Interaction of a Novel Signaling Protein (AHI-1) with BCR-ABL Modulates BCR-ABL Transforming Activity and Imatinib Response of Chronic Myeloid Leukemia Progenitor Cells.

Ahi-1 (Abelson helper integration site 1) is a novel gene that was initially identified by provirus insertional mutagenesis in v-abl-induced murine pre-B cell lymphoma as a candidate cooperate oncogene. The Ahi-1 protein has a SH3 domain, multiple SH3 binding sites and WD-repeat domains, suggesting novel signaling activities. We recently demonstrated that Ahi-1/AHI-1 transcript levels are highly deregulated in human leukemic cells, particularly in highly enriched populations of BCR-ABL⁺ leukemic stem cells from patients with chronic myeloid leukemia (CML) where levels of BCR-ABL transcripts are also elevated. Interestingly, overexpression of Ahi-1 alone in primitive murine hematopotic cells confers a proliferative advantage in vitro and induces a lethal leukemia in vivo; these effects can be enhanced by BCR-ABL, a fusion oncogene that drives CML pathogenesis. To investigate whether deregulated expression of AHI-1 may directly contribute to BCR-ABL-mediated transformation and altered signaling pathways in human CML, knockdown of AHI-1 expression in K562 cells, a cell line that was derived from a patient with CML and that is characterized by highly increased expression of AHI-1, was performed using retroviral-mediated RNA interference. Retroviral-mediated suppression specifically inhibited endogenous AHI-1 expression in transduced cells by 70% as evaluated by Q-RT-PCR and Western blot analyses. It further caused a significant reduction in their growth factor independence in semi-solid cultures (up to 5-fold, P< 0.01) and in single cell cultures (2-fold, P< 0.05) by comparison to cells transduced with a control vector. Importantly, lentiviral-mediated suppression of AHI-1 in BCR-ABL transduced primitive human cord blood (CB) cells (lin⁻CD34⁺) further showed a significant reduction of up to 10-fold (P< 0.01) in colony-forming cell (CFC) output and up to 5-fold (P< 0.01) in liquid suspension cultures as compared to BCR-ABL transduced cells alone. In addition, suppression of AHI-1 expression also resulted in a reduction in CFC output in lin⁻CD34⁺ stem/progenitor cells from CML patients (n=3). Strikingly, co-expression of Ahi-1 in BCR-ABL inducible BaF3 cells immediately reverses growth deficiencies exhibited by down-regulation of BCR-ABL in both semi-solid cultures (>50-fold) and in liquid suspension cultures in the absence of IL-3 (>30-fold), and is associated with sustained phosphorylation of BCR-ABL and enhanced activation of JAK2/STAT5. Moreover, a direct physical interaction between AHI-1 and BCR-ABL at endogenous levels has now been identified in CML cells; this interaction complex is also associated with a 120 kDa tyrosine phosphorylated protein. In addition, significant changes in imatinib (IM, an inhibitor of the BCR-ABL) response have been further demonstrated in Ahi-1 transduced BCR-ABL inducible BaF3 cells (P< 0.01) and in AHI-1 suppressed CML stem/progenitor cells (n=3). These findings provide strong evidence that Ahi-1/AHI-1 interacts with BCR-ABL to form an interaction complex and that this interaction complex further modulates altered BCR-ABL signaling and transforming activities of CML progenitor cells, including their response to IM.