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Discovery of a first-in-class CDK2 selective degrader for AML differentiation therapy

Nature Chemical Biology volume 17, pages 567–575 (2021)Cite this article

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Abstract

The discovery of effective therapeutic treatments for cancer via cell differentiation instead of antiproliferation remains a great challenge. Cyclin-dependent kinase 2 (CDK2) inactivation, which overcomes the differentiation arrest of acute myeloid leukemia (AML) cells, may be a promising method for AML treatment. However, there is no available selective CDK2 inhibitor. More importantly, the inhibition of only the enzymatic function of CDK2 would be insufficient to promote notable AML differentiation. To further validate the role and druggability of CDK2 involved in AML differentiation, a suitable chemical tool is needed. Therefore, we developed first-in-class CDK2-targeted proteolysis-targeting chimeras (PROTACs), which promoted rapid and potent CDK2 degradation in different cell lines without comparable degradation of other targets, and induced remarkable differentiation of AML cell lines and primary patient cells. These data clearly demonstrated the practicality and importance of PROTACs as alternative tools for verifying CDK2 protein functions.

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Fig. 1: The design of PROTAC compounds and their potencies in vitro.
Fig. 2: Potent and selective CDK2 degradation induced by CPS2.
Fig. 3: CDK2 degradation and AML cell differentiation triggered by CPS2.
Fig. 4: The antileukemic effects of CPS2 are mediated by CDK2 degradation.
Fig. 5: Transcriptome changes in AML cells following CPS2 and shCDK2 treatment.
Fig. 6: Effects of CPS2 on primary AML cell and HSC differentiation.

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Data availability

All raw gels are included in source data files. The source structure of CDK2 and CRBN can be accessed via PDB IDs-4CI3 and 3PY0 (http://www.rcsb.org/). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository46 with the dataset identifier PXD021592. The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive47 in National Genomics Data Center, Beijing Institute of Genomics, Chinese Academy of Sciences48, under accession number CRA003240, which is publicly accessible at https://bigd.big.ac.cn/gsa. Source data are provided with this paper.

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Acknowledgements

We acknowledge and thank the patients who provided the primary samples for this research. The laboratory of Q. Wang, Beijing Institute Genomics, Chinese Academy of Science kindly donated MV-4-11, OCI-AML2, OCI-AML3 and Kasumi-1 cell lines. MOLT-4 cells were provided by Stem Cell Bank, Chinese Academy of Sciences. NB4 cells were provided by L. Wu, University of Southern California. We acknowledge them for their support for this work. We acknowledge Q. Tao, Z. Yang, H. Yang, W. Lv, S. Zhu, C. Yang, X. Yang, Y. Su, J. Wang, Y. Zhang and C. Qian for their help. This work was supported by the National Natural Science Foundation of China (nos. 81573277, 81622042 and 81773567), National Major Scientific and Technological Special Project for ‘Significant New Drugs Development’ (nos. SQ2017ZX095003 and 2018ZX09711001) and ‘National Key R&D Program of China’ (no. 2020YFE0202200).

Author information

Author notes

  1. These authors contributed equally: Liguo Wang, Xuejing Shao, Tianbai Zhong, Yue Wu, Aixiao Xu.

Authors and Affiliations

  1. MOE Key Laboratory of Protein Sciences, School of Pharmaceutical Sciences, MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing, China

    Liguo Wang, Yue Wu, Xiuyun Sun, Hongying Gao, Yongbo Liu, Tianlong Lan, Yan Tong & Yu Rao

  2. Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China

    Xuejing Shao, Aixiao Xu, Wenxin Du, Wei Wang, Yingqian Chen, Bo Yang, Qiaojun He & Meidan Ying

  3. Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing, China

    Tianbai Zhong

  4. Department of Research, Beijing Rehabilitation Hospital, Capital Medical University, Beijing, China

    Xue Tao

  5. MOE Key Laboratory of Bioinformatics, Centre for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Ting Li & Haiteng Deng

  6. National Center for Protein Science, Tsinghua University, Beijing, China

    Ting Li, Xianbin Meng & Haiteng Deng

  7. School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, China

    Yu Rao

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Contributions

L.W., X. Shao, T.Z., Y.W. and A.X. contributed equally to this work. Y.R. and M.Y. designed the project. L.W., X.Shao, Y.R. and M.Y. wrote the paper. X. Sun helped organize the paper. L.W., Y.L. and Y.W. designed, simulated and synthesized the molecules. Y.W. and T. Lan helped synthesize the molecules. L.W., T.Z. and X.T. performed western blot procedures, cell proliferation experiments and acute toxicity experiments in vivo. X. Shao and A.X. examined the western blot, cell proliferation, cell differentiation effect and cell apoptosis in AML. Y.T. and H.G. helped prepare the samples for western blot. X. Shao, A.X., W.D., W.W. and Y.C. performed and analyzed RNA-sequencing data, collected patient primary cell samples and performed HSC toxicity experiments. X. Shao, A.X., W.D. and W.W. performed the target validation experiment. T. Li, X.M. and H.D. helped to prepare samples for proteomics and results analysis. B.Y. and Q.H. provided support for some experiments.

Corresponding authors

Correspondence to Meidan Ying or Yu Rao.

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Extended data

Extended Data Fig. 1 Ternary complexes model of a POI, PROTAC and E3 ligase.

Ternary complexes model of a POI, PROTAC and E3 ligase. Ternary complex formation is mediated by PROTAC technology. Apo CDK2 protein (PDB ID: 3PY0) and CRBN (PDB ID: 4CI3) are correspondingly shown as the ribbon and surface in the model. The ternary complex model was based on docking and molecular dynamics simulations with Schrodinger and Desmond software.

Extended Data Fig. 2 Acute toxicity and cell proliferation inhibition of CPS2.

Acute toxicity and cell proliferation inhibition of CPS2. a, Acute toxicity experiment. Vehicle, 400 mg/kg CPS2 and 400 mg/kg J2 were independently intraperitoneally administered into mice at a single dose. The survival time and body weight were recorded (n = 6 for each group). For body weight, ***: p<0.001; J2 vs. Control. #: p<0.05, ##: p<0.01; CPS2 vs. Control. b, Cell proliferation assay of NB4 cells treated with several concentrations of CPS2 and J2 for the indicated times. **: p<0.01, ***: p<0.001; CPS2 or J2 vs. Control. c, Cells apoptosis experiment. PI/Annexin V apoptosis assay of the NB4 cells treated with several concentrations of CPS2 and J2 for the indicated times. *: p<0.05, **: p<0.01, ***: p<0.001; CPS2 or J2 vs. Control. b-c, All experiments were conducted with three replicas. a-c, The error bars represent the means±s.d. The significance analysis was conducted by two-tailed unpaired Student’s t-test.

Source data

Extended Data Fig. 3 Synergetic effect of CPS2 with conventional inhibitors.

Synergetic effect of CPS2 with conventional inhibitors. a-c, In MV-4-11 cell line, CPS2 could enhance the effect of Copanlisib and Palbociclib. a, Cell viability assessment under CPS2 and Copanlisib treatment. For the combo curve, CPS2 was used as indicated concentration with 100 nM Copanlisib. CPS2 and Copanlisib independently were used as control. 5000 MV-4-11 cells were seeded into 96-wells plates. The cells were treated for 3 days. b, Cell viability assessment under CPS2 and Palbociclib treatment. For the combo curve, CPS2 was used as indicated concentration with 120 nM Palbociclib. CPS2 and Palbociclib independently were used as control. 5000 MV-4-11 cells were seeded into 96-wells plates. The cells were treated for 3 days. c, FA-CI curve of CPS2 combo treatment. FA-CI is calculated by CompuSyn (Ver. 1.0) based on the data in (a) and (b). The curve is draw by GraphPad prism 7, nonlinear regression (curve fit). d, Cell viability assessment under CPS2 and Palbociclib treatment in Kasumi-1 cells. For the combo curve, CPS2 was used as indicated concentration with 100 nM Palbociclib. CPS2 and Palbociclib independently were used as control. 5000 Kasumi-1 cells were seeded into 96-wells plates. The cells were treated for 3 days. a-d, The data are individual replicate values and shown as means±s.d. (n= 3 for biologically independent samples per group).

Extended Data Fig. 4 The effect of CPS2 on CDK2 degradation and AML cell differentiation.

The effect of CPS2 on CDK2 degradation and AML cell differentiation. a, Protein expression levels of different CDKs after CPS2 treatment for the indicated times in U937 and HL60 cells. b, Effects of CPS2 on the protein level of CDK2 in AML cells. NB4 and U937 cells were treated with the indicated concentrations of CPS2 for 3 h and 6 h. **: p<0.01, ***: p<0.001; CPS2 vs. the Control. c-d, Cell differentiation analysis of HL60 cells treated with different concentrations of CPS2 for the indicated times. c, CD11b expression. **: p<0.01, ***: p<0.001; CPS2 vs. the Control. d, Cell morphological analysis (RG) and NBT-reducing activity assay (NBT). Left: Representative images of RG and NBT assays. Right: The statistical data from the NBT assay. ***: p<0.001; CPS2 vs. the Control. a-d, The western-blot and images are representative of minimally three independent experiments. The data are individual replicate values and shown as means±s.d. (n= 3 for biologically independent samples per group).

Source data

Extended Data Fig. 5 Effects of AURKA and IKZF1 inhibition on AML cells differentiation.

Effects of AURKA and IKZF1 inhibition on AML cells differentiation. NB4 cells were infected with lentivirus-shAURKA (#1 or #2) or shIKZF1 (#1 or #2) for indicated times. shCDK2 #2 as positive group. a, The silencing efficiency of shRNA against AURKA for 3 days. **: p<0.01, ***: p<0.001; shRNA vs. shCtrl. b, The silencing efficiency of shRNA against IKZF1 for 3 days. ***: p<0.001; shRNA vs. shCtrl. c, CD11b expression. **: p<0.01, ***: p<0.001; shRNA vs. shCtrl. d, NBT-reducing activity assay (NBT). Left: the representative images of NBT assays. Right: The statistical data of NBT assay. *, p<0.05; ***: p<0.001; shRNA vs. shCtrl. e, Cell morphological analysis. f, The overexpression of IKZF1 in NB4 cells. ***, p<0.001; pCLS vs. IKZF1. All data was shown as mean±s.d., n = 3. Other data are representative of at least three individual experiments and one representative image is shown. The significance analysis was conducted by two-tailed unpaired Student’s t test.

Source data

Extended Data Fig. 6 The protein level of CDK2 in AML cells following CPS2 and shCDK2 treatment.

The protein level of CDK2 in AML cells following CPS2 and shCDK2 treatment. a, Protein expression levels of CDK2 after CPS2 treatment for the indicated times in NB4 cells. b, Western-blot analysis of CDK2 expression in NB4 cells transduced with different shRNAs (#1 and #2) against CDK2 for the indicated times. a-b, The gels are representative of minimally three biologically independent experiments. The error bars represent the mean±s.d. of n=3 in biologically duplicates. The significance analysis was conducted by two-tailed unpaired Student’s t-test. (a) ***: p<0.001. Each time point group vs. 0 h group. (b) ***: p<0.001. Each shCDK2 group as indicated vs. shCtrl.

Source data

Extended Data Fig. 7 Schematic representation of comparing gene expression profiles of between CDK2 decline induced by CPS2, shCDK2 and CDK2 inhibition driven by J2.

Schematic representation of comparing gene expression profiles of between CDK2 decline induced by CPS2, shCDK2 and CDK2 inhibition driven by J2. Red circles reflect the differential genes between CPS2 and DMSO, blue circles reflect the overlay differential genes between shCDK2 (#1 & #2) and shCtrl, green circles reflect the differential genes between J2 and DMSO. The overlay shows the shared differential genes between (a) CPS2 (12 h) & shCDK2 (D2.5) & J2 (12 h) and (b) CPS2 (24 h) & shCDK2 (D2.5) & J2 (24 h). The yellow box showed the genes responding to CPS2 and shCDK2, but not J2; The green box showed the genes responding to CPS2, shCDK2 and J2. The differentiation-related genes are highlighted with red.

Extended Data Fig. 8 CPS2 induces primary AML cell differentiation.

CPS2 induces primary AML cell differentiation. a-c, Primary AML samples collected at diagnosis from the peripheral blood of patients were treated with CPS2 for 7 days. a, CD11b expression. b, The protein level of CDK2. c, Cell morphological analysis.

Source data

Supplementary information

Supplementary Information

Supplementary Notes 1 and 2, Figs. 1–8 and Tables 1–5.

Reporting Summary

Supplementary Data 1

The affinity of CPS2 with representative targets.

Supplementary Data 2

Protein level change after CPS2 treatment.

Supplementary Data 3

List of overlapped genes in NB4 cells in response to CPS2

Supplementary Data 4

Three clusters from Metascape pathway enrichment analysis of the changed genes in response to CPS2 and lentivirus-shRNA-CDK2.

Supplementary Data 5

P value of each supplementary figure.

Source data

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Wang, L., Shao, X., Zhong, T. et al. Discovery of a first-in-class CDK2 selective degrader for AML differentiation therapy. Nat Chem Biol 17, 567–575 (2021). https://doi.org/10.1038/s41589-021-00742-5

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