Abstract 4133: Cyclin-dependent kinase signaling in oncology: assessing the effectiveness of a CDK9-targeting using no-wash immunoassays

Abstract Cyclins are regulatory subunits that bind to a cyclin dependent kinase (CDK), thus activating the kinase and controlling its activity. Most CDKs form a complex with a particular cyclin and are involved in the regulation of either cell cycle or cell transcription. Dysregulation of CDKs has been found to be a primary driver in tumorigenesis. Thus, CDK9 is an attractive target for cancer therapeutics due to its crucial role in transcription regulation, particularly of short-lived anti-apoptotic proteins such as MCL-1 and XIAP. Among the investigated CDK9-based therapeutic strategies, the degradation of CDK9 using specific compounds like proteolysis targeting chimera (PROTAC) is of high interest. The purpose of our study was to determine whether homogeneous time-resolved fluorescence (HTRF) and a bead-based homogeneous immunoassay platform (AlphaLISA Surefire Ultra, ALSU) assays are effective solutions for identifying and characterizing PROTAC degradation of CDK9. Both assays were used to monitor the expression levels of CDK9 or GAPDH in cell lysates from HeLa or MCF-7 cells. Assessing CDK9 levels allowed the identification of a PROTAC degradation of CDK9 while the determination of GADPH levels was used as an internal control for potential changes to global protein levels in drug treatment. Cells were treated for 4h either with 1µM of a reference CDK9 degrader, Thal-SNS-032 or with the same concentration of its two individual chemical components, Thalidomide and SNS-032 (as negative controls). Cells were then lysed, and the corresponding lysates were assessed in either CDK9 or GAPDH assays. Thal-SNS6032 induced a robust degradation of CDK9 (80 to 95%) while Thalidomide and SNS-032 did not affect the expression level of CDK9. Moreover, none of the tested compounds affected the GAPDH expression level suggesting that the changes of CDK9 levels is not due to changes in the global protein concentration. To further characterize the effect of Thal-SNS-032, a dose-response treatment was carried out on HeLa and MCF-7 cells. CDK9 and GAPDH levels were assessed in the corresponding cell lysates and a dose-dependent degradation of CDK9 was observed with half-maximal degradation values (DC50) ranging from 60 to 166nM while GADPH levels remained unchanged. These data suggest that HTRF and ALSU assays are efficient solutions to identify and characterize PROTAC targeting of CDK9. Results obtained for CDK9 can be translated to other CDK targets of interest in oncology research as a broad portfolio of total CDK detection kits is available using the two assay platforms. Citation Format: Najim Douayry, Milind Rajopadhye, Olivia J. Kelada. Cyclin-dependent kinase signaling in oncology: assessing the effectiveness of a CDK9-targeting using no-wash immunoassays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4133.