The NEDD8-activating enzyme (NAE) initiates a protein homeostatic pathway essential for cancer cell growth and survival. MLN4924 is a selective inhibitor of NAE currently in clinical trials for the treatment of cancer. Here, we show that MLN4924 is a mechanism-based inhibitor of NAE and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme. The NEDD8-MLN4924 adduct resembles NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be further utilized in subsequent intraenzyme reactions. The stability of the NEDD8-MLN4924 adduct within the NAE active site blocks enzyme activity, thereby accounting for the potent inhibition of the NEDD8 pathway by MLN4924. Importantly, we have determined that compounds resembling MLN4924 demonstrate the ability to form analogous adducts with other ubiquitin-like proteins (UBLs) catalyzed by their cognate-activating enzymes. These findings reveal insights into the mechanism of E1s and suggest a general strategy for selective inhibition of UBL conjugation pathways.

Recent studies suggest that targeting transcriptional machinery can lead to potent and selective anticancer effects in cancers dependent on high and constant expression of certain transcription factors for growth and survival. Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the CDK-activating kinase complex. Its function is required for both cell-cycle regulation and transcriptional control of gene expression. CDK7 has recently emerged as an attractive cancer target because its inhibition leads to decreased transcript levels of oncogenic transcription factors, especially those associated with super-enhancers. Here, we describe a selective CDK7 inhibitor SY-1365, which is currently in clinical trials in populations of patients with ovarian and breast cancer (NCT03134638). In vitro, SY-1365 inhibited cell growth of many different cancer types at nanomolar concentrations. SY-1365 treatment decreased MCL1 protein levels, and cancer cells with low BCL2L1 (BCL-XL) expression were found to be more sensitive to SY-1365. Transcriptional changes in acute myeloid leukemia (AML) cell lines were distinct from those following treatment with other transcriptional inhibitors. SY-1365 demonstrated substantial antitumor effects in multiple AML xenograft models as a single agent; SY-1365-induced growth inhibition was enhanced in combination with the BCL2 inhibitor venetoclax. Antitumor activity was also observed in xenograft models of ovarian cancer, suggesting the potential for exploring SY-1365 in the clinic in both hematologic and solid tumors. Our findings support targeting CDK7 as a new approach for treating transcriptionally addicted cancers. Significance: These findings demonstrate the molecular mechanism of action and potent antitumor activity of SY-1365, the first selective CDK7 inhibitor to enter clinical investigation.

CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.

CDK7 has emerged as an exciting target in oncology due to its roles in two important processes that are misregulated in cancer cells: cell cycle and transcription. This report describes the discovery of SY-5609, a highly potent (sub-nM CDK7 Kd) and selective, orally available inhibitor of CDK7 that entered the clinic in 2020 ( Identifier: NCT04247126). Structure-based design was leveraged to obtain high selectivity (>4000-times the closest off target) and slow off-rate binding kinetics desirable for potent cellular activity. Finally, incorporation of a phosphine oxide as an atypical hydrogen bond acceptor helped provide the required potency and metabolic stability. The development candidate SY-5609 displays potent inhibition of CDK7 in cells and demonstrates strong efficacy in mouse xenograft models when dosed as low as 2 mg/kg.

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.