PROJECT 2 ABSTRACT GI GVHD remains the major cause of morbidity and non-relapse mortality (NRM) after HCT. We discovered that a major cause of GI crypt destruction is the accelerated death of intestinal stem cells (ISCs), which is mediated by activation of receptor interacting protein kinase 1 (RIPK1). Our data show that inhibition of RIPK1 with two compounds specific for RIPK1, Necrostatin-1s and GNE684, prevent ISC damage in organoid culture and in two experimental models of GVHD. We have also shown that the Paneth cell protein regenerative 3γ (REG3γ) that is lost from the crypt during GVHD is a critical anti- apoptotic protein and inhibits the activation of RIPK1, rescuing ISCs from GVHD and reducing mortality. These findings support the central hypothesis of Project 2 that inhibition of RIPK1 activation prevents ISC loss and allows regeneration of crypts, thus reducing GVHD. Our three specific aims will investigate this hypothesis. Specific Aim 1: To determine the role of RIPK1 activation in ISC damage and GVHD. We will first use systemic administration of pharmacological inhibitors of RIPK1 in well-defined mouse models to treat and prevent GVHD, with detailed analyses of GVHD severity of the GI tract, correlating metrics of crypt histopathology, RIPK1 expression and activation. We will complement these analyses using mutant HCT recipient mice with inactive forms of RIPK1 and RIPK3 proteins. We will also analyze the effect of RIPK1 inhibitors on GVL effects using two leukemic cell lines. In human GI biopsies, we will analyze phospo-RIPK1 expression as a biomarker of GVHD severity and as a predictor of long term outcomes. These studies will complement and expand the preclinical data that form the rationale for the Phase 2 trial of RIPK1 inhibition in high risk patients in Project 3. Specific Aim 2: To analyze the effect of REG3γ on RIPK1 activation. This specific aim will investigate the role of REG3γ on the expression of Complex I/II components and the role of TNFα and RIPK1 on REG3γ expression. In summary, these studies will illuminate the key mechanisms by which inhibition of RIPK1 prevents the inflammatory cell death that causes GI crypt damage during GVHD and thus will lead to novel strategies to control this major cause of NRM after allogeneic BMT.