SY-5609, an orally available selective CDK7 inhibitor demonstrates broad anti-tumor activity in vivo

Previously, we reported on a series of highly potent, selective, and non-covalent CDK7 inhibitors that demonstrated antiproliferative activity against triple-negative breast cancer (TNBC) and ovarian cancer (OVA) cell lines and tumor growth inhibition in cell line-derived (CDX) and patient-derived (PDX) mouse xenograft models. Here, we report on the in vitro and in vivo profile of our development candidate, SY-5609. Methods: Kinase inhibition assays at both Km and 2 mM [ATP] were used to assess inhibition of CDK2, CDK7, CDK9, and CDK12. SPR was used to determine the Kd, kon, and koff binding characteristics of SY-5609 to immobilized CDK7/Cyclin H dimer. CDK7 compound occupancy was determined using a biotinylated small molecule probe to pull down free CDK7 following incubating of HL60 cells with SY-5609. Inhibition of tumor cell line growth was assessed following 72 hrs of incubation with SY-5609. Flow cytometry was used to assess apoptosis and cell cycle modulation after 48 hrs of treatment. Effects on DNA damage and repair were assessed by immunofluorescence staining for γH2AX and RAD51 proteins. To assess in vivo effects, mice were implanted subcutaneously and randomized for treatment when tumors reached 150-200 mm3 and dosed orally for 3 weeks by both QD and BID dosing regimens. Collected tumor tissue samples were analyzed for protein levels of MCL1, pCDK2, MYC, and RNA Pol II CTD pSer5 by western blot. Results: SY-5609 bound CDK7/Cyclin H with a Kd of 0.059 nM and occupied CDK7 in HL60 cells with an EC50 of 33 nM. Cell growth inhibition EC50 values were 6-17 nM in a panel of solid tumor cell lines. Selectivity of SY-5609 over CDK12, CDK9, and CDK2 was 2492-, 2508-, and 8068-fold, respectively. SY-5609 led to induction of apoptosis, cell cycle arrest, and inhibition of DNA damage repair in tumor cell lines. Dose-dependent tumor growth inhibition was observed in a panel of CDX and PDX solid tumor models with both QD and BID dosing of SY-5609 with resulting decreases in direct (pCDK2, RNA Pol II CTD pSer5) and indirect (MCL1, MYC) protein biomarkers. In summary, we describe SY-5609, an orally available, potent, and selective CDK7 inhibitor that drives strong responses in CDX and PDX tumor models. These data support the rationale for advancing SY-5609 into IND-enabling studies. Citation Format: Shanhu Hu, Jason Marineau, Kristin Hamman, Michael Bradley, Anneli Savinainen, Sydney Alnemy, Nisha Rajagopal, David Orlando, Claudio Chuaqui, Eric Olson. SY-5609, an orally available selective CDK7 inhibitor demonstrates broad anti-tumor activity in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4421.