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 #
5R01CA129528-05
Application #
8212586
Study Section
Gene and Drug Delivery Systems Study Section (GDD)
Program Officer
Howcroft, Thomas K
Project Start
2008-04-01
Project End
2014-01-31
Budget Start
2012-02-01
Budget End
2014-01-31
Support Year
5
Fiscal Year
2012
Total Cost
$275,034
Indirect Cost
$92,212
Name
University of Utah
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
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
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
Dixon, Andrew S; Miller, Geoffrey D; Bruno, Benjamin J et al. (2012) Improved coiled-coil design enhances interaction with Bcr-Abl and induces apoptosis. Mol Pharm 9:187-95
Dixon, Andrew S; Constance, Jonathan E; Tanaka, Tomoyuki et al. (2012) Changing the subcellular location of the oncoprotein Bcr-Abl using rationally designed capture motifs. Pharm Res 29:1098-109
Woessner, David W; Lim, Carol S; Deininger, Michael W (2011) Development of an effective therapy for chronic myelogenous leukemia. Cancer J 17:477-86
Dixon, Andrew S; Pendley, Scott S; Bruno, Benjamin J et al. (2011) Disruption of Bcr-Abl coiled coil oligomerization by design. J Biol Chem 286:27751-60
Mossalam, Mohanad; Dixon, Andrew S; Lim, Carol S (2010) Controlling subcellular delivery to optimize therapeutic effect. Ther Deliv 1:169-93
Dixon, Andrew S; Lim, Carol S (2010) The nuclear translocation assay for intracellular protein-protein interactions and its application to the Bcr coiled-coil domain. Biotechniques 49:519-24

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