Cbl and Cbl-b Ubiquitin Ligases are Essential for Intestinal Epithelial Stem Cell Maintenance

Abstract

SUMMARYAmong the signaling pathways that control the stem cell self-renewal and maintenance vs. acquisition of differentiated cell fates, those mediated by receptor tyrosine kinase (RTK) activation are well established as key players. CBL family ubiquitin ligases are negative regulators of RTKs but their physiological roles in regulating stem cell behaviors are unclear. While hematopoieticCbl/Cblbknockout (KO) leads to a myeloproliferative disease due to expansion and reduced quiescence of hematopoietic stem cells, mammary epithelial KO led to stunted mammary gland development due to mammary stem cell depletion. Here, we examined the impact of inducibleCbl/Cblbdouble-KO (iDKO) selectively in the Lgr5-defined intestinal stem cell (ISC) compartment.Cbl/CblbiDKO led to rapid loss of theLgr5HiISC pool with a concomitant transient expansion of theLgr5Lotransit amplifying population. LacZ reporter-based lineage tracing showed increased ISC commitment to differentiation, with propensity towards enterocyte and goblet cell fate at the expense of Paneth cells. Functionally,Cbl/CblbiDKO impaired the recovery from radiation-induced intestinal epithelial injury.In vitro,Cbl/CblbiDKO led to inability to maintain intestinal organoids. Single cell RNAseq analysis of organoids revealed Akt-mTOR pathway hyperactivation in iDKO ISCs and progeny cells, and pharmacological inhibition of the Akt-mTOR axis rescued the organoid maintenance and propagation defects. Our results demonstrate a requirement forCbl/Cblbin the maintenance of ISCs by fine tuning the Akt-mTOR axis to balance stem cell maintenance vs. commitment to differentiation.