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Summary: Mouse embryonic stem cells (mESCs) can be maintained in vitro in defined N2B27 medium supplemented with two chemical inhibitors for GSK3 and MEK (2i) and the cytokine leukemia inhibitory factor (LIF), which act synergistically to promote self-renewal and pluripotency. Here, we find that genetic deletion of the four genes encoding the TCF/LEF transcription factors confers mESCs with the ability to self-renew in N2B27 medium alone. TCF/LEF quadruple knockout (qKO) mESCs display dysregulation of several genes, including Aire, Dnmt3l, and IcosL, located adjacent to each other within a topologically associated domain (TAD). Aire, Dnmt3l, and IcosL appear to be regulated by TCF/LEF in a β-catenin independent manner. Moreover, downregulation of Aire and Dnmt3l in wild-type mESCs mimics the loss of TCF/LEF and increases mESC survival in the absence of 2iL. Hence, this study identifies TCF/LEF effectors that mediate exit from the pluripotent state.

Targeting of Wnt signaling represents a promising anti-cancer therapy. However, the consequences of systemically attenuating the Wnt pathway in an adult organism are unknown. Here, we globally prevent Wnt secretion by genetically ablating Wntless. We find that preventing Wnt signaling in the entire body causes mortality due to impaired intestinal homeostasis. This is caused by the loss of intestinal stem cells. Reconstitution of Wnt/β-catenin signaling via delivery of external Wnt ligands prolongs the survival of intestinal stem cells and reveals the essential role of extra-epithelial Wnt ligands for the renewal of the intestinal epithelium. Wnt2b is a key extra-epithelial Wnt ligand capable of promoting Wnt/β-catenin signaling and intestinal homeostasis. Wnt2b is secreted by subepithelial mesenchymal cells that co-express either Gli1 or Acta2. Subepithelial mesenchymal cells expressing high levels of Wnt2b are predominantly Gli1 positive.

BCL9/9L proteins enhance the transcriptional output of the β-catenin/TCF transcriptional complex and contribute critically to upholding the high WNT signaling level required for stemness maintenance in the intestinal epithelium. Here we show that a BCL9/9L-dependent gene signature derived from independent mouse colorectal cancer (CRC) models unprecedentedly separates patient subgroups with regard to progression free and overall survival. We found that this effect was by and large attributable to stemness related gene sets. Remarkably, this signature proved associated with recently described poor prognosis CRC subtypes exhibiting high stemness and/or epithelial-to-mesenchymal transition (EMT) traits. Consistent with the notion that high WNT signaling is required for stemness maintenance, ablating Bcl9/9l-β-catenin in murine oncogenic intestinal organoids provoked their differentiation and completely abrogated their tumorigenicity, while not affecting their proliferation. Therapeutic strategies aimed at targeting WNT responses may be limited by intestinal toxicity. Our findings suggest that attenuating WNT signaling to an extent that affects stemness maintenance without disturbing intestinal renewal might be well tolerated and prove sufficient to reduce CRC recurrence and dramatically improve disease outcome.