Capture of BCR-ABL for induction of apoptosis in chronic myelogenous leukemia: from inhibition to nuclear escort

The primary cause of chronic myelogenous leukemia (CML) is the presence of the Philadelphia chromosome, resulting in the oncoprotein Bcr-Abl. The constitutive tyrosine kinase activity of Bcr-Abl, localized intracellularly in the cytoplasm, activates numerous oncogenic signaling pathways. Conversely, Bcr-Abl trapped in the cell nucleus induces apoptosis. This work is aimed at turning the oncogenic Bcr-Abl signaling into apoptotic signaling through redirecting Bcr-Abl, found only in the cancerous cells, to the nucleus. First, this work validated that Bcr-Abl could be directed to the nucleus with nuclear localization signals, and that nuclear Bcr-Abl does induce apoptosis. Ectopic expression of a nuclear localized Bcr-Abl construct in CML cells activated apoptotic signaling. Next, this worked focused on the design and isolation of Bcr-Abl binding domains. The first approach was the rational design of the Bcr-Abl coiled-coil domain which led to identification of mutations that could be made to enhance the oligomerization properties with Bcr-Abl. A nuclear translocation assay was then developed for studying protein interactions inside a live cell, and confirmed the modifications to the coiled-coil domain improved the binding to the wild-type coiled-coil domain representative of Bcr-Abl. In addition to the application to translocating Bcr-Abl iv to the nucleus, the improved oligomerization domain (CCmut2) was found to function as an inhibitor of Bcr-Abl. A second generation modification to the Bcr-Abl coiled-coil domain, CCmut3, was found to exhibit even greater binding than CCmut2 and also inhibited Bcr-Abl. As an alternative binding approach we used the intracellular antibody capture (IAC) methodology to isolate single domain antibodies (iDabs). Two Bcr-Abl regions with potential implications in the cytoplasmic retention of Bcr-Abl were targeted in the iDabs screens, the actin binding domain and Dbl homology/Pleckstrin homology domains. These screens produced ABI7, and when co-expressed with Bcr-Abl, caused a redistribution of the normal Bcr-Abl localization pattern. Both CCmut3 and ABI7 were tested for their ability to translocate Bcr-Abl into the nucleus and together were found to efficiently redirect Bcr-Abl to the nucleus. The culmination of this work is an established method to efficiently redirect Bcr-Abl from the cytoplasm into the nucleus where it is known to induce apoptosis.