ENHANCED AND SELECTIVE KILLING OF CHRONIC MYELOGENOUS LEUKEMIA CELLS WITH AN ENGINEERED BCR-ABL BINDING PROTEIN AND IMATINIB

摘要

The oncoprotein Bcr-Abl stimulates pro-survival pathways and suppresses apoptosis from its exclusively cytoplasmic locale, but when targeted to the mitochondrial compartment of leukemia cells, Bcr-Abl was potently cytotoxic. Therefore, we designed a protein construct to act as a mitochondrial chaperone to move Bcr-Abl to the mitochondria. The chaperone (i.e., the 43.6 kDa intracellular cryptic escort (iCE)) contains an EGFP tag and two previously characterized motifs: 1) An optimized Bcr-Abl binding motif that interacts with the coiled-coil domain of Bcr (ccmut3; 72 residues), and 2) A cryptic mitochondrial targeting signal (cMTS; 51 residues) that selectively targets the mitochondria in oxidatively stressed cells (i.e., Bcr-Abl positive leukemic cells) via phosphorylation at a key residue (T193) by protein kinase C. While the iCE colocalized with Bcr-Abl it did not re-localize to the mitochondria. However, the iCE was selectively toxic to Bcr-Abl positive K562 cells as compared to Bcr-Abl negative Cos-7 fibroblasts and 1471.1 murine breast cancer cells. The toxicity of the iCE to leukemic cells was equivalent to 10μM imatinib at 48 hours and the iCE combined with imatinib potentiated cell death beyond imatinib or the iCE alone. Substitution of either the ccmut3 or the cMTS with another Bcr-Abl binding domain (derived from Ras/Rab interaction protein 1 (RIN1; 295 residues)) or MTS (i.e., the canonical IMS derived from Smac/Diablo; 49 residues) did not match the cytotoxicity of the iCE. Additionally, a phosphorylation null mutant of the iCE also abolished the killing effect. The mitochondrial toxicity of Bcr-Abl and the iCE in Bcr-Abl positive K562 leukemia cells was confirmed by flow cytometric analysis of 7-AAD, TUNEL, and annexin-V staining. DNA segmentation and cell viability were assessed by microscopy. Subcellular localization of constructs was determined using confocal microscopy (including statistical colocalization analysis). Overall, the iCE was highly active against K562 leukemia cells and the killing effect was dependent upon both the ccmut3 and functional cMTS domains.