Inhibition of the Receptor Interactive Protein Kinase 1 (RIP1) Pathway Prevents Acute Gvhd

Acute graft-versus-host disease (GVHD) causes apoptosis of intestinal stem cells (ISCs) that are key targets in the gastrointestinal (GI) tract (Zeiser and Blazar, NEJM 2917; 377:2167). The receptor interactive protein kinase 1 (RIP1) is an important mediator of apoptosis and we investigated the role of the (RIP1) pathway in GI tract and ISC damage in both human and mouse GVHD models. Immunohistochemical staining of human GI biopsies from GVHD patients showed strong phospho-RIP1 staining in biopsies with Lerner grades 2/3 compared to grades 0/1 (12/17 v 0/8, p < 0.01). We then established in vitro models of ISC apoptosis (at least 50% loss) by adding 10 ng/ml of both IFNg and TNFa to organoid cultures from both ileum and colon (organoids cultures with either IFNg or TNFa alone showed no change). Addition to cultures of 0.1 mcg/ml GNE684, a well-characterized small molecule RIP inhibitor (Patel et al, Cell Death Diff 2020; 27:161), dramatically reversed ISC loss in both human and mouse ileal organoids as measured by cross sectional area (Fig 1). WE observed an identical pattern in colon cultures where GNE684 reversed loss of human ISCs (70.0 + 7.7 % v 29.3 + 2.5 %, p < 0.001) and mouse ISCs (100 + 6.6 % v 41 + 6.6 % p < 0.001). In vivo, 14 daily ip injections of GNE684 prevented mortality in a GVHD mouse model (C3H.SW → B6) in a dose dependent fashion (Fig 2). Systemic GNE administration also improved survival in a second model (Balb/c → B6: day 30 survival 7/10 v 0/10, p < 0.001). In the C3H.SW → B6 model there were dramatic reductions in CD4+ lamina propria effector lymphocytes secreting either IFNg (56 + 11 v 9 + 2, p < 0.001) or IL-17 ( 22 + 10 v 7 + 4, p < 0.01) and in CD8+ effector cells secreting IFNg (39 + 11 v 8 + 3, p < 0.001) or IL-17 ( 19 + 8 v 7 + 2, p < 0.001) on day 10 after HCT. Importantly, GVHD was dramatically reduced when administration of GNE684 was used as treatment for GVHD beginning on day +7 after GVHD was established (day 40 survival: 8/12 v 2/12, p = 0.02). Additionally, survival dramatically improved in RIP1 kinase-dead recipients compared to wild-type B6 controls in both the C3H.SW → B6 model (13/16 v 4/16, p < 0.001) and in the Balb/c →B6 model (13/20 v 1/20, p < 0.001). IFNg secreting CD4+ and CD8+ effector lymphocytes that infiltrated the lamina propria were significantly reduced in RIP1 kinase-dead recipients compared to wild-type controls (58 + 13 v 11 + 7 and 39 + 11 v 14 + 4, respectively [p < 0.001 for both]). Infiltrating lymphocytes secreting IL-17 were also reduced four fold. Importantly, administration of GNE684 preserved beneficial GVL effects when lethal doses of myeloid tumor C1498 cells that caused 100% mortality by day 28 in syngeneic BMT recipients were added to the donor inoculum (day 42 survival: 7/10 v 4/13, p = 0.03). We conclude that RIP1 is a key mediator of GI damage during GVHD. In vitro RIP1 inhibition prevents ISC apoptosis in both mouse and human models of GI GVHD. RIP1 inhibition, either through genetic alteration of the RIP1 kinase domain in transplant recipients or through systemic administration of the RIP1 inhibitor GNE684, protects the GI tract from acute GVHD damage as measured both clinically and histologically in preclinical models while preserving beneficial GVL effects. These data suggest that GNE684 a clinical trial of GNE684 is warranted in patients with gastrointestinal GVHD.

Ferrara:Equillium: Consultancy, Research Funding; Genentech: Research Funding; Incyte: Research Funding; Mesoblast: Research Funding; Alexion: Consultancy; Bo Fu Rui: Consultancy; Eurofins Viracor: Consultancy, Patents & Royalties; Nimbus Discovery: Consultancy; Physicians Education Resource: Consultancy; Xenikos: Consultancy. Levine:Biogen: Research Funding; Equillium: Consultancy, Research Funding; Incyte: Research Funding; MaaT Pharma: Research Funding; Mesoblast: Consultancy, Research Funding; Bluebird Bio: Consultancy; Jazz: Consultancy; Mallinckrodt/Therakos: Consultancy; X4 Pharmaceuticals: Consultancy; Eurofins Viracor: Patents & Royalties. Webster:Genentech-Roche: Current Employment. Vucic:Genentech-Roche: Current Employment.