Engineering Oncogenic Kinases to Activate Apoptotic Machinery: A Novel Rewiring of Bcr-Abl and PKCβII
Authors: Hoekstra David Arthur , Carlson Louise M , Lee Kelvin P 
Published: October 2019

Oncogenic kinases, such as Bcr-Abl, EGFR, and Src, are crucial for the initiation, maintenance and progression several cancers. Accordingly, these kinases have become a popular target for small molecule based inhibition. However, despite the development of 2nd and 3rd generation inhibitors, resistance to this therapy remains a significant clinical obstacle. Instead of attempting to develop a new inhibitor to block kinase signaling, we hypothesized that we could hijack the oncogenic kinase to activate a rewired ‘pro-drug’, leading to the death or differentiation of the cancerous cell. As a model for this rewiring, we selected Bcr-Abl, an oncogenic kinase which is constitutively expressed and critical for chronic myeloid leukemia (CML) cell survival. For our pro-drug, we chose protein kinase C, isoform βII (PKCβII) whose activation has been shown to increase apoptosis and dendritic cell (DC) differentiation in CML cells. PKCβII is a member of the PKC family of serine/threonine kinases and is in an inactive state in the cytosol due to interactions between its pseudosubstrate and kinase domains. Upon activation, the pseudosubstrate domain releases the kinase domain, and PKCβII translocates from the cytoplasm to the plasma membrane. It has been previously reported that substitution of the alanine at position 25 in the pseudosubstrate domain for a phosphomimetic glutamic acid leads to the constitutive activation of PKCβII. To create a pro-drug from PKCβII, we substituted the alanine at position 25 for a phosphorylatable tyrosine (A25Y) along with the corresponding Bcr-Abl kinase target motif into the pseudosubstrate domain of PKCβII. This allows the Bcr-Abl tyrosine kinase to phosphorylate the introduced tyrosine on PKCβII, thereby activating it through an incorporation of a negative charge on the pseudosubstrate domain. We hypothesize that this PKCβII activation induces apoptosis, inhibits growth, and spurs the differentiation in transformed Bcr-Abl+ leukemic cells, while leaving Bcr-Abl-cells unaffected.

Using molecular modeling techniques, we designed four mutated PKCβII constructs that contain elements of both the native PKCβII pseudosubstrate sequence as well as the Bcr-Abl kinase target motif yet do not affect the native folding of the protein. To determine whether these mutated constructs were activated by Bcr-Abl, we transfected them into both Bcr-Abl+ and Bcr-Abl- cells and used both confocal microscopy and ImageStream technology to assess their location. Transfection of both WT-PKCβII and mutant A25Y-PKCβII into Bcr-Abl- cells resulted in no significant difference in activation and membrane translocation, however when these constructs were transfected into Bcr-Abl+ cells, we observed a significant increase in activation and membrane translocation of mutant A25Y-PKCβII when compared to WT-PKCβII constructs (p<0.05). When Bcr-Abl was expressed in cells that did not previously have it, we saw that there was a significant increase in activation and membrane translocation for mutant A25Y-PKCβII when compared to WT-PKCβII transfected cells (p<0.05). Cells treated with the Bcr-Abl inhibitor imatinib resulted in a significant decrease in activation of A25Y-PKCβII mutant constructs, suggesting that this activation is dependent upon Bcr-Abl activity (p<0.05). We found that this rewiring produced no effect on the constitutive signaling of Bcr-Abl, as there were no significant differences in the levels of phospho-Stat5, phospho-Bcr, and phospho-CRKL between mutant A25Y-PKCβII and WT-PKCβII transfected cells. This rewiring lead to a significant decrease five days post transfection in the number of Bcr-Abl+ cells, with no differences in Bcr-Abl- cells when transfected with A25Y-PKCβII compared to WT-PKCβII (p<0.05). This decrease was found to be due to both a significant increase in apoptosis (p<0.05) and a decrease in proliferation (p<0.05) in A25Y-PKCβII transfected Bcr-Abl+ cells. Lastly, inducible expression of A25Y-PKCβII mutant constructs resulted in a significant decrease in tumor growth in a murine model of CML (p<0.05). These findings demonstrate that Bcr-Abl mediated activation of PKCβII is feasible and induces death in leukemic cells, and provides a novel mechanism by which oncogenic kinases can be re-wired to activate pro-death agents and can have broad therapeutic applications.

Disclosures

No relevant conflicts of interest to declare.