Genome-wide CRISPR screens reveal a Wnt–FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors

Steinhart, Zachary; Pavlovic, Zvezdan; Chandrashekhar, Megha; Hart, Traver; Wang, Xiaowei; Zhang, Xiaoyu; Robitaille, Mélanie; Brown, Kevin R.; Jaksani, Sridevi; Overmeer, René M.; Boj, Sylvia F.; Adams, Jarrett; Pan, James; Clevers, Hans; Sidhu, Sachdev S.; Moffat, Jason; Angers, Stéphane

doi:10.1038/nm.4219

Cited by 267 publications

(256 citation statements)

References 36 publications

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“…This result is in contrast to the induction of noncanonical Wnt signaling by Wnt7a via FZD10 (57). In agreement with our results, it has been recently demonstrated that pancreatic cancer cells that mainly secrete Wnt7b are dependent on FZD5 (58). The focus of this study was the canonical Wnt signaling pathway.…”

Section: Discussionsupporting

confidence: 93%

Mapping of Wnt‐Frizzled interactions by multiplex CRISPR targeting of receptor gene families

et al. 2017

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Signaling pathway modules are often encoded by several closely related paralogous genes that can have redundant roles and are therefore difficult to analyze by loss-of-function analysis. A typical example is the Wnt signaling pathway, which in mammals is mediated by 19 Wnt ligands that can bind to 10 Frizzled (FZD) receptors. Although significant progress in understanding Wnt-FZD receptor interactions has been made in recent years, tools to generate systematic interaction maps have been largely lacking. Here we generated cell lines with multiplex mutant alleles of FZD1, FZD2, and FZD7 and demonstrate that these cells are unresponsive to canonical Wnt ligands. Subsequently, we performed genetic rescue experiments with combinations of FZDs and canonical Wnts to create a functional ligand–receptor interaction map. These experiments showed that whereas several Wnt ligands, such as Wnt3a, induce signaling through a broad spectrum of FZD receptors, others, such as Wnt8a, act through a restricted set of FZD genes. Together, our results map functional interactions of FZDs and 10 Wnt ligands and demonstrate how multiplex targeting by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 can be used to systematically elucidate the functions of multigene families.—Voloshanenko, O., Gmach, P., Winter, J., Kranz, D., Boutros, M. Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families.

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Section: Discussionsupporting

confidence: 93%

Mapping of Wnt‐Frizzled interactions by multiplex CRISPR targeting of receptor gene families

et al. 2017

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“…Moreover, high FZD receptor expression at the cell surface contributes to overactive Wnt signaling in subsets of pancreatic, ovarian, gastric, and colorectal tumors (16)(17)(18)(19). Recently, antibody-mediated inhibition of FZD5 demonstrated a role for this receptor in pancreatic ductal adenocarcinoma and colorectal carcinoma models (20). Altogether, these findings highlight the importance of FZD receptors in Wnt-dependent stem cell and cancer biology.…”

mentioning

confidence: 88%

Unsaturated fatty acyl recognition by Frizzled receptors mediates dimerization upon Wnt ligand binding

Nile¹,

Mukund²,

Stanger³

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

105

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Frizzled (FZD) receptors mediate Wnt signaling in diverse processes ranging from bone growth to stem cell activity. Moreover, high FZD receptor expression at the cell surface contributes to overactive Wnt signaling in subsets of pancreatic, ovarian, gastric, and colorectal tumors. Despite the progress in biochemical understanding of Wnt-FZD receptor interactions, the molecular basis for recognition of Wnt cis-unsaturated fatty acyl groups by the cysteine-rich domain (CRD) of FZD receptors remains elusive. Here, we determined a crystal structure of human FZD7 CRD unexpectedly bound to a 24-carbon fatty acid. We also report a crystal structure of human FZD5 CRD bound to C16:1 cis-Δ9 unsaturated fatty acid. Both structures reveal a dimeric arrangement of the CRD. The lipid-binding groove exhibits flexibility and spans both monomers, adopting a U-shaped geometry that accommodates the fatty acid. Re-evaluation of the published mouse FZD8 CRD structure reveals that it also shares the same architecture as FZD5 and FZD7 CRDs. Our results define a common molecular mechanism for recognition of the cis-unsaturated fatty acyl group, a necessary posttranslational modification of Wnts, by multiple FZD receptors. The fatty acid bridges two CRD monomers, implying that Wnt binding mediates FZD receptor dimerization. Our data uncover possibilities for the arrangement of Wnt-FZD CRD complexes and shed structural insights that could aide in the identification of pharmacological strategies to modulate FZD receptor function.nt signaling is evolutionarily conserved from metazoans to humans (1). It is critical for tissue homeostasis and during development (2, 3). On the other hand, derangements in Wnt signaling are associated with severe pathologies in mammals, including cancer (4-6). Signaling is initiated at the cell surface where the secreted, lipid-modified Wnt glycoprotein interacts with the extracellular N-terminal cysteine-rich domain (CRD) of the frizzled (FZD) receptor (7). This high-affinity interaction occurs at two distinct contact sites, one comprising a protein-fatty acyl interface and another a protein-protein interaction interface (8-10). Sequence analysis revealed that there are 10 human frizzled genes grouped into four clusters (SI Appendix, Fig. S1A).Among their multiple roles in physiology, FZD receptors have emerged as critical regulators of Wnt-dependent stem cell processes. For example, FZD7 is a critical component of the stem cell niche and was shown by genetic knockdown experiments to be essential for maintaining human embryonic and epithelial limbal stem cells in an undifferentiated state (11,12). Of the 10 mammalian frizzleds, FZDs 1, 2, and 7 are enriched at the base of mammalian adult intestinal crypts where the stem cells reside (13,14), and FZD7 is required for stem cell-mediated regeneration of the intestinal epithelium (15). Moreover, high FZD receptor expression at the cell surface contributes to overactive Wnt signaling in subsets of pancreatic, ovarian, gastric, and colorectal tumors (16)(17)(18)(19)...

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“…FZD6 upregulation in colorectal cancer, neuroblastoma and triple-negative breast cancer is involved in stem-like features, EMT and drug resistance (193–196). Based on FZD5 upregulation in solid tumors, including RNF43 -mutated pancreatic cancer (197,198), FZD7 upregulation in breast cancer, colorectal cancer, glioma and hepatocellular carcinoma (199–203) and FZD10 upregulation in breast cancer, colorectal cancer and synovial sarcoma (204–207), anti-FZD5 IgG, anti-FZD7 mAb and anti-FZD10 mAbs have been developed for cancer therapy. Vantictumab (OMP-18R5), initially isolated as an FZD7-binding antibody, is a broad-spectrum anti-FZD mAb that reacts with FZD1, FZD2, FZD5, FZD7 and FZD8 (208), which all belong to the FZD1/2/7 or FZD5/8 subfamily among the FZD family (204).…”

Section: Therapeutics Targeting Wnt Signaling Cascadesmentioning

confidence: 99%

Molecular genetics and targeted therapy of WNT-related human diseases (Review)

Katoh¹,

2017

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Canonical WNT signaling through Frizzled and LRP5/6 receptors is transduced to the WNT/β-catenin and WNT/stabilization of proteins (STOP) signaling cascades to regulate cell fate and proliferation, whereas non-canonical WNT signaling through Frizzled or ROR receptors is transduced to the WNT/planar cell polarity (PCP), WNT/G protein-coupled receptor (GPCR) and WNT/receptor tyrosine kinase (RTK) signaling cascades to regulate cytoskeletal dynamics and directional cell movement. WNT/β-catenin signaling cascade crosstalks with RTK/SRK and GPCR-cAMP-PKA signaling cascades to regulate β-catenin phosphorylation and β-catenin-dependent transcription. Germline mutations in WNT signaling molecules cause hereditary colorectal cancer, bone diseases, exudative vitreoretinopathy, intellectual disability syndrome and PCP-related diseases. APC or CTNNB1 mutations in colorectal, endometrial and prostate cancers activate the WNT/β-catenin signaling cascade. RNF43, ZNRF3, RSPO2 or RSPO3 alterations in breast, colorectal, gastric, pancreatic and other cancers activate the WNT/β-catenin, WNT/STOP and other WNT signaling cascades. ROR1 upregulation in B-cell leukemia and solid tumors and ROR2 upregulation in melanoma induce invasion, metastasis and therapeutic resistance through Rho-ROCK, Rac-JNK, PI3K-AKT and YAP signaling activation. WNT signaling in cancer, stromal and immune cells dynamically orchestrate immune evasion and antitumor immunity in a cell context-dependent manner. Porcupine (PORCN), RSPO3, WNT2B, FZD5, FZD10, ROR1, tankyrase and β-catenin are targets of anti-WNT signaling therapy, and ETC-159, LGK974, OMP-18R5 (vantictumab), OMP-54F28 (ipafricept), OMP-131R10 (rosmantuzumab), PRI-724 and UC-961 (cirmtuzumab) are in clinical trials for cancer patients. Different classes of anti-WNT signaling therapeutics are necessary for the treatment of APC/CTNNB1-, RNF43/ZNRF3/RSPO2/RSPO3- and ROR1-types of human cancers. By contrast, Dickkopf-related protein 1 (DKK1), SOST and glycogen synthase kinase 3β (GSK3β) are targets of pro-WNT signaling therapy, and anti-DKK1 (BHQ880 and DKN-01) and anti-SOST (blosozumab, BPS804 and romosozumab) monoclonal antibodies are being tested in clinical trials for cancer patients and osteoporotic post-menopausal women. WNT-targeting therapeutics have also been applied as reagents for in vitro stem-cell processing in the field of regenerative medicine.

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Genome-wide CRISPR screens reveal a Wnt–FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors

Cited by 267 publications

References 36 publications

Mapping of Wnt‐Frizzled interactions by multiplex CRISPR targeting of receptor gene families

Mapping of Wnt‐Frizzled interactions by multiplex CRISPR targeting of receptor gene families

Unsaturated fatty acyl recognition by Frizzled receptors mediates dimerization upon Wnt ligand binding

Molecular genetics and targeted therapy of WNT-related human diseases (Review)

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