Project Summary/Abstract: Human herpesvirus(HHV)-8-negative, idiopathic multicentric Castleman disease (iMCD) is a deadly hematologic illness involving polyclonal lymphoproliferation and multiple organ system dysfunction. iMCD is diagnosed in approximately 1,500 individuals annually in the USA; 35% die within 5 years. Limited options exist beyond cytotoxic chemotherapies for the 66% of patients refractory to interleukin-6 (IL-6) blockade with siltuximab; relapse is common. The etiology, pathological cell types, and dysregulated signaling pathways are poorly understood. Improved understanding of disease mechanisms is necessary to identify new treatments. In the previous funding cycle, we demonstrated that iMCD patients display increased mTOR signaling in lymph nodes and activated T cells in circulation. Inhibiting mTOR signaling with sirolimus led to a decrease in activated T cells and clinical improvement in a subset of patients. However, the signals leading to increased mTOR activation and alternative pathways involved in treatment-refractory patients remain unknown. We propose that dysregulated cytokine signaling could be responsible for the increased mTOR signaling in immune cells and hyperinflammation in iMCD. Our recent preliminary data suggest that T cells from iMCD patients in remission display increased pS6 expression, a read-out of mTOR activation, upon stimulation with Type I interferon (IFN-I) and IL-6 compared to healthy controls. Given that IFN-I and IL-6 signal through JAK1 and JAK2, we hypothesized that JAK1/2 inhibition could abrogate the increased mTOR activation induced by IL-6 and IFN- I. Indeed, in vitro JAK1/2 inhibition by ruxolitinib abrogated the increased mTOR activation in iMCD patient T cells. Based on these in vitro results and proteomics data indicating enrichment of JAK-STAT3 signaling across iMCD patients, we administered ruxolitinib to a highly treatment refractory and critically ill pediatric iMCD patient, who has been in complete remission for 24 months, 20-fold longer than her previous average remission duration. We hypothesize that JAK1/2 signaling is a central mediator of mTOR activation and iMCD pathogenesis, JAK-mediated hypersensitivity to cytokine stimulation is the mechanistic basis, and ruxolitinib interrupts iMCD by inhibiting JAK1/2, T cell activation, and mTOR. In Aim 1, we will study the activation of JAK1/2 and mTOR signaling in iMCD patient samples during flare. In Aim 2, we will rigorously evaluate how iMCD patient T cells respond to cytokine stimulation with IFN-I and IL-6 in vitro and establish the mechanistic link between JAK1/2 and mTOR signaling. Based on our preliminary data, we hypothesize that IRS1 phosphorylation downstream of JAK1/2 can lead to PI3K/AKT and mTOR activation. Aim 3 outlines a mechanistic study of ruxolitinib in iMCD patients to investigate JAK1/2 signaling in vivo. The proposed studies will advance understanding of dysregulated signaling pathways and cell types in iMCD a...