Nuclear receptor-binding SET domain-containing 2 (NSD2) is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two proline-tryptophan-tryptophan-proline (PWWP) domains and five plant homeodomains (PHDs) believed to serve as chromatin reading modules. Here, we report a chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1, antagonizes PWWP1 interaction with nucleosomal H3K36me2 and selectively engages endogenous NSD2 in cells. UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 that result from translocations prevalent in multiple myeloma (MM). Mutations of other NSD2 chromatin reader domains also increase NSD2 nucleolar localization and enhance the effect of UNC6934. This chemical probe and the accompanying negative control UNC7145 will be useful tools in defining NSD2 biology.
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The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE54 partner repository with the data set identifier PXD017641. Additionally, the code used to analyze the proteomics data has been posted to Zenodo at https://doi.org/10.5281/zenodo.5153406. The structures of NSD2-PWWP1 in complex with MRT866 and UNC6934 were deposited to the PDB with accession numbers 7MDN and 6XCG respectively. Source data are provided with this paper.
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The Structural Genomics Consortium is a registered charity (no. 1097737) that receives funds from AbbVie, Bayer AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genentech, Genome Canada through Ontario Genomics Institute (OGI-196), EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant 875510), Janssen, Merck KGaA (also known as EMD in Canada and the United State), Merck & Co (also known as MSD outside Canada and the United States), Pfizer, Takeda and Wellcome (106169/ZZ14/Z). We acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC) for a postdoctoral fellowship awarded to D.D. This work was supported by the National Cancer Institute, NIH (grant R01CA242305) to L.I.J. M.S. gratefully acknowledges NSERC (grant RGPIN-2019-04416). Research in EpiCypher was supported by NIH grants R44GM117683 and R44GM116584. This work was supported by Cancer Research Society operating grant (25418) to D.B.-L. We thank T. Hajian for purifying proteins and L. Halabelian for providing fluorescein-labeled dsDNA. We thank the University of North Carolina’s Department of Chemistry Mass Spectrometry Core Laboratory, especially D. Wallace, for their assistance with MS analysis. The Mass Spectrometry Core Laboratory is supported by the National Science Foundation under grant number CHE-1726291. Research reported in this publication was supported, in part, with funding by the University of North Carolina’s School of Medicine Office of Research. We thank F. Potjewyd for reviewing the primary synthesis data supporting this manuscript. Receptor-, channel- and transporter-binding profiles were generously provided by the National Institute of Mental Health’s Psychoactive Drug Screening Program, contract number HHSN-271-2018-00023-C (NIMH PDSP). The NIMH PDSP is directed by B.L. Roth at the University of North Carolina at Chapel Hill and Project Officer J. Driscoll at NIMH, Bethesda MD, USA. For experimental details, please refer to the PDSP web site at https://pdsp.unc.edu/ims/investigator/web/.
M.R.M., I.K.P., N.W.H., M.J.M., M.A.C. and M.-C.K. are employees of EpiCypher, a commercial developer and supplier of reagents and platforms used in this study: recombinant semi-synthetic dNucs and the dCypher binding assay. The remaining authors declare no competing interests.
Peer review information Nature Chemical Biology thanks Tomasz Cierpicki, Jonathan Licht and 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.
Left: Representative sensorgram (green) with the kinetic fit (black dots). Right: Steady-state response (black circles) with the steady-state 1:1 binding model fitting (red dashed line).
The binding of both compounds to NSD2-PWWP1 F266A mutant in parallel to the wild-type (wt) was assessed by DSF in quadruplicate at 100 µM of each compound. Assays were performed in 100 mM HEPES, 150 mM NaCl, pH 7.5, as previously described (https://doi.org/10.1038/nprot.2007.321). Tm (°C) values are presented as mean ± SD in the above table. High fluorescence background and weak transitions were observed in the presence of F266A mutant protein. The Tm of the F266A mutant (37.6 ± 0.1 °C) was much lower than the wt-NSD2-PWWP1 (45 ± 0.1 °C).
Inhibitory activity of 1 and 10 µM (red and green resp.) UNC6934 (a) or UNC7145 (b) on a panel of 33 protein methyltransferases. Experiments run as three independent measurements. Constructs used for NSD1, NSD2 and NSD3 correspond to the catalytic domain (residues 1810-2120, 934-1241 and 1014-1323 respectively).
Binding profile of NSD2-PWWP1 against a panel of methylated designer nucleosomes (top). Results shown are two independent experiments showing mean ± sd. Binding of full-length NSD2 to unmethylated or H3K36me2 nucleosomes with and without an excess of salmon sperm DNA (bottom). n = 2 independent experiments.
A fluorescence polarization (FP)-based DNA displacement assay was used to test the ability of (a) UNC6934 and (b) UNC7145 to displace dsDNA from NSD2-PWWP1. Maximum FP signal was generated using 500 nM of NSD2-PWWP1 and 5 nM fluorescein-labelled dsDNA (32 bp) as described in materials and methods and was considered as 100%. Various concentrations of each compound up to 25 µM was tested for displacement of the dsDNA. UNC6934 and UNC7145 showed no significant effect on dsDNA binding to NSD2-PWWP1. In a complementary experiment, (c) binding of dsDNA to NSD2-PWWP1 in the presence of 20 µM (●) UNC6934, and 20 µM (■) UNC7145 or (▲) no compound was tested at various concentrations of NSD2-PWWP1 up to 2.5 µM. No significant difference in the pattern of dsDNA binding to NSD2-PWWP1 was observed in the presence of either compound, indicating UNC6934 and UNC7145 are not binding to the dsDNA binding site. In addition, (d) the unlabeled dsDNA (32 bp) was tested as a control for displacing the fluorescein-labelled dsDNA. Unlabeled dsDNA was able to compete with the labelled dsDNA for the dsDNA binding site with a displacement constant (Kdisp) of 300 nM. The final DMSO concentration was 0.5% in all experiments. Experiments were performed in triplicate (n = 3).
SPR sensorgram (solid green) is shown with the kinetic fit (black dots), and Kd values were generated from kinetic fitting; The steady state responses (black circles) were shown with the steady state 1:1 binding model fitting (red dashed line). NSD2-PWWP1 domain was immobilized on the flow cell of an CM5 sensor chip in 1x HBS-EP buffer, yielding ~4000 RU. Using buffer with 0.5% DMSO and single cycle kinetics with 60 s contact time and a dissociation time of 120 s at a flow rate of 75 µL/min.
a, Effect of UNC6934 and UNC7145 on cell viability after a 72-hour treatment. Cells (HCT116, HEK293, HT1080, MCF7 and U2OS lines) were treated with indicated concentrations of either UNC7145 or UNC6934 in 96-well plates and nuclei counted by staining Vybrant™ DyeCycle™ Green Stain and imaging on an IncuCyte live-cell analysis system. Each point represents the average number of cells across fields relative to DMSO treated control (n = 3 independent experiments). Plots show each experiment as a point. Bars represent the mean relative cell number ± sem. b, Effect of UNC6934 and UNC7145 on cell viability after 6- and 12-days treatment. Cells (KMS-11, MM1S, RS4;11, TKO and UTMC2 lines) were treated with indicated concentrations of either UNC7145 or UNC6934 in 12-well plates and percentage of viable cells was measured by CellTiter-Glo® luminescent cell viability assay. Bar plots show the mean relative viability ± sem (n = 3 or 4 experiments).
Extended Data Fig. 8 Effect of UNC6934 & nucleolar perturbation on NSD2 localization and rRNA expression.
a, Confocal microscopy of U2OS cells treated for 4 hours with 5 µM UNC6934 or UNC7145 stained for NSD2 (green) and DNA (blue; Hoechst 33342), scale bar = 15 µm. Experiments were repeated on three separate occasions with similar results. b, UNC6934 has no significant effect on pre-rRNA expression. 5-EU incorporation assay to measure nascent rRNA synthesis in cells pre-treated for 1 hour with 5 µM UNC6934 or UNC7145. On the left, violin and boxplots generated from three independent experiments show the log2 fluorescence intensity of 5-EU signal within nucleolar regions defined by staining with a nucleolin antibody per nucleus. On the right, representative microscopy images captured on an EVOS FL Auto 2 microscope from three independent experiments are shown (scale bar = 60 µm). c, pre-rRNA expression levels measured by RT-qPCR show no major effect on ribosome transcription in response to 5 µM UNC6934 or UNC7145 at 2, 4, or 16 hours. Expression levels of pre-rRNA shown are relative to DMSO control and normalized to the beta-2-microglobulin housekeeping gene. Each time point represents an independent experiment with four technical replicates. Data shown as mean ± sd. Included in each time point is a control treatment known to disrupt rRNA expression51,55,56, controls include at 2 hours - 250 nM actinomycin D, at 4 hours - 1 µM doxorubicin, and at 16 hours - glucose starvation. Primer sequences are shown in Supplementary Table 3. d, Nucleolar perturbation alters the localization of NSD2. Representative confocal microscopy images of NSD2 and fibrillarin staining in U2OS cells treated for four hours with DMSO control, 50 nM actinomycin D, or 1 µM doxorubicin (scale bar = 15 µm). Experiment was repeated on three separate occasions as quantified in e, Quantification of co-localization between NSD2 and the nucleolar marker fibrillarin as determined by Pearson correlation coefficient (PCC). The median Pearson Correlation Coefficient is shown for three independent biological replicates (n = 3). The mean ± sd is shown as a single black point with error bars.
a, Cellular fractionation experiments in KMS-11 cells show increased displacement of NSD2 from chromatin in the S3 fraction containing 150 mM NaCl relative to DMSO or UNC7145 treated cell lysates. On the left, a schematic of the fractionation protocol. On the right, western blot analysis of NSD2 in each fraction. 1:5 indicates a fifth of the sample was run on the SDS-PAGE relative to other samples. Experiment was repeated on two separate occasions with similar results. b, Western blot analysis of global H3K36me2 levels in KSM-11 cells treated for 72 hours with several doses of either UNC7145 or UNC6934 shows no significant change in response to compound. A well-characterized isogenic line harboring a deletion of exon 7 in the mutated KMS-11 NSD2 allele is included as a control (TKO2). Experiment was repeated on two separate occasions with similar results. c, Western blot analysis of global H3K36me1, H3K36me2, H3K36me3 and H3K27me3 levels in KSM-11 cells treated for 72 hours with 5 µM of UNC6934. Histone H3 is included as a loading control. Experiment was repeated on two separate occasions with similar results. d, UNC6934 does not affect the proliferation of KMS-11 cells on bone marrow stroma in vitro. Stable GFP expressing KMS-11 and isogenic TKO lines were pre-treated with 5 µM of either UNC7145 or UNC6934 for 10 days prior to plating on a confluent layer of the OP9 murine bone stromal cell line. Proliferation was monitored by measuring GFP+ cells over the course of seven days on an IncuCyte live cell analysis system. Representative images are shown on left (scale bar = 90 µm). On the right, the average cell count per image is shown for two independent experiments, each with at least six technical replicates. Data shown as mean ± sd. e, Impact of compound treatment on H3K36me2 in KMS-11 cells. H3K36me2 ChIP-qPCR is shown at selected NSD2 target genes and (f) rDNA loci and positive and negative control regions. Each color represents a different treatment or cell type. KMS-11 cells were treated with DMSO (red bars), 5 µM UNC6934 (blue bars), or 5 µM UNC7145 (green bars). NSD2 translocated allele knockout (TKO, purple bars) control cells are shown. Each bar represents the mean percentage input of three independent samples (n = 3) and error bars indicate standard deviation. Target genes that are significantly different compared to DMSO control are indicated (*, Two-way ANOVA with Tukey post-hoc test adjusted P < 0.05; specific P values are as follows: ChIP-qPCR target genes- BACE2 P = 0.002373; CA2 P = 4.01e-06; CDH2 P = 3.31e-09; ITGB1 P = 0.008311; NEO1 P = 0.000298; PDN2 P = 0.000139; SERPINE2 P = 0.000110; SNTB1 P = 1.03e-08; TGFA P = 6.85e-09; WASF3-(UNC6934) P = 0.037971; WASF3-(TKO) P = 0.004125; ATF7 P = 0.000929; HMNC1 P = 7.80e-13 SKAP1 P = 0.000110. rDNA loci target genes- ITS1 P = 0.00126; SKAP1 P = 5.92e-06). g, UNC6934 has no significant effect on mRNA levels of NSD2 targets. mRNA levels measured by RT-qPCR show no significant effects on transcription of selected NSD2 targets in response to compound treatment. KMS-11 cells were treated for 72 hours with 5 µM UNC6934 or UNC7145. NSD2 translocated allele knockout (TKO) cells were cultured for 72 hours and were used as control. Transcription levels shown are relative to DMSO control and normalized to the TATA-box-binding protein housekeeping gene. Each bar represents the mean value of three independent samples (n = 3) and error bars indicate standard error. Target genes that are significantly different compared to DMSO control are indicated (*, One-way ANOVA with Tukey post-hoc test adjusted P < 0.05; specific P values are as follows: RT-qPCR target genes- BACE2 P = 0.007717; CA2 P = 0.007717; PNF2 P = 0.007717; CEACAM21 P = 0.007182; ZYX P = 0.028332).
Extended Data Fig. 10 Deletion of the Nucleolar Localization Sequences significantly reduces NSD2 nucleolar localization driven by UNC6934.
a, Representative confocal microscopy of U2OS cells co-expressing GFP-tagged NSD2 wild-type/mutants and RFP-tagged fibrillarin (scale bar = 10 µm). Transfected cells were treated for four hours with DMSO control, 5 µM UNC6934 or 5 µM UNC7145 and co-localization was measure by Pearson correlation coefficient (PCC). b, Quantification of co-localization between RFP-tagged fibrillarin and NSD2 wild-type or mutants by median PCC per biological replicate between NSD2 and fibrillarin signal across three independent experiments (n = 3; P-values derived from a two-way ANOVA to DMSO control for each panel are indicated as *P = 0.022). For each experiment, PCC/nucleus was measured across ten fields of view. Data shown as mean ± sd.
SPR data, PWWP selectivity DSF data, nucleosome-binding data.
NanoBRET binding data for UNC6934 and UNV7145 to NSD2-PWWP1 and NanoBRET selectivity data for UNC6934 and UNC7145 binding to NSD3-PWWP1, NSD2-PWWP1 and NSD2-PWWP1 (F266A mutant).
NSD2 immunoblot analysis of UNC7096–streptavidin bead pulldowns.
Quantification of colocalization between NSD2 and the nucleolar marker fibrillarin as determined by PCC.
Quantification of colocalization between RFP-tagged fibrillarin and NSD2 WT or mutants. Assessing domain cooperativity of NSD2–GFP point mutants. Computational prediction of NoLSs in NSD2.
Triplicate SPR analysis of the binding of UNC6934 to NSD2-PWWP1.
Quadruplicate binding measured by DSF of UNC6934, UNC7145 or DMSO to NSD2-PWWP1 or NSD2-PWWP1 (F266A mutant).
Selectivity of UNC6934 and UNC7145 on 33 methyltransferases measured in triplicate at 1 and 10 μM.
AlphaScreen counts for binding of NSD2-PWWP1 to nucleosomes containing different post-translational modifications. Binding of fl-NSD2 to H3K36me2 nucleosomes in the presence or absence of SSD.
Binding of dsDNA to NSD2-PWWP1 measured by fluorescence polarization in the presence of increasing concentrations of UNC6934 or UNC7145. Binding of dsDNA to NSD2-PWWP1 measured by fluorescence polarization with increasing concentrations of NSD2-PWWP1 in the presence of UNC6934, UNC7145 or DMSO.
SPR binding of UNC7096 to NSD2-PWWP1.
Cell viability on five cell lines after 72-h dosing with UNC6934 or UNC7145. Cell viability on an additional five cell lines after 6 and 12 d of dosing with UNC6934 or UNC7145.
5-EU incorporation assay to measure nascent rRNA synthesis in cells pretreated for 1 h with 5 µM UNC6934 or UNC7145. pre-rRNA expression levels measured by RT–qPCR in response to 5 µM UNC6934 or UNC7145 at 2, 4 or 16 h. Quantification of colocalization between NSD2 and the nucleolar marker fibrillarin.
Cellular fractionation experiments in KMS11 cells. Western blot analysis of NSD2 in each fraction. Western blot analysis of global H3K36me2 levels in KSM-11 cells. Western blot analysis of global H3K36me1, H3K36me2, H3K36me3 and H3K27me3 levels in KSM-11 cells. Proliferation of KMS-11 cells on bone marrow stroma in vitro. H3K36me2 ChIP–qPCR is shown at selected NSD2 target genes and rDNA loci. mRNA levels measured by RT–qPCR.
Western blot analysis of NSD2 in each fraction. Western blot analysis of global H3K36me2 levels in KSM-11 cells. Western blot analysis of global H3K36me1, H3K36me2, H3K36me3 and H3K27me3 levels in KSM-11 cells.
Quantification of colocalization between RFP-tagged fibrillarin and NSD2 WT or mutants.
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Dilworth, D., Hanley, R.P., Ferreira de Freitas, R. et al. A chemical probe targeting the PWWP domain alters NSD2 nucleolar localization. Nat Chem Biol 18, 56–63 (2022). https://doi.org/10.1038/s41589-021-00898-0
Cellular and Molecular Life Sciences (2022)