Enhanced and Selective
Killing of Chronic Myelogenous Leukemia Cells with an Engineered BCR-ABL
Binding Protein and Imatinib
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Abstract
The oncoprotein Bcr-Abl stimulates prosurvival 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 relocalize
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 10 μM imatinib at 48 h 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