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.