Article | Published:

AZD9291 inactivates the PRC2 complex to mediate tumor growth inhibition


Deregulated Polycomb repressive complex 2 (PRC2) is intimately involved in tumorigenesis and progression, making it an invaluable target for epigenetic cancer therapy. Disrupting the EZH2–EED interaction, which is required for PRC2 enzymatic activity, is a promising strategy for cancer treatment. However, this kind of inhibitors are still limited. The in-cell protein–protein interaction screening was conducted for approximately 1300 compounds by NanoBRET technology. Co-immunoprecipitation (Co-IP), protein thermal shift assay (PTSA), and cellular thermal shift assay (CETSA) were performed to investigate the regulation of PRC2 by AZD9291. The anti-tumor effects of AZD9291 on breast cancer (BC) cells and diffuse large B-cell lymphoma (DLBCL) cells were detected. MicroRNA array assay, luciferase reporter assay, and qRT-PCR were conducted to identify the interaction and regulation among AZD9291, EZH2, and miR-34a. We discovered that, AZD9291, a potent and selective EGFR inhibitor, disrupted the interaction of EZH2–EED, leading to impairment of PRC2 activity and downregulation of EZH2 protein. In addition, AZD9291 declined EZH2 mRNA expression via upregulating the expression of a tumor suppressor, miR-34a. Our results suggest that AZD9291 can serve as a lead compound for further development of antagonist of PRC2 protein–protein interactions and EZH2 mRNA may be a direct target of miR-34a through non-canonical base pairing.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    Lewis EB. A gene complex controlling segmentation in Drosophila. Nature. 1978;276:565–70.

  2. 2.

    Ntziachristos P, Tsirigos A, Van Vlierberghe P, Nedjic J, Trimarchi T, Flaherty MS, et al. Genetic inactivation of the polycomb repressive complex 2 in T cell acute lymphoblastic leukemia. Nat Med. 2012;18:298–301.

  3. 3.

    De Raedt T, Beert E, Pasmant E, Luscan A, Brems H, Ortonne N, et al. PRC2 loss amplifies Ras-driven transcription and confers sensitivity to BRD4-based therapies. Nature. 2014;514:247–51.

  4. 4.

    Nikoloski G, Langemeijer SM, Kuiper RP, Knops R, Massop M, Tonnissen ER, et al. Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes. Nat Genet. 2010;42:665–7.

  5. 5.

    Morin RD, Johnson NA, Severson TM, Mungall AJ, An J, Goya R, et al. Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin. Nat Genet. 2010;42:181–5.

  6. 6.

    Bodor C, O’Riain C, Wrench D, Matthews J, Iyengar S, Tayyib H, et al. EZH2 Y641 mutations in follicular lymphoma. Leukemia. 2011;25:726–9.

  7. 7.

    McCabe MT, Graves AP, Ganji G, Diaz E, Halsey WS, Jiang Y, et al. Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27). Proc Natl Acad Sci USA. 2012;109:2989–94.

  8. 8.

    Di Croce L, Helin K. Transcriptional regulation by Polycomb group proteins. Nat Struct Mol Biol. 2013;20:1147–55.

  9. 9.

    Kim W, Bird GH, Neff T, Guo G, Kerenyi MA, Walensky LD, et al. Targeted disruption of the EZH2–EED complex inhibits EZH2-dependent cancer. Nat Chem Biol. 2013;9:643–50.

  10. 10.

    Qi W, Zhao K, Gu J, Huang Y, Wang Y, Zhang H, et al. An allosteric PRC2 inhibitor targeting the H3K27me3 binding pocket of EED. Nat Chem Biol. 2017;13:381–8.

  11. 11.

    He Y, Selvaraju S, Curtin ML, Jakob CG, Zhu H, Comess KM, et al. The EED protein–protein interaction inhibitor A-395 inactivates the PRC2 complex. Nat Chem Biol. 2017;13:389–95.

  12. 12.

    Lu X, Yu L, Zhang Z, Ren X, Smaill JB, Ding K. Targeting EGFRL858R/T790M and EGFRL858R/T790M/C797S resistance mutations in NSCLC: current developments in medicinal chemistry. Med Res Rev. 2018;38:1550–81.

  13. 13.

    Machleidt T, Woodroofe CC, Schwinn MK, Mendez J, Robers MB, Zimmerman K, et al. NanoBRET—a novel BRET platform for the analysis of protein–protein interactions. ACS Chem Biol. 2015;10:1797–804.

  14. 14.

    Kong X, Chen L, Jiao L, Jiang X, Lian F, Lu J, et al. Astemizole arrests the proliferation of cancer cells by disrupting the EZH2–EED interaction of polycomb repressive complex 2. J Med Chem. 2014;57:9512–21.

  15. 15.

    Lucet IS, Murphy JM. Characterization of ligand binding to pseudokinases using a thermal shift assay. Methods Mol Biol. 2017;1636:91–104.

  16. 16.

    Jafari R, Almqvist H, Axelsson H, Ignatushchenko M, Lundback T, Nordlund P, et al. The cellular thermal shift assay for evaluating drug target interactions in cells. Nat Protoc. 2014;9:2100–22.

  17. 17.

    Denisenko OS, Suzuki M, Bomsztyk H. Point mutations in the WD40 domain of Eed block its interaction with Ezh2. Mol Cell Biol. 1998;18:5634–42.

  18. 18.

    Zingg D, Debbache J, Schaefer SM, Tuncer E, Frommel SC, Cheng P, et al. The epigenetic modifier EZH2 controls melanoma growth and metastasis through silencing of distinct tumour suppressors. Nat Commun. 2015;6:6051.

  19. 19.

    Li Z, Hou P, Fan D, Dong M, Ma M, Li H, et al. The degradation of EZH2 mediated by lncRNA ANCR attenuated the invasion and metastasis of breast cancer. Cell Death Differ. 2017;24:59–71.

  20. 20.

    Kim KH, Kim W, Howard TP, Vazquez F, Tsherniak A, Wu JN, et al. SWI/SNF-mutant cancers depend on catalytic and non-catalytic activity of EZH2. Nat Med. 2015;21:1491–6.

  21. 21.

    Lee ST, Li Z, Wu Z, Aau M, Guan P, Karuturi RK, et al. Context-specific regulation of NF-kappaB target gene expression by EZH2 in breast cancers. Mol Cell. 2011;43:798–810.

  22. 22.

    Xu K, Wu ZJ, Groner AC, He HH, Cai C, Lis RT, et al. EZH2 oncogenic activity in castration-resistant prostate cancer cells is Polycomb-independent. Science. 2012;338:1465–9.

  23. 23.

    Ambros V. The functions of animal microRNAs. Nature. 2004;431:350–5.

  24. 24.

    Ito Y, Inoue A, Seers T, Hato Y, Igarashi A, Toyama T, et al. Identification of targets of tumor suppressor microRNA-34a using a reporter library system. Proc Natl Acad Sci USA. 2017;114:3927–32.

  25. 25.

    Shi Y, Liu C, Liu X, Tang DG, Wang J. The microRNA miR-34a inhibits non-small cell lung cancer (NSCLC) growth and the CD44hi stem-like NSCLC cells. PLoS One 2014;9:e90022.

  26. 26.

    Adams BD, Parsons C, Slack FJ. The tumor-suppressive and potential therapeutic functions of miR-34a in epithelial carcinomas. Expert Opin Ther Targets. 2016;20:737–53.

  27. 27.

    Cebria F, Kobayashi C, Umesono Y, Nakazawa M, Mineta K, Ikeo K, et al. The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature. 2002;419:624–9.

  28. 28.

    Bachmann IM, Halvorsen OJ, Collett K, Stefansson IM, Straume O, Haukaas SA, et al. EZH2 expression is associated with high proliferation rate and aggressive tumor subgroups in cutaneous melanoma and cancers of the endometrium, prostate, and breast. J Clin Oncol. 2006;24:268–73.

  29. 29.

    Gan L, Yang Y, Li Q, Feng Y, Liu T, Guo W. Epigenetic regulation of cancer progression by EZH2: from biological insights to therapeutic potential. Biomark Res. 2018;6:10.

  30. 30.

    Bracken AP. EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. EMBO J. 2003;22:5323–35.

  31. 31.

    Gardner EE, Lok BH, Schneeberger VE, Desmeules P, Miles LA, Arnold PK, et al. Chemosensitive relapse in small cell lung cancer proceeds through an EZH2-SLFN11 axis. Cancer Cell. 2017;31:286–99.

  32. 32.

    Fan TY, Wang H, Xiang P, Liu YW, Li HZ, Lei BX, et al. Inhibition of EZH2 reverses chemotherapeutic drug TMZ chemosensitivity in glioblastoma. Int J Clin Exp Pathol. 2014;7:6662–70.

  33. 33.

    Mayr C, Wagner A, Stoecklinger A, Jakab M, Illig R, Berr F, et al. 3-Deazaneplanocin A may directly target putative cancer stem cells in biliary tract cancer. Anticancer Res. 2015;35:4697–705.

  34. 34.

    Fabian MR, Sonenberg N, Filipowicz W. Regulation of mRNA translation and stability by microRNAs. Annu Rev Biochem. 2010;79:351–79.

  35. 35.

    Wang P, Li Z, Liu H, Zhou D, Fu A, Zhang E. MicroRNA-126 increases chemosensitivity in drug-resistant gastric cancer cells by targeting EZH2. Biochem Biophys Res Commun. 2016;479:91–6.

  36. 36.

    Zhu Z, Tang J, Wang J, Duan G, Zhou L, Zhou X. MiR-138 acts as a tumor suppressor by targeting EZH2 and enhances cisplatin-induced apoptosis in osteosarcoma cells. PLoS One 2016;11:e0150026.

  37. 37.

    Zhang D, Ni Z, Xu X, Xiao J. MiR-32 functions as a tumor suppressor and directly targets EZH2 in human oral squamous cell carcinoma. Med Sci Monit. 2014;20:2527–35.

  38. 38.

    Zhang T, Qian H, Hu C, Lu H, Li JB, Wu YF, et al. MiR-26a mediates ultraviolet B-induced apoptosis by targeting histone methyltransferase EZH2 depending on Myc expression. Cell Physiol Biochem. 2017;43:1188–97.

  39. 39.

    Qian K, Liu G, Tang Z, Hu Y, Fang Y, Chen Z, et al. The long non-coding RNA NEAT1 interacted with miR-101 modulates breast cancer growth by targeting EZH2. Arch Biochem Biophys. 2017;615:1–9.

  40. 40.

    Zhang L, Liao Y, Tang L. MicroRNA-34 family: a potential tumor suppressor and therapeutic candidate in cancer. J Exp Clin Cancer Res. 2019;38:53.

  41. 41.

    Xia W, Hou M. Mesenchymal stem cells confer resistance to doxorubicin-induced cardiac senescence by inhibiting microRNA-34a. Oncol Lett. 2018;15:10037–46.

  42. 42.

    Di Bari M, Bevilacqua V, De Jaco A, Laneve P, Piovesana R, Trobiani L, et al. miR-34a-5p mediates cross-talk between M2 muscarinic receptors and Notch-1/EGFR pathways in U87MG glioblastoma cells: implication in cell proliferation. Int J Mol Sci. 2018;19:E1631.

  43. 43.

    Kim JS, Kim EJ, Lee S, Tan X, Liu X, Park S, et al. miR-34a and miR-34b/c have distinct effects on the suppression of lung adenocarcinomas. Exp Mol Med. 2019;51:9.

  44. 44.

    Misso G, Di Martino MT, De Rosa G, Farooqi AA, Lombardi A, Campani V, et al. miR-34: a new weapon against cancer? Mol Ther Nucleic Acids. 2014;3:e194.

  45. 45.

    Li CH, Xiao Z, Tong JH, To KF, Fang X, Cheng AS, et al. EZH2 coupled with HOTAIR to silence MicroRNA-34a by the induction of heterochromatin formation in human pancreatic ductal adenocarcinoma. Int J Cancer. 2017;140:120–9.

  46. 46.

    Kwon H, Song K, Han C, Zhang J, Lu L, Chen W. Epigenetic silencing of miRNA-34a in human cholangiocarcinoma via EZH2 and DNA methylation: impact on regulation of Notch pathway. Am J Pathol. 2017;187:2288–99.

Download references

Author information

KLZ, QQS, and YMS performed experiments and analyzed data. KLZ, YFF, JD, and YC  designed the research, drafted and edited the manuscript. YC and JD supervised the research.

Conflict of interest

The authors declare no competing interests.

Correspondence to Jian Ding or Yi Chen.

Supplementary information

Supplementary Information(We want a replacement . Please kindly check the attachment which we provide again. Thanks.)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark


  • PRC2
  • EZH2–EED
  • AZD9291
  • miR-34a
  • non-canonical base pairing
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5