Displacement of WDR5 from Chromatin by a WIN Site Inhibitor with Picomolar Affinity

Abstract

The chromatin-associated protein WDR5 is a promising target for pharmacological inhibition in cancer. Drug discovery efforts center on the blockade of the "WIN site" of WDR5, a well-defined pocket that is amenable to small molecule inhibition. Various cancer contexts have been proposed to be targets for WIN site inhibitors, but a lack of understanding of WDR5 target genes and of the primary effects of WIN site inhibitors hampers their utility. Here, by the discovery of potent WIN site inhibitors, we demonstrate that the WIN site links WDR5 to chromatin at a small cohort of loci, including a specific subset of ribosome protein genes. WIN site inhibitors rapidly displace WDR5 from chromatin and decrease the expression of associated genes, causing translational inhibition, nucleolar stress, and p53 induction. Our studies define a mode by which WDR5 engages chromatin and forecast that WIN site blockade could have utility against multiple cancer types.

Keywords: MLL; WDR5; cancer; cancer therapy; chromatin; gene expression; nucleoar stress; p53; ribosomal proteins; small molecule inhibitor.

Figure 1.. Discovery of Small Molecule WIN Site Inhibitors

(A) Elaboration of fragment hit C1 into our first-generation chemical probe C3. (B–D) X-ray co-crystal structures of WDR5 bound to (B) the fragment hit C1 (PDB: 6DY7), (C) C2 (PDB: 6E1Y), and (D) C3 (PDB: 6E22). (E) Elaboration of fragment hit C4 into our second-generation chemical probe C6. (F–H) X-ray co-crystal structures of WDR5 bound to (F) the fragment hit C4 (PDB: 6E1Z), (G) C5 (PDB: 6DYA), and (H) C6 (PDB: 6E23). See also Figure S1 and Tables S1 and S2.

Figure 2.. WIN Site Inhibition Induces Apoptosis and Kills Cells in a p53-Dependent Manner

(A) MV4:11 cells were treated with DMSO, 2 μM C6, or C6nc; samples collected at the indicated time points; and live cells quantified via trypan blue exclusion. To prevent culture overgrowth, the DMSO- and C6nc-treated samples were replated at day 3 (“replate”) to the original starting density and treatment continued. (B) Stacked bar graph showing the distribution of cell-cycle phases, determined by flow cytometry, in MV4:11 cells treated with DMSO, 2 μM C6nc, or C6 for the indicated times. (C) Western blot for cleaved PARP-1 (P-cl), or histone H3, in lysates from MV4:11 cells treated with DMSO, 2 μM C6nc, or C6 for the indicated times. (D) Bar graph showing the percentage of annexin V⁺ and propidium iodide-negative (PI⁻) cells in MV4:11 cells treated with DMSO, 2 μM C6nc, or C6 for the indicated days. Camptothecin (CPT) is a positive control for the induction of apoptosis. (E) Western blot, showing the effects of DMSO, 2 μM C6, or C6nc (24 h), on p53 and p21 protein levels in MV4:11 cells. Histone H3 is a loading control. (F) Western blot, showing p53 levels in MV4:11 cells treated with 2 μM C6nc or C6 for 24 h, and expressing either (1) a scrambled shRNA control (scr), (2) shRNA 427 against p53 (sh_427), or (3) shRNA 941 against p53 (sh_941). (G) Dose-response curves for compound C6 and its negative control, C6nc, in MV4:11 cells expressing the indicated shRNAs. GI₅₀ results from 3-day treatment with C6 are shown to the right of the appropriate curves. (H) Western blot, showing the effects of DMSO, 3 μM C6, or C6nc (24 h), on p53 and p21 protein levels in Molm13 cells. (I) Western blot, showing p53 levels in the clones of Molm13 cells that were CRISPR targeted with either an EGFP (clones 2e and 3e) or a p53 (clones 2p and 4p) guide RNA (gRNA). Cells were treated with C6 or C6nc at 3 μM for 24 h before analysis. (J) Dose-response curves for compound C6 (3-day treatment) in the modified Molm13 cells described in (I). The GI₅₀ values are 7 μM for clones 2e and 3e, 18 μM for clone 2p, and 15 μM for clone 4p. (A, D, G, and J) Error bars represent SEM. See also Figure S3.

Figure 3.. WIN Site Blockade Inhibits WDR5-Bound Genes Linked to Protein Synthesis

(A) Table shows the number of transcripts significantly (FDR < 0.05) altered by 3 days of treatment of MV4:11 cells with 2 μM C6 or C6nc, compared to DMSO control. (B) Gene Ontology (GO) enrichment clusters for genes significantly repressed by C6 treatment of MV4:11 cells, as determined by RNA-seq. Numbers in italics represent the number of repressed genes in each category. (C) Highly significantly enriched (FDR q = 0.0) Reactome gene sets (defined in the Molecular Signatures Database), determined by gene set enrichment analysis (GSEA) of RNA-seq from MV4:11 cells treated with 2 μM C6. (D) Overlap of genomic binding sites for WDR5 in MV4:11 cells, as determined by ChIP-seq with either the D9E1I or 429A anti-WDR5 antibodies. (E) Top eight GO enrichment categories for genes bound by WDR5 in DMSO-treated MV4:11 cells. (F) Distribution of WDR5 binding sites in MV4:11 cells, binned according to the distance from the annotated TSS. Output includes either all of the WDR5 binding events (Set-wide) or only those at RPGs (Ribosome). RGA, region-gene association. (G) Venn diagram, showing overlap of genes bound by WDR5 (ChIP-seq), with genes induced by C6 treatment (RNA-seq) in MV4:11 cells. (H) As in (G), except overlap is between the genes bound by WDR5 and those repressed by C6 treatment. See also Figure S4.

Figure 4.. WIN Site Blockade Induces Translational Stress and Stimulates p53 Synthesis

(A) Representative flow cytometry histograms showing Alexa Fluor 647-labeled OPP incorporation into nascent polypeptides of MV4:11 cells treated with DMSO, 2 μM C6nc, or C6. As a control for the inhibition of translation, CHX was added to cells 30 min before the addition of OPP. (B) Quantification of the nucleolus:nuclear ratio of NPM1 in MV4:11 cells treated with 4 μM C6 (or C6nc) for 3 days. ns, no significant difference (p = 0.1). ****p < 0.0001. Data are shown as a box and whisker plot. Box extends from the 25th to 75th percentile with median marked by the middle line, whiskers extend from minimum to maximum point. (C) MV4:11 cells were treated with DMSO, 2 μM C6, or 2 μM nutlin-3 for 24 h, after which CHX was added and proteins sampled at the indicated time points (CHX; in min). p53 and histone H3 (loading control, shown here for the C6 treatment) were detected by Western blotting. (D) RT-qPCR analysis of the indicated mRNA levels on polysomal or monosomal fractions collected following the treatment of MV4:11 cells with 5 μM C6 or DMSO for 24 h. Data are shown as a box and whisker plot of relative mRNA changes of indicated genes in each fraction, relative to DMSO. Box extends from the 25th to the 75th percentile, with the median marked by the middle line; whiskers extend from minimum to maximum point; n = 3 experiments. See also Figure S5.

Figure 5.. WIN Site Inhibitors Act Rapidly to Displace WDR5 from Chromatin and Inhibit Transcription at WDR5-Bound Genes

(A) Heatmap listing genes with significant changes in gene body-associated polymerases in MV4:11 cells treated with 36 μM compound C3, as determined by PRO-seq. The orange bar indicates whether WDR5 is bound to the locus. RPS17 (*) and RPL17(***) are listed twice, because two distinct National Center for Biotechnology Information Reference Sequence Database (RefSeq) IDs were called for those loci. LOC100506548 (**) is the read-through transcription from RPL37. (B) Heatmaps displaying log₂-transformed fold change of PRO-seq read counts in 200 bp bins ± 5 kb around the TSS of loci showing gene body changes after C3 treatment. (C) ChIP-PCR analysis of WDR5 binding at 5 loci in MV4:11 cells treated with DMSO (DM), or 36 μM of C3 (or C3nc) for 4 h. Chromatin samples were immunoprecipitated with an anti-WDR5 antibody (D9E1l) or an immunoglobulin G (IgG) control. Data are presented as a percentage of the signal for the same amplicon in the input chromatin. (D) Western blot, showing WDR5 distribution in the soluble (S2), soluble nuclear (S3), or insoluble chromatin (P3) fractions of MV4:11 cells treated for 4 h with DMSO or 36 μM of C3. Histone H3 is a control for the specificity of the insoluble chromatin fraction. (E) ChIP-PCR analysis of FLAG (FL)-tagged WDR5, wild-type (WT), or a WIN site mutant (F133A), stably expressed in HEK293 cells. Vector cells are the negative control; ChIP was performed with an anti-FLAG antibody. (F) Scatterplot of normalized average read counts for WDR5 binding peaks in DMSO-, C6nc-, and C6-treated MV4:11 cells. Peaks are ranked based on read counts in DMSO-treated cells. (C and E) Error bars represent SEM. See also Figure S6.