Abstract:

VPS34, the sole class III phosphatidylinositol 3-kinase, is a central regulator of autophagy and endosomal trafficking. Despite its established roles in multiple tissues, its function in the intestinal epithelium remains unexplored. The intestinal epithelium undergoes continuous renewal driven by Lgr5⁺ intestinal stem cells (ISC), and its integrity depends on tightly coordinated programs of proliferation, differentiation and cell death. As ISC are at the origin of colorectal cancer, it is essential to understand the mechanisms that regulate their proliferation and differentiation. To investigate VPS34 function in the intestinal epithelium, we generated an inducible, epithelium-specific knockout mouse model (VilCreERT2Vps34fl/fl). Vps34 deletion causes rapid-onset fatal enteritis within eight days of tamoxifen injection, characterized by extensive cell death and compensatory crypt hyperproliferation, loss of differentiated epithelial lineages, junctional disruption and immune cell infiltration. Mechanistically, VPS34 loss impairs both autophagic flux, evidenced by p62 accumulation and increase LC3-I/LC3-II ratio, and endosomal-lysosomal trafficking, as indicated by the altered distribution of Rab5⁺ and Rab7⁺ endosomes and LAMP1+ lysosomes. Consistent with the role of VPS34 in cell division, we identify major mitotic abnormalities and midbody accumulation. Single-cell RNA-seq uncovers a global loss of intestinal epithelial identity and marked depletion of the Lgr5+/OLFM4+ ISC pool, accompanied by the amplification of the fetal-like revival stem cell (RevSC) population, characterized by nuclear YAP accumulation and activation of a YAP-driven regenerative program. Bulk RNA-seq of small intestine further reveals extensive reprogramming, including downregulation of metabolic pathways and epithelial transporters, and activation of inflammatory and immune responses. Ultrastructural analyses show loss of microvilli and severe barrier defects, associated with infiltration of immune cells. Intestinal organoids derived from in vivo depletion of Vps34 are nonviable, whereas those with ex vivo deletion, although viable, exhibit strong cell death that results in their death after 2 passages following 4OH-TAM treatment, demonstrating a cell-autonomous requirement for VPS34 in epithelial survival. Investigation of cell death mechanisms identified combined activation of three regulated cell death pathways: extrinsic apoptosis, disulfidptosis and necroptosis. Cell death is p53-independent, as demonstrated by the absence of phenotypic rescue in Vps34;Trp53 double-knockout mice. Joint inhibition of apoptosis and necroptosis in vitro partially rescues organoid viability. These findings uncover a previously unrecognized role for VPS34 as a central regulator of homeostasis and cell fate in the intestinal epithelium, identifying a convergent multi-pathway cell death response that overwhelms the endogenous RevSC-mediated regenerative program.