The stability of the Wnt pathway transcription factor beta-catenin is tightly regulated by the multi-subunit destruction complex. Deregulated Wnt pathway activity has been implicated in many cancers, making this pathway an attractive target for anticancer therapies. However, the development of targeted Wnt pathway inhibitors has been hampered by the limited number of pathway components that are amenable to small molecule inhibition. Here, we used a chemical genetic screen to identify a small molecule, XAV939, which selectively inhibits beta-catenin-mediated transcription. XAV939 stimulates beta-catenin degradation by stabilizing axin, the concentration-limiting component of the destruction complex. Using a quantitative chemical proteomic approach, we discovered that XAV939 stabilizes axin by inhibiting the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2. Both tankyrase isoforms interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin-proteasome pathway. Thus, our study provides new mechanistic insights into the regulation of axin protein homeostasis and presents new avenues for targeted Wnt pathway therapies.

Inductive signals and transcription factors involved in motor neuron generation have been identified, raising the question of whether these developmental insights can be used to direct stem cells to a motor neuron fate. We show that developmentally relevant signaling factors can induce mouse embryonic stem (ES) cells to differentiate into spinal progenitor cells, and subsequently into motor neurons, through a pathway recapitulating that used in vivo. ES cell-derived motor neurons can populate the embryonic spinal cord, extend axons, and form synapses with target muscles. Thus, inductive signals involved in normal pathways of neurogenesis can direct ES cells to form specific classes of CNS neurons.

R-spondin proteins strongly potentiate Wnt signalling and function as stem-cell growth factors. Despite the biological and therapeutic significance, the molecular mechanism of R-spondin action remains unclear. Here we show that the cell-surface transmembrane E3 ubiquitin ligase zinc and ring finger 3 (ZNRF3) and its homologue ring finger 43 (RNF43) are negative feedback regulators of Wnt signalling. ZNRF3 is associated with the Wnt receptor complex, and inhibits Wnt signalling by promoting the turnover of frizzled and LRP6. Inhibition of ZNRF3 enhances Wnt/β-catenin signalling and disrupts Wnt/planar cell polarity signalling in vivo. Notably, R-spondin mimics ZNRF3 inhibition by increasing the membrane level of Wnt receptors. Mechanistically, R-spondin interacts with the extracellular domain of ZNRF3 and induces the association between ZNRF3 and LGR4, which results in membrane clearance of ZNRF3. These data suggest that R-spondin enhances Wnt signalling by inhibiting ZNRF3. Our study provides new mechanistic insights into the regulation of Wnt receptor turnover, and reveals ZNRF3 as a tractable target for therapeutic exploration.

Hedgehog (Hh) proteins comprise a family of secreted signaling molecules essential for patterning a variety of structures in animal embryogenesis. During biosynthesis, Hh undergoes an autocleavage reaction, mediated by its carboxyl-terminal domain, that produces a lipid-modified amino-terminal fragment responsible for all known Hh signaling activity. Here it is reported that cholesterol is the lipophilic moiety covalently attached to the amino-terminal signaling domain during autoprocessing and that the carboxyl-terminal domain acts as an intramolecular cholesterol transferase. This use of cholesterol to modify embryonic signaling proteins may account for some of the effects of perturbed cholesterol biosynthesis on animal development.

Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.