Abstract 4088: The functional mechanism of Clk2 supporting the development of chemoresistance in non-BRCA1 deficient ovarian cancer

Abstract

Abstract Background: Platinum is the most commonly used drug for the treatment of ovarian cancer. However, resistance to platinum has also become a serious clinical problem. The mutation of BRCA (BRCA1/2) in ovarian cancer, is the major cause of the occurrence of tumors. Our previous microarray data showed that expression level of CDC-like kinase (Clk2) was higher in ovarian cancer tissues than the compared normal tissues. As a protein kinase, Clk2 phosphorylates and activates BRCA1. Methods: 1.Quantitative PCR and IHC were conducted to verify the expression of Clk2 in normal ovarian and tumor tissues.2.The WB was used to examine the correlation between Clk2 protein level and the time and concentration of cisplatin, as well as the protein expression of Clk2 in the platinum sensitive and resistant cell strains of ovarian cancer.3.After knocking down Clk2, the IC50 of cisplatin, the DNA damage level and the apoptosis ratio of drug-resistant cell were examined.4.Tumor formation model was made in nude mice to test the tumor size of drug-resistant cell.5.Protein profiling was analysed to screen proteins that bind to endogenous Clk2 in ovarian cancer cells. Co-IP was used to prove the binding of Clk2 to BRCA1 and to detect the relationship between their binding levels and the time and concentration gradient of platinum.6.The effect of Clk2 overexpression on the activation of DNA damage-related proteins was detected by WB.7.IHC was used to observe of changes of p-BRCA1 and formation of DNA damage repair focus after knockout of Clk2.8.The correlation between p-BRCA1 expression and Clk2 in ovarian cancer was demonstrated via IHC.9.The recombinant kinase experiment was conducted to analyze the ability of the Clk2 protein kinase to activate BRCA1.10.The related pathway inhibitors were used to preliminarily screen for the signaling pathways in which cisplatin induced upregulation of Clk2 and validate.11.The "synergistic lethal effect" of the combination of Clk2 with PARP inhibitors was explored preliminarily. Conclusions: 1.Compared with normal ovarian tissues, Clk2 is up-regulated in ovarian cancer tissue, especially in chemo-resistant ovarian cancer tissues, and high expression of Clk2 indicates poor prognosis. 2.Clk2 enhances DNA damage repair and promotes drug resistance in ovarian cancer. 3.Clk2 activates BRCA1 in chemo-resistant ovarian cancer cells. 4.Platinum activates p38 signal, which contributes to the phosphorylation of T343 in Clk2 Activation Loop, then Clk2 protein is stabilized. And Clk2 can activate BRCA1 in chemo-resistant ovarian cancer cells. 5.A preliminary study shows that combining Clk2 and PARP inhibitors can form a "synergistic lethal effect" and increase DNA damage in ovarian cancer platinum-resistant cells. Citation Format: Shuting Huang, Yinan Jiang, Min Zheng. The functional mechanism of Clk2 supporting the development of chemoresistance in non-BRCA1 deficient ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4088.