Disease-associated KBTBD4 mutations in medulloblastoma elicit neomorphic ubiquitylation activity to promote CoREST degradation

Abstract

Medulloblastoma is the most common malignant brain tumour in children. Genomic studies have identified distinct disease subgroups: wnt/wingless (WNT), sonic hedgehog (SHH), and non-WNT/non-SHH, comprising group 3 and group 4. Alterations in WNT and SHH signalling form the pathogenetic basis for their subgroups, whereas those for non-WNT/non-SHH tumours remain largely elusive. Recent analyses have revealed recurrent in-frame insertions in the E3 ubiquitin ligase adaptor Kelch Repeat and BTB Domain Containing 4 (KBTBD4) in cases of group 3/4 medulloblastoma. Critically, group 3/4 tumours with KBTBD4 mutations typically lack other gene-specific alterations, such as MYC amplification, indicating KBTBD4 insertion mutations as the primary genetic driver. Delineating the role of KBTBD4 mutations thus offers significant opportunities to understand tumour pathogenesis and to exploit the underpinning mechanisms therapeutically. Here, we show a novel mechanism in cancer pathogenesis whereby indel mutations in KBTBD4 drive its recognition of neo-substrates for degradation. We observe that KBTBD4 mutants promote the recruitment and ubiquitylation of the REST Corepressor (CoREST), which forms a complex to modulate chromatin accessibility and transcriptional programmes. The degradation of CoREST promoted by KBTBD4 mutation diverts epigenetic programmes inducing significant alterations in transcription to promote increased stemness of cancer cells. Transcriptional analysis of >200 human group 3 and 4 medulloblastomas by RNA-seq, highlights the presence of CoREST and stem-like signatures in tumours with KBTBD4 mutations, which extend to a further sub-set of non-mutant tumours, suggesting CoREST alterations as a novel pathogenetic mechanism of wide relevance in groups 3 and 4. Our findings uncover KBTBD4 mutation as a novel driver of epigenetic reprogramming in non-WNT/non-SHH medulloblastoma, establish a novel mode of tumorigenesis through gain-of-function mutations in ubiquitin ligases (neo-substrate recruitment) and identify both mutant KBTBD4 and CoREST complexes as new druggable targets for improved tumour-specific therapies.

Fig. 1. KBTBD4 mutations elicit neomorphic activity promoting the recognition and ubiquitylation of CoREST.

A Domain organisation of KBTBD4. The Kelch domain contains 6 Kelch repeat motifs (I-VI) that form the six blades of a β-propeller fold. Recurrent indel mutations KBTBD4^R313PRR and KBTBD4^P311PP map to the second Kelch repeat. B HEK293T cells transfected with empty vector (EV), Flag-KBTBD4^WT, Flag-KBTBD4^R313PRR or Flag-KBTBD4^P311PP were immunoprecipitated and eluted with Flag-peptide. Eluates were digested and analysed by LC-MS/MS. Cells were treated with 100 nM MLN4924 for 4 h before harvesting. The table highlights interactors from the CRL3^KBTBD4 and CoREST complexes (red and light blue shading, respectively). C Structural model of the KBTBD4 Kelch domain (left). Red star indicates the substrate-binding pocket. Indel mutations are predicted to lengthen the substrate-binding loop in blade II to support neo-substrate interaction (right). The inserted mutant amino acids (denoted by an arrow) lie on the outer edge of the loop that faces away from the central substrate pocket. N-terminal wild-type residues preceding the mutation site are therefore repositioned into the substrate pocket (Ile310 Cα atoms in each model are shown as spheres for reference). D HEK293T cells were co-transfected with HA-tagged CoREST and an Empty Vector, Flag-KBTBD4^WT, Flag-KBTBD4^R313PRR or Flag-KBTBD4^R311PP and were treated with MLN4924. Whole-cell extracts were immunoprecipitated (IP) with anti-FLAG resin, and immunoprecipitates were immunoblotted as indicated. E HEK293T cells were co-transfected with HA-CoREST along with Flag‐KBTBD4^WT, Flag-KBTBD4^R313PRR or Flag-KBTBD4^R312RG as indicated (+). After treatment with MLN4924 cell lysates were immunoprecipitated by anti‐FLAG resin and immunoblotted as indicated. F HEK293T cells were co-transfected with MYC‐tagged ubiquitin and HA-CoREST along with Flag‐KBTBD4^WT, Flag-KBTBD4^R313PRR or Flag-KBTBD4^P311PP as indicated (+). HA‐CoREST was immunoprecipitated with anti‐HA resin under denaturing conditions and immunoblotted. Cells were treated with 5 μM MG132 for 4 h before harvesting. The poly-ubiquitylated CoREST (indicated by brackets) was detected by immunoblot with anti-Myc antibody.

Fig. 2. Expression of KBTBD4 mutants in medulloblastoma cells promotes CoREST ubiquitylation and modifies epigenetic marks.

A D283Med cells expressing Flag‐KBTBD4^WT, Flag-KBTBD4^R313PRR or Flag-KBTBD4^P311PP after doxycycline induction (1 µg/mL for 40 h) were treated with 100 nM MLN4924 for 4 h as indicated (+). After lysis, extracts were immunoprecipitated with Flag-resin and immunoblotted as indicated. B D283Med cells expressing Flag‐KBTBD4^WT, Flag-KBTBD4^R313PRR or Flag-KBTBD4^P311PP were treated with doxycycline (1 µg/mL) for 16 h and lysed. Endogenous ubiquitylated proteins were isolated using GST fused ubiquitin binding domain UBA^Ubq and separated by SDS-PAGE. Isolated proteins were detected by immunoblot as indicated. All samples were treated with MG132 for 6 h. C D283Med cells containing Flag‐KBTBD4^WT, Flag-KBTBD4^R313PRR or Flag-KBTBD4^P311PP were treated with doxycycline (1 µg/mL) for the indicated time (h = hours). Samples were separated by SDS-page and immunoblotted as indicated. D, E Flow cytometry analysis. D (Top three panels) Representative images showing the gating procedure where Negative and Positive gates were determined using 5 controls: Compensation beads (green), unstained cells (orange), cells stained only with secondary antibody (not shown), cells treated with LSD1 inhibitor followed by incubation with either isotype (Blue) or H3K4me2 (red) primary antibodies and secondary antibody. The remaining panels are representative images showing the results related to flow cytometry analysis of H3K4me1 and H3K4me2 in D283Med cells expressing Flag‐KBTBD4^WT, Flag-KBTBD4^R313PRR or Flag-KBTBD4^P311PP after doxycycline induction (1 µg/mL for 4 days). E Bar charts showing the quantification of the analysis in D, plotted as percentage increase of the Mean Fluorescence Intensity (MFI) of positive cells where controls (- doxy) were set as 100% (dashed line). Data are presented as mean ±standard deviation (SD), with at least three independent experiments. P values (*P < 0.05 and **P < 0.005) were calculated by paired and two‐tailed t‐test.

Fig. 3. Expression of KBTBD4 mutants in medulloblastoma promotes the de-repression of CoREST target genes activating pro-tumorigenic programmes.

Volcano plots showing the differential gene expression analysis from D283Med cells expressing (A) Flag‐KBTBD4^WT, (B) Flag-KBTBD4^R313PRR or (C) Flag-KBTBD4^P311PP treated with doxycycline (1 µg/mL) for four days compared to their untreated controls. Coloured dots represent differentially expressed genes between two groups with P-adjust <0.05. KBTBD4 (indicated in Purple) and known CoREST target genes (yellow) are highlighted. ChIP analysis of LSD1 occupancy on gene promoters in (D) D283Med and (E) D425Med cells expressing Flag‐KBTBD4^WT or Flag-KBTBD4^R313PRR upon doxycycline (1 µg/mL) treatment for 3 days. Data are expressed as percentage of input. F, G Dotplot of KEGG pathway enrichment analysis of genes differentially upregulated comparing either KBTBD4^R313PRR or KBTBD4^P311PP treated with doxycycline versus untreated controls. The X axis shows the gene ratio which is the percentage of genes enriched in a KEGG term. The Y axis represents the enriched pathways; the size of the node represents the number of enriched genes within a particular term.

Fig. 4. Mutant KBTBD4 enhances stemness of medulloblastoma cells.

Volcano plot showing upregulated ES-like signature genes (yellow dots). RNA-seq upregulated genes (P-adjust <0.05) between (A) KBTBD4^R313PRR and (B) KBTBD4^P311PP treated with doxycycline compared to untreated samples. C Tumorsphere-forming capacity of D283Med cells in which KBTBD4^WT, KBTBD4^R313PRR or KBTBD4^P311PP expression was induced with doxycycline (doxy+) compared to untreated controls (doxy−). Quantification of 3 independent experiments (bar chart) and representative pictures of doxycycline-induced cells (scale bar denotes 100 µm). D Aldehyde dehydrogenase (ALDH) activity, measured as percentage of ALDH positive cells, of D425Med parental cells or, D425Med cells expressing KBTBD4^WT, KBTBD4^R313PRR or KBTBD4^P311PP+/− LSD1i treatment (0.5 µM for 24 h). [N,N-diethylaminobenzaldehyde (DEAB), ALDH inhibitor]. P values (**P < 0.005 and ****P < 0.00005) were calculated by paired and two‐tailed t‐test.

Fig. 5. KBTBD4 transcriptional signatures define a specific cluster of medulloblastomas.

A Heatmap showing single sample Gene Set Enrichment Analysis (ssGSEA) scores for D283Med cell-based models and human primary medulloblastomas (Group3; n = 67, Group4; n = 156). Custom genesets represented are dysregulated by doxocycline-induced expression of KBTBD4 mutants (R313PRR and P311PP) in D283Med cell lines. + indicates doxycycline treatment, - indicates untreated controls. B Stacked bar plots representing percentage frequency of gene signature clusters across different subgroups (Group3 and Group4) and subtypes (I-VIII) of primary medulloblastomas.

Fig. 6. Neomorphic KBTBD4 mutations enable this E3 ubiquitin ligase to recruit, ubiquitylate and degrade CoREST favouring proliferation of neuronal progenitors and tumorigenesis.

During normal development, CoREST complexes containing LSD1, HDAC1 or 2, and CoRESTs (also known as RCORs) silence chromatin by deacetylating and demethylating histone H3 at position Lys4 (K4). In medulloblastoma, mutations allow KBTBD4 to destabilise CoREST complexes thereby preventing chromatin repression leading to the increased transcription of CoREST targets and stemness genes. The activation of stemness genes is a potential driver of medulloblastoma tumorigenesis.