Wnt-regulated lncRNA discovery enhanced by in vivo identification and CRISPRi functional validation

Wnt signaling is an evolutionarily conserved developmental pathway that is frequently hyperactivated in cancer. While multiple protein-coding genes regulated by Wnt signaling are known, the functional lncRNAs regulated by Wnt signaling have not been systematically characterized. We comprehensively mapped lncRNAs from an orthotopic Wnt-addicted pancreatic cancer model, identifying 3,633 lncRNAs, of which 1,503 were regulated by Wnt signaling. We found lncRNAs were much more sensitive to changes in Wnt signaling in xenografts than in cultured cells. To functionally validate Wnt-regulated lncRNAs, we performed CRISPRi screens to assess their role in cancer cell proliferation. Consistent with previous genome-wide lncRNA CRISPRi screens, around 1% (13/1,503) of the Wnt-regulated lncRNAs could modify cancer cell growth in vitro. This included CCAT1 and LINC00263, previously reported to regulate cancer growth. Using an in vivo CRISPRi screen, we doubled the discovery rate, identifying twice as many Wnt-regulated lncRNAs (25/1,503) that had a functional effect on cancer cell growth. Our study demonstrates the value of studying lncRNA functions in vivo, provides a valuable resource of lncRNAs regulated by Wnt signaling and establishes a framework for systematic discovery of functional lncRNAs.