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Discovery and resistance mechanism of a selective CDK12 degrader

Nature Chemical Biology volume 17, pages 675–683 (2021)Cite this article

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Abstract

Cyclin-dependent kinase 12 (CDK12) is an emerging therapeutic target due to its role in regulating transcription of DNA-damage response (DDR) genes. However, development of selective small molecules targeting CDK12 has been challenging due to the high degree of homology between kinase domains of CDK12 and other transcriptional CDKs, most notably CDK13. In the present study, we report the rational design and characterization of a CDK12-specific degrader, BSJ-4-116. BSJ-4-116 selectively degraded CDK12 as assessed through quantitative proteomics. Selective degradation of CDK12 resulted in premature cleavage and poly(adenylation) of DDR genes. Moreover, BSJ-4-116 exhibited potent antiproliferative effects, alone and in combination with the poly(ADP-ribose) polymerase inhibitor olaparib, as well as when used as a single agent against cell lines resistant to covalent CDK12 inhibitors. Two point mutations in CDK12 were identified that confer resistance to BSJ-4-116, demonstrating a potential mechanism that tumor cells can use to evade bivalent degrader molecules.

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Fig. 1: Development and characterization of CDK12 degrader BSJ-4-116.
Fig. 2: CDK12 degradation preferentially leads to premature cleavage and poly(adenylation) of long genes enriched with DDR genes.
Fig. 3: BSJ-4-116 inhibits the growth of T-ALL cells and sensitizes them to PARP inhibition.
Fig. 4: Chronic exposure leads to acquired resistance to BSJ-4-116 mediated by G-loop mutations.
Fig. 5: Chronic exposure leads to acquired resistance to BSJ-4-116 mediated by G-loop mutations.

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Data availability

Dataset of exonic nonsynonymous variants excluding loss-of-function mutations in Jurkat-resistant cells is available in Supplementary Dataset 1. KINOMEscan data are provided in Supplementary Dataset 2. A complete GSEA result is provided in Supplementary Dataset 3. Whole-exome sequencing data of parental and resistant cell lines to BSJ-4-116 have been deposited at the National Center for Biotechnology Information (NCBI) Sequence Read Archive with BioProject accession no. PRJNA634900. Poly(A) 3′-sequencing data have been deposited at the NCBI’s Gene Expression Omnibus (accession no. GSE161650). Crystal structure of human CRBN in complex with DDB1 and lenalidomide has a PDB accession no. 4TZ4. Crystal structures of the human CDK12–cyclin K complex have PDB accession nos. 5ACB, 6CKX and 6B3E. Crystal structure of the human CDK13–cyclin K complex has PDB accession no. 5EFQ. Source data are provided with this paper.

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Acknowledgements

We thank M. Kostic for her editing of this manuscript. This work was supported by a National Institutes of Health grant (nos. PO1 CA154303 to N.S.G. and U24-DK116204 to P.K.S.) and Deutsche Forschungsgemeinschaft GE 976/9-2 (to M.K.).

Author information

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  1. These authors contributed equally: Baishan Jiang, Yang Gao, Jianwei Che.

Authors and Affiliations

  1. Department of Cancer Biology, Dana–Farber Cancer Institute, Boston, MA, USA

    Baishan Jiang, Yang Gao, Jianwei Che, Wenchao Lu, Jie Jiang, Inchul You, Nicholas Kwiatkowski, Tinghu Zhang & Nathanael S. Gray

  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    Baishan Jiang, Yang Gao, Jianwei Che, Wenchao Lu, Jie Jiang, Inchul You, Nicholas Kwiatkowski, Tinghu Zhang & Nathanael S. Gray

  3. Institute of Structural Biology, University of Bonn, Bonn, Germany

    Ines H. Kaltheuner & Matthias Geyer

  4. Department of Hematology and Oncology, Boston University, Boston, MA, USA

    Ruben Dries

  5. Department of Computational Medicine, Boston University, Boston, MA, USA

    Ruben Dries

  6. Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA, USA

    Marian Kalocsay, Matthew J. Berberich & Peter K. Sorger

  7. Promega Corporation, Madison, WI, USA

    Kristin M. Riching & Danette L. Daniels

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Contributions

N.S.G. and T.Z. conceived the project. B.J. performed the compound synthesis and structure determination with help from I.Y. Y.G. and W. L. executed cellular biological experimental research with help from J.J. K.R and D.D. performed the NanoBRET ternary complex assays. J.C. executed computational modeling, whole-exome sequencing analysis and mutational experiments design. R.D. and Y.G. performed genomic data analysis. I.K and M.G. executed CDK12 in vitro kinase assay. M.B. M.K and P.K.S. performed proteomic analysis. T.Z. Y.G. J.C. B.J. and N.S.G. co-wrote the paper. All authors edited the manuscript.

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Correspondence to Tinghu Zhang or Nathanael S. Gray.

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Competing interests

N.S.G. is a founder, science advisory board member and equity holder in Gatekeeper, Syros, Petra, C4, B2S, Aduro and Soltego. The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield, Ephiphanes and Sanofi. J.C. is a consultant to Soltego, Jengu and Allorion, and equity holder for Soltego, Allorion and M3 bioinformatics & technology Inc. P.K.S. is a member of the science advisory board or board of directors of Merrimack Pharmaceutical, Glencoe Software, Applied Biomath and RareCyte Inc., and has equity in these companies. B.J., J.C., Y.G., N.K., T.Z. and N.S.G. are inventors on CDK12 degrader patents. All remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Development of CDK12 degraders BSJ-4-23 and BSJ-4-116.

a, Chemical structures of THZ531 and its 3 fragments with ligand efficiency values. b, In vitro CDK12 kinase assay. Assays were performed at an ATP concentration of 30 μM (apparent Km). Data are presented as mean ± s.d. of n = 3 biologically independent samples. c, Preliminary screening immunoblots for CDK12, CDK13 and β-Actin in Jurkat cells after 6 h treatment with DMSO or different CDK12 degraders at the indicated concentrations. d, Binding groove for BSJ-4-23 in modeled ternary complex of CDK12/BSJ-4-23/CRBN (CDK12 in blue, PDB ID: 5ACB, CRBN in orange, PDB ID: 4TZ4, BSJ-4-23 carbons in light grey). e, Time-dependent effect of BSJ-4-116 (50 nM) on CDK12, CDK13 and cyclin K protein levels after 2 h, 4 h, 8 h, 16 h and 24 h treatment in Jurkat cells. f, Left: Immunoblots for CDK12, CRBN and β-Actin in WT and CRBN null Jurkat cells after 6 h treatment with DMSO, BSJ-4-23 (250 nM) and BSJ-4-116 (50 nM); Right: Immunoblots for CDK12 and α-tubulin in Jurkat cells following 2 h pre-treatment with DMSO, Carfizomib (400 nM), MLN4924 (1000 nM), Thalidomide (1000 nM) and THZ531 (250 nM) followed by 6 h co-treatment with DMSO or BSJ-4-116 (50 nM). g, KinomeScan kinase selectivity profile for BSJ-4-116. BSJ-4-116 was profiled at a concentration of 1 μM against a panel of 468 human kinases. The results for the binding interactions are reported as a percent of the DMSO control (% control), where larger red circles indicate stronger binding hits. The selectivity score was defined as the ratio of the number of kinases inhibited to a specified percentage versus the total number of kinases. For this experiment, specified percent inhibition was set at 10%, resulting in S(10) value of 0.017 for BSJ-4-116. h, Degradation effect of BSJ-4-116 and BSJ-4-116NC at indicated doses prechecked by western blots for the proteomics experiment in Jurkat cells. Data in (c), (e), (f) and (h) represent n=2 independent experiments. i, NanoBRET live cell ternary complex assays performed in MOLT-4 cells co-expressing HaloTag-CRBN and one of the following C-terminal NanoLuc fusions: CDK12, CDK12 (K745R), CDK12 (L752M), CDK12 (K745R/L752M) or CDK13, CDK13 (R723K), CDK13 (M730L), CDK13 (R723K/M730L). The fold increase in NanoBRET signal relative to BSJ-4-116NC was plotted after 3 h treatment with the indicated compounds with n=6 biologically independent samples.

Source data

Extended Data Fig. 2 CDK12 degradation preferentially leads to premature cleavage and polyadenylation (PCPA) of long genes enriched with DDR genes.

a, Genome-wide correlation analysis for replicates from each condition showing significant correlation between BSJ-4-116 vs THZ531, and DMSO vs BSJ-4-116NC. b, Immunoblots for CDK12 and α-tubulin in Jurkat cells treated with DMSO or BSJ-4-116 (50 nM) for indicated hours. Data are representative of n=2 independent experiments. c, Fisher exact test showing significant overlap in genes downregulated by BSJ-4-116 vs THZ531 (p=0). There was also significant overlap in the small numbers of gene upregulated (p=1.42e-136). d, GSEA of downregulated genes in Jurkat cells treated with BSJ-4-116 and THZ531. e, Additional enriched GSEA signatures enriched by BSJ-4-116 treatment. f, Left: qRT-PCR analysis of the indicated DDR gene expression in Jurkat and MOLT4 cells treated with BSJ-4-116 (50 nM) or BSJ-4-116NC (100 nM) for 10 h. Data were normalized to GAPDH and compared to DMSO-treated controls (n=3). Right: Immunoblots for indicated DDR and cell death markers in Jurkat and MOLT4 cells treated with DMSO or BSJ-4-116 (50 nM) for indicated hours. Data are representative of n=2 independent experiments. g, Bar plot showing the frequency of retrieved polyadenylation site (PAS) motifs 100bp upstream of the poly(A) 3’-seq peaks. h, Average metagene profiles of normalized poly(A) 3’-seq reads over gene bodies and extending –2 to +2 kb of all detected genes in Jurkat cells treated with BSJ-4-116 (50 nM) or THZ531 (250 nM) vs DMSO for 8 h. Sense and antisense reads are depicted by solid and dashed lines, respectively. i, Boxplots showing the differential usage (log2 fold-change) of polyadenylation sites at three different genomic locations. The comparison BSJ-4-116 vs. BSJ-4-116NC is shown in red and THZ531 vs. DMSO is shown in green. j, Schematic illustration of PCPA caused by CDK12 inhibition or degradation.

Source data

Extended Data Fig. 3 BSJ-4-116 inhibits the growth of T-ALL cells and sensitizes them to PARP inhibition.

a, Cell-cycle analysis of Jurkat and MOLT4 cells treated with BSJ-4-116 (50 nM) and BSJ-4-116NC (100 nM) for 24 h. DNA was stained with propidium iodide (PI) before flow cytometry analysis. G/M% values are presented as mean ± s.d. of n=3 biologically independent samples and are representative of n=2 independent experiments. b, Excess over Bliss synergy plots for serial dilutions of BSJ-4-116 in combination with Olaparib in CRBN null Jurkat (top) and MOLT4 (bottom) cells. n=3 replicates.

Extended Data Fig. 4 Chronic exposure leads to acquired resistance to BSJ-4-116 mediated by G-loop mutations.

a, Dose response curves for parental and resistant Jurkat and MOLT4 cells treated with BSJ-4-23 at indicated dose range for 72 h. Percent cell growth relative to DMSO-treated was analyzed using growth rate inhibition assay method. Data are presented as mean ± s.d. of n=3 biologically independent samples. b, Detection of heterozygous G739S mutation in Jurkat resistant cells. DNA chromatograms of sanger sequencing shows region of mutation from PCR-amplified CDK12 cDNA. c, Immunoblots for CDK12, CDK9 and GAPDH in parental and resistant Jurkat and MOLT4 cells treated with DMSO, BSJ-4-23 (250 nM), BSJ-4-116 (50 nM) or THAL-SNS-032 (250 nM) for 8 h. Data represent n=2 independent experiments. d, CDK12 kinase domain structure (PDB code: 5ACB) showing the locations of G-loop mutations I733 and G739.

Source data

Supplementary information

Supplementary Information

Supplementary Note Synthetic procedures.

Reporting Summary

Supplementary Data 1

Exonic nonsynonymous variants excluding loss of function mutations in Jurkat-resistant cells.

Supplementary Data 2

Full KINOMEscan dataset.

Supplementary Data 3

Complete GSEA result.

Supplementary Data 4

Compound structures in Fig. 1a.

Source data

Source Data Fig. 1

Uncropped western blots for Fig. 1.

Source Data Fig. 3

Uncropped western blots for Fig. 3.

Source Data Fig. 4

Uncropped western blots for Fig. 4.

Source Data Extended Data Fig. 1

Uncropped western blots for Extended Data Fig. 1.

Source Data Extended Data Fig. 2

Uncropped western blots for Extended Data Fig. 2.

Source Data Extended Data Fig. 4

Uncropped western blots for Extended Data Fig. 4.

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Jiang, B., Gao, Y., Che, J. et al. Discovery and resistance mechanism of a selective CDK12 degrader. Nat Chem Biol 17, 675–683 (2021). https://doi.org/10.1038/s41589-021-00765-y

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