Abstract 5692: Identification of novel CDK12 inhibitors that synergize with PARP inhibition through induction of ‘BRCAness’ phenotype
Authors: Ainscow Edward K. , Bahl Ashwani , Thompson Stuart , Flatt Luke , Crepin Damien , Stevenson Brett 
Published: September 2020

Abstract
CDK12 has emerged as potential target in a range of cancers through its ability to regulate the transcription and mRNA processing of DNA repair genes. Inhibition or knockdown of CDK12 leads to a ‘BRCAness' phenotype caused by low expression of BRCA1/2, ATM, ATR and genes of the FANC group proteins1. CDK12 inhibition has therefore been proposed as a potential approach to expand the clinical utility of PARP inhibitors in triple negative breast and ovarian cancer2. Furthermore, CDK12 inhibition may improve the efficacy of immunomodulators by increasing mutation rates in cancer cells and driving the emergence of neo-antigens3. Here we describe the activity of a novel class of orally bioavailable, non-covalent CDK12 inhibitors. The compounds show selectivity for CDK12 over other members of the CDK family such as CDK1, 2, 4, 6, 7 and 9 (n.b. cross reactivity with CDK13 is predicted) in biochemical assays. In cells, selective, potent inhibition of phosphorylation of Ser2 of the RNA polymerase II c-terminal domain repeat, a known site of CDK12 activity, is observed Consistent with CDK12 inhibition, the cells showed a highly potent (IC50 &lt; 10 nM) ability to induce cell death in the A673 cell line. A673 is a Ewing's sarcoma cell line that expresses the EWS-FLI fusion protein and is therefore sensitive to CDK12 inhibition. In contrast, the osteosarcoma cell line U2-OS showed low sensitivity to the compounds (IC50 &gt; 1 µM). Inhibition of transcription by the compounds leads to downregulation of the expression of a range of genes associated with the DNA damage repair pathways, including RAD51, ATR, ATM and NBN. Impairment of the DNA repair capacity of the cells was reflected by an increase in histone γ-H2AX, a marker of DNA strand breaks, upon incubation with the compounds. In combination with Olaparib, a clinically approved PARP inhibitor, we observed a synergistic effect on cell viability in the OV-90 ovarian cancer cell line. OV-90 display a BRCA-wt phenotype and are insensitive to single agent activity of PARP inhibitors. However, the synergistic response with our CDK12 inhibitors indicated the induction of a ‘BRCAness' phenotype in these cells. Overall the compounds demonstrate a phenotype consistent with selective CDK12 inhibition and cellular potency that compares favorably with other published compounds. The compounds have a high potential for being developed as once-daily oral compounds for use in combination with PARP inhibition, immunomodulators and as single agent therapy. 1 Dudbury SJ et al. (2018), Nature, 564 (7734), 141-145. 2 Joshi PM et al. (2014), J. Biol. Chem., 289, 9247-9253. 3 Antonarakis ES (2018), N. Eng. J. Med., 379, 1087-1089.
Citation Format: Edward K. Ainscow, Ashwani Bahl, Stuart Thompson, Luke Flatt, Damien Crepin, Brett Stevenson. Identification of novel CDK12 inhibitors that synergize with PARP inhibition through induction of ‘BRCAness’ phenotype [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5692.