Discovery and Characterization of SY-1365, a Selective, Covalent Inhibitor of CDK7 Shanhu Hu1, Jason J
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Published OnlineFirst May 7, 2019; DOI: 10.1158/0008-5472.CAN-19-0119 Cancer Translational Science Research Discovery and Characterization of SY-1365, a Selective, Covalent Inhibitor of CDK7 Shanhu Hu1, Jason J. Marineau1, Nisha Rajagopal1, Kristin B. Hamman1, Yoon Jong Choi1, Darby R. Schmidt1, Nan Ke1, Liv Johannessen1, Michael J. Bradley1, David A. Orlando1, Sydney R. Alnemy1, Yixuan Ren1, Stephane Ciblat2, Dana K. Winter2, Anzhelika Kabro2, Kevin T. Sprott1, J. Graeme Hodgson1, Christian C. Fritz1, John P. Carulli1, Emmanuelle di Tomaso1, and Eric R. Olson1 Abstract Recent studies suggest that targeting transcriptional machin- more sensitive to SY-1365. Transcriptional changes in acute ery can lead to potent and selective anticancer effects in cancers myeloid leukemia (AML) cell lines were distinct from those dependent on high and constant expression of certain tran- following treatment with other transcriptional inhibitors. SY- scription factors for growth and survival. Cyclin-dependent 1365 demonstrated substantial antitumor effects in multiple kinase 7 (CDK7) is the catalytic subunit of the CDK-activating AML xenograft models as a single agent; SY-1365–induced kinase complex. Its function is required for both cell-cycle growth inhibition was enhanced in combination with the regulation and transcriptional control of gene expression. BCL2 inhibitor venetoclax. Antitumor activity was also CDK7 has recently emerged as an attractive cancer target observed in xenograft models of ovarian cancer, suggesting because its inhibition leads to decreased transcript levels of the potential for exploring SY-1365 in the clinic in both oncogenic transcription factors, especially those associated hematologic and solid tumors. Our findings support targeting with super-enhancers. Here, we describe a selective CDK7 CDK7 as a new approach for treating transcriptionally inhibitor SY-1365, which is currently in clinical trials in addicted cancers. populations of patients with ovarian and breast cancer (NCT03134638). In vitro, SY-1365 inhibited cell growth of Significance: These findings demonstrate the molecular many different cancer types at nanomolar concentrations. SY- mechanism of action and potent antitumor activity of SY- 1365 treatment decreased MCL1 protein levels, and cancer 1365, the first selective CDK7 inhibitor to enter clinical cells with low BCL2L1 (BCL-XL) expression were found to be investigation. Introduction activates CDK9 via T-loop phosphorylation, providing another layer of regulation on transcription initiation and elongation (7, The role of cyclin-dependent kinase 7 (CDK7) in regulating 8). Roles in DNA repair, transcription termination, RNA capping, cell-cycle progression and transcription has been described in and chromatin modification have also been described (1, 9–11). both yeast (Kin28) and human cells (1–3). In cell-cycle regula- On the basis of findings in nonclinical models, CDK7 inhibi- tion, CDK7 complexes with cyclin H and MNAT1 to form the tion has emerged as a therapeutic approach in cancer. Although CDK-activating kinase, (CAK), and phosphorylates CDK1, 2, 4, the downstream events of CDK7 inhibition have not been fully and 6 to promote cell-cycle progression (2, 3). As part of the elucidated, decreased expression of oncogenes, including aber- multisubunit transcription factor II human (TFIIH), CDK7 phos- rantly expressed transcription factors associated with super- phorylates the C-terminal domain (CTD) of the RNA polymerase enhancers (SE), has been described (12). Treatment with the II (RNAPII) subunit Rbp1, with preference for the Ser5 and Ser7 small-molecule CDK7 inhibitor THZ1 resulted in apoptosis and residues of the YSPTSPS heptapeptide repeat. This CTD phos- inhibition of tumor growth in several human tumor murine phorylation plays a regulating role in the ability of RNAPII to xenograft models, including T-cell acute lymphoblastic leuke- initiate a productive transcription cycle (1, 4–6). Moreover, CDK7 mia (13), high-grade glioma (14), melanoma (15), neuroblasto- ma (12), medullary thyroid carcinoma (16), hepatocellular car- 1Syros Pharmaceuticals, Inc., Cambridge, Massachusetts. 2Paraza Pharma, Inc., cinoma (17, 18), esophageal squamous cell carcinoma (19), Quebec City, Canada. nasopharyngeal carcinoma (20), small-cell lung cancer (SCLC; ref. 21), triple-negative breast cancer (TNBC; ref. 22), and ovarian Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). cancer (23). Notably, these animal models had minimal weight loss, suggesting the potential for a therapeutic margin based on Corresponding Author: Shanhu Hu, Syros Pharmaceuticals Inc., 620 Memorial enhanced sensitivity of tumor cells. Furthermore, THZ1 is Dr., Suite 300, Cambridge, MA 02139. Phone: 617-744-1340; Fax: 617-744-1377; E-mail: [email protected] reported to suppress adaptive resistance development toward several targeted cancer therapies (24). Cancer Res 2019;79:3479–91 Transcriptionally, THZ1 treatment recapitulated the effects of doi: 10.1158/0008-5472.CAN-19-0119 selective inhibition of an analog-sensitive CDK7 variant with a Ó2019 American Association for Cancer Research. bulky ATP analog that does not inhibit wild-type CDK7. Both www.aacrjournals.org 3479 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst May 7, 2019; DOI: 10.1158/0008-5472.CAN-19-0119 Hu et al. pharmacologic agents affected RNAPII pausing and the expres- ML-2 (ACC-15) was purchased from DSMZ. All cell lines were sion of a common set of genes (1, 9). cultured according to manufacturer's instructions at 37Cin5% Given that THZ1 lacked several features desired in a clinically CO2 in a humidified incubator. All cell lines used in the study were applicable molecule, we executed medicinal chemistry on the under passage 15. Mycoplasma test was not performed. THZ1 scaffold to identify a CDK7 inhibitor suitable for clinical investigation. We describe a new molecule, SY-1365, as more Antiproliferation assay potent, selective, and metabolically stable than THZ1. SY-1365 is A total of 386 cell lines were treated with SY-1365 at following in clinical development as a single agent and in combination with concentrations: 0.0005, 0.0015, 0.0046, 0.014, 0.041, 0.12, 0.37, standard-of-care agents in multiple ovarian and breast cancer and 1.1 mmol/L. Cells were incubated with compound for populations (NCT03134638). 72 hours or at least two doubling times if they were slow growing. CellTiter-Glo (G7570, Promega) signal was measured before treatment on day0 and then a minimum of 3 or maximum of Materials and Methods 6 days after treatment depending on the cell line. Simultaneously, Chemicals the signal was measured from the cell line treated with DMSO for JQ1 (S7110), flavopiridol (S2679), and venetoclax (S8048) the same duration. were purchased from Selleck Chemicals, LLC. NVP2 (HY-12214A) The CellTiter-Glo signals after treatment with drug at increasing was purchased from MedChemExpress, LLC. concentrations were normalized to DMSO to obtain the relative Detailed synthetic protocols for SY-314, SY-351, SY-1365, cell count. A dose–response curve was fit to these relative counts biotinylated SY-1365, and SY-1365 covalent docking to CDK7 using the GRcalculate and GRlogisticFit functions from the R can be found in Supplementary Materials and Methods. package GRmetrics (26). Response metrics calculated from the cell line data are in Supplementary Table S2 and described in Kinase activity assay Supplementary Materials and Methods. On the basis of these Compound potencies were determined for each CDK by mea- response metrics, we developed an algorithm for classifying cell suring loss of activity of the kinase on a peptide substrate. All lines into two categories, "low-sensitive/insensitive" or "high- assays included active CDK protein purified with cyclin protein sensitive" to SY-1365 (Supplementary Table S2; Supplementary (cyclin H and Mat1 with CDK7), ATP, substrate peptide (5-FAM- Materials and Methods). YSPTSPSYSPTSPSYSPTSPSKKKK with CDK7), and compound or DMSO. After incubation, the amount of phosphorylated peptide Apoptosis assay product was measured using Caliper/LabChip EZ Reader. Percent For TNBC and immortalized normal cell lines, logarithmically inhibition by the compound was determined by comparing the growing cells were seeded the day before treatment and grown to amount of substrate converted to product with compound versus 60%–80% confluence. For acute myeloid leukemia (AML) cell DMSO control. Further details can be found in Supplementary lines, cells were seeded at 0.5 Â 106/mL and dosed 2 hours later. Materials and Methods. For all cell lines, cells were dosed with either DMSO or the indicated dosages of SY-1365 for 48 hours before staining with Determination of KI and kinact Annexin V and propidium iodide (10010-02, Southernbiotech, The CDK7 inhibition kinase assay was performed as above; ApoScreen Annexin V Apoptosis Kit) following manufacturer's however, compound and CDK7/cyclin H/MNAT1 complex were recommendations. Samples were analyzed by flow cytometry and preincubated for 0, 5, 10, 20, 30, and 60 minutes prior to reaction results were analyzed on FlowJo V10. initiation with ATP and substrate. CDK7/cyclin H/MNAT1 con- centration was increased to 30 nmol/L with a 5-minute reaction Occupancy assay time. The time-dependent decay of enzyme activity due to cova- To determine the ratio of SY-1365–occupied to unoccupied lent inhibition