CTP-OD-HA Fusion Peptide Reversed The Malignant Phenotypes Of CML Cells Through Inhibition Of BCR-ABL Homodimerization

Chronic myeloid leukemia (CML) is characterized by the persistentexpression of the BCR-ABL fusion protein with constitutive tyrosine kinase activity. The BCR-ABL oncoprotein can activate multiple oncogenic signal transduction pathways and plays a critical role in the pathogenesis and progression of CML through inhibition of cellular differentiation and apoptosis, as well as promotion of proliferation and deregulated adhesion that eventually lead to malignant transformation. In the recent years, with the advent of the era of small molecular inhibitors, Imatinb, Dasatinib and Nilotnib have greatly advanced the therapeutic effects on CML, however, drug resistance and disease relapse inevitablly emerged due to their inability to kill the malignant clones in CML. As a result, further exploration for new targeted therapeutic targets in CML is urgently needed.The BCR-ABL homodimerization mediated by its N-terminal oligomerization (OD) domain is the critical factor for both its tyrosine autophosphorylation, molecular configulation transformation, as well as the deregulated Abl tyrosine kinase activity. We thus speculated that transduction of an exogenous OD domain into the CML cells may be a novel CML treatment strategy through inhibition of BCR-ABL tyrosine kinase by intefering with the its homodimerization. Recently, protein drugs have been finding its way into clinical practice, however, protein drug-based cellular therapeutic strategy is being limited by the fact that macromolecules can not penetrate cellular membranes and that many proteins need accurate subcelluar localization to exert their functions.Cytoplasmic transduction peptide (CTP) is a recently reported novel transduction peptide that can carry macromolecules to penetrate the cellular membranes with a preference to localize into the cytoplasmic compartment, and is therfore applicable for the transduction of cytoplasmic protein peptides. Given that the designed OD peptide in the present study is aimed to competitively inhibit the homodimerization of the cytoplasmic BCR-ABL oncoprotein, the CTP system fulfills an appropriate transduction system for the OD domian.The present study was launched to construct, express and purify the cytoplasmic localizing CTP-OD-HA fusion peptide (HA tag was introduced to further facilitate immunoblot detection), and to further investigate its potential effects on cellular growth, proliferation and apoptosis in both the various in vitro cultured BCR-ABL positive CML cells and the in vivo BP210-Babl/C CML model mice. It is hoped that the CTP-OD-HA fusion peptide will exert its anti-CML effect through its inhibition of both the homodimerization of the BCR-ABL oncoprotein and the resultant inhibition of tyrosine kinase activity 'and that it can be exploited as a novel targeted molecular therapeutic strategy for CML. The main experiments arepresented as follows:1. The cloning, prokaryotic expression, purification and biological activity analysis for the CTP-OD-HA fusion protein: The OD, HA and CTPfragments were sequentially cloned into the prokaryotic expression vector pET32a(+) followed by double enzyme digestions and bi-directional sequencing. The correctly constructed pCTP-OD-HA recombinant plasmid was further transduced into the E.coli. BL-21(DE3) expression bacteria and IPTG was added subsequently to induce the expression of the CTP-OD-HA fusion protein. The expression products were further identified by bothSDS-PAGE and western blot; The histidine affinity chromatography wasemployed to purify the target protein; The transduction potential of the FITC-CTP-OD-HA in CML cells was detected under the fluorescence microscopy after it was desalted, cleaved by enterokinase, reclaimed by asecond affinity purification process and labelled by FITC ;Immunocytochemical staining and confocal microscopy were performed to observe the subcellular localization of the transduced fusion protein in CML cells; Western blot was employed to further evaluate the transduction potential of the CTP-OD-HA fusion protein in CML cells in service of the goal of exploration of the therapeutic effect of the CTP-OD-HA fusion protein in CML.2. To observe the potential specific biological effects of CTP-OD-HA, such as proliferation inhibiting and apotosis inducing effects on CML cells and to further probe into its molecular mechanisms.3. To construc the Balb/C CML model mice transplanted by the BCR-ABL transformed BP210 hematological cells, and to further evaluate and validate its features in respect of its hemetological and organ infiltration characteristics as well as the repeatability of model construction followed by the observation of the protective effect of its transduction on CML model mice.All the following results and conclusions were reached after the aboveexperiments:1. CTP-OD-HA fusion protein can be induced to express in a soluble form at a high efficency by 1mM IPTG at 37℃for 4h; High purity CTP-OD-HA fusion protein was acquired after Ni-NTA affinity purification; CTP-OD-HA fusion protein was proved to have potently transduced into CML cells after it was desalted, enterokinase cleaved and FITC labelled ; The cytoplasmic localization preference of the CTP-OD-HA fusion protein was validated by Immunocytochemical staining and confocal microscopy; It's potent transduction potential in various CMLcells were further confirmed by Western blot. All the above results indicated that the CTP-OD-HA fusion gene can be induced to express in a soluble form at high efficiency in the pET expression systems and the resultant fusion protein showed excellent biological activities, thus laying a sound foundation for further evaluation of the biological functions of the CTP-OD-HA fusion peptide.2. The purified CTP-OD-HA fusion protein was demonstrated to have BCR-ABL specific proliferation inhibiting and apotosis inducing effects onCML cells after its transduction; Further exploration into the underlyingmechanisms revealed that CTP-OD-HA inhibited both the homodimerization, tyrosine phosphorylation and oncogenic tyrosine kinase pathway triggered by the BCR-ABL oncoprotein through its competitive binding to the BCR-ABL oncoprotein after it was transduced into CML cells. The transduced CTP-OD-HA peptide was also shown to induce the expression of proapototic genes without altering the cytoplasmic localization of the BCR-ABL oncoprotein. All the above results indicated that the inhibition of homodimerization of the BCR-ABL oncoprotein through exogenous transduction of the OD domain can be developed as a potential anti-CML therapeutic strategy. 3. The Balb/C CML model mice transplanted by the BCR-ABL transformed BP210 hematological cells were successfully constructed and validated, and further in vivo experiments showed that the pre-transduction of CTP-OD-HA fusion protein could not only significantly inhibit the leukemogenic potential of CML cells in the recipient mice, but also prolong the survival time of the CML model mice.