Abstract 3144: shRNA library screening by dual shRNA technique identifies CHEK1 as a synthetic lethal gene in the absence of IKKε in ovarian cancer.
Authors: Kim Marianne K H , Wright George , Staudt Louis , Annunziata Christina 
Published: August 2014

Background: IKKε was previously identified as an oncogene in breast cancer and was associated with poor clinical outcome in ovarian cancer. Recently, we demonstrated that IKKε is a key regulator of invasion and metastasis programs in ovarian cancer. Therefore, we developed a rapid and robust dual shRNA technique to create IKKε-matched cell line pairs and performed synthetic lethality screens to identify the key molecular targets influenced by the loss of IKKε expression in ovarian cancer.

Material and methods: To create an IKKε-matched cell line pair, we utilized a retroviral bicistronic vector containing the transmembrane and extracellular domains of the mouse T-cell surface glycoprotein CD8 alpha, co-expressed with either IKKε or negative control shRNAs. The positively transduced cells were isolated using mouse CD8a-specific microbeads. After recovery from beads purification, the human kinome shRNA library, containing a puromycin selectable marker fused to GFP, was introduced. To identify shRNAs that sensitized ovarian cancer cells to IKKε depletion, we harvested each shRNA library infected control and IKKε- depleted cells at two time points. This screening was performed in two different ovarian cancer cell lines, one with and one without p53 mutation.

Results: We prioritized the genes consistently depleted at two different time points, depending on their doubling times (A2780 at Days 4 and 7, Ovcar5 at Days 7 and 14). In order to generalize this synthetic lethality to ovarian cancers expressing a high level of IKKε, we first focused on the genes identified in both ovarian cancer cell lines, yielding 29 candidate genes. We examined the expression levels of these 29 candidate genes in The Cancer Genome Atlas data portal containing more than 500 ovarian serous cystadenocarcinoma. Most of these 29 genes were overexpressed in a subset of TCGA ovarian cancers. Strikingly, CHEK1 was overexpressed by more than 2 fold in 98% of TCGA ovarian cancers. The combination knockdown with IKKε and CHEK1 resulted in a further decrease in cellular proliferation and a further increase in G0/G1 arrest compared to individual depletion of either gene. Consistently, we observed the same biological effect using pharmacological inhibitors of IKKε (BX795) and CHEK1 (PF00477736).

Conclusion: Our dual shRNA technique efficiently identified CHEK1 as an IKKε synthetic lethal gene in ovarian cancer. Identification of IKKε dependent signaling will lead to development of context-specific therapeutics in the poor prognostic group of patients with a high level of IKKε expression.

Citation Format: Marianne K H Kim, George Wright, Louis Staudt, Christina Annunziata. shRNA library screening by dual shRNA technique identifies CHEK1 as a synthetic lethal gene in the absence of IKKε in ovarian cancer. [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 3144. doi:10.1158/1538-7445.AM2013-3144