Targeting the immunoproteasome as a novel therapeutic strategy for hemophagocytic lymphohistiocytosis

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PROJECT SUMMARY Primary hemophagocytic lymphohistiocytosis (pHLH) comprises an expanding array of inherited disorders of the immune system characterized by severe hyperinflammation. Despite use of the glucocorticoid dexamethasone, chemotherapeutic agent etoposide, specific cytokine-targeting agents, and allogeneic hematopoietic stem cell transplantation, up to 40% of pHLH patients die due to uncontrolled disease or the complications of its treatment. Accordingly, there is a pressing need to develop new and more effective therapies. To date, much of what we have learned about the pathogenesis of pHLH has come from the study of humans and mice lacking expression of perforin, a pore forming protein essential for the cytotoxic function of CD8 T cells and natural killer (NK) cells. Following infection with Lymphocytic Choriomeningitis virus (LCMV), perforin-deficient mice develop a fatal HLH-like illness characterized by organomegaly, anemia, thrombocytopenia, hypercytokinemia, hyperferritinemia, and fulminant tissue inflammation. Due to their cytolytic defects, perforin- deficient CD8 T and NK cells cannot eliminate LCMV-infected antigen presenting cells (APCs). It has been proposed that the persistence of these activated APCs results in a feed-forward loop to further drive T cell proliferation and proinflammatory cytokine production, ultimately culminating in the signs and symptoms of HLH. Within APCs, the immunoproteasome processes endogenous and exogenous proteins, including viral proteins, into peptides that are loaded onto Major Histocompatibility Complex class I (MHCI) molecules for presentation to other cells of the immune system. Given the persistence of activated APCs in pHLH and the central role for antigen presentation in driving immune cell activation, we hypothesize that inhibiting the immunoproteasome will serve as a rational and potentially beneficial therapeutic maneuver. Indeed, using an in vitro model of LCMV infection, we observe that the immunoproteasome inhibitors ONX-914 and KZR-616 significantly reduce LCMV-specific CD8 T cell proliferation and interferon-gamma (IFNg) production. Similarly, treatment of LCMV-infected perforin-deficient mice with KZR-616 significantly diminishes the manifestations of HLH, including splenomegaly, CD8 T cell expansion, tissue immunopathology, and serum IFNg levels. In this R21 Exploratory Research Grant, which is being submitted in response to Notice of Special Interest: Investigations on Inborn Errors of Immunity/Primary Immunodeficiencies (NOT-AI-21-032), we will further explore the therapeutic effects and mechanisms of action of immunoproteasome inhibition using the perforin- deficient mouse model. In Aim 1, we will examine the extent to which immunoproteasome inhibition ameliorates clinical disease, testing specific drug schedules and combinations. Aim 2 will explore the mechanistic basis using complementary in vivo and in vitro assays. If successful, these studies will elucidate whether immunoproteasome inhibition lessens inflammation in pHLH and set the stage for future clinical trials aimed at improving the overall outcome for individuals with these often-fatal immune system disorders.