HuR降解通过BRAF剪接抑制突变型结直肠癌

Fig. 1 | Identification of dHuRs. a, HTRF-based primary screen of a compound library (1.6 µM) for CRBN binders. Cpds, compounds. b, Schematic of the proteomic screen in CRBN WT versus CRBN-KO cells treated with compound pools (left), and volcano plots showing protein abundance changes in JHH-7 CRBN WT (middle) or CRBN-KO (right) cells. LC–MS/MS, liquid chromatography– tandem mass spectrometry; TMT, tandem mass tag. c, Chemical structure and molecular weight (MW) of dHuR-1 (1). d, Immunoblot of HuR in JHH-7 cells treated with dimethyl sulfoxide (DMSO) or dHuR-1. e, Immunoblot of HuR in JHH-7 cells treated with dHuR-1 (10 µM) with or without MLN4924 (1 µM; a NEDD8 inhibitor) or MG132 (10 µM; a proteasome inhibitor). f, Quantified immunoblot of HuR in JHH-7 CRBN WT and CRBN-KO cells treated with cycloheximide (CHX; 100 µg ml−1) in the presence or absence of dHuR-1 (10 µM) over time (0–8 h). g, Schematic of the chemical structures of 2–7 generated by appending the dHuR-1 tail moiety to the core scaffold of Poma or lenalidomide (Lena; left). HTRF-measured binding affinity of the indicated compounds to CRBN (left y axis) and HiBiT-based quantification of HuR degradation (right y axis) are also shown (right). h, Chemical structure and MW of dHuR-2 (8), an optimized degrader. i, Volcano plots of protein abundance changes in KP4 cells treated with dHuR-1 or dHuR-2 (10 µM, 2 h). P values were determined by two-tailed, unpaired Student’s t-test (b,i). Grey dotted lines indicate cut-off of fold-change (FC) and P value (b,i). For gel source data, see Supplementary Fig. 2.

Fig. 2 | Characterization of CRBN–MGD–HuR ternary complex formation. a, NanoBRET assay comparing ternary complex formation between CRBN and either WT HuR or the HuR(G58N) mutant induced by 10 μM dHuR-1. Data are presented as mean ± s.d.; n = 3 biologically independent samples. P values were determined by two-tailed, unpaired Student’s t-test. BRET, bioluminescence resonance energy transfer. b, TR-FRET assay showing ternary complex formation between His–DDB1–CRBN and GST–HuR induced by various concentrations (8 to 0.0005 μM, fourfold dilutions) of dHuR-1 or dHuR-2. c, Western blot detection of polyubiquitinated HuR after incubation with recombinant E1, E2 and E3 enzymes ± 100 μM dHuR-2 (2 h at 30 °C). For gel source data, see Supplementary Fig. 2. Ub, ubiquitin. d, Cryo-EM map of DDB1–CRBN bound to HuR and dHuR-2 (the DDB1 BPB domain was masked out). e, Binding pocket of dHuR-2 in the ternary complex. Key interacting residues are shown in stick representation, and Cα of G58 is shown in sphere representation. Hydrogen bonds are depicted as dashed lines. f, dHuR-2 (green) induced a tight protein– protein interaction between CRBN (grey) and HuR (pink). DDB1 was omitted for clarity. g, Binding of dHuR-2 created an additional buried surface area at the protein–protein interface between CRBN and HuR. The buried surface from the binding of dHuR-2 (light brown) was compared with the buried surface between CRBN and HuR (purple).

Fig. 3 | HuR degradation exhibited selective efficacy in BRAF-mutant CRC and suppressed MAPK signalling. a, DepMap analysis revealed preferential sensitivity of BRAF-mutant tumour cells to HuR deletion. Data represent the difference in HUR CRISPR scores between mutant and WT cell lines with case numbers indicated. P values were calculated by two-tailed, unpaired Student’s t-test. Data were from DepMap (DepMap_Public_23Q2+Score and Mutation_22Q2_Public; https://depmap.org/portal/). Dashed lines indicate cut-off for effect of gene aberration and P value. b, Growth inhibition of dHuR-2 in BRAF-mutant and WT cell lines over 8 days. Data were normalized to DMSO control. Data are presented as mean ± s.d.; n = 3 biologically independent samples. c, 3D spheroid growth assay of Colo205 cells stably expressing control single-guide RNA (sgRNA; sgCtrl) or HUR-targeting sgRNA (sgHuR) with or without doxycycline (Dox; 200 ng ml−1) induction (left). Data are presented as mean ± s.d.; n = 3 biologically independent samples. P values were determined by two-tailed, unpaired Student’s t-test. Immunoblot confirmed HuR depletion post-induction (right). d, Immunoblot analysis of HuR degradation in parental Colo205, CRBN−/− and HURG58A cells treated with dHuR-2 for 24 h. Vinculin served as a loading control. e, Corresponding 3D spheroid inhibition by dHuR-2 (0.001–1 μM) in Colo205 parental versus HURG58A cells over 8 days. f, Tumour growth curves in Colo205 xenografts (n = 6 per group) treated with vehicle or dHuR-2 (6.25–25 mg kg−1, b.i.d.) for 28 days. Data show mean tumour volume ± s.e.m. mpk, mg kg−1; p.o., oral administration. g, KEGG pathway enrichment analysis of downregulated genes in Colo205 cells treated with dHuR-2 (1 μM, 96 h) versus DMSO control. The top ten enriched pathways are shown (false discovery rate < 0.05). h, Immunoblot analysis of MAPK pathway components in Colo205 parental and HURG58A cells treated with dHuR-2 (0.1 or 1 μM, 6 days). For gel source data, see Supplementary Fig. 2.

Fig. 4 | HuR regulated BRAF splicing through direct RNA binding. a, Immunoblot analysis of HuR, BRAF and EGFR protein levels in BRAF-mutant and WT CRC cell lines treated with DMSO or dHuR-2 for 8 days. Vinculin served as a loading control. b, RNA-seq tracks of the BRAF exon 17–19 region with percent spliced in (top). RT–PCR analysis of BRAF transcripts with exon 18 included or skipped in Colo205 cells treated with 1 μM dHuR-2 over time (6–24 h) (bottom). Red shading highlights the region (around BRAF exon 18) with number of reads altered notably upon dHuR-2 treatment. c, RT–PCR analysis of the abundance of BRAF transcripts with exon 18 included or skipped in HURG58A (top) or HUR-KO (bottom) Colo205 cells. sgNT, non-targeting sgRNA. d, Quantitative PCR (qPCR) analysis of bound RNAs from IgG or HuR pull-downs in Colo205 cells treated with or without dHuR-2. RIP, RNA immunoprecipitation. e, Immunoblot of HuR pull-downs using biotinylated BRAF intron 17 RNA (containing U-rich element) versus scrambled control. f, Sensorgrams of HuR binding to a U-rich element derived from BRAF RNA (3.84 to 0.03 μM, twofold dilutions). RU, response unit. g, BRAF minigene diagram (top), and RT–PCR analysis of BRAF minigene variants (WT versus U-rich mutant) in 293T cells treated with 1 μM dHuR-2 (24 h; bottom). For the U-rich mutant, the 20-bp U-rich sequence upstream of the 3′ splice site (SS) was mutated to UCUCUCUCUCUCUCUCUCUC. h, Schematic of the BRAF 3′ region showing exon 18-included (X1) and exon 18-skipped (X2) isoforms. Skipping of the 154-bp exon 18 shifts the stop codon by 127 bp, yielding a 27-bp shorter BRAF-X2. i, Schematic of FLAG–BRAF (X1 or X2)–IRES–eGFP reporter constructs (top). Immunoblot analysis of 293T cells expressing BRAF isoforms, showing FLAG–BRAF and p-ERK levels (bottom). j, Dual-fluorescence reporters of the BRAF C terminus (exons 17–19) for X1 or X2 (top). Flow cytometry of mCherry normalized to eGFP in cells treated with DMSO, CQ (10 μM, 24 h) or MG132 (10 μM) is also shown (bottom). k, Polysome profiling of Colo205 cell lysates (10–50% sucrose). The absorbance (optical density at 260 nm (OD260)) trace shows peaks of 40S, 60S and 80S monosomes and polysomes. Fractions analysed in subsequent panels are indicated along the x axis. Schematic created in BioRender; Wang, X. https://biorender.com/prnw9m6 (2026). l, Distribution of BRAF-X1 and BRAF-X2 mRNAs across polysome fractions quantified by RT–qPCR. ACTB mRNA served as a positive control for translationally active transcripts. Data are presented as the percentage of total mRNA in each fraction. m, RT–qPCR of BRAF isoform switch (X1/X2) in Colo205 cells ± dHuR-2 (1 µM, 24 h). Data are presented as mean ± s.d.; n = 3 biologically independent samples (d,j,l,m). P values were determined by two-tailed, unpaired Student’s t-test. For gel source data, see Supplementary Fig. 2.

Fig. 5 | HuR degradation overcomes BRAFi resistance through sustained pathway suppression. a, Dose–response curves for the cell viability of parental and BRAFi-resistant WiDr cells treated with dabrafenib (0.1–100 nM), encorafenib (0.1–100 nM) or dHuR-2 (0.01–10 μM) for 6 days. b, Immunoblot of HuR, BRAF and p-ERK levels in Colo205 cells treated with 1 μM dHuR-2 from 3 h to 8 days. HSP70 served as a loading control. c, Colony formation assay of parental and BRAFi-resistant WiDr cells transduced with sgCtrl or sgHuR after 14-day culture (left), and immunoblot confirmation of HUR-KO efficiency (right). d, Relative cell viability of Colo205 cells treated with dabrafenib (0.1−10 nM) ± dHuR-2 (0.1 or 0.3 μM) for 6 days. e, Tumour growth curves of BRAFV600E patient-derived xenograft (PDX) treated with vehicle, dHuR-3 (25 mg kg−1, q.d.), encorafenib (30 mg kg−1, q.d.) or a combination for 25 days. q.d., once daily. n = 6 animals pre group. Data show mean tumour volume ± s.e.m. f, KEGG signature score of the MAPK pathway in Colo205 cells treated for 96 h with DMSO, 1 μM dHuR-2, 3 nM dabrafenib or both. Sequencing was performed for biological duplicates. The boxplots show the median (centre line), interquartile range (box) and full data range (whiskers). Individual data points are overlaid. g, Immunoblot of MAPK pathway components in Colo205 cells treated with 1 μM dHuR-2, 3 nM dabrafenib or both. Vinculin served as a loading control. h, Scatter plot of synthetic lethal genes to dHuR-2. The x axis shows the gene essentiality score shown as log2 fold change (log2FC; DMSO day (D) 12 versus DMSO D0) and the y axis indicates the sensitive score shown as log2FC (dHuR-2 D12 versus DMSO D12). Synthetic lethal genes to dHuR-2 are marked with red dots. i, Colony formation in HT29 cells treated with dHuR-2 (1 μM), gefitinib (3 μM), MEK162 (10 nM) or both for 14 days. j, Diagram illustrating the mechanism of action of dHuRs synergy with BRAFi, EGFRi or MEKi via mediating BRAF exon 18 skipping. Diagram created in BioRender; Wang, X. https://biorender.com/wk9d06q (2026). For gel source data, see Supplementary Fig. 3.