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Distinct CDK6 complexes determine tumor cell response to CDK4/6 inhibitors and degraders

Nature Cancer volume 2, pages 429–443 (2021)Cite this article

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Abstract

Cyclin-dependent kinases (CDKs) 4 and 6 inhibitors (CDK4/6is) are effective in metastatic breast cancer, but they have been only modestly effective in most other tumor types. Here we show that tumors expressing low CDK6 rely on CDK4 function and are exquisitely sensitive to CDK4/6is. In contrast, tumor cells expressing both CDK4 and CDK6 have increased reliance on CDK6 to ensure cell cycle progression. We discovered that CDK4/6is and CDK4/6 degraders potently bind and inhibit CDK6 selectively in tumors in which CDK6 is highly thermo-unstable and strongly associated with the HSP90–CDC37 complex. In contrast, CDK4/6is and CDK4/6 degraders are ineffective in antagonizing tumor cells expressing thermostable CDK6, due to their weaker binding to CDK6 in these cells. Thus, we uncover a general mechanism of intrinsic resistance to CDK4/6is and CDK4/6i-derived degraders and the need for new inhibitors targeting the CDK4/6i-resistant, thermostable form of CDK6 for application as cancer therapeutics.

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Fig. 1: Intrinsic resistance to CDK4/6is is associated with incomplete inhibition of RB–E2F and expression of CDK6.
Fig. 2: Low expression of CDK6 predicts for sensitivity to CDK4/6is in NSCLC.
Fig. 3: Tumor cells expressing both CDK4 and CDK6 depend selectively on CDK6.
Fig. 4: Development of MS140, a potent and selective CDK4/6 degrader (PROTAC).
Fig. 5: CDK4/6-directed degradation is more effective than CDK4/6is in CDK4/6i-S tumor cells.
Fig. 6: In CDK4/6-R cells, CDK4/6 degraders fail to degrade CDK6 due to weak binding of compound.
Fig. 7: CDK4/6i-R cells express CDK6 as a thermostable, weak HSP90 client protein.

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Data availability

MS raw files for CDK6 complex analysis are available at the Mass Spectrometry Interactive Virtual Environment (https://massive.ucsd.edu) under ID MSV000086571.

MS files for global protein degradation have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD023137.

Normalized protein quantification data and cell line omics data can be downloaded at DepMap (https://depmap.org/portal/).

Reagents generated in this study will be made available on request, but we may require a payment and/or a completed Materials Transfer Agreement if there is potential for commercial application. Source data are provided with this paper.

Code availability

Data analysis was performed in R v.3.6.3 using custom-made or publicly available R packages. The code is available from GitHub (https://github.com/lijin0303/CDK46_expression_dependency). Figure 3e and Extended Data Fig. 3b were generated with R 3.6.3 (Packages taigr, dplyr, tidyr, ggplot2, ggpubr) and corresponding code can be found in https://github.com/lijin0303/CDK46_expression_dependency. Source data are provided with this paper.

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Acknowledgements

We thank A. Lujambio, S. Parekh, R. Parsons, E.P. Reddy, R. Sachidanandam, (Icahn School of Medicine at Mount Sinai), I. Aifantis (New York University), S.M. Buckley (University of Nebraska), C.A. Pratilas (Johns Hopkins University) and W.G. Kaelin Jr. (Harvard Medical School) for sharing cell lines and reagents. P.I.P. is supported by the US National Institutes of Health (NIH)/NCI (R01CA204314, R01 CA240362 and R01CA238229), the Irma T. Hirschl Trust, the Manhasset Women’s Coalition against Breast Cancer, the Breast Cancer Alliance, the Melanoma Research Foundation, the Melanoma Research Alliance and Tisch Cancer Institute developmental awards. T.A.A. has been supported by grant T32CA078207 and Z.K. would like to acknowledge the 2017 Robin Chemers Neustein Postdoctoral Fellowship. W.R.S. acknowledges support from the NIH (1R01CA233626) and the Ludwig Center at Harvard, T.I. is supported by the Department of Defense Peer Reviewed Cancer Research Program Horizon Award (W81XWH-19-1-0271). X.C. is supported by the NIH (R01-GM133107). S.A.A. acknowledges support from a grant from the Breast Cancer Research Foundation. This work was supported in part by the P30CA196521 grant (to J.J.) from the NIH and an endowed professorship from the Icahn School of Medicine at Mount Sinai (to J.J.). This work utilized the AVANCE NEO 600 MHz NMR Spectrometer System that was upgraded with funding from an NIH SIG grant 1S10OD025132-01A1.

Author information

Authors and Affiliations

  1. Department of Oncological Sciences, The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Xuewei Wu, Tamer A. Ahmed, Zoi Karoulia, Christos Adamopoulos, Stuart A. Aaronson & Poulikos I. Poulikakos

  2. Department of Pharmacological Sciences, The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Xiaobao Yang, Yan Xiong, Jing Liu & Jian Jin

  3. The Broad Institute of Harvard and MIT, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA

    Ruitong Li, Takahiro Ito & William R. Sellers

  4. Eli Lilly and Company, Indianapolis, IN, USA

    Hong Wang & Sean G. Buchanan

  5. Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, USA

    Li Wang, Ling Xie & Xian Chen

  6. Department of Biochemistry and Molecular Pharmacology, New York University, New York, NY, USA

    Beatrix Ueberheide

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  1. Xuewei Wu
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Contributions

X.W. conceived of the study, performed most of the experiments, analyzed the data and helped write the manuscript. X.Y. and Y.X. synthesized chemical compounds. T.A.A, Z.K. and C.A. performed experiments and analyzed data. S.A.A designed research, analyzed data and helped write the manuscript. J.L. and J.J. designed chemical compounds and analyzed data. L.X. carried out MS analysis. B.U. and X.C. supervised MS analysis. R.L., L.W., T.I. and H.W. performed data analysis. S.G.B. and W.R.S. designed the research, analyzed the data and helped to write the manuscript. P.I.P conceived of the study, designed research, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Poulikos I. Poulikakos.

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Competing interests

X.W., X.Y., Y.X., T.A., Z.K., J.L., J.J. and P.I.P. hold a patent on CDK4/6-directed degradation (WO/2018/106870). The Jian Jin laboratory received research funds from Celgene Corporation, Levo Therapeutics and Cullgen, Inc. J.J. is an equity shareholder and consultant of Cullgen, Inc. H.W. and S.G.B. are employees of Eli Lilly. All other authors declare no competing interests.

Additional information

Peer review information Nature Cancer thanks John Brognard, Keiran Smalley and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Intrinsic resistance to CDK4/6i is associated with incomplete inhibition of Rb/E2F and expression of CDK6.

a, Cell growth crystal violet assay for the indicated cell lines treated with increasing concentrations of PB for 10-16 days and stained with crystal violet. CDK4/6i-sensitive cell lines were highlighted in blue, CDK4/6i-resistant cell lines were in red. b, MCF7 and HCT116 were treated with 1 µM PB for 24, 48 and 72 hr and lysates were immunoblotted with the indicated antibodies. c, Colo205 cells were treated with 1 µM PB at the indicated time points. Cell lysates were immunoblotted with the indicated antibodies.

Source data

Extended Data Fig. 2 Low expression of CDK6 predicts for sensitivity to CDK4/6i in NSCLC.

a, The indicated cell lines were treated with increasing concentrations of PB for 24 hr. Lysates were immunoblotted with the indicated antibodies. b, GI90 values of PB and CDK4/6 dependency in NSCLC cell lines.

Source data

Extended Data Fig. 3 Tumors expressing both CDK4 and CDK6 depend selectively on CDK6.

a, A673 and TC-71 cells were transfected with non-targeting control or siCDK4 or siCDK6 for 72 hr. Lysates were immunoblotted with the indicated antibodies. b, Relationship between CDK4 and CDK6 expression (CCLE RNA-seq) and DepMap CRISPR–Cas9 single-gene knockout scores (CERES; 20Q1 public dataset). All expression values are in log2(TPM +1). Cell lines harboring COSMIC hotspot mutations to RB1 are annotated in orange. P-values were calculated based on linear regression analysis.

Source data

Extended Data Fig. 4 Development of MS140, a potent and selective CDK4/6-degrader (PROTAC).

a. IC50 of in vitro kinase activity assays for PB and MS140 against CDK4/cyclin D1 and CDK6/cyclin D1. b, T47D cells were pretreated with either the proteasome inhibitor 100 nM bortezomib (BOR), 10 µM PB, 10 µM pomalidomide (POM) or 1 µM MLN4924 (MLN) for 4 hr, followed by treatment with MS140 (100 nM/3 hr). Lysates were subjected to immunoblotting with the indicated antibodies. c, Chemical structure of the MS140 negative control (MS140-ve) that does not bind CRBN.

Source data

Extended Data Fig. 5 CDK4/6-directed degradation is more effective than CDK4/6i in CDK4/6i-S tumor cells.

a, MCL cell lines were treated with 0.1 µM PB or MS140 at different time points. Lysates were immunoblotted with the indicated antibodies. b, Colo205 cells expressing Dox-inducible shCDK4 or shCDK6 were treated with or without 0.1 µg/ml doxycycline for 72 hr and cell lysates were subjected to immunoblotting with the indicated antibodies. c, Colo205 cells expressing Dox-inducible shCDK4 or shCDK6 were treated with or without 0.1 µg/ml doxycycline for 10 days followed by crystal violet staining. d, Dependency score of CDK4 and CDK6 from cancer cell line encyclopedia (CCLE) and Depmap portal database. e, GI50 values of PB and MS140 in hematologic cancer cell lines. f, Growth curve for an efficacy assay in JeKo-1 tumor xenografts in nude mice treated with vehicle or MS140 (25 mg/kg, b.i.d) or PB (50 mg/kg, q.d.) for 21 days. Each treatment contained 8 animals (n=8). Data represent mean ± SEM. g, Body weight in mice bearing JeKo-1 tumors treated with vehicle (n=8) or PB (50 mg/kg, q.d., n=8) or MS140 (25 mg/kg, b.i.d., n=8) in the course of the experiment (21 days). Data are presented as mean ± S.D. h, White blood cell, lymphocytes and red blood cell counts in C57BL/6 mice before treatment and post treatment with PB (50 mg/kg, q.d., n=8) or MS140 (25 mg/kg, b.i.d., n=7) for 21 days. Data are presented as mean ± S.D. Statistical significance was determined by paired two-tailed Student’s t-test.

Source data

Extended Data Fig. 6 In CDK4/6-R cells, CDK4/6 degraders fail to degrade CDK6 due to weak binding of compound.

a, Calu6 cells transiently expressing pcDNA3 (Ev) or pcDNA3-Flag-CRBN were treated with increasing concentrations of MS140 for 24 hr. Lysates were subjected to immunoblotting with the indicated antibodies. b, MV4-11 and A375 were treated MS140 (3 nM) or YKL-06-102 (3 nM) or BSJ-02-162 (3 nM) at different time points. Lysates were subjected to immunoblotting with the indicated antibodies. c, KMS-12-PE and Calu6 were treated MS140 (3 nM) or YKL-06-102 (3 nM) or BSJ-02-162 (3 nM) at different time points. Lysates were subjected to immunoblotting with the indicated antibodies.

Source data

Extended Data Fig. 7 CDK4/6i-resistant cells express CDK6 as a thermostable, weak HSP90 client protein.

a, Comparison of total peptide-spectrum match (PSM) for CDK6-interacting proteins by mass spectrometry in KMS-12-PE and Calu6. b, Cell lysates from Colo205 and Calu6 were either subjected to Co-IP with a CDK6 antibody followed by immunoblotting with HSP90, CDC37 and CDK6, or immunoblotted with the indicated antibodies. c, The indicated cell lines were treated with increasing concentrations of Ganetespib (GAN) for 24 hr. Lysates were subjected to immunoblotting with the indicated antibodies. d, The indicated cell lines were treated with 40 nM Luminespib (LUM) at the indicated time points. Lysates were subjected to immunoblotting with the indicated antibodies. e. Calu6 cell line expressing Dox-inducible shCDC37 were treated with or without 0.1 µg/ml doxycycline for 72 hr and cell lysates were subjected to immunoblotting with the indicated antibodies. f, CDK4-dependent cell lines were treated with 30 nM GAN for the indicated time points. Lysates were subjected to immunoblotting with the indicated antibodies. g, KMS-12-PE and Calu6 were treated with increasing concentrations of GAN for 24 hr. Lysates were subjected to immunoblotting with the indicated antibodies. h, Lysates from A375 cells ectopically expressing V5-CDK6 or V5-CDK6 S178p were immunoprecipited with a V5 antibody. The immunoprecipitates were subjected to kinase assay with recombinant Rb protein as substrate.

Source data

Supplementary information

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Wu, X., Yang, X., Xiong, Y. et al. Distinct CDK6 complexes determine tumor cell response to CDK4/6 inhibitors and degraders. Nat Cancer 2, 429–443 (2021). https://doi.org/10.1038/s43018-021-00174-z

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