Abstract 2170: Development of a "dual-hit" combination therapy for CML.
Authors: Miller Geoffrey D. , Woessner David W. , Lim Carol S. 
Published: August 2014

Functioning as a constitutively active tyrosine kinase, the oncoprotein Bcr-Abl, the causative agent of chronic myeloid leukemia (CML), requires homo-oligomerization via a coiled-coil domain present on the N-terminus of the Bcr portion of the protein. Currently, small molecule therapeutics that target the tyrosine kinase domain (tyrosine kinase inhibitors, or TKIs) are administered as the gold standard treatment for CML. However, evidence shows that the acquisition of mutations in the tyrosine kinase domain against these small molecules is inevitable, rendering many of the therapeutics useless against mutated forms of Bcr-Abl. The most recently discovered TKI, ponatinib (AP24534), has shown to combat nearly all of the commonly seen mutations, including the previously uncontrollable T315I mutation. But, ponatinib is not without limitations, as it has been shown to cause dose-dependent pancreatitis and thrombocytopenia as adverse side effects. While many small molecules have been created to target the tyrosine kinase domain, our recent work has shown that disruption of Bcr-Abl oligomerization can also induce apoptosis and decrease the oncogenic potential of the disease. This work was done using a modified coiled-coil domain, termed CCmut3, which was rationally designed and mutated to bind to the endogenous Bcr-Abl coiled-coil domain, inhibiting oligomerization and thus preventing Bcr-Abl signaling. The purpose of this work is to exploit a “dual-hit” approach to inhibit Bcr-Abl using two therapeutics with different mechanisms of action. Here, we show that combining CCmut3 with ponatinib not only increases the potency of the therapy but also lowers the required therapeutic dose of ponatinib in vitro using K562 cells (Bcr-Abl-positive, human CML cells). Western blot analysis shows that both Bcr-Abl phosphorylation and phosphorylation of downstream signaling targets STAT5 and CrkL can be decreased using the combination at a ponatinib dose of 100 pM. In addition, transformative ability (via colony forming assay) very significantly declines when using a 1 nM dose in combination as opposed to simply administering 1 nM ponatinib only. Finally, caspase 3/7, 7AAD, and DNA segmentation assays were performed to show enhanced apoptosis induction upon combination treatment when compared with either CCmut3 or ponatinib treatment alone. Currently, this combination is being tested in a Bcr-Abl-positive Ba/F3 cell line containing the T315I mutation in the Abl tyrosine kinase domain. Lastly, attempts to truncate and cap the CCmut3 helix to provide stability and more simple therapeutic delivery are underway, in addition to elucidating the individual intracellular mechanisms of action of both CCmut3 and ponatinib.

Citation Format: Geoffrey D. Miller, David W. Woessner, Carol S. Lim. Development of a "dual-hit" combination therapy for CML. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2170. doi:10.1158/1538-7445.AM2013-2170