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Small-molecule inhibitors targeting Polycomb repressive complex 1 RING domain

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.

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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.

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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

Affiliations

Authors

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.

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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.

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

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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

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