Abstract 3852: Improved coiled-coil design enhances interaction with Bcr-Abl and induces apoptosis

Abstract

Abstract The fusion protein Bcr-Abl responsible for the oncogenic activity in chronic myelogenous leukemia (CML) depends heavily on oligomerization for function. Formation of Bcr-Abl oligomers occurs through a coiled-coil domain existing at the N-terminal portion of the Bcr segment. Previously, we have shown that disruption of this oligomerization utilizing a modified coiled-coil domain (CCmut2) both decreases the proliferation of CML cells and results in the induction of apoptosis. An important contributing factor to these results stems from the increased destabilization of CCmut2 homo-dimers. This phenomenon occurs due to the rationally mutated residues in the modified coiled-coil which allow for favorable interactions with the wild-type Bcr-Abl, but destabilizing interactions with other CCmut2 molecules, thus discouraging the formation of CCmut2 homo-dimers. In turn, this increases the availability and affinity for the modified coiled-coil to interact with and inhibit endogenous Bcr-Abl in CML cells. An additional mutation (K39E) was included that could potentially further destabilize the mutant homo-dimer. Added to the other mutations found in CCmut2 (C38A, S41R, L45N, E48R, Q60E), the new construct was termed CCmut3. To show that CCmut3 results in enhanced binding to a wild-type coiled-coil domain (representative of endogenous Bcr-Abl), 2 separate assays were performed. Colocalization studies were used to show the degree to which CCmut3 localizes intracellularly with Bcr-Abl. Additionally, a mammalian two-hybrid assay was conducted to show the relative selective dimerization between CCmut3 and the Bcr-Abl coiled-coil. Next, caspase 3/7 assays were performed and showed the ability of CCmut3 to induce apoptosis. The CCmut 3 also showed nuclear segmentation (a measurement of late-stage apoptosis) and decrease in oncogenic proliferation (via the colony forming assay). Taken together, these results provide strong evidence for using CCmut3 as a therapeutic for CML. Lastly, attempts to truncate the CCmut3 to find the smallest possible sequence needed (e.g., helix 2) to bind and kill cells effectively is currently underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3852. doi:1538-7445.AM2012-3852