Abstract 5980: Identification of CDK15 activating cyclins by a recombinant co-expression approach

Abstract

Cyclin dependent kinases (CDK) form a family of 20 different CDKs with an additional 6 CDK-like proteins. Members of this family have been shown to be involved in the regulation of many critical cellular processes, including the regulation of cell cycle progression, gene transcription and neuronal function. Deregulated CDK activity has been observed frequently in human cancer and other diseases. Enzymatic kinase activity of CDKs has been found to depend on the formation of complexes with regulatory proteins of the Cyclin family and requires in some cases the presence of additional complex partners, like MAT1 for CDK7/Cyclin H. To date, at least 15 different Cyclins have been described. For some CDKs it has been shown that they can also be activated by binding to proteins distinct from classical Cyclins. However, even while the 20 canonical CDKs share significant structural homology, they regulate very different functions of cell growth and development, partially due to their inherent substrate specificity but also regulated by their respective Cyclin partners. During the past 30 years several CDKs have been subject to extensive study resulting in a multitude of publications. This includes specifically CDK1, CDK2 and CDK4/6 and to a lesser extend CDK7 and CDK9. Other CDKs have been studied much less extensively and consequently the accumulated knowledge about their biological function and regulation is very limited. CDK15 (also known as PFTK2 or ALS2CR7) is one of these understudied members of the CDK family, with less than 20 publications in PubMed and the search term "CDK15" in contrast to more than 8600 hits for "CDK2". Its biological function is largely obscure even while evidence was presented that CDK15 is involved in the regulation of breast and colorectal cancer. Comparable to all other members of the CDK family, recombinantly overexpressed purified CDK15 does not exhibit detectable in-vitro activity and to date no Cyclin or other activating complex partner for CDK15 has been published. Since CDK inhibitors have been in the focus of interest at least since the approval of the first candidate (Palbociclib) for the treatment of ER+/HER+ breast cancer, testing the selectivity of other drug candidates within the complete group of CDKs is critically important to avoid unwanted off-target effects and toxicity. In order to enable in-vitro activity studies with CDK15 we evaluated different CDK15 constructs in combination with a comprehensive panel of different Cyclins using the baculo virus expression system. CDK15 and the respective Cyclins were co-expressed in insect cells, purified by affinity chromatography and tested for in-vitro activity using a panel of generic, broad-spectrum protein kinase substrates. We will present data on the identification of CDK15 activating Cyclins and the biochemical characterization of the in-vitro kinase activity of such CDK15/Cyclin complexes including a panel of kinase inhibitors. Citation Format: Daniel Mueller, Constance Ketterer, Diane Kraemer, Carolin Heidemann-Dinger, Lena Pilgermayer, Thomas Weber, Andreas Gericke, Frank Totzke, Michael H. Kubbutat. Identification of CDK15 activating cyclins by a recombinant co-expression approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5980.