Cbl and Cbl-b ubiquitin ligases are essential for intestinal epithelial stem cell maintenance

Receptor tyrosine kinases (RTKs) control stem cell maintenance vs. differentiation decisions. Casitas B -lineage lymphoma (CBL) family ubiquitin ligases are negative regulators of RTKs, but their stem cell reg ulatory roles remain unclear. Here, we show that Lgr5+ intestinal stem cell (ISC)-specific inducible Cbl knockout (KO) on a Cblb null mouse background (iDKO) induced rapid loss of the Lgr5 Hi ISCs with tran sient expansion of the Lgr5 Lo transit -amplifying population. LacZ-based lineage tracing revealed increased ISC commitment toward enterocyte and goblet cell fate at the expense of Paneth cells. Func tionally, Cbl/Cblb iDKO impaired the recovery from radiation -induced intestinal epithelial injury. In vitro , Cbl/Cblb iDKO led to inability to maintain intestinal organoids. Single -cell RNA sequencing in organoids identified Akt-mTOR (mammalian target of rapamycin) pathway hyperactivation upon iDKO, and pharmacological Akt-mTOR axis inhibition rescued the iDKO defects. Our results demonstrate a require ment for Cbl/Cblb in the maintenance of ISCs by fine-tuning the Akt-mTOR axis to balance stem cell main tenance vs. commitment to differentiation.