Abstract C74: Reassessing IKKε as a novel oncology target

Abstract

Abstract Recent years have seen increasing efforts to employ integrative genomic approaches to identify novel oncogenes. In this manner, IKBKE coding for IKKε was recently proposed as a novel oncogene, aberrantly activating the NF-κB signaling pathway in breast cancers (Boehm et al. Cell. 2007). We pursued a series of experiments that delve more deeply into the hypothesis of IKKε as a novel oncology target; the resulting evidence challenges the proposed oncogenic mechanism. We conducted RNA interference studies on mutiple breast cancer cell lines with multiple IKBKE siRNA motifs to define a general on-target phenotype. Consistent with published findings, we confirmed that although 4 IKBKE siRNA motifs tested significantly reduced IKKε protein expression (78–90% silencing) only 2 of these motifs significantly inhibited cell growth. We then engineered ‘non-silenceable’ IKBKE constructs packaged in BacMam virus and were able to simultaneously silence the endogenous IKBKE and exogenously express the non-silenceable IKKε at protein levels below, equivalent to, and significantly above the endogenous levels. We demonstrated that the antiproliferative effects of these 2 siRNAs could not be rescued by functional, exogenously expressed IKBKE, indicating likely off-target toxicity. We further explored the correlations between IKBKE gene amplification, IKKε protein overexpression, and NF-κB pathway activation using RNAi in conjunction with an NF-κB -luciferase reporter assay. We observed that IKBKE amplification correlated poorly with both IKKε protein expression and NF-κB pathway activation. Furthermore, silencing downstream components of the NF-κB signaling pathway had little impact on the IKBKE-amplified cells; and conversely, silencing IKBKE had no impact on NF-κB -reporter activity in the IKBKE-amplified cells. In addition, we over-expressed the kinase-dead IKKε (K38A), and observed no effect on tumor cell growth. Finally, using BacMam virus to serially titrate IKK , we confirmed that the level of exogenous IKKε needed to achieve a modest pathway activation (2-fold increase in NF-κB -luciferase reporter assay) was >10 times the endogenous protein level found in an IKBKE-amplified breast cancer line. We also undertook a limited medicinal chemistry effort to arrive at several potent small molecule tool inhibitors of both IKK and TBK1 including GSK2292978A (8nM & 1nM respectively). GSK2292978A confirmably inhibited IKKε activity in a cellular mechanistic assay (IC50∼170nM). Using a 3-day proliferation assay, we observed no evident selectivity of GSK2292978A for IKBKE-amplified or IKKε overexpressing cell lines compared to non-amplified cell lines. These findings resulted in our conclusion that IKKε likely does not represent a true oncogene in breast cancer and suggest the vital need for well-designed controls in cases where RNA interference significantly defines target validation. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C74.