Inhibition of native and mutant BCR-ABL using a rationally designed coiled-coil

Chronic myeloid leukemia (CML) is caused by the constitutive kinase activity of the fusion oncoprotein BCR-ABL. Conventional therapy in CML utilizes tyrosine kinase inhibitors (TKIs), small molecules that target the ATP-binding pocket in the BCR-ABL kinase domain. Despite their success in treating this disease, continued use of TKIs can lead to drug resistance due to point mutations in the kinase domain. Additionally, non-specific (non-BCR-ABL) kinase inhibition by these TKIs can cause toxic off-target effects. To function as an aberrant kinase, BCR-ABL must first homo-oligomerize via a coiled-coil (CC) domain located at its N-terminus. Thus, inhibiting BCR-ABL oligomerization abolishes its function as an oncoprotein. Designing an inhibitor of the 72-amino acid BCR-ABL CC domain is the focus of this dissertation. To engineer a construct capable inhibiting oligomerization, strategically designed mutations were incorporated into an isolated BCR-ABL CC domain with the goal of promoting higher affinity binding to endogenous BCR-ABL while at the same time disfavoring binding to our isolated CC construct. The designed construct, called CCmut3, was tested in vitro in leukemia cells containing both wild-type and mutant BCR-ABL. Overall, in vitro treatment with CCmut3 resulted in a decrease in BCR-ABL kinase activity, induction of apoptosis, and a reduction in the proliferation and transformative ability of CML cells. Next, combining CCmut3 with ponatinib, a recently approved BCR-ABL TKI, was also explored. This combination resulted in improved BCR-ABL inhibition and a lowering of the dose of ponatinib necessary for efficacy. Finally, the later chapters in this dissertation focus on possible methods in which the deliverability and stability of CCmut3 can be improved. Truncation and helical capping were both attempted, however, neither provided an inhibitory advantage over the full-length CCmut3 construct. Thus, current designs are focusing on creating a hydrocarbon stapled and truncated CCmut3 peptide, expected to result in a translatable product for wild-type and therapy-resistant CML.