Background 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. Methods We comprehensively mapped Wnt-regulated lncRNAs from an orthotopic Wnt-addicted pancreatic cancer model and examined the response of lncRNAs to Wnt inhibition between in vivo and in vitro cancer models. We further annotated and characterized these Wnt-regulated lncRNAs using existing genomic classifications (using data from FANTOM5) in the context of Wnt signaling and inferred their role in cancer pathogenesis (using GWAS and expression data from the TCGA). To functionally validate Wnt-regulated lncRNAs, we performed CRISPRi screens to assess their role in cancer cell proliferation both in vivo and in vitro. Results We identified 3633 lncRNAs, of which 1503 were regulated by Wnt signaling in an orthotopic Wnt-addicted pancreatic cancer model. These lncRNAs were much more sensitive to changes in Wnt signaling in xenografts than in cultured cells. Our analysis suggested that Wnt signaling inhibition could influence the co-expression relationship of Wnt-regulated lncRNAs and their eQTL-linked protein-coding genes. Wnt-regulated lncRNAs were also implicated in specific gene networks involved in distinct biological processes that contribute to the pathogenesis of cancers. Consistent with previous genome-wide lncRNA CRISPRi screens, around 1% (13/1503) of the Wnt-regulated lncRNAs were found to 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 (25/1503) that had a functional effect on cancer cell growth. Conclusions 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.

20Background: Wnt signaling is an evolutionarily conserved developmental pathway that 21 is frequently hyperactivated in cancer. While multiple protein-coding genes regulated by 22Wnt signaling are known, the functional lncRNAs regulated by Wnt signaling have not 23 been systematically characterized. 24Results: We comprehensively mapped lncRNAs from an orthotopic Wnt-addicted 25 pancreatic cancer model, identifying 3,633 lncRNAs, of which 1,503 were regulated by 26 Wnt signaling. We found lncRNAs were much more sensitive to changes in Wnt 27 signaling in xenografts than in cultured cells. To functionally validate Wnt-regulated 28 lncRNAs, we performed CRISPRi screens to assess their role in cancer cell proliferation. 29Consistent with previous genome-wide lncRNA CRISPRi screens, around 1% (13/1,503) 30 of the Wnt-regulated lncRNAs could modify cancer cell growth in vitro. This included 31 CCAT1 and LINC00263, previously reported to regulate cancer growth. Using an in vivo 32CRISPRi screen, we doubled the discovery rate, identifying twice as many Wnt-33 regulated lncRNAs (25/1,503) that had a functional effect on cancer cell growth. 34 Conclusions:Our study demonstrates the value of studying lncRNA functions in vivo, 35 provides a valuable resource of lncRNAs regulated by Wnt signaling and establishes a 36 framework for systematic discovery of functional lncRNAs. 37 38