Objective: Major limitation of successful implementation of targeted therapy in esophageal adenocarcinoma (EAC) is low frequency of target specific biomarker alteration and intratumoral heterogeneity. Cyclin dependent kinase 9 (CDK9) is a ubiquitous serine threonine kinase whoseinhibition has been used as a therapeutic option in other solid tumors. However, critical in vitro and in vivo information about the efficacy and mechanism of CDK9 inhibition in EAC is lacking and needs to be generated before a clinical trial is proposed.
Design: We assessed the CDK9 protein expression in EAC cell lines and in patient tissue samples of Barrett's esophagus and EAC. Subsequently, we studied effects of genetic downregulation (shCDK9) in SKGT4 and chemical inhibition of CDK9 by CDK9 inhibitor II, a specific, potent competitive inhibitor of CDK9 (Santa Cruz Biotechnology, CA) on proliferation, apoptosis, and cell cycle of SKGT4, FLO-1 and OE33. In vivo effects of shCDK9 and CDK9 inhibitor II was analyzed in xenografts experiments performed on nude mice after injection of FLO-1 or shCDK9-SKGT 4 and control cells in right flank or abdomen. We also assessed the effects of shCDK9 and CDK9 inhibitor II on expression of MCL-1 and phosphorylation of RNA polymerase II in EAC cells.
Results: Higher expression of CDK9 was observed in all EAC cell lines and in tumor samples of patients with EAC as compared to normal squamous epithelium and Barrett's Esophagus. shCDK9 in SKGT4 cells induced significant reduction in proliferation, increase in apoptosis and G1 arrest as compared to control cells in vitro. In vivo experiment with SKGT4-shCDK9 demonstrated no tumor growth in 4 of 10 mice and tumor volume was significantly smaller in SKGT4-shCDK9 as compared to control SKGT4 cells (72.89 ± 12.88 mm3 v.270 ± 64.07 mm3, p< 0.01) in other mice. Treatment with CDK9 inhibitor II demonstrated dose dependent reduction in proliferation, increased apoptosis and G1 arrest in all three cell lines in vitro and more than 50% reduction in tumor volume in xenografts as compared to control. CDK9 inhibitor II reduced RNA Pol II phosphorylation and expression of MCL-l and up-regulation of MCL-1 expression induced resistance to CDK9 inhibitor II in all three EAC cell lines..
Conclusions: CDK9 inhibition has strong anti-EAC properties in vitro and in vivo. These properties along with strong diffuse expression of CDK9 in human EAC support further exploration of CDK9 as a potential therapeutic target for EAC.
Citation Format: Zhimin Tong, Devkumar Chatterjee, Defeng Deng, Omkara Veeranki, Alicia Mejia, Jaime Rodriguez, Jaffer Ajani, Wayne Hofstetter, Steven Lin, Sushovan Guha, Scott Kopetz, Sunil Krishnan, Dipen Maru. Cyclin-dependent kinase 9: a potential therapeutic target for esophageal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3815.