Changing the subcellular localization of a signal transducing protein involved in disease is a novel approach for therapeutic intervention. The subcellular location of some proteins plays a critical role in the etiology of disease. A precise example of this is Bcr- Abl protein, the causative agent of chronic myelogenous leukemia (CML). When Bcr-Abl is in the cytoplasm of cells, it behaves as an oncogene, but if forced to the nucleus, it becomes an apoptotic factor. CML is a myeloproliferative disorder characterized by increased proliferation of granulocytes and their immature precursors;with a median survival time of 4 to 6 years. The goal of this study is to use our ligand responsive protein switch constructs to control the subcellular location of Bcr-Abl, and convert Bcr- Abl from an oncogene to an apoptotic factor. It has been shown that depletion of Bcr- Abl from the cytoplasm by nuclear trapping of Bcr-Abl can result in apoptosis. If Bcr-Abl can be directed to the nucleus, it can be converted from an oncogene to an apoptotic factor. Since Bcr-Abl oligomerizes with itself to form tetramers, nuclear trapping could be achieved by introducing exogenously localization-controllable Bcr-Abl. Upon ligand induction, localization controllable Bcr-Abl will oligomerize with wt Bcr-Abl and will undergo transport to the nucleus, followed by cellular apoptosis.
In Aim 1 we will subclone localization controllable versions of Bcr-Abl (Bcr-Abl protein switch, PS) with a fluorescent tag and show oligomerization with wild-type (wt) Bcr-Abl, translocate to the nucleus, and cause apoptosis of Bcr-Abl positive K562 cells. Localization of Bcr-Abl PS will be monitored by fluorescence microscopy, and apoptosis will be tested using standard cell death assays. Interaction of wt Bcr-Abl with Bcr-Abl PS will be determined using an in vivo oligomerization between wt Bcr-Abl and Bcr-Abl protein switch and mammalian two-hybrid assay.
Aim 2 will test the Bcr-Abl PS in Gleevec.-resistant leukemic cells similarly.
Aim 3 will test and use specific promoters that allow preferential expression of Bcr-Abl PS in leukemia cells only.
Aim 4 will test if Bcr-Abl PS will eradicate/diminish leukemia in a human xenograft model using Balb/C nude mice injected with human leukemia cells. Our goal is to use ligand responsive protein switch constructs to control the subcellular location of Bcr-Abl, and convert Bcr-Abl from an oncogene to an apoptotic factor. Our long-term, ultimate goal is to use localization controllable versions of Bcr-Abl (as gene therapy) for treatment of CML.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA129528-04
Application #
8016679
Study Section
Gene and Drug Delivery Systems Study Section (GDD)
Program Officer
Howcroft, Thomas K
Project Start
2008-04-01
Project End
2013-01-31
Budget Start
2011-02-01
Budget End
2012-01-31
Support Year
4
Fiscal Year
2011
Total Cost
$275,147
Indirect Cost
Name
University of Utah
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Bruno, Benjamin J; Lim, Carol S (2015) Inhibition of bcr-abl in human leukemic cells with a coiled-coil protein delivered by a leukemia-specific cell-penetrating Peptide. Mol Pharm 12:1412-21
Woessner, D W; Eiring, A M; Bruno, B J et al. (2015) A coiled-coil mimetic intercepts BCR-ABL1 dimerization in native and kinase-mutant chronic myeloid leukemia. Leukemia 29:1668-75
Velusamy, Thirunavukkarasu; Palanisamy, Nallasivam; Kalyana-Sundaram, Shanker et al. (2013) Recurrent reciprocal RNA chimera involving YPEL5 and PPP1CB in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 110:3035-40
Woessner, David W; Lim, Carol S (2013) Disrupting BCR-ABL in combination with secondary leukemia-specific pathways in CML cells leads to enhanced apoptosis and decreased proliferation. Mol Pharm 10:270-7
Miller, Geoffrey D; Woessner, David W; Sirch, Monika J et al. (2013) Multidomain targeting of Bcr-Abl by disruption of oligomerization and tyrosine kinase inhibition: toward eradication of CML. Mol Pharm 10:3475-83
Bruno, Benjamin J; Miller, Geoffrey D; Lim, Carol S (2013) Basics and recent advances in peptide and protein drug delivery. Ther Deliv 4:1443-67
Davis, James R; Mossalam, Mohanad; Lim, Carol S (2013) Controlled access of p53 to the nucleus regulates its proteasomal degradation by MDM2. Mol Pharm 10:1340-9
Constance, Jonathan E; Despres, Samuel D; Nishida, Akemi et al. (2012) Selective targeting of c-Abl via a cryptic mitochondrial targeting signal activated by cellular redox status in leukemic and breast cancer cells. Pharm Res 29:2317-28
Davis, James R; Mossalam, Mohanad; Lim, Carol S (2012) Utilizing the estrogen receptor ligand-binding domain for controlled protein translocation to the insoluble fraction. Pharm Res 29:3455-63
Constance, Jonathan E; Woessner, David W; Matissek, Karina J et al. (2012) Enhanced and selective killing of chronic myelogenous leukemia cells with an engineered BCR-ABL binding protein and imatinib. Mol Pharm 9:3318-29

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