (TKI) developed for cancer therapy is a Bcr-Abl kinase inhibitor, imatinib mesylate. Although imatinib has benefited CML patients and showed extraordinary initial remission response in patients, problems including drug resistance and residual disease have been noticed. It has been postulated that TKI resistance is caused by secondary mutation in Abl kinase and cell cycle accelerating surrogate pathways through JAK2, Syk, and Src family kinases. Current strategies of resolving drug resistance are increasing imatinib dose and altering therapy. Although both have been shown to increase the likelihood of improving clinical outcomes, decisions are made on the basis of one-dose-for-all studies. In addition, the complexity of signal transduction suggests the necessity of monitoring signaling proteins in a specific, systemic, high-throughput manner. However, present methodologies of studying molecular sources of resistance and measuring the treatment response either requires a large amount of patient samples or lacks the sensitivity or the dynamic range for indicating protein expression levels and activities. Here we introduce a new approach for investigating and quantifying the kinase activity using synthetic peptide 'biosensors'. Sequences that are substrates of Abl, Syk, JAK2, and Src family kinases were synthesized, each with a cell-penetrating peptide at the C terminus. These biosensors were used to probe activity of multiple intracellular kinases simultaneously in a real-time manner. In this thesis, we reported use of the Abl biosensor and the detection of Bcr-Abl activity and inhibition by imatinib in the human CML cell line K562 using Western blot and mass spectrometry. We improved the reproducibility and limit of detection (to fmol levels) by incorporating the multiple reaction monitoring technique (MRM). We also examine the degradation of the Abl biosensor in cells and showed that the biosensor is degraded from the C terminus with the N terminal `reporting module' remaining intact. We investigated the role of the `targeting module' in the Abl biosensor by measuring the kinetics parameters of the biosensors with and without the SH3 targeting sequence using a label-free quantification mass spectrometry. Although the difference in Km is not dramatic, results suggested that the phosphorylation levels are significantly different in the biosensor that contains SH3 binding sequence and in the biosensors without an SH3 binding sequence. We proceed to develop a multiplexed biosensor monitoring techinique by applying the Abl, Syk, JAK2, and Src family biosensors to peripheral blood mononuclear cells (PBMCs) and bone marrow primary cells to obtain a sub-kinome view. In patient PBMCs and bone marrow cells, both Abl and JAK2 biosensors reported high phosphorylation levels (≥69%). In cells isolated from a patient who is in remission, Abl biosensor reported zero phosphorylation while JAK2 biosensor reported a relatively higher phosphorylation. On the other hand, in the 'non-cancerous' human PBMCs, low phosphorylation in Abl, Syk, and JAK2 biosensors was detected, and a higher phosphorylation level (~67%) was detected in the Src family kinase biosensor. While it is necessary to characterize ionization/detection efficiency of the biosensors and to further refine experimental procedures before drawing a biological conclusion, our work showed the proof-of-concept that the multiplexed biosensor assay can be used for monitoring activity of kinases in primary samples. The biosensor technique has the potential of providing complementary information of intracellular signaling that is not revealed by other techniques, and may contribute to CML disease management by serving as a drug response/disease progression indicator.

Degree

Ph.D.

Advisors

Parker, Purdue University.

Subject Area

Biology|Pharmacology|Biochemistry|Oncology

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