Parkinson's disease (PD) is commonly associated with dysfunctional mitochondrial homeostasis. PINK1, a S/T kinase mutated in early-onset PD, generates phosphoserine 65 ubiquitin (pS65Ub) on damaged mitochondria facilitating their removal. Here, we show that pS65Ub translocates into the nucleus after generation at damaged mitochondria and is directly attached to substrates by resident E3 ligases. Histone H2A is a major substrate and is modified at lysine 119 (H2AK119) by the polycomb silencer, E3 ligase RING1B. At nucleosomes, pS65Ub simultaneously suppresses RING1B and potentiates H2A deubiquitinases USP16 and USP21. Epigenetic profiling and RNA sequencing reveal that pS65Ub is enriched at the promoters of poorly expressed yet dynamically regulated genes and is associated with H2AK119ub depletion. Functionally, we show that pS65Ub enrichment drives polycomb target gene expression, which accelerates the maturation of dopaminergic neurons. Importantly, post-mortem PD brains exhibit elevated nuclear pS65Ub, potentially linking nuclear pS65Ub accumulation with disease pathogenesis. Together, these data indicate that pS65Ub generated at damaged mitochondria regulates fundamental cellular processes at distant sites.

Modification of the VHL-binding fragments contained in proteolysis targeting chimeras (PROTACs) that potently degrade the BRM protein (also known as SMARCA2) improved degradation selectivity over the closely-related paralog protein BRG1 (SMARCA4). In particular, replacement of the phenyl-thiazole entity commonly employed in the generation of VHL-dependent PROTACs with pyridyl-thiazole, phenyl imidazole, and phenyl-nitrile moieties consistently improved the BRG1/BRM degradation selectivity ratios of multiple, structurally-diverse degrader compounds. Crystal structures of these new VHL-binding fragments in complex with the VHL protein were obtained to better understand their interactions. Some of these VHL alterations, the phenyl-nitrile substitution in particular, afforded molecules that displayed strong antiproliferative activities against BRM-dependent (BRG1-mutant) cancers but minimal potency toward wild-type cell lines. One such compound (21, G-9293) was profiled in detailed broad proteomics and chromatin accessibility experiments, and its biological properties were clearly differentiated from a less-selective BRM-degrader (5, A947) in the latter assessment. The highly selective molecule (21, G-9293) was also extensively profiled in vitro using a panel of lung cancer cell lines (defined by BRG1 or BRM status) along with several prostate cancer lines. It exhibited similar antiproliferation activity relative to the less-selective BRM-degrader (5, A947) against the lung lines but significantly diminished potency toward the prostate cancer cells.

Expression of TGF-β family receptors and SMAD signaling activity in MIB1- and FKBP1A-dependent cell lines.

MIB1 negatively regulates BMPR2 protein levels. A, Global proteomic analysis by mass spectrometry of MIB1-iKO cells treated with Dox (0.5 μg/mL) for 3 days (n = 3). Volcano plot displays proteins with >2-fold change and statistical significance based on −log10 (P value) for MIB1 KO (+Dox) vs. MIB1 WT (−Dox). A total of 7,860 proteins were quantified. Orange points indicate proteins with >2-fold change and meeting the significance threshold; blue points indicate statistically significant proteins with <2-fold change; gray points did not meet the significance criteria. B, RNA-seq expression (RPKM) of selected genes from MIB1-iKO cells treated with Dox (0.5 μg/mL) for 3 days under basal BMP signaling conditions. BMPR2 and ACVR1 mRNA levels do not show notable changes. Each plot shows two biological replicates per condition (n = 2). FC was calculated from RPKM values comparing +Dox, KO vs. −Dox, WT. No statistical analysis was performed. RPKM, normalized reads per kilobase gene model per million total reads. C, Immunoblot analysis of BMP pathway proteins in MIB1-iKO cells treated with Dox (0.5 μg/mL) for 3 days. β-Actin served as the loading control. D, Immunoblot analysis of biotinylated cell surface proteins from MIB1-iKO cells treated with or without Dox (0.5 μg/mL) for 4 days, showing BMPR2 at the plasma membrane in the absence of MIB1. Immunoblot (E) and qPCR (F) analyses for HUVECs transfected with siRNA targeting MIB1, HPRT (positive control), or a nontargeting control (NTC) for 72 hours. RNA expression normalized to GAPDH. Experiments were repeated with at least three donors. P values were calculated using an unpaired Student t test; ****, P < 0.0001; and “ns,” not significant.

Abstract Non-canonical HLA-presented peptides are promising therapeutic targets, but their low abundance makes them difficult to reproducibly identify and quantify, particularly in multiplexed immunopeptidomics workflows. Here we present MIRA-MS (Model-Informed Real-time Acquisition for Mass Spectrometry), a real-time acquisition strategy that combines fragment ion-indexed database searching with artificial intelligence-based prediction of peptide fragmentation and retention time to guide quantitative scan acquisition. In a clear cell renal cell carcinoma model, MIRA-MS increased the number of quantified non-canonical immunopeptides by 97-107% relative to standard acquisition methods while also improving recovery of canonical peptides by 45-89%. These results establish real-time AI-guided acquisition as a powerful approach for deeper and more reproducible immunopeptidome profiling.

The repair of DNA double-strand breaks by homologous recombination is essential for genomic integrity, and its dysregulation is a hallmark of cancer1. Central to homologous recombination is the RAD51 recombinase, whose assembly into a nucleoprotein filament is governed by five RAD51 paralogues (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3)2. Mutations in any of these proteins predispose individuals to multiple cancers or genetic disorders3-6. These paralogues are thought to form two functionally separate complexes RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) and RAD51C-XRCC3 (CX3), that act independently at different stages of homologous recombination7-11. Here we demonstrate that all five paralogues can assemble into a single, ATP-dependent BCDX2-CX3-RAD51 supercomplex. The architecture of this assembly bound to single-stranded DNA reveals a contiguous filament where the CX3 module stacks atop BCDX2, creating a protofilament template for RAD51 filament formation. We further identify a novel, RAD51B-independent DX2-CX3 complex (RAD51D-XRCC2-RAD51C-XRCC3) functioning as a stable RAD51 anchor on single-stranded DNA, and we capture it in multiple states, including capping RAD51 filament segment. These distinct assemblies are differentially regulated by ATPase activity, defining a dynamic BCDX2-CX3 'loader' and a stable DX2-CX3 'anchor' that provide functional modularity to the homologous recombination machinery. This work provides a unifying mechanism for human RAD51 paralogue function and delivers an atomic blueprint for interpreting disease-causing mutations.

BMP ligands, but not TGF-β, inhibit growth of cell lines dependent on both MIB1 and FKBP1A. A, Gene dependency scores for MIB1 and FKBP1A plotted for 1,164 cancer cell lines in DepMap (Public 22Q4 Chronos). Cell lines dependent on MIB1 or FKBP1A were defined by a dependency score ≤ −0.5. B, MIB1- and FKBP1A-dependent cell lines were pretreated with BMP receptor inhibitor LDN (50 nmol/L) for 15 minutes followed by treatment with a cocktail of BMP ligands (top) or pretreated for 15 minutes with TGF-β receptor inhibitor SB431542 (SB, 5 μmol/L) followed by treatment with a TGF-β ligand cocktail (bottom). After 6 days, a CellTiter-Glo luminescent assay was performed. Statistical significance shown for the comparison between cytokine-treated conditions (+BMP4 or +TGF-β1) and the control group (two-way ANOVA with Tukey test, 4-group comparison). C, IncuCyte live-cell analysis over 6 days for NCIH838 cells treated with the BMP (left) or TGF-β (right) cocktails in the presence or absence of 50 nmol/L LDN or 5 μmol/L SB, respectively. D, CellTiter-Glo assay of NCIH838 cells treated with a titration of the indicated ligands over 6 days. All conditions were included in the two-way ANOVA with Dunnett’s test; statistical significance is indicated only for comparisons of +BMP4 vs. −BMP4 without LDN. E, IncuCyte live-cell analysis for NCIH838 cells treated with 50 nmol/L LDN, 10 ng/mL of BMP4, or both. F, NCIH838 cells were treated with 50 nmol/L LDN, 10 ng/mL of BMP4 or both for 7 days and then assayed by CellTiter-Glo. The results are represented as the mean ± SD; n = 3 (two-way ANOVA with Tukey correction). G and H, Colony formation assays for NCIH838 cells treated with titrations of BMP4 in the absence (control) or presence of LDN (G) or TGF-β1 in the absence (control) or presence of SB (H) for 7 days; scale bars, 5 mm. Representative wells shown; the experiment was repeated independently at least three times. I, IF staining of NCI-H838 cells for phosphorylated SMAD proteins (orange). pSMAD1/5/9 staining under control (untreated) conditions or after treatment with BMP4 (11 ng/μL), with or without 50 nmol/L LDN. pSMAD2/3 staining under control conditions or after treatment with TGF-β1 (11 ng/μL), with or without 5 μmol/L SB. Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. J, Immunoblot of cytoplasmic (C) and nuclear (N) fractions from NCI-H838 cells pretreated with 1 μmol/L LDN or 5 μmol/L SB for 24 hours and then stimulated with 10 ng/mL BMP4 or TGF-β1 for 1.5 hours to detect pSMAD proteins. CREB and HSP90 were used as loading controls for nuclear and cytoplasmic fractions, respectively. For all panels, *, P < 0.05; **, P < 0.01 and ***, P < 0.001; ****, P < 0.0001; “ns,” not significant.

Caspase-1, -4, -5 and -11 activate gasdermin D (GSDMD) pores, causing pyroptotic cell death and the release of the interleukins IL-1β and IL-18 (ref. 1). Blocking this pathway holds therapeutic promise for the treatment of inflammatory disorders, but cell-permeable caspase inhibitors have not proved successful in clinical trials2. Here we describe covalent caspase inhibitors that selectively block pyroptosis and IL-1β secretion despite being excluded from healthy cells. These inhibitors did not prevent caspase-driven apoptosis, implying that GSDMD pores facilitated their uptake. Membrane-impermeable dyes entered the cells rescued from pyroptosis, consistent with transient membrane permeabilization by GSDMD pores. Caspase inhibition prevented rather than delayed cell death, consistent with membrane repair mechanisms neutralizing the initial GSDMD pores. Inhibiting caspase-1 and caspase-11 suppressed IL-1β and IL-18 production in a mouse model of endotoxic shock, underscoring the therapeutic potential of exploiting GSDMD pores for targeted caspase inhibition in inflammatory diseases.

Loss of MIB1 enhances BMP signaling activity. A, Immunoblot analysis of pSMAD1/5/9 levels in MIB1-iKO cells treated with Dox (0.5 μg/mL) and BMP inhibitor LDN (50 nmol/L) for 3 days. β-Actin served as the loading control. B, IF analysis of pSMAD1/5/9 (green) with DAPI (blue) nuclear staining in cells treated as described in A; scale bars, 50 μm. C, Phospho-SMAD1/5/9 (orange) or pSMAD2/3 (orange) were evaluated by IF in NCI-H838 cells transfected with MIB1 or nontargeting control (NTC) siRNA for 48 hours, with DAPI nuclear staining (blue); scale bar, 50 μm. D, Immunoblot analysis of pSMAD1/5/9 in additional MIB1- and FKBP1A-dependent cancer cell lines transfected with MIB1 or NTC siRNA for 48 to 72 hours. β-Actin served as the loading control. E, Top, schematic of the dTAG-MIB1 construct that expresses a FKBP12F36V–MIB1 fusion protein (dTAG-MIB1, Cas9-resistant) that is degraded upon addition of dTagV-1 ligand. E, Bottom, proof-of-concept experiment: NCI-H838 MIB1-iKO cells engineered to express dTAG-MIB1 and were treated with Dox (0.5 μg/mL), dTagV-1 (100 nmol/L), or a combination of both. After 24 hours, MIB1 protein levels were analyzed by IB. β-Actin served as the loading control. F, Time-course analysis using IB to assess MIB1 and pSMAD1/5/9 protein levels after different durations of dTagV-1 (100 nmol/L) in dTAG-MIB1 cells. β-Actin served as the loading control. G, pSMAD1/5/9 protein level in dTAG-MIB1 cells grown in 0.4%, 2%, or 10% serum 18 hours prior to dTagV-1 ligand treatment. β-Actin served as the loading control. Data are representative of three independent experiments. [E (top), Created in BioRender. Cottonham, C. (2026) https://BioRender.com/jbpsnmi.]

Alternative mRNA splicing and post-transcriptional RNA modification are key mechanisms that regulate transcript function; however, their role in neuronal activity and neurodegenerative disease remains poorly defined. In this study, we evaluated two nanopore-based long-read sequencing (LR-seq) formats: cDNA-PCR sequencing (CPS) and direct RNA sequencing (DRS). We then applied DRS to profile both full-length isoforms and RNA modifications in major brain cell types derived from induced pluripotent stem cells (iPSCs) and post-mortem Alzheimer's disease (AD) brains. Relative to CPS, DRS achieved higher accuracy and sensitivity for transcript quantification, de novo transcript model construction, and open reading frame (ORF) annotation across neuropathological gene sets. Focusing on iPSC-derived neurons, we built a multi-omic atlas to connect transcriptional output with translational engagement and protein abundance, by integrating DRS-based mRNA abundance, N 6 -methyladenosine (m6A) status and poly(A) tail length with ribosome profiling (Ribo-seq) and mass spectrometry (MS). The combination of DRS and Ribo-seq data demonstrated synergism in predicting protein abundance. This analysis also uncovered a significant inverse relationship between m6A modification and mRNA abundance, which was dependent on the engagement of the ribosomal A-site. Lastly, we applied DRS to the epitranscriptomic analysis of AD brain samples, demonstrating that m6A profiles can be used to distinguish early-versus late-stage disease.

Dysregulated microglia are intimately involved in neurodegeneration, including Alzheimer's disease (AD) pathogenesis, but the mechanisms controlling pathogenic microglial gene expression remain poorly understood. The transcription factor CCAAT/enhancer binding protein beta (c/EBPβ) regulates pro-inflammatory genes in microglia and is upregulated in AD. We show expression of c/EBPβ in microglia is regulated post-translationally by the ubiquitin ligase COP1 (also called RFWD2). In the absence of COP1, c/EBPβ accumulates rapidly and drives a potent pro-inflammatory and neurodegeneration-related gene program, evidenced by increased neurotoxicity in microglia-neuronal co-cultures. Antibody blocking studies reveal that neurotoxicity is almost entirely attributable to complement. Remarkably, loss of a single allele of Cebpb prevented the pro-inflammatory phenotype. COP1-deficient microglia markedly accelerated tau-mediated neurodegeneration in a mouse model where activated microglia play a deleterious role. Thus, COP1 is an important suppressor of pathogenic c/EBPβ-dependent gene expression programs in microglia.

Protein phosphatase 2A (PP2A) is a Ser/Thr phosphatase that regulates the phosphorylation of almost all cellular processes, including cell division and proliferation1,2. PP2A forms heterotrimeric holoenzyme complexes comprising a catalytic subunit (PP2Ac), a scaffolding subunit (PP2Aa) and variable B regulatory subunits that exert precise control over enzyme substrate specificity and prevent indiscriminate dephosphorylation of phosphoproteins3. However, the mechanisms that control the activity of uncomplexed catalytic subunits have remained relatively unclear. Here we find that the E3 ligase SKP1-CUL1-F-box (SCF) complex containing F-box other protein 42 (FBXO42, also known as JFK; hereafter, SCFFBXO42) degrades holoenzyme-free PP2Ac in a complex with the coiled-coil protein CCDC6 to maintain cancer cell fitness. The cryo-electron microscopy structure of the FBXO42-CCDC6-PP2Ac assembly reveals a pseudosymmetric architecture in which CCDC6 forms a central dimeric template that recruits multiple copies of PP2Ac and creates a substrate for FBXO42. Both the quaternary structure of this CCDC6-PP2Ac heterodimer and the post-translationally methylated tail of PP2Ac are recognized by FBXO42 for ubiquitination. The multivalent structure facilitated by CCDC6 enables the assembly of multiple degradation complexes along a single coiled coil, leading to the turnover of free phosphatases and downregulation of catalytic activity. Together, our findings define a mechanism for PP2A control through the ubiquitin-proteosome system and establish a paradigm for cullin-RING ligase-substrate interactions.

Kinetochores are essential macromolecular complexes anchoring chromosomes to the mitotic spindle, ensuring faithful cell division. Despite their critical role, the structural organization of kinetochores across diverse species remains poorly understood. We present the inner kinetochore constitutive centromere-associated network (CCAN) structure of the silkmoth Bombyx mori , an insect that lacks the canonical centromere-specifying histone variant CENP-A and exhibits chromosome-wide centromeric activity (holocentric). The B. mori CCAN incorporates four previously uncharacterized centromeric subunit proteins that are structurally related to the Dam1/DASH complex but function in scaffolding the inner kinetochore rather than in microtubule binding. Similar to the yeast and human systems, the B. mori CCAN also entraps DNA within its central closed chamber. However, unlike these systems, the B. mori CCAN can also assemble in vitro into a self-contained head-to-head dimer via atypical histone-fold protein dimerization. On the basis of our findings, we propose that the holocentric organization may emerge from the modular arrangement of discrete kinetochore units.

The ligase domains of MIB1 are essential for its regulatory function in BMP. A, Top, schematic (modified from (20)) of the endogenous MIB1 coding region, with exons shown as boxes and protein domains indicated. A, Bottom, Cas9-resistant MIB1 cDNA constructs, containing mutated PAM sequences (asterisks), express either WT MIB1 cDNA (MIB1 WT, blue) or MIB1 cDNA with all three RING domains deleted (MIB1ΔR1-3, orange). B, Immunoblot analysis of MIB1 protein levels in MIB1-iKO cells that were engineered to stably express Cas9-resistant MIB1 WT or MIB1 ΔR1-3 after 3 days of Dox (0.5 μg/mL) treatment. β-Actin served as the loading control. C–E, MIB1-iKO cells overexpressing MIB1 WT or MIB1ΔR1–3 were treated with Dox (0.5 μg/mL) for the indicated time points. C, Colony formation assay after 14 days of Dox treatment; scale bars, 10 mm. Images are representative of three independent experiments. D, CellTiter-Glo assay after 7 days of Dox treatment. Data represent three independent experiments. Two-way ANOVA with Tukey test; *, P < 0.05. “ns” denotes nonsignificant differences between no MIB1 cDNA cells and those expressing MIB1 constructs. E, Immunoblot analysis of BMPR2 immunoprecipitates from MIB1-iKO cells. Lane labels indicate MIB1 WT (no cDNA, −Dox), MIB1 KO (no cDNA, +Dox; Dox-induced KO of endogenous MIB1), MIB1 WT OE (MIB1-mNeonGreen expressed in MIB1 KO cells), and MIB1ΔR1-3 OE (MIB1ΔR1-3-IRES-mNeonGreen expressed in MIB1 KO cells). Cell lysates were subjected to IP with anti-BMPR2 antibody or IgG control, followed by IB with the indicated antibodies. Input lysates are shown for comparison. A longer exposure of the MIB1 immunoblot is included to facilitate detection of MIB1 in BMPR2 immunoprecipitates. Blue and orange arrowheads indicate BMPR2-associated MIB1 WT OE and MIB1ΔR1-3 OE, respectively. β-Actin is shown as an input and specificity control. F, Densitometric quantification of MIB1 and BMPR2 protein levels from the input immunoblots shown in E. Signals were normalized to β-Actin and expressed relative to the MIB1 WT (no cDNA, −Dox) control.

MIB1 and BMPR2 associate in cancer and endothelial cells. A, Co-IP of endogenous BMPR2 and MIB1 from NCIH838 cells treated with CLQ (25 μmol/L) or MG132 (10 μmol/L) for 6 hours prior to lysis to stabilize BMPR2 protein levels. IB confirmed co-precipitation of MIB1. Dox, used to induce KO of MIB1. B, Co-IP of endogenous BMPR2 and MIB1 from HUVECs transfected with control or BMPR2 siRNA for 72 hours. IP and IB were performed as described in A. C, Schematic (top) illustrates the experimental workflow for pull-down of endogenous MIB1 from HUVEC lysates using recombinant His-tagged BMPR2. Association of MIB1 with BMPR2 was verified by IB.

MIB1 is a strongly selective cancer dependency linked to negative regulation of TGF-β family signaling. A, Gene dependency scores (gene effect score) for a subset of cancer cell lines in the CRISPR (DepMap Public 22Q2, Chronos) dataset exhibiting selective MIB1 dependency. For context, dependency scores for an essential gene (RPL14) and a nonessential gene (UCP1) are also shown. B, MIB1 dependency scores from DepMap (Public 22Q2, Chronos) for 1,086 cancer cell lines grouped by lineage. Each circle represents an individual cell line; box-and-whisker plots summarize the distribution across lineages. C, Top 10 MIB1 correlates based on DepMap (Public 24Q2 Chronos and Expression). Expression correlates (#): genes whose transcript levels correlate with MIB1 dependency; codependency correlates (^): genes whose presence or absence correlates with MIB1 dependency. MIB1 anticorrelates are shown on the left (green shaded boxes, solid outlines); correlates are on the right (red shaded boxes, dashed outlines). D, Simplified overview of the TGF-β family signal pathway with MIB1 correlates highlighted at their points of impact. Data are presented as the mean ± SD; n = 3 (two-way ANOVA with Tukey correction). [D, Created in BioRender. Cottonham, C. (2026) https://BioRender.com/ydae6kh.]

MIB1 KO mirrors BMP-mediated suppression of cell proliferation and cell-cycle progression. A, Overview of the all-in-one CRISPR/Cas9 expression construct to generate MIB1-iKO. MIB1 is targeted by Cas9 following 0.5 μg/mL Dox addition. B, MIB1 protein was evaluated by IB after a time course of 0.5 μg/mL Dox treatment in clone 2.1. C–E, MIB1-iKO cells were treated with 0.5 μg/mL (Dox), 10 ng/mL BMP4, or both, with or without 50 nmol/L LDN. E, includes 5 nmol/L LDN. C, Cell viability was assessed using CellTiter-Glo on day 12 (n = 3). Statistical analysis was performed using two-way ANOVA with Tukey post hoc test. D, Cell growth was monitored by IncuCyte live-cell imaging over 6 days (n = 3). Two-way ANOVA with Tukey test was used for statistical analysis. E, Colony formation assay performed on day 7. Representative wells shown from three independent experiments. Scale bars, 5 mm. F and G, MIB1-iKO cells were treated with the BMP inhibitor LDN (50 nmol/L), TGF-β inhibitor SB (5 μmol/L), or Notch inhibitor Compound E (CompE, 1 μmol/L) and stimulated with 10 ng/mL BMP4 or 10 ng/mL TGF-β1 with or without Dox (0.5 μg/mL). F, A CellTiter-Glo assay was performed on day 14. The results are represented as the mean ± SD; n = 3 (two-way ANOVA with Tukey correction). G, Colony formation assay performed on day 14. Representative images shown from three independent experiments. Scale bars, 5 mm. H, MIB1-iKO cells were treated with combinations of Dox (0.5 μg/mL), BMP4 (10 ng/mL), and LDN (50 nmol/L), as indicated. After 4 days, EdU was incorporated for 1 hour and detected by flow cytometry to assess cell-cycle distribution. Data are from three independent experiments. Statistical significance was determined for all cell-cycle phases using two-way ANOVA with Tukey post hoc test; P values are shown for G0/G1. I, Immunoblot of MIB1 and p21 protein levels after 3 days of 0.5 μg/mL Dox treatment. J, IF to detect the subcellular localization of p21 (red). DAPI staining was used to visualize nuclei (blue). Scale bars, 50 μm. Representative images from three independent experiments are shown. For all panels, *, P < 0.05; **, P < 0.01 and ***, P < 0.001; ****, P < 0.0001. [A, Created in BioRender. Cottonham, C. (2026) https://BioRender.com/9nig4ft.]

The synthesis and biological characterization of multiple degrader antibody conjugates (DACs) bearing a heterobifunctional VHL-dependent proteolysis targeting chimera (PROTAC) payload is described. The conjugated molecule (A515) potently and extensively degrades the BRM protein (also known as SMARCA2) in cell-based assessments and exhibits moderate degradation selectivity for BRM over the closely related paralog protein BRG1 (SMARCA4). A CD71-targeting DAC that utilizes A515 as a payload and employs a disulfide-based linker affords strong, antigen-dependent efficacy in an H1944 xenograft model. Similarly, a Trop2-targeting DAC-bearing A515 that incorporates a protease-cleavable linker provides potent BRM degradation outcomes in HCC515 in vitro assessments along with encouraging antigen-dependent antitumor activity in several HCC515-based xenograft experiments. Structure-activity relationship information based on in vitro BRM degradation results and single-dose in vivo pharmacodynamic experiments is also provided for several additional CD71 and Trop2-targeting DACs in which the nature of the linkers and the A515 attachment sites are varied.

Loss of MIB1 amplifies BMP target gene expression. A, Overview of the BMP inhibitor washout experiment for RNA-seq. MIB1 KO was induced by Dox (0.5 μg/mL) under BMP-off conditions (+LDN, 50 nmol/L). After 3 days, cells were split equally into medium containing BMP4 (10 ng/mL) with or without continued LDN (50 nmol/L) treatment, generating groups 1 to 4 as indicated. RNA was harvested 24 hours later for sequencing analysis. B, In parallel with RNA collection, BMP pathway activity was assessed by IB detection of SMAD1/5/9 phosphorylation 24 hours after washout of the BMP inhibitor. This confirmed effective pathway suppression and reactivation. C, RNA-seq analysis of gene expression changes following BMP inhibitor washout (n = 3 per group; 16,400 genes analyzed). The dashed line indicates genes with equivalent FC upon BMP reactivation (BMP-on/BMP-off) in both MIB1 KO and MIB1 WT cells. Potentiated BMP target genes are labeled in bold orange; other BMP target genes and pathway components are indicated with blue labels. D, Zoomed-in view of the region in C highlighting potentiated genes (orange), with BMP target genes in bold orange. E, GSEA of RNA-seq data from the BMP inhibitor washout experiment (C and D), comparing group 4 (MIB1 KO, BMP-on) with group 2 (MIB1 WT, BMP-on). Gene sets were derived from the Hallmark database. [A, Created in BioRender. Cottonham, C. (2026) https://BioRender.com/5qnb2jv.]

Abstract Mind bomb 1 (MIB1), an E3 ubiquitin ligase required for NOTCH activation, has been identified as a strongly selective cancer dependency in genome-wide loss-of-function screens in more than 1,000 cancer cell lines. However, MIB1-correlating dependencies in these screens unexpectedly linked MIB1 not to Notch but rather to the family of transforming growth factor-β and bone morphogenic protein (BMP) signaling molecules. In this study, using genetic and pharmacologic methods, we show that MIB1 loss phenocopies BMP tumor-suppressive function. MIB1-dependent cancer cell lines were sensitive to BMP ligands, and MIB1 loss selectively enhanced BMP signaling, as evidenced by SMAD1/5/9 phosphorylation and transcriptional responses that correlated with increased BMP receptor protein levels. Growth inhibition and enhanced signaling were both reversed by inhibitors of BMP type 1 receptors, demonstrating that these MIB1 effects directly reflect a function for MIB1 as a negative regulator of BMP signaling. Unlike wild-type MIB1, reexpression of RING domain–deleted MIB1 failed to rescue growth inhibition in MIB1-depleted cells, suggesting that negative regulation depends on E3 ligase activity. Supporting this regulatory role, we found that MIB1 and the BMP type II receptor BMPR2 physically associate in both cancer and endothelial cells. By revealing this previously unknown role for MIB1 as a Notch-independent negative regulator of BMP signaling, our study expands the significance of MIB1 in cancer biology, with implications for targeting BMP signaling in cancer and other diseases. Significance: This study identifies MIB1 as a Notch-independent, E3 ligase–dependent regulator of BMP signaling through control of BMPR2 protein abundance, revealing a mechanistic vulnerability in BMP-responsive cancers and a potential therapeutic target.