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.
| Original language | English |
|---|---|
| Article number | 365 |
| Journal | Nature Communications |
| Volume | 10 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 1 Dec 2019 |
Funding
We thank members of the laboratory of M.M.M. for experimental support, helpful discussions and suggestions. We thank the European Synchrotron Radiation Facility (ESRF) for the provision of synchrotron radiation facilities; and beamline scientists of the ESRF for assistance. We thank U-protein Express for the provision of protein expression facilities. We thank Guangyun Yu and Ronald Pieters (Utrecht University, Department of Pharmaceutical sciences - Molecular Pharmacy, The Netherlands), for providing access to the MicroCal Auto ITC200. This work is part of the Oncode Institute, which is partly financed by the Dutch Cancer Society. This work was supported by European Research Council Starting Grant 242958, the Netherlands Organization for Scientific Research NWO VICI Grant 91815604, ECHO Grant 711.013.012 (to M.M.M.), European Union Grant FP7 Marie Curie ITN 608180 “WntsApp” (to M.M.M.), Fondazione Michelangelo for the Advancement of the Study and Treatment of Cancer (to N.F.), BioStruct-X Grant 283570 and NWO Spinoza Grant 01.80.104.00 (to P.G.).