An oral and selective CDK12 inhibitor demonstrates robust anti-tumor activity

Abstract Background: CDK12 has emerged as an attractive cancer target due to its role in transcription and DNA damage repair regulation. In pre-clinical models, small molecule CDK12 inhibitors have demonstrated promising tumor killing effect, especially in combination with DNA damaging agents. Here we profiled new oral and selective CDK12 inhibitors. Material and methods: ADP-glo࣪ assays: inhibition of recombinant CDK12/CyclinK, CDK2/CyclinE, CDK7/CyclinH/MAT1 and CDK9/CyclinT2 was measured by assessing ADP converted from ATP via luminescent signal at Km and 20x Km ATP. Kinome screen: 1 µM of Compound A was tested in duplicate against 370 kinases at 10 µM ATP in the radiometric HotSpot™ assay. Cellular assays: p-Ser2, p-Ser5 and total RNA pol II signals were assessed 4hrs after compound treatment while ɣH2AX and BRCA1 signals were measured after 24-48hr compound treatment via Immunofluorescence (IF) assays. Cell proliferation was determined using cell titer glo after 48-72hrs compound treatment. Apoptosis was measured by annexin V/PI staining and flow cytometry analysis after 48-72hrs of treatment. Cell cycle profile was evaluated with Click-iT-EdU and FxCycle violet stain and flow cytometry analysis following 24-48hrs of treatment. Mouse xenograft: balb/c mice were implanted subcutaneously with H1048 or MDA-MB-468 cells and randomized for treatment with test drug or vehicle when tumors reached 150-200mm3. Mice were dosed BID through oral administration for 4 weeks. Combination effect of Compound A and lurbinectedin was tested in vivo. Lurbinectedin and Compound A were dosed QW and BID respectively for 28 days in H1048 CDX model. Results: A series of CDK12 inhibitors were designed and profiled in biochemical and cellular assays. A representative member of the class, Compound A, exhibited selectivity over CDK2, CDK7, and CDK9 of 46-, 27-, and 9-fold, respectively. Compound A inhibited proliferation in a panel of cell lines with EC50 in the low nanomolar range. Compound A treatment led to dose dependent apoptosis in multiple cancer cell lines and induced G2/M arrest in cancer cell lines. In vitro, combination treatment with Compound A and lurbinectedin lead to increased DNA damage accumulation, decreased homologous recombination repair, and overall enhanced antiproliferation. Dose dependent tumor growth inhibition in SCLC and TNBC CDX models was observed with Compound A treatment. Enhanced and durable antitumor effect was observed with lurbinectedin and Compound A combination compared to single agent treatment in vivo. Conclusions: We designed and profiled orally available, CDK12 selective inhibitors with potent activity as single agent in vitro and in vivo in multiple cancer models. Compound A demonstrated enhanced antitumor effect in vitro and in vivo when combined with DNA damaging agents. These data support the rationale for advancing one or more members of this class toward clinical development. Citation Format: Shan hu, David Moebius, Wojciech Dworakowski, Elliott Cooper, Derek LaPlaca, Sydney Alnemy, Phone Perera, Jason Marineau, Claudio Chuaqui, John P. Carulli, Eric Olson. An oral and selective CDK12 inhibitor demonstrates robust anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5393.