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Background: CDK12 regulates the expression of multiple genes in the DNA damage response (DDR) pathway by phosphorylating the C-terminal domain of RNA polymerase II (CTD pSer2), and it is a potential therapeutic target to exacerbate DNA damage and cell death in DDR-addicted, HER2-amplified, MYC-overexpressing, or EWS-FLI1-positive cancers. PARP inhibitor resistance is often driven by dysregulation of DDR signaling, and CDK12 inhibition may induce a state of DDR deficiency and increase the effectiveness of PARP inhibition. Materials and methods: Biochemical activity was assayed by ADP-glo (Promega), cellular proliferation was quantified by CellTiter-Glo (Promega), and CTD pSer2 was quantified by MSD immunoassay (Meso Scale Discovery). Kinase panels and in vivo studies are described in figure legends. Results: OKI-1546 has a biochemical IC50 of 15 nM against CDK12 and is highly selective for CDK12 and CDK13 when compared to other closely related CDKs; it is 649-, 81-, 11-, and 64-fold selective over CDKs 1, 2, 7, and 9, respectively. This selectivity was further confirmed via testing OKI-1546 against an affinity-based kinase panel. OKI-1546 potently inhibited proliferation of multiple cancer cell lines, with IC50 values that correlated with 50% inhibition of CTD pSer2. In CD-1 mice, OKI-1546 showed 77% oral bioavailability (%F) with a half-life of 2.7 hrs, and SD rats had 81 %F. Furthermore, a single 10 mg/kg dose reached plasma concentrations that surpassed the plasma protein binding-adjusted IC50 of CTD pSer2, which was confirmed in tumor-bearing mice showing 60% CTD pSer2 inhibition in tumors 4 hours after a single oral dose. That same dose translated into 119% tumor growth inhibition following 21 days of daily administration in the BRCA1 wild-type MDA-MB-231 tumor model and 121% tumor growth inhibition following 21 days of daily administration in the BRCA1-mutant MDA-MB-436 tumor model in mice. Four different PARP inhibitors synergistically combined with OKI-1546 to inhibit cell growth in multiple in vitro cancer models. Conclusion: OKI-1546 is a novel, covalent inhibitor of CDK12 and CDK13 that is potent, specific, orally available, and shows in vivo target engagement and tumor growth inhibition in preclinical cancer models. OKI-1546 showed activity in vivo as a single agent and cellular data suggest that inhibition of CDK12 has the potential to enhance the effects of other DDR-targeting therapeutic agents. Citation Format: Alexander A. Strait, David A. Mareska, Jyothi Mahadevan, Christie A. Schulte, Yevgeniy Izrayelit, Mark L. Boys, Richard Woessner. OKI-1546 is a selective, orally bioavailable inhibitor of CDK12 that causes tumor regression in multiple preclinical cancer models [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 1635.