Wsb2 and Cul5 mutants have high levels of BimEL, BimL and BimS.

A. Competitive growth assay comparing cells expressing sgRNAs against Wsb2 (red line) or control sgRNAs (blue line) in Cas9 positive HAP1 cells in the presence of 25 µM TTFA, over an 8-day period. B. Co-dependencies data from the DepMap consortium and Gene Ontology (GO) classification of the top 100 Wsb2’s co-dependencies. C. Schematic representation of the intrinsic apoptotic pathway. D. Immunoblot analysis of apoptotic proteins in clonal RPE1 Wsb2 or Cul5 mutant cells. E. qPCR analysis of transcript levels of Mcl1, Bcl-xl, BimEL, and Noxa in Wsb2 or Cul5 mutant RPE1s cells normalized to WT. F. 5xHA-Wsb2 or an untagged Wsb2 allele were induced with DOX overnight and samples were immunoprecipitated with anti-HA antibodies.

Wsb2 regulates Bim through Bcl-xl and Mcl1

A. Schematic representation of the three main Bim splicing isoforms, highlighting their domains and regulatory sites and showing how phosphorylation events mediated by the MAPK pathway influence their stability and function. Specifically, BimEL is targeted for degradation by the SCFβTrCP complex following phosphorylation by Erk1/2 and Rsk1/2. Green lollipops indicate ubiquitination sites. Pink, orange, and purple lollipops represent Rsk1/2, Erk1/2, and Jnk/P38 phospho-sites, respectively. B. HA immunoprecipitation of extract from untagged or 5xHA-tagged Wsb2 cells co-expressing 3xFLAG tagged BimEL, BimL, or BimS after 3 hours DOX induction. C. HA immunoprecipitation of untagged or 5xHA-tagged Wsb2 in cells co-expressing or not expressing 3xFLAG tagged BimEL. D. HA immunoprecipitation from cells co-expressing 5xHA-Wsb2, 3xFLAG tagged BimEL or a truncated 3xFLAG-Bim lacking the C-terminal domain (3xFLAG-BimE2A-E4), as indicated. E. Same as Figure 2B, except that half the cells were treated with the pan-BH3 mimetic ABT-263 (Navitoclax) for 3 hours. F. Co-immunoblot analysis of 3xFLAG-BimL in the presence of the BH3 mimetics ABT-263, ABT-199, S63845, or A1331852. Immunoprecipitation of 3xFlag-Bim was used to confirm the efficacy of each BH3 mimetic in disrupting its interaction (Fig. S2B) with the respective anti-apoptotic proteins. Dox (to induce 3Flag-Bim) and drugs were added for 3 hours before harvesting.

Wsb2 regulates Bim independently of the ßTrCP1 pathway

A. WT and Wsb2 mutant RPE1 cells were treated with cycloheximide and samples were harvested as indicated. B. As in panel A, except that PMA was added to one set of samples at the same time as cycloheximide. Quantitation (Fig. S3A), was normalized to Vinculin loading control and subsequently normalized to the t=0 signal. Phosphoforms of each Bim isoform were combined for normalization. C. As in panel B, comparing cells treated with Cycloheximide and ABT-263 to cells additionally treated with MLN4924. All drugs were added at t=0. D. WT and dox inducible 3xFLAG-BimEL RPE1 cells were DOX induced 3 hours and treated with Z-VAD-FMK (to block apoptosis) before treatment with cycloheximide and samples harvested as indicated. E. Flag-Ubiquitin and 13Myc-BimL were co-induced with DOX in wild type or Wsb2 mutant RPE1 cells as indicated, and extracts were immunoprecipitated with anti-Myc antibodies.

Bim is targeted through a direct Wsb2-Bcl-xl interaction

A. Alphafold 2 multimer predicted Wsb2 dimerization with BCL2 protein family members. The four metrics iPTM (Confidence of protein-protein interaction), PTM (Accuracy of the relative positioning of domains within a multi-domain protein), pLDDT (Confidence in the local structure) and PAE (Expected positional error in the distance between pairs of residues) are shown for dimer prediction between Wsb2 and apoptosis proteins. The Cul5 adaptors EloB and EloC were used as a positive control. B. Top panel represent Alphafold2 predicted structure of the Wsb2/Bcl-xl complex with pLDDT coloring. Bottom panel with arbitrary coloring represent Alphafold2 predicted interaction between Wsb2 and Bcl-xl through the yellow and light blue residues. C. Alignment of Bcl-xl, Bcl2, Bcl-w, Mcl1 and Bim showing conservation of the interaction motif between Bcl-xl, Bcl2 and Bcl-w, framed in red. D. In vivo validation of Alphafold2 dimer predictions was performed using co-immunoprecipitation assays. A 5xHA-tagged Wsb2 allele was co-expressed with either V5-tagged wild-type Bcl-xl or Bcl-xlK157A/E158A/M159A as indicated. In all cell lines, a 3xFlag-tagged Bim EL allele was co-expressed. E. HA immunoprecipitation in cell lines expressing a 5HA-tagged WT or Wsb2R157E/R174E/R297E allele and a V5-tagged wild-type Bcl-xl, as indicated. In all cell lines, a 3xFlag-tagged BimEL allele was co-expressed. F. HA immunoprecipitation in cells expressing an untagged Wsb2, 5HA-tagged Wsb2 or 5HA-tagged Wsb2R157E/R174E/R297E allele. In all cell lines, a 3xFlag-tagged BimEL allele was co-expressed.

Wsb2 is essential in Neuroblastoma

A. GTEx portal analysis of transcript levels in bulk tissue RNA sequencing for Wsb2, Wsb1, EloB, and EloC. B. DepMap data of tumor sensitivity to Wsb2 depletion across multiple cancer type. C. DepMap data correlation of tumor sensitivity to Wsb2 and Bcl2l2 depletion. D. Time course competitive growth assay of CHP134 or TGW cells expressing shRNA against Wsb2. Wild-type (GFP-) cells were mixed at an equal ratio with cells expressing either a shRNA scramble control (GFP+) or a shRNA targeting Wsb2 (GFP+). The ratio of GFP-positive to GFP-negative cells was measured over 3 days and normalized to a GFP-negative wild-type control signal. Data are represented as mean ± SD E. Immunoblot analysis in CHP134 cells expressing Wsb2 shRNA, showing that Wsb2 depletion leads to increased levels of cleaved Parp (c-Parp). F. Promega Caspase-Glo assay luminescence signal normalized to 1000 cells and uninfected control, after transfection with shRNAs against WSB2 or Control over a 2-day time course. Data are represented as mean ± Range.