Investigating the Cellular Responses and Mitigation of Injury in the Small Intestinal Epithelia After High Dose Radiation

Abstract

Ionizing radiation induces acute toxicity in tissues with rapid cellular turnover, as these cells are highly susceptible to pre-mitotic apoptosis and mitotic catastrophe. When vulnerable tissues are affected, such as the epithelial lining of the gastrointestinal tract, this can result in an often-lethal condition known as gastrointestinal acute radiation syndrome (GI-ARS). GI-ARS arises from radiation-induced destruction of intestinal stem cells responsible for continuous renewal of the intestinal mucosal barrier, leading to breakdown of the gut epithelial lining, nausea, vomiting, diarrhea, and an increased risk of infection and sepsis, with symptoms and mortality typically manifesting within 10 days after exposure. This dissertation interrogates the identity and function of the intestinal stem cells at homeostasis and following radiation injury, incorporates multiple proposed models of intestinal regeneration, and evaluates a novel pharmacological intervention utilizing the Hippo signaling pathway to ameliorate GI-ARS. In this dissertation, I employ novel in vivo lineage tracing techniques to define the role of Lgr5+ intestinal stem cells (ISCs), identifying Lgr5+ ISCs as the primary source of intestinal self-renewal, and Lgr5+ ISCs as the dominant stem cell population responsible for reconstituting the irradiated small intestine. Furthermore, I integrate the contributions of canonical ISCs, reserve ISCs (rISCs), and revival stem cells (RevSCs) into a unified model of epithelial regeneration, highlighting their collective roles in repairing the irradiated intestinal epithelium. I identify a novel mechanism in which Bmi1+ cells, generally known as rISCs, act as a transitional cell population that is induced transiently before the maturation of Clu+ RevSCs and during the conversion of Clu+ RevSCs to new Lgr5+ ISCs in response to radiation injury. Furthermore, I show that the tumor suppressor p53 functions in Bmi1+ cells to promote the emergence of Clu+ RevSCs in the damaged intestinal epithelium. Taken together, these data support a dynamic, unified model of intestinal regeneration, in which multiple stem-like and damage-induced epithelial cell populations act cooperatively to regenerate the intestinal epithelium. In addition to studying intestinal regenerative dynamics, I investigate the roles of the Hippo pathway effectors Yap and Taz in regulating intestinal homeostasis and regeneration following high-dose radiation injury. I address a previously overlooked and undefined question, where I demonstrate that Yap, not Taz, is the critical effector driving intestinal epithelial regeneration following irradiation (IR). Specifically, Yap IEC-null mice fail to produce Clu+ RevSCs after high-dose IR, whereas Taz IEC-null mice exhibit a normal Clu+ RevSC emergence phenotype. Building upon these findings, I characterize a novel small molecule inhibitor of Yap’s upstream regulators, Lats1/2, NCGC-023. I demonstrate that transient inhibition of Lats1/2 by NCGC-023 is sufficient to ameliorate GI-ARS in vivo in a murine model, promoting regeneration and protecting crypt transit amplifying cells from mitotic catastrophe. Furthermore, I show that NCGC-023 protects human enteroids from high-dose radiation-induced death, while not promoting oncogenesis in a pre-malignant murine enteroid model. Together, these findings highlight a therapeutically actionable mechanism to enhance intestinal regeneration after radiation injury while minimizing the risk of long-term adverse effects. In summary, this dissertation demonstrates the multi-functionality of various intestinal stem cell pools, including canonical, reserve, and revival populations in orchestrating epithelial regeneration. It further establishes that pharmacological modulation of a key regenerative pathway, the Hippo-Yap pathway, can be harnessed to ameliorate GI-ARS, offering a promising therapeutic strategy to promote the repair of irradiated intestinal epithelium while maintaining long term safety.