Journal: bioRxiv

Article Title: Phosphorylation-driven Targeted Protein Degradation of Oncogenic β-catenin

doi: 10.64898/2026.03.16.712096

Figure Lengend Snippet: A) Schematic depiction of induced proximity between eGFP-FRB*-β-catenin and HA- and FKBP12 F36V - tagged Open Reading Frames (ORF) using the small molecules Rapamycin or the Rapalog A/C Heterodimeriser. B, C) Rapamycin or A/C Heterodimeriser induced dimerisation of eGFP-FRB*-β-catenin and CSNK1D- or SIAH2-FKBP12 F36V leads to β-catenin degradation in DLD-1 colorectal cancer cells. Double tagged DLD-1 CTNNB1 eGFP-FRB*/eGFP-FRB* cells (clone #43) were infected with virus expressing the indicated -FKBP12 F36V -HA fusion proteins, selected with puromycin for 2 days and then treated with Rapamycin (0.5 µM), A/C Heterodimeriser (1 µM) or equivalent volumes of DMSO for 24 h. Then, eGFP-FRB*-β-catenin abundance was quantified by flow cytometry. B) Histograms of eGFP intensity of one representative biological replicate are shown normalised to mode. C) Median fluorescence intensity of eGFP in n = 4 or 7 biological replicates with mean and standard deviation. A minimum of 8,800 cells per condition were recorded in the final gate. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparisons test comparing treatment groups to DMSO. * Padj ≤ 0.05, *** Padj ≤ 0.0001, ns = not significant. D) Double tagged DLD-1 CTNNB1 eGFP-FRB*/eGFP-FRB* cells (clone #43) expressing CSNK1D-FKBP12 F36V exhibit a strong growth defect when treated with Rapamycin (0.5 µM) or A/C Heterodimeriser (1 µM) over the course of 14 days. Medium with drugs was refreshed every 2 days. Confluence was analysed using an Incucyte time-lapse microscope. Representative of n = 3 biological replicates. FKBP12 - 12-kDa FK506-Binding Protein; FRB* - FKBP12-Rapamycin Binding Domain, T2098L; a.u. - arbitrary unit

Article Snippet: Then, 500 000 cells were seeded in a 12 well plate and treated with 1 μM A/C Heterodimeriser (Takara Bio USA, Inc., 635056) or 0.2 % EtOH as vehicle control for 8 h or 16 h. RNA was isolated using the PureLink RNA Mini Kit (Invitrogen, Thermo Fisher Scientific, 12183018A) and on-column PureLinkTM DNase Set (Invitrogen, Thermo Fisher Scientific, 12185010) following the manufacturer’s instructions as described above.

Techniques: Infection, Virus, Expressing, Flow Cytometry, Fluorescence, Standard Deviation, Microscopy, Binding Assay

Journal: bioRxiv

Article Title: Phosphorylation-driven Targeted Protein Degradation of Oncogenic β-catenin

doi: 10.64898/2026.03.16.712096

Figure Lengend Snippet: A) Experimental outline of bulk RNA-sequencing (RNAseq) experiment: double tagged DLD-1 CTNNB1 eGFP-FRB*/eGFP-FRB* cells (clone #43) were infected with virus expressing CSNK1D-FKBP12 F36V -HA and selected with puromycin for 2 days. Then, cells were treated with A/C Heterodimeriser (1 µM) or equivalent volumes of EtOH for 8 h or 16 h before RNA isolation for differential gene expression analysis. B) Principal component analysis (PCA) plot confirmed that variance between RNAseq samples of three biological replicates stemmed mainly from type and time of treatment. C) 8 h and 16 h treatment with the A/C Heterodimeriser induced a robust downregulation of Wnt/β-catenin target genes. Heatmap of scaled expression of genes associated with the β-catenin dependent Wnt signalling pathway in control (EtOH treated) or A/C Heterodimeriser treated samples. D, E) Volcano plots depicting differentially expressed genes at 8 h or 16 h after A/C vs. EtOH control treatment. Significantly regulated genes are shown in dark grey with Padj ≤ 0.01 and fold change > 2. F) Gene Ontology (GO) biological processes (BP) differentially downregulated in A/C Heterodimersier vs. EtOH control treated samples at 8 h and 16 h included ‘Canonical Wnt signalling pathway’ as a driver term. G) Enrichment plots depict depletion of genes contained in the human gene set ‘HALLMARK_WNT_BETA_CATENIN_SIGNALING’ (M5895) in A/C Heterodimeriser vs. EtOH control treated samples at 8 h and 16 h. ES - Enrichment score; NES – normalised enrichment score; FKBP12 - 12-kDa FK506-Binding Protein; FRB* - FKBP12-Rapamycin Binding Domain, T2098L

Article Snippet: Then, 500 000 cells were seeded in a 12 well plate and treated with 1 μM A/C Heterodimeriser (Takara Bio USA, Inc., 635056) or 0.2 % EtOH as vehicle control for 8 h or 16 h. RNA was isolated using the PureLink RNA Mini Kit (Invitrogen, Thermo Fisher Scientific, 12183018A) and on-column PureLinkTM DNase Set (Invitrogen, Thermo Fisher Scientific, 12185010) following the manufacturer’s instructions as described above.

Techniques: RNA Sequencing, RNA sequencing, Infection, Virus, Expressing, Isolation, Gene Expression, Control, Binding Assay

Journal: bioRxiv

Article Title: Phosphorylation-driven Targeted Protein Degradation of Oncogenic β-catenin

doi: 10.64898/2026.03.16.712096

Figure Lengend Snippet: A, B) CSNK1D-dependent degradation of β-catenin is kinase activity and Ubiquitin-Proteasome System (UPS) dependent. DLD-1 CTNNB1 eBFP2/eGFP cells (clone #28) were infected with virus expressing the indicated -vhhGFP fusion proteins, selected with puromycin for 2 days and then analysed or further treated. A) Kinase-activity dead mutants of CSNK1D do not degrade eBFP2/eGFP-β-catenin. Representative Western Blot of n = 3 biological replicates. B) Inhibitors of the UPS rescue CSNK1D-dependent degradation of β-catenin and lead to accumulation of T41/S45 phosphorylated β-catenin. After selection, cells were treated with DMSO (0.1 %), the proteasome inhibitor MG132 (10 µM), the E1 inhibitor TAK-243 (1 µM), the inhibitor of neddylation MLN-4924 (10 µM) or Bafilomycin (1 µM) for 18 h before harvest and analysis by Western Blot. C, D, E, F) Genome-wide CRISPR/Cas9 knock-out fitness screen identifies regulators of CSNK1D-dependent β-catenin degradation. Highlighted are genes involved in cell-cell contacts (green), transcriptional regulation (light green), neddylation (pink), the SCF β-TrCP E3 ubiquitin ligase complex (red, FBXW11 is also known as BTRC2), and others (black). C) Schematic of positive selection screen in double tagged DLD-1 CTNNB1 eGFP-FRB*/eGFP-FRB* cells (clone #43) with stable expression of CSNK1D-FKBP12 F36V -HA. After puromycin selection, cells were split into three replicates per condition and treated with A/C Heterodimeriser (1 µM) or equivalent volumes of EtOH. Media and compounds were refreshed every 2 days. D) Rank order plot depicting the gene level summary of sgRNAs that regulate CSNK1D-dependent β-catenin degradation identified at Endpoint (T16). Black dots represent regulators with FDR < 0.01. E) overall sgRNA distribution and individual sgRNA enrichment at T16 of the highlighted genes, summarised in F) . G, H, I) GSK3 and AXIN are dispensable for CSNK1D-induced degradation of β-catenin. G) HEK293 Flp-In T-REx GFP-CTNNB1 cells were transfected with CRISPR ribonucleoproteins (RNPs) to generate GSK3A/GSK3B or AXIN1/AXIN2 double knock-out (KO) cell lines. H) Western blot confirms gene knock-out. I) HEK293 Flp-In T-REx cells stably expressing eGFP-β-catenin under a TET-inducible promoter were transfected with the indicated - vhhGFP fusion proteins and eGFP expression was measured by flow cytometry as read-out for β-catenin abundance. Individual data points of four or five independent experiments are shown with mean and standard deviation. A minimum of 4,500 cells per condition were recorded in the final gate. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparisons test comparing all -vhhGFP groups to NLuc-vhhGFP. * Padj ≤ 0.05, ** Padj ≤ 0.0001, ns = not significant. For Western blots: * signal from previous staining; kDa = Kilodalton; GAPDH served as loading control; RLuc - Renilla luciferase; NLuc - NanoLuc luciferase; vhhGFP - nanobody binding to eGFP and eBFP2; a.u. - arbitrary unit

Article Snippet: Then, 500 000 cells were seeded in a 12 well plate and treated with 1 μM A/C Heterodimeriser (Takara Bio USA, Inc., 635056) or 0.2 % EtOH as vehicle control for 8 h or 16 h. RNA was isolated using the PureLink RNA Mini Kit (Invitrogen, Thermo Fisher Scientific, 12183018A) and on-column PureLinkTM DNase Set (Invitrogen, Thermo Fisher Scientific, 12185010) following the manufacturer’s instructions as described above.

Techniques: Activity Assay, Ubiquitin Proteomics, Infection, Virus, Expressing, Western Blot, Selection, Genome Wide, CRISPR, Knock-Out, Transfection, Stable Transfection, Flow Cytometry, Standard Deviation, Staining, Control, Luciferase, Binding Assay