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Functional and structural study of the AHI-1 SH3 domain, characterization of the BCR-ABL-AHI-1-Dynamin-2 protein complex and investigation of oncogenic roles of dynamin-2 in chronic myeloid leukemia

University of British Columbia

Tyrosine kinase inhibitor (TKI) therapies have been introduced into clinical practice with remarkable effects on chronic myeloid leukemia (CML). However, early relapse, acquired drug resistance and persistence of leukemic stem cells (LSCs) remain problematic. Improved treatments specifically targeting key molecular elements active in CML LSCs are needed. One candidate is the oncoprotein AHI-1 (Abelson helper integration site-1), which is highly deregulated in LSCs. It harbors two key domains, SH3 and WD40-repeat, which are known important mediators of protein-protein interactions. An AHI-1-mediated protein complex containing BCR-ABL and JAK2 has been shown to modulate transforming activity and TKI-response/resistance of CML LSCs. In this study, I investigated the functional roles of the AHI-1 SH3 domain in regulation of cellular resistance of primitive CML cells to TKIs. I showed that deletion of the SH3 domain of Ahi-1 significantly enhanced apoptotic response of BCR-ABL⁺ cells to TKIs compared to cells expressing full-length Ahi-1. I solved the crystal structure of the AHI-1 SH3 domain and identified several unique features, providing potential target sites for designing specific drugs. Using immunoprecipitation/mass spectrometry, I identified a novel protein interaction between AHI-1 and Dynamin-2 (DNM2), a GTPase, through the AHI-1 SH3 domain. I showed that DNM2 expression was significantly upregulated in CML stem/progenitor cells compared to normal bone marrow cells. I also determined that the AHI-1 SH3 domain and the proline rich domain of DNM2 were mainly responsible for their interaction. Most importantly, I identified a novel protein complex in CML cells, containing BCR-ABL, AHI-1 and DNM2. Furthermore, I demonstrated an oncogenic role of DNM2 in primitive CML cells by showing that knockdown of DNM2 greatly impaired the survival of CML stem/progenitor cells and sensitized them to TKI treatments. Lastly, I illustrated that DNM2 might be involved in deregulation of endocytosis, ROS production and autophagy in TKI-insensitive CML stem/progenitor cells. This study detailed the identification and characterization of the newly-identified BCR-ABL-AHI-1-DNM2 protein complex and described the oncogenic functions of DNM2 in primitive CML cells. It further suggested that targeting DNM2 may facilitate eradication of LSCs as a new treatment option in CML.

Text

2017-01-21

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/60191

Experimental Medicine

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/