Identification of AHI-1 Involvement in Mediating Cellular Resistance to Imatinib Mesylate in Chronic Myeloid Leukemia Cells

Chronic myeloid leukemia (CML) is a clonal multilineage myeloproliferative disorder arising from the neoplastic transformation of a pluripotent hematopoietic stem cell that acquires a unique BCR-ABL fusion gene. The BCR-ABL oncoprotein displays constitutively elevated tyrosine kinase activity that deregulates cellular proliferation and apoptosis control through effects on several common signal transduction cascades, including the PI3K/AKT, JAK2/STAT5, and NF-kB pathways. The current first line treatment for CML involves administration of the tyrosine kinase inhibitor imatinib mesylate (IM) that has shown promise in treating chronic phase CML patients. However, early relapses and IM-resistant disease have emerged and are frequently associated with mutations in the BCR-ABL kinase domain that affect inhibitor binding. AHI-1 (Abelson helper integration site 1) is a recently discovered oncogene that has been demonstrated to be highly deregulated in a CML cell line (K562) and in primary leukemic stem/progenitor cells from CML patients. AHI-1 contains several unique domains that are indicative of signalling functions, including both an SH3 and a WD40-repeat domain. We have recently demonstrated that overexpression of murine Ahi-1 is able to transform IL-3 dependent Baf3, resulting in cells able to grow in the absence of growth factors. When these transduced cells were injected into sublethally irradiated NOD/SCID immunodeficient mice, the mice developed leukemia, demonstrating the oncogenic properties of Ahi-1. Interestingly, these in vitro and in vivo effects can be enhanced by co-transduction of BCR-ABL in these cells. In addition, a direct interaction between AHI-1 and BCR-ABL at endogenous levels was identified in K562 cells and this protein interaction complex further mediated IM response/resistance in CML stem/progenitor cells. To further investigate AHI-1’s involvement in mediating this cellular resistance to IM, AHI-1 was either stably overexpressed in K562 cells by transduction of EF1a-AHI-1-IRES-YFP lentivirus or was suppressed in K562 cells using a lentiviral-mediated RNA interference approach. Interestingly, overexpression of AHI-1 in K562 cells significantly increased cellular survival in the presence of 1, 5 and 10 uM of IM as measured by a viability assay; survival of these cells was similar to that observed in an IM resistant K562 cell line reported to be highly resistant to IM in vitro. Furthermore, suppression of AHI-1 had the opposite effect, with cells displaying heightened sensitivity to IM at concentrations as low as 1 uM. Phosphorylation and protein expression levels of several proteins known to be involved in BCR-ABL signalling, including JAK2, STAT5, AKT and NF-kB (P105, P50, and P65 subunits), were then quantified by Western blot analysis. Interestingly, elevated phosphorylation and protein expression levels of JAK2, and STAT5 and total protein expression levels NF-kB p105/p55 subunits were observed in both the AHI-1 overexpressing K562 cells and IM resistant K562 cells, while reduced phosphorylation and protein expression of these same proteins was observed in AHI-1 suppressed K562 cells. Differential expression of phosphorylated NF-kB p65 subunit at serine 536 was observed, while total protein expression levels did not significantly differ. Phosphorylated AKT expression levels were only affected in AHI-1 suppressed K562 cells, and total AKT protein expression was not affected in AHI-1 overexpressed or suppressed cells. Interestingly, AHI-1 protein expression was highly elevated at endogenous levels in the IM resistant K562 cells relative to a parental K562 cell line. These findings suggest that AHI-1 may play an important role in mediating cellular resistance to IM through activation of several signalling proteins involved in BCR-ABL signalling pathway, including JAK2 and STAT5.