Abstract 2855: CDK12 inhibition remodels DNA replication to drive selective gene amplification
Authors: Shivani Yalala, Julia Kutz, Helmut Pospiech, Harri M. Itkonen
Journal: Cancer Research (2025)

Abstract Background and Objective: Transcription is controlled by phosphorylation of the C-terminal domain (CTD) of RNA Polymerase II (RNA Pol II). Cyclin-dependent transcriptional kinase 12 (CDK12) phosphorylates the CTD to sustain productive transcription elongation of long genes. Unexpectedly, because cancer cells typically exhibit high levels of transcription, CDK12 inactivation defines a subset of aggressive prostate cancers, which have a high number of gene amplifications in the absence of simpler mutations. Defects in transcription of specific genes and collisions between transcription and DNA replication machineries have been proposed as the source of this mutational signature. We hypothesize that DNA replication machinery and program are directly impacted by CDK12 inactivation, and some of the genes selectively amplified in the mutant tumors are required to sustain proliferation of these cells. Methods: To assess the effects of CDK12 inactivation on DNA synthesis, we have measured nucleotide incorporation and relied on DNA combing assays. ChIP-seq experiments against the DNA replication machinery were used to probe the DNA replication program. Finally, proteomic-profiling of DNA replication machinery in the presence and absence of CDK12 inhibitor was used to evaluate the effects on its composition. Results: CDK12 inhibition leads to defects in DNA replication as measured by nucleotide incorporation, which are explained, at least in part, due to replication fork stalling as determined using DNA combing assays. Integration of ChIP-seq data against the DNA replication machinery with data from CDK12 mutant prostate cancer patients revealed accumulation of the machinery on a specific sub-set of genes amplified in the patient samples. We termed this gene set as replication-defective and CDK12 mutant amplified (ReDeAmp). Mass spectrometry-based profiling of DNA replication machinery revealed significant remodeling in response to CDK12 inhibition. Currently, we are probing if the genes of the ReDeAmp-signature or the proteins accumulating to DNA replication machinery are essential for the proliferation of the CDK12-mutant cells. Conclusions: Our data show that decrease in CDK12 activity disrupts DNA replication, which we propose explains the unique mutational signature observed clinically when CDK12 is inactivated. These findings further pose that selective gene amplifications are required in the CDK12-deficient cells. If this was the case, CDK12 inactivation would serve as a biomarker of sensitivity for the acquired synthetic lethality - this is currently under intense investigation in our laboratory. Citation Format: Shivani Yalala, Julia Kutz, Helmut Pospiech, Harri M. Itkonen. CDK12 inhibition remodels DNA replication to drive selective gene amplification [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 2855.