Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Small-molecule inhibitors targeting Polycomb repressive complex 1 RING domain

Nature Chemical Biology volume 17, pages 784–793 (2021)Cite this article

Subjects

Abstract

Polycomb repressive complex 1 (PRC1) is an essential chromatin-modifying complex that monoubiquitinates histone H2A and is involved in maintaining the repressed chromatin state. Emerging evidence suggests PRC1 activity in various cancers, rationalizing the need for small-molecule inhibitors with well-defined mechanisms of action. Here, we describe the development of compounds that directly bind to RING1B–BMI1, the heterodimeric complex constituting the E3 ligase activity of PRC1. These compounds block the association of RING1B–BMI1 with chromatin and inhibit H2A ubiquitination. Structural studies demonstrate that these inhibitors bind to RING1B by inducing the formation of a hydrophobic pocket in the RING domain. Our PRC1 inhibitor, RB-3, decreases the global level of H2A ubiquitination and induces differentiation in leukemia cell lines and primary acute myeloid leukemia (AML) samples. In summary, we demonstrate that targeting the PRC1 RING domain with small molecules is feasible, and RB-3 represents a valuable chemical tool to study PRC1 biology.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Development and structural characterization of the PRC1 inhibitor RB-2.
Fig. 2: RB-3 inhibits PRC1 and disrupts the interaction with nucleosomes.
Fig. 3: Treatment with RB-3 leads to a reduction in H2Aub and differentiation in TEX cells.
Fig. 4: RB-3 reduces H2Aub via disruption of PRC1 binding to target genes.
Fig. 5: Treatment with RB-3 reduces colony forming capacity of MLL–ENL cells.
Fig. 6: RB-3 reduces global H2Aub levels and induces differentiation in primary human AML samples.

Data availability

RNA-seq and ChIP–seq data for TEX cells treated with RB-3 have been submitted to the Gene Expression Omnibus (GEO) database under accession codes GSE123490 and GSE123930, respectively. Structures of RING1B–BMI1f and RING1B–BMI1f cocrystalized in the presence of RB-2 were deposited in the PDB under the accession codes 6WI7 and 6WI8, respectively. The structure of the RING1B–BMI1f–RB-2 complex determined using a hybrid approach was deposited in the PDB under the accession code 7ND1. PDB structures 3RPB and 4R8P were used for data analysis. Source data are provided with this paper.

References

  1. Simon, J. A. & Kingston, R. E. Mechanisms of Polycomb gene silencing: knowns and unknowns. Nat. Rev. Mol. Cell Biol. 10, 697–708 (2009).

    Article  CAS  PubMed  Google Scholar 

  2. Chittock, E. C., Latwiel, S., Miller, T. C. & Muller, C. W. Molecular architecture of Polycomb repressive complexes. Biochem. Soc. Trans. 45, 193–205 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Gao, Z. et al. PCGF homologs, CBX proteins, and RYBP define functionally distinct PRC1 family complexes. Mol. Cell 45, 344–356 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Taherbhoy, A. M., Huang, O. W. & Cochran, A. G. BMI1–RING1B is an autoinhibited RING E3 ubiquitin ligase. Nat. Commun. 6, 7621 (2015).

    Article  PubMed  Google Scholar 

  5. Scelfo, A. et al. Functional landscape of PCGF proteins reveals both RING1A/B-dependent- and RING1A/B-independent-specific activities. Mol. Cell 74, 1037–1052 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Buchwald, G. et al. Structure and E3-ligase activity of the Ring–Ring complex of Polycomb proteins Bmi1 and Ring1b. EMBO J. 25, 2465–2474 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Tamburri, S. et al. Histone H2AK119 mono-ubiquitination is essential for Polycomb-mediated transcriptional repression. Mol. Cell 77, 840–856 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Blackledge, N. P. et al. PRC1 catalytic activity is central to Polycomb system function. Mol. Cell 77, 857–874 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. McGinty, R. K., Henrici, R. C. & Tan, S. Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome. Nature 514, 591–596 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Blackledge, N. P. et al. Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and Polycomb domain formation. Cell 157, 1445–1459 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Rose, N. R. et al. RYBP stimulates PRC1 to shape chromatin-based communication between Polycomb repressive complexes. eLife 5, e18591 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Fursova, N. A. et al. Synergy between variant PRC1 complexes defines Polycomb-mediated gene repression. Mol. Cell 74, 1020–1036 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Bonnet, D. & Dick, J. E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 3, 730–737 (1997).

    Article  CAS  PubMed  Google Scholar 

  14. Kreso, A. et al. Self-renewal as a therapeutic target in human colorectal cancer. Nat. Med. 20, 29–36 (2014).

    Article  CAS  PubMed  Google Scholar 

  15. Martin-Perez, D., Piris, M. A. & Sanchez-Beato, M. Polycomb proteins in hematologic malignancies. Blood 116, 5465–5475 (2010).

    Article  CAS  PubMed  Google Scholar 

  16. Sauvageau, M. & Sauvageau, G. Polycomb group proteins: multi-faceted regulators of somatic stem cells and cancer. Cell Stem Cell 7, 299–313 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Lessard, J. & Sauvageau, G. Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature 423, 255–260 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Shima, H. et al. Ring1A and Ring1B inhibit expression of Glis2 to maintain murine MOZ–TIF2 AML stem cells. Blood 131, 1833–1845 (2018).

    Article  CAS  PubMed  Google Scholar 

  19. Rizo, A. et al. Repression of BMI1 in normal and leukemic human CD34+ cells impairs self-renewal and induces apoptosis. Blood 114, 1498–1505 (2009).

    Article  CAS  PubMed  Google Scholar 

  20. van den Boom, V. et al. Non-canonical PRC1.1 targets active genes independent of H3K27me3 and is essential for leukemogenesis. Cell Rep. 14, 332–346 (2016).

    Article  PubMed  CAS  Google Scholar 

  21. Alchanati, I. et al. The E3 ubiquitin-ligase Bmi1/Ring1A controls the proteasomal degradation of Top2α cleavage complex—a potentially new drug target. PLoS ONE 4, e8104 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Su, W. et al. The Polycomb repressor complex 1 drives double-negative prostate cancer metastasis by coordinating stemness and immune suppression. Cancer Cell 36, 139–155 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Li, Z. et al. Structure of a Bmi-1–Ring1B Polycomb group ubiquitin ligase complex. J. Biol. Chem. 281, 20643–20649 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. van Zundert, G. C. P. et al. The HADDOCK2.2 web server: user-friendly integrative modeling of biomolecular complexes. J. Mol. Biol. 428, 720–725 (2016).

    Article  PubMed  CAS  Google Scholar 

  25. Bhatnagar, S. et al. TRIM37 is a new histone H2A ubiquitin ligase and breast cancer oncoprotein. Nature 516, 116–120 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kalb, R., Mallery, D. L., Larkin, C., Huang, J. T. & Hiom, K. BRCA1 is a histone-H2A-specific ubiquitin ligase. Cell Rep. 8, 999–1005 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Horn, V. et al. Structural basis of specific H2A K13/K15 ubiquitination by RNF168. Nat. Commun. 10, 1751 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Dixon, A. S. et al. NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells. ACS Chem. Biol. 11, 400–408 (2016).

    Article  CAS  PubMed  Google Scholar 

  29. Warner, J. K. et al. Direct evidence for cooperating genetic events in the leukemic transformation of normal human hematopoietic cells. Leukemia 19, 1794–1805 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. McDermott, S. P. et al. A small molecule screening strategy with validation on human leukemia stem cells uncovers the therapeutic efficacy of kinetin riboside. Blood 119, 1200–1207 (2012).

    Article  CAS  PubMed  Google Scholar 

  31. van Rhenen, A. et al. High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival. Clin. Cancer Res. 11, 6520–6527 (2005).

    Article  PubMed  CAS  Google Scholar 

  32. Pulikkan, J. A., Tenen, D. G. & Behre, G. C/EBPα deregulation as a paradigm for leukemogenesis. Leukemia 31, 2279–2285 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Yu, M. et al. Direct recruitment of Polycomb repressive complex 1 to chromatin by core binding transcription factors. Mol. Cell 45, 330–343 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Barbour, H., Daou, S., Hendzel, M. & Affar, E. B. Polycomb group-mediated histone H2A monoubiquitination in epigenome regulation and nuclear processes. Nat. Commun. 11, 5947 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Cales, C. et al. Inactivation of the Polycomb group protein Ring1B unveils an antiproliferative role in hematopoietic cell expansion and cooperation with tumorigenesis associated with Ink4a deletion. Mol. Cell. Biol. 28, 1018–1028 (2008).

    Article  CAS  PubMed  Google Scholar 

  36. Nishida, Y. et al. Preclinical activity of the novel B-cell-specific Moloney murine leukemia virus integration site 1 inhibitor PTC-209 in acute myeloid leukemia: implications for leukemia therapy. Cancer Sci. 106, 1705–1713 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Barabe, F., Kennedy, J. A., Hope, K. J. & Dick, J. E. Modeling the initiation and progression of human acute leukemia in mice. Science 316, 600–604 (2007).

    Article  CAS  PubMed  Google Scholar 

  38. Jeusset, L. M. & McManus, K. J. Developing targeted therapies that exploit aberrant histone ubiquitination in cancer. Cells 8, 165 (2019).

    Article  CAS  PubMed Central  Google Scholar 

  39. Park, I. K. et al. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature 423, 302–305 (2003).

    Article  CAS  PubMed  Google Scholar 

  40. Su, W. J. et al. RNF2/Ring1b negatively regulates p53 expression in selective cancer cell types to promote tumor development. Proc. Natl Acad. Sci. USA 110, 1720–1725 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Ardley, H. C. Ring finger ubiquitin protein ligases and their implication to the pathogenesis of human diseases. Curr. Pharm. Des. 15, 3697–3715 (2009).

    Article  CAS  PubMed  Google Scholar 

  42. Duffy, M.J., Synnott, N.C., O’Grady, S. & Crown, J. Targeting p53 for the treatment of cancer. Semin. Cancer Biol. https://doi.org/10.1016/j.semcancer.2020.07.005 (2020).

  43. Watt, G. F., Scott-Stevens, P. & Gaohua, L. Targeted protein degradation in vivo with proteolysis targeting chimeras: current status and future considerations. Drug Discov. Today Technol. 31, 69–80 (2019).

    Article  PubMed  Google Scholar 

  44. Gray, F. et al. BMI1 regulates PRC1 architecture and activity through homo- and hetero-oligomerization. Nat. Commun. 7, 13343 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Jaatinen, T. et al. Global gene expression profile of human cord blood-derived CD133+ cells. Stem Cells 24, 631–641 (2006).

    Article  CAS  PubMed  Google Scholar 

  46. Gal, H. et al. Gene expression profiles of AML derived stem cells; similarity to hematopoietic stem cells. Leukemia 20, 2147–2154 (2006).

    Article  CAS  PubMed  Google Scholar 

  47. Sheffield, P. J., Derewenda, U., Taylor, J., Parsons, T. J. & Derewenda, Z. S. Expression, purification and crystallization of a BH domain from the GTPase regulatory protein associated with focal adhesion kinase. Acta Crystallogr. D Biol. Crystallogr. 55, 356–359 (1999).

    Article  CAS  PubMed  Google Scholar 

  48. Brzovic, P. S. et al. Binding and recognition in the assembly of an active BRCA1/BARD1 ubiquitin-ligase complex. Proc. Natl Acad. Sci. USA 100, 5646–5651 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Wiener, R. et al. E2 ubiquitin-conjugating enzymes regulate the deubiquitinating activity of OTUB1. Nat. Struct. Mol. Biol. 20, 1033–1039 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    Article  CAS  PubMed  Google Scholar 

  51. Tugarinov, V. & Kay, L. E. Quantitative NMR studies of high molecular weight proteins: application to domain orientation and ligand binding in the 723 residue enzyme malate synthase G. J. Mol. Biol. 327, 1121–1133 (2003).

    Article  CAS  PubMed  Google Scholar 

  52. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  PubMed  Google Scholar 

  53. Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22–25 (2010).

    Article  CAS  PubMed  Google Scholar 

  54. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Tugarinov, V. & Kay, L. E. Ile, Leu, and Val methyl assignments of the 723-residue malate synthase G using a new labeling strategy and novel NMR methods. J. Am. Chem. Soc. 125, 13868–13878 (2003).

    Article  CAS  PubMed  Google Scholar 

  58. Huang, H. et al. Covalent inhibition of NSD1 histone methyltransferase. Nat. Chem. Biol. 16, 1403–1410 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Egan, B. et al. An alternative approach to ChIP–seq normalization enables detection of genome-wide changes in histone H3 lysine 27 trimethylation upon EZH2 inhibition. PLoS ONE 11, e0166438 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  60. Grembecka, J. et al. Menin–MLL inhibitors reverse oncogenic activity of MLL fusion proteins in leukemia. Nat. Chem. Biol. 8, 277–284 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was funded by the National Institute of Health (NIH) R01 grants CA207272, CA226759 and CA240514 to T.C., CA201204, CA244254 and CA160467 to J.G. and LLS Scholar grants (1340-17) to T.C. and (1215-14) to J.G. This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract number DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant 085P1000817). TEX and MLL–ENL cells were received from J. Dick, University Health Network, Toronto, Canada. We thank M. Carroll and G. Danet-Desnoyers from the Stem Cell and Xenograft Core at the University of Pennsylvania for providing a human AML primary sample.

Author information

Author notes

  1. Jingya Wang

    Present address: MedImmune, LLC, Gaithersburg, MD, USA

  2. These authors contributed equally: S. Shukla, W. Ying, F. Gray, Y. Yao, M. L. Simes.

  3. These authors jointly supervised this work: J. Grembecka, T. Cierpicki.

Authors and Affiliations

  1. Department of Pathology, University of Michigan, Ann Arbor, MI, USA

    Shirish Shukla, Weijiang Ying, Felicia Gray, Yiwu Yao, Miranda L. Simes, Qingjie Zhao, Hongzhi Miao, Hyo Je Cho, Paula González-Alonso, Alyssa Winkler, George Lund, Trupta Purohit, EunGi Kim, Xiaotian Zhang, Joshua M. Ray, Shihan He, Caroline Nikolaidis, Juliano Ndoj, Jingya Wang, Russell J. H. Ryan, Jolanta Grembecka & Tomasz Cierpicki

  2. Biological and Environmental Science and Engineering (BESE), King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia

    Łukasz Jaremko & Mariusz Jaremko

  3. Division of Hematology and Medical Oncology, Leukemia Program, Weill Cornell Medicine/New York-Presbyterian Hospital, New York, NY, USA

    Monica L. Guzman

Authors
  1. Shirish Shukla
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Weijiang Ying
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Felicia Gray
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yiwu Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Miranda L. Simes
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Qingjie Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hongzhi Miao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hyo Je Cho
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Paula González-Alonso
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Alyssa Winkler
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. George Lund
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Trupta Purohit
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. EunGi Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Xiaotian Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Joshua M. Ray
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Shihan He
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Caroline Nikolaidis
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Juliano Ndoj
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Jingya Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Łukasz Jaremko
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Mariusz Jaremko
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Russell J. H. Ryan
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Monica L. Guzman
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Jolanta Grembecka
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Tomasz Cierpicki
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.C. and J.G. were responsible for initiating and supervising the project. S.S., F.G., H.M., P.G.-A., M.L.S., A.W., T.P., S.H., C.N., J.N., J.W., X.Z., J.M.R. and E.K. performed screenings, testing of biochemical activity, cell-based studies and animal studies; H.J.C., F.G., G.L., Ł.J. and M.J. performed the structural biology studies; W.Y., Y.Y. and Q.Z. synthesized compounds; M.L.G. and R.J.H.R. provided reagents and advised the study. All authors contributed to data analysis and writing of the manuscript.

Corresponding authors

Correspondence to Jolanta Grembecka or Tomasz Cierpicki.

Ethics declarations

Competing interests

W.Y., Y.Y., F.G., Q.Z., J.G. and T.C. are co-inventors on a patent application for PRC1 inhibitors.

Additional information

Peer review information Nature Chemical Biology thanks Joshua Plotnik, Chi Wai Eric So 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 Identification and characterization of fragment hit RB-1.

a, 1H-15N HSQC spectrum of 80 μM 15N RING1B-BMI1f (blue) superimposed onto 80 μM 15N RING1B-BMI1f with 2 mM RB-1 (red). Several residues with the largest chemical shift perturbations are labeled. b, In vitro ubiquitination assay showing inhibition of RING1B-BMI1f E3 ligase activity with RB-1. Purified HeLa nucleosomes were incubated with E1 (UBE1), E2 (UBCH5C), E3 (RING1B-BMI1f), ATP and Flag-tagged ubiquitin. Blots were probed with antibody against Flag. RING1B (K85E) corresponds to the assay with RING1B(K85E)-BMI1 mutant. Assay was repeated 2 times (top and bottom immunoblots). c, Mapping of chemical shift perturbations ΔHN (ppm) determined upon binding of 2 mM RB-1 to 80 μM RING1B-BMI1f. Residues are colored as follows: ΔHN < 0.01 (yellow); 0.01 ≤ ΔΗΝ < 0.04 (orange); ΔΗΝ ≥ 0.04 (red). BMI1 residues are in black. Location of K85 is labeled.

Source data

Extended Data Fig. 2 Structural characterization of the RING1B-BMI1f interaction with RB-2.

a, Superposition of the crystal structures of RING1B-BMI1f (colored gray) and RING1B-BMI1 complex (PDB 2CKL; RING1B is colored in magenta and BMI1 is blue). Positions of N- and C-termini are shown. b, Crystal structure determined for RING1B-BMI1f cocrystalized with RB-2. Electron density is contoured at 1 σ (blue) and selected residues are labeled. c, Crystal structure of RING1B-BMI1f cocrystalized with RB-2 shown in surface representation. Opened site exposing hydrophobic side chains of L80 and L100 (both in pale green) is shown. Surrounding positively charged residues are colored in pale blue. d, Analysis of chemical shift perturbations determined for 120 μM DCN RING1B-BMI1f upon binding with 500 μM RB-2. ΔHN has been calculated as \(\sqrt {({\Delta}\delta _{HN}^2 + 0.1 \ast {\Delta}\delta _N^2)}\) in ppm (top) and ΔCO is a difference in CO chemical shifts in ppm (bottom). e, Strips from 3D 1H-13C HSQC-NOESY spectra for 290 μM ILV 2H,13C,15N RING1B-BMI1f (black) and 120 μM ILV 2H,13C,15N RING1B-BMI1f with 500 μM RB-2 (blue). Assignment indicates NOEs between RING1B-BMI1f protein and RB-2). f, Labeling of the RB-2 protons. g, 20 lowest energy conformers of RING1B-BMI1f with bound RB-2. RING1B residues are in pale green and BMI1 are in gray. RB-2 is shown with magenta carbons. h, Binding of RB-2 to the wild-type RING1B-BMI1 and RING1B(L94A)-BMI1f point mutant. 1H-15N HSQC spectra for 60 μM 15N RING1B-BMI1f or 60 μM 15N RING1B(L94A)-BMI1f (shown in red) are titrated with 100 μM RB-2 (shown in blue).

Extended Data Fig. 3 Validation of the binding of RB-3 to RING1B-BMI1f and profiling the selectivity of RB-3.

a, Assigned 1H-13C HSQC spectrum of 60 μM 13C,15N RING1B-BMI1f (red) showing methyl group region superimposed onto the spectrum of 60 μM 13C,15N RING1B-BMI1f with 60 μM RB-3 (blue). b, Comparison of the binding of RB-3 and RB-nc using NMR. 1H-15N HSQC spectra of 60 μM 15N RING1B-BMI1f (red) titrated with 100 μM RB-nc (green), 400 μM RB-nc (blue) and 100 μM RB-3 (black). Of note, 100 μM RB-3 results in almost complete saturation of chemical shift perturbations with 60 μM 15N RING1B-BMI1f. c, In vitro ubiquitination assay showing effect of RB-nc on RING1B-BMI1 E3 ligase activity. H2Aub was detected using Western blot and H3 was used as loading control. Lane with no E3 depicts sample without RING1B-BMI1f serving as negative control. Assay was repeated two times. d, 1H-15N HSQC spectrum of 60 μM 15N RING1B-BMI1f with 5% DMSO (black) superimposed onto 60 μM 15N RING1B-BMI1f with 60 μM (blue) and 120 μM RB-3 (red). e, 1H-15N HSQC spectrum of 60 μM 15N RING1A-BMI1f with 5% DMSO (black) superimposed onto 60 μM 15N RING1A-BMI1f with 60 μM (blue) and 120 μM RB-3 (red). f, In vitro ubiquitination assay showing the effect of RB-3 on RING1A-BMI1f and RING1B-BMI1f activity. H2Aub was detected using Western blot and H3 was used as loading control. Assays were repeated two times. g, In vitro ubiquitination assays showing no activity of RB-3 on BRCA1-BARD1, TRIM37 and RNF168. H2Aub is detected using Western blot and H3 or H2A was used as loading controls. Lanes with no E3 depicts samples without BRCA1-BARD1, TRIM37 or RNF168, respectively, serving as negative controls. Assays were repeated two times.

Source data

Extended Data Fig. 4 Development and characterization of biotinylated RING1B probe compounds.

a, structures of RB-3-biot and RB-nc-biot. b, 1H-15N HSQC spectra of 60 μM 15N RING1B-BMI1f (green) titrated with 60 μM RB-3-biot or RB-nc-biot (red) and 120 μM RB-3-biot or RB-nc-biot (blue). c, Pull-down from HEK293T cells with RB-3-biot or RB-nc-biot followed by detection of RING1B or BMI1 using Western Blot analysis. Two controls were used in the pull-down assay, DMSO or biotinylated histone H3 peptide (biotin-H3), to assess non-specific binding. Assays were repeated two times.

Source data

Extended Data Fig. 5 RB-3 inhibits H2Aub in cancer cell lines.

a, Evaluation of BMI1 and RING1B knockdown, and treatment with RB-3 in K562 cells. Left western blot analysis in K562 cells transfected with BMI1/RING1B siRNAs for 4 d. Right immunoblots of K562 cells treated with indicated doses of RB-3 for 4 d. Representative blots of two independent experiments are shown. b, Evaluation of BMI1 and RING1B knockdown, and treatment with RB-3 in HeLa cells. Left Western blot in HeLa cells transfected with BMI1/RING1B siRNAs for 4 d. Right western blot analysis in HeLa cells treated with indicated doses of RB-3 for 4 d. Representative blots of two independent experiments are shown. c, Activity of RB-3 and RB-nc in MOLM13 cells treated for 4 d with indicated doses of compounds. Representative blots of two independent experiments are shown. d, Activity of RB-3 in MV4;11 cells treated for 4 d. Representative blots of two independent experiments are shown.

Source data

Extended Data Fig. 6 Long-term effect of treatment of TEX cells with RB-3.

a, Western blot detection of H2Aub in TEX cells treated with increasing doses of RB-3 and RB-nc for 21 days. Representative blot out of two replicates. b, Western blot detection of total cellular ubiquitination levels using ubiquitin specific antibody in TEX cells treated with RB-3 for 21 days. Representative blot out of three replicates. c, Flow cytometry analysis of CD34 in TEX cells treated with RB-3 for 7 days. On day 7, cells were stained with APC/CY7-conjugated human anti-CD34 antibody and analyzed by FACS. Representative histograms of two independent experiments. d, Flow cytometry analysis of CD34 and CD38 in TEX cells treated with RB-3 (top panel) and RB-nc (bottom panel) for 21 days. On day 21, cells were stained with APC/CY7-conjugated human CD34 and PE-conjugated CD38 antibodies and analyzed by FACS. Representative histograms of two independent experiments. e,f, Flow cytometry analysis of myeloid differentiation marker CD11b/ITGAM in TEX cells treated with RB-3 for 7 days (e) and with RB-3 (f, top panel) and RB-nc (f, bottom panel) for 21 days. Cells were stained with Pacific Blue human CD11b antibody and analyzed by FACS. Representative histograms of two independent experiments. g, Flow cytometry analysis of dendritic cells differentiation marker CD86/B7-2 in TEX cells treated with RB-3 (top panel) and RB-nc (bottom panel) for 21 days. On day 21, cells were stained with Super Bight 436 conjugated human CD86 antibody and analyzed by FACS. Representative histograms of two independent experiments.

Source data

Extended Data Fig. 7 RB-3 regulates expression of target genes and impairs enrichment of H2Aub and RING1B on target gene promoter regions in TEX cells.

a, qRT–PCR showing time dependent changes in transcript levels of CD34, C/EBPα, CD-86 upon treatment with 25 μM RB-3 and RB-nc. Representative data out of two replicates. b, Analysis of the H2Aub and H3 levels, and binding of RING1B to the two promoter regions of C/EBPα in TEX cells treated with RB-nc and DMSO for 8 days using ChIP assay. Representative data out of two replicates. c, Analysis of the H2Aub and H3 levels, and binding of RING1B to the two promoter regions of CD34 in TEX cells treated with RB-3 (red), RB-nc (blue) and DMSO (black) for 8 days using ChIP assay. Representative data out of two replicates. d, Analysis of the H2Aub and H3 levels, and binding of RING1B to the two promoter regions of ITGAM in TEX cells treated with RB-3 (red), RB-nc (blue) and DMSO (black) for 8 days using ChIP assay. Representative data out of two replicates. Two promoter region primers between 1 to 2 kb upstream of the transcription start site (TSS) for each of CD34, C/EBPα and ITGAM promoter were selected and analyzed (panels b, c, d). e, Average genome-wide occupancy of H2Aub around the transcription start sites (TSS) and genomic regions in TEX cells determined from ChIP-seq experiment. TEX cells were treated with DMSO and 25 μM RB-3 for 6 days.

Extended Data Fig. 8 Comparison of the activity of PTC209 and RB-3 in TEX cells.

a, Graph showing effect of PTC209 on TEX cells proliferation. TEX cells were treated with the indicated concentrations of PTC209 for 4 days and processed for cell proliferation analysis. Experiment was performed 2 times, GI50 is mean ± s.d. b, western blot showing levels of H2Aub, H2Bub, H2A, H3 and BMI1 in TEX cells treated for 4 days with indicated doses of RB-3 (left) and PTC209 (right). Representative blots from two independent experiments. c, qRT–PCR indicating levels of C/EBPα upon 4 d treatment with indicated doses of RB-3 and PTC209. Representative data from two replicates. d, qRT–PCR indicating levels of CD34 upon 4 d treatment with indicated doses of RB-3 and PTC209. Representative data from two replicates.

Source data

Extended Data Fig. 9 RB-3 has no effect on normal CD34+ cells.

a, Quantitation of colony numbers (CFU) upon treatment of cord blood CD34+ cells from healthy donors with RB-3. Experiments were performed two times and representative data are mean ± s.d. and analyzed by two-tailed t-test; ns – not significant. b, Population of CD34+ cells upon treatment with RB-3. Experiments were performed two times and representative data are mean ± s.d. and analyzed by two-tailed t-test; ns – not significant. c, Flow cytometry analysis of CD34 and CD38. Representative histograms of two independent experiments. d, Effect of RB-3 on various populations of mature cells with representative colony pictures. CFU-GM, granulocyte–macrophage progenitors; CFU-GEMM, oligopotential progenitors; BFU-E, burst-forming unit-erythroid cells; CFU-E, colony-forming unit-erythroid cells. Experiments were performed two times and representative data are mean ± s.d. and analyzed by two-tailed t-test; ns – not significant. e, Cell morphology of CD34+ cells treated with RB-3 analyzed by Wright Giemsa staining. Representative slides are shown from two independent experiments.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Figs. 1–7 and Note.

Reporting Summary

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Shukla, S., Ying, W., Gray, F. et al. Small-molecule inhibitors targeting Polycomb repressive complex 1 RING domain. Nat Chem Biol 17, 784–793 (2021). https://doi.org/10.1038/s41589-021-00815-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-021-00815-5

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing