Molecular and Structural Characterization of the SH3 Domain of AHI-1 in Regulation of Cellular Resistance of BCR-ABL⁺ Chronic Myeloid Leukemia Cells to Tyrosine Kinase Inhibitors

Abstract 966

Chronic myeloid leukemia (CML) is a clonal multilineage myeloproliferative disorder characterized by the presence of the fusion gene BCR-ABL with increased tyrosine kinase activity. Imatinib mesylate (IM) and other BCR-ABL tyrosine kinase inhibitors (TKIs), including dasatinib (DA) and nilotinib (NL), have been introduced into clinical practice with remarkable effects on chronic phase CML. However, early relapses, acquired drug resistance, and persistence of leukemic stem cells remain problematic. Improved treatment approaches to target other key molecular elements active in CML stem/progenitor cells are needed. One candidate is AHI-1 (Abelson helper integration site 1), an oncogene that is highly deregulated in CML leukemic stem cells. It harbors two key domains, SH3 and WD40-repeat, which are known important mediators of protein-protein interactions. We recently demonstrated that AHI-1 physically interacts with BCR-ABL and JAK2 in CML cells and this interaction complex mediates transforming activity and TKI response/resistance of CML stem/progenitor cells. We have also shown that AHI-1 interacts independently with JAK2 and BCR-ABL via different binding sites to mediate their activities. In this study, we have characterized the biological and structural functions of the SH3 domain of AHI-1. To determine roles of the SH3 domain in regulation of cell proliferation and TKI response/resistance, several mutant forms, including SH3 domain deletion (SH3^Δ), double WD40-repeat and SH3 domain deletion (SH3WD40^Δ) and N-terminal deletion (N-ter^Δ, containing SH3 and WD40-repeat domains) were generated and stably transduced into BCR-ABL inducible BaF3 cells, in which the level of expression of BCR-ABL can be down-regulated by exposure to doxycycline. Overexpression of full-length Ahi-1 in BCR-ABL inducible cells resulted in fewer Annexin V+ apoptotic cells with doxycyclin (suppression of BCR-ABL) compared to BCR-ABL inducible cells (3 and 29% v.s.10 and 60% after 24 or 48 hours). Cells expressing the SH3^Δ mutant and the SH3WD40^D mutant displayed dramatically increased Annexin V+ cells (10, 77% and 34, 90% v.s.3 and 29%), while cells expressing the N-ter^Δ mutant had similar numbers of Annexin V+ cells compared to BCR-ABL inducible cells (6 and 41% v.s.10 and 60%). Similarly, BCR-ABL⁺ cells transduced with SH3^Δ and SH3WD40^D mutants displayed significantly increased apoptotic cells compared to cells transduced with full-length Ahi-1 in the presence of 2 μM IM (57, 87 vs. 26%), 2μM NL (65, 87 vs. 25%) and 150 nM DA (63, 96 vs. 34%) after 24 hour treatment. BCR-ABL⁺ cells transduced with the N-ter^δ mutant also showed more sensitivity to the drug treatments compared to the cells with the full-length Ahi-1(36% for IM, 40% for NL and 40% for DA), but with lower sensitivity than cells carrying the Ahi-1 SH3 domain deletion mutants, indicating that the SH3 domain of Ahi-1 plays a role in the mediation of TKI resistance. The crystal structure of the AHI-1 SH3 domain at 1.32-Å resolution revealed that the AHI-1SH3 domain adopts a canonical SH3 folding, but with an unusual C-terminal α helix. There are three large negatively charged patches, which are constructed by the n-Src loop, the end of the RT loop and the C-terminal helix, and this special feature may be involved in binding selectivity and specificity. PD1R peptide, known to interact with the PI3K SH3 domain, was used to model the binding pattern between AHI-1 SH3 domain and its ligands, and there may be formation of an “Arg-Arg-Trp” stack within the binding interface, which could be a targeting site for designing specific drugs. Moreover, using the AHI-1 SH3 domain as protein ‘bait' in immunoprecipitation/mass spectrometry, Dynamin-2 was identified as a potential interacting partner of AHI-1; both AHI-1 and Dynamin-2 are involved in trafficking and signaling processes. In conclusion, the investigation of the structure of AHI-1 SH3 domain and its interacting proteins will thus provide invaluable insight in identification of key interaction sites in regulation of drug resistance and may be utilized for development of small molecule inhibitors for CML.