Abstract
Wnt-induced β-catenin-mediated transcription is a driving force for stem cell self-renewal during adult tissue homeostasis. Enhanced Wnt receptor expression due to mutational inactivation of the ubiquitin ligases RNF43/ZNRF3 recently emerged as a leading cause for cancer development. Consequently, targeting canonical Wnt receptors such as LRP5/6 holds great promise for treatment of such cancer subsets. Here, we employ CIS display technology to identify single-domain antibody fragments (VHH) that bind the LRP6 P3E3P4E4 region with nanomolar affinity and strongly inhibit Wnt3/3a-induced β-catenin-mediated transcription in cells, while leaving Wnt1 responses unaffected. Structural analysis reveal that individual VHHs variably employ divergent antigen-binding regions to bind a similar surface in the third β-propeller of LRP5/6, sterically interfering with Wnt3/3a binding. Importantly, anti-LRP5/6 VHHs block the growth of Wnt-hypersensitive Rnf43/Znrf3-mutant intestinal organoids through stem cell exhaustion and collective terminal differentiation. Thus, VHH-mediated targeting of LRP5/6 provides a promising differentiation-inducing strategy for treatment of Wnt-hypersensitive tumors.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Binding Sites
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Cell Differentiation
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Cell Line, Tumor
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Cell Proliferation
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Crystallography, X-Ray
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Fibroblasts / cytology
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Fibroblasts / drug effects
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Fibroblasts / metabolism
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Gene Expression Regulation
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HEK293 Cells
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Humans
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Intestine, Small / cytology
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Intestine, Small / drug effects
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Intestine, Small / metabolism
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Low Density Lipoprotein Receptor-Related Protein-5 / antagonists & inhibitors
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Low Density Lipoprotein Receptor-Related Protein-5 / chemistry*
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Low Density Lipoprotein Receptor-Related Protein-5 / genetics
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Low Density Lipoprotein Receptor-Related Protein-5 / metabolism
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Low Density Lipoprotein Receptor-Related Protein-6 / antagonists & inhibitors
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Low Density Lipoprotein Receptor-Related Protein-6 / chemistry*
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Low Density Lipoprotein Receptor-Related Protein-6 / genetics
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Low Density Lipoprotein Receptor-Related Protein-6 / metabolism
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Mice
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Models, Molecular
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Organoids / cytology
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Organoids / drug effects*
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Organoids / metabolism
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Protein Binding
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Protein Conformation, beta-Strand
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Protein Interaction Domains and Motifs
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Single-Domain Antibodies / chemistry*
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Single-Domain Antibodies / genetics
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Single-Domain Antibodies / metabolism
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Stem Cells / cytology
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Stem Cells / drug effects*
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Stem Cells / metabolism
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Transcription, Genetic
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Ubiquitin-Protein Ligases / genetics
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Ubiquitin-Protein Ligases / metabolism
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Wnt3A Protein / genetics*
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Wnt3A Protein / metabolism
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beta Catenin / genetics
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beta Catenin / metabolism
Substances
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CTNNB1 protein, mouse
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Low Density Lipoprotein Receptor-Related Protein-5
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Low Density Lipoprotein Receptor-Related Protein-6
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Lrp5 protein, mouse
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Lrp6 protein, mouse
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Single-Domain Antibodies
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Wnt3A Protein
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Wnt3a protein, mouse
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beta Catenin
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RNF43 protein, mouse
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Ubiquitin-Protein Ligases