Abstract B172: Identification of a novel RNA processing mechanism of intronic readthrough to a transcriptional stop leading to truncated transcript expression, including FANCI and ATM, upon CDK12 inhibition
Authors: Denz Christopher , Johannes Jeffrey , Yao Yi , Kulkarni Meghana , Dulak Austin , Guan Nin , Su Nancy , Lamb Michelle , Fawell Stephen , Guichard Sylvie 
Published: August 2018

Cyclin-dependent kinase 12 (CDK12), a key regulator of transcriptional processing along with its binding partner cyclin K (CCNK), has been implicated in the modulation of genes involved with the DNA damage response (DDR) including, but not limited to, homologous recombination repair (HRR). However, the exact mechanism by which CDK12 is having this effect has been unclear. To identify differentially expressed transcripts between wild-type and mutant CDK12 that may indicate alternative or mis-splicing events and help understand the mechanism of CDK12 loss of function, we analyzed RNA-Seq data from TCGA ovarian cancer comparing transcripts in loss of function mutated CDK12 (~3% of ovarian) vs wild-type samples. We identified a number of transcripts that were at least twofold higher in CDK12 mutant vs wild-type samples. Deeper analysis showed an enrichment in truncated transcripts, a subset of which may arise from readthrough into an intron containing a transcriptional stop, including the DDR genes FANCI and ATM. This potential mechanism by which CDK12 may regulate DDR genes was tested in vitro using Taqman probes upstream and downstream of the introns with the stop signal for both ATM and FANCI. To validate the differential expression of FANCI and ATM transcripts observed in TCGA samples, we treated the ovarian cells lines OVCAR8 and OV90 with CDK12 siRNA for 72hrs. CDK12 knockdown resulted in truncated transcript expression and a reduction in the full-length transcripts of DDR genes FANCI and ATM. Reduction in levels of full-length transcripts also leads to decreased levels of FANCI and ATM protein. Knockdown of the closely related homolog CDK13 by siRNA did not lead to expression of truncated FANCI and ATM transcripts. Sensitization to olaparib was observed for CDK12 but not CDK13 siRNA treated ovarian cells in a 14-day clonogenic assay indicating a loss of HRR proficiency due to CDK12 depletion. A biochemical screen was developed and a synthetic chemistry plan initiated to identify CDK12 selective probe compounds to try to confirm the target biology, DDR gene modulation, and RNA processing effects seen with siRNA. Potent and selective inhibitors of CDK12/13 were identified and used to treat ovarian cancer cells. Differential expression of full-length vs truncated FANCI and ATM transcripts was seen by 4-8 hrs with CDK12/13 selective compounds. However, at higher concentrations and longer time points, effects on general transcription were observed as indicated by a decrease in full-length ATM and FANCI, potent effects on p-Ser2 of RNApolII, and growth inhibition in a 48-hr proliferation assay. The small-molecule inhibitors partially recapitulate the RNA processing effects on ATM and FANCI; however, effect on CDK13 or the retention of CCNK in an inactive complex may lead to broader transcriptional effects. To our knowledge, these are the first data linking CDK12 inhibition to a RNA processing mechanism of intronic readthrough to a transcriptional stop leading to modulation of a subset of transcripts, including the DDR genes ATM and FANCI.

Citation Format: Christopher Denz, Jeffrey Johannes, Yi Yao, Meghana Kulkarni, Austin Dulak, Nin Guan, Nancy Su, Michelle Lamb, Stephen Fawell, Sylvie Guichard. Identification of a novel RNA processing mechanism of intronic readthrough to a transcriptional stop leading to truncated transcript expression, including FANCI and ATM, upon CDK12 inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B172.