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Cytokine storm syndromes (CSS) are severe hyperinflammatory conditions characterized by excessive immune system activation leading to organ damage and death. Hemophagocytic lymphohistiocytosis (HLH), a disease often associated with inherited defects in cell-mediated cytotoxicity, serves as a prototypical CSS for which the 5-year survival is only 60%. Frontline therapy for HLH consists of the glucocorticoid dexamethasone (DEX) and the chemotherapeutic agent etoposide. Many patients, however, are refractory to this treatment or relapse after an initial response. Notably, many cytokines that are elevated in HLH activate the JAK/STAT pathway, and the JAK1/2 inhibitor ruxolitinib (RUX) has shown efficacy in murine HLH models and humans with refractory disease. We recently reported that cytokine-induced JAK/STAT signaling mediates DEX resistance in T cell acute lymphoblastic leukemia (T-ALL) cells, and that this could be effectively reversed by RUX. On the basis of these findings, we hypothesized that cytokine-mediated JAK/STAT signaling might similarly contribute to DEX resistance in HLH, and that RUX treatment would overcome this phenomenon. Using ex vivo assays, a murine model of HLH, and primary patient samples, we demonstrate that the hypercytokinemia of HLH reduces the apoptotic potential of CD8 T cells leading to relative DEX resistance. Upon exposure to RUX, this apoptotic potential is restored, thereby sensitizing CD8 T cells to DEX-induced apoptosis in vitro and significantly reducing tissue immunopathology and HLH disease manifestations in vivo. Our findings provide rationale for combining DEX and RUX to enhance the lymphotoxic effects of DEX and thus improve the outcomes for patients with HLH and related CSS.

β-Thalassemia is a common, frequently debilitating, inherited anemia caused by HBB gene mutations that reduce or eliminate the expression of the β-globin subunit of adult hemoglobin (HbA, α2β2). Consequently, excess free α-globin forms toxic precipitates in red blood cells (RBCs) and their precursors, leading to ineffective erythropoiesis and hemolytic anemia. Previously, we showed that free α-globin is eliminated by protein quality-control pathways, including the ubiquitin-proteasome system and autophagy (Khandros et al., Blood 2012;119:5265). In β-thalassemic mice, disruption of the Unc-51-like autophagy activating kinase gene (Ulk1) increased α-globin precipitates and worsened the pathologies of β-thalassemia. Treatment of β-thalassemic mice with rapamycin to inhibit mTOR (an ULK1 inhibitor) reduced α-globin precipitates, lessened ineffective erythropoiesis, and increased the lifespan of circulating RBCs in an Ulk1-dependent fashion. To investigate the therapeutic potential of rapamycin in human β-thalassemia, we treated erythroid precursors generated by in vitro differentiation of patient-derived CD34+ hematopoietic stem and progenitor cells. Reverse-phase high-performance liquid chromatography (HPLC) analysis of hemoglobinized erythroblasts generated from transfusion-dependent (TD, n = 5) or non-transfusion-dependent (NTD, n = 5) β-thalassemia patients revealed α-chain excesses (α-chain/β-like [β + γ + δ] chain) of approximately 40% and 15%, respectively (compared to 7 normal donors; P < 0.001). Rapamycin (10µM or 20µM) or the proteasome inhibitor MG132 (2.5µM) was added to day 13 cultures, which contained mid- to late-stage erythroblasts, and α-globin accumulation was determined by HPLC 2 days later. As expected, proteasome inhibition by MG132 raised free α-globin levels in thalassemic erythroblasts (P < 0.01) and induced cell death (P < 0.01). In contrast, rapamycin reduced free α-globin in a dose-dependent manner by 40% and 85% in TD (P < 0.0001) and NTD β-thalassemia (P < 0.001), respectively, but had no effect on erythroblasts derived from normal CD34+ cells (figure). We also observed decreases in the accumulation of autophagic markers, such as SQSTM1/p62 protein, by Western blotting. We observed no negative effects of rapamycin on the survival of patient-derived erythroblasts. Also of note, under our experimental conditions, rapamycin treatment of erythroblasts did not induce fetal hemoglobin production, as has been previously reported, thereby excluding this potential mechanism for reducing globin chain imbalances. Overall, rapamycin treatment significantly reduced the accumulation of free α-globin in TD β-thalassemia and almost fully corrected the imbalance in NTD β-thalassemia cells. Our findings identify a new drug-regulatable pathway for ameliorating β-thalassemia. Rapamycin is approved and well studied, and it has a generally manageable toxicity profile. Moreover, there are additional pharmacologic approaches to activating ULK via mTOR inhibition or other pathways. These approaches may lead to effective drug therapies for β-thalassemia, particularly NTD or intermittently TD forms of the disease. Disclosures Cappellini: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Vifor: Membership on an entity's Board of Directors or advisory committees; Sanofi/Genzyme: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria.

The Hemophagocytic lymphohistiocytoses (HLH) comprise a heterogeneous group of disorders characterized by abnormal and severely damaging immune responses. Patients with HLH experience episodes of inflammation associated with the hyper-activation of CD8+ T cells and macrophages, which overproduce pro-inflammatory cytokines including interferon (IFN)-γ, interleukins (IL)-12 and -18, -6 and Tumor Necrosis Factor-α. Current treatments include immunosuppressive medications (corticosteroids, anti-thymocyte globulin) with or without cytotoxic chemotherapeutic agents (etoposide). Despite the use of these drugs, >50% of HLH patients die from unbridled inflammation. To develop more effective treatments for HLH, our laboratory has focused on blocking the effects of cytokines, which drive inflammation and mediate morbidity and mortality in HLH. We recently demonstrated that targeting the Janus Kinases (JAK1/2) with the JAK inhibitor ruxolitinib (INCB018424) significantly ameliorates the clinical and laboratory manifestations of HLH in preclinical mouse models (R. Das et al., Blood, 2016). Despite its beneficial effects, ruxolitinib did not completely abrogate disease. We therefore sought to understand its mechanisms of action and further improve upon these results. In mice, IFN-γ is central to disease pathogenesis, as its neutralization reduces anemia and prolongs survival. To examine whether ruxolitinib might be acting primarily through the inhibition of this cytokine, we modeled HLH in perforin-deficient (Prf1-/-) mice that were infected with lymphocytic choriomeningitis virus (LCMV). Beginning on day 4 post infection (p.i.), mice were treated or not with ruxolitinib (90 mg/kg by oral gavage twice daily) or with an IFN-γ blocking antibody (0.5 mg intraperitoneally [i.p.] on days 4 and 7). On day 9 p.i., the end point for these studies, mice were examined for the manifestations of HLH. Compared to untreated LCMV-infected mice, which lost on average 16% of starting body weight, mice treated with ruxolitinib or IFN-γ blockade exhibited only 7% weight loss. Ruxolitinib and IFN-γ blockade also ameliorated LCMV-induced anemia and thrombocytopenia, with platelet counts with ruxolitinib treatment reaching levels comparable to those observed in uninfected animals. Of note, ruxolitinib treatment decreased splenomegaly by over 50% (p=0.0001), while IFN-γ blockade had no effect. When compared to mice that received either no treatment or IFN-γ blockade, mice treated with ruxolitinib also exhibited a reduction in inflammation, which was manifested as a significantly lower area of liver encompassed by inflammatory foci, as well as a marked reduction in the absolute number of total and gp33-tetramer reactive splenic and intrahepatic CD8+ T cells. Together, these results suggest that ruxolitinib exerts its effects through mechanisms that are in part independent of the inhibition of IFN-γ signaling. We next examined whether combining ruxolitinib with dexamethasone, a gold standard medication in HLH treatment, might further diminish inflammation. To this end, LCMV-infected Prf1-/- mice were treated or not with ruxolitinib (as above), dexamethasone (15mg/kg i.p. daily) or ruxolitinib and dexamethasone. All treatments resulted in a similar reduction in weight loss. Thrombocytopenia was abrogated with ruxolitinib treatment, either alone or in combination with dexamethasone. Mice treated with both drugs, however, did not develop splenomegaly and were less anemic than mice treated with either single agent. Strikingly, when compared to the livers of infected untreated mice, livers from mice treated with combination therapy exhibited a 99.7% decrease in the area encompassed by inflammatory foci. This was in contrast to mice treated with ruxolitinib or dexamethasone, which showed a 66.8% or 87% reduction, respectively. Consistent with these findings, combination therapy was also more potent in decreasing the numbers and to a lesser extent the frequencies of total and antigen-specific CD8+ T cells. These findings suggest that treatment with ruxolitinib and dexamethasone further limits inflammation and lessens the manifestations of HLH, which represents a serious clinical problem for which there has been limited progress in treatment. The results of these studies support the incorporation of ruxolitinib and dexamethasone into future clinical trials to improve the cure rate for HLH. Disclosures No relevant conflicts of interest to declare.

We are studying a novel mechanism by which prototypical erythrocyte proteins also act in endothelial cells to regulate vascular tone. Previously, we reported that the alpha subunit of hemoglobin (αHb) is expressed in the myoendothelial junction of endothelial cells in resistance arterioles where it binds endothelial nitric oxide (NO) synthase (eNOS) and degrades NO, thereby stimulating vasoconstriction. To extend this observation, we examined arterioles of mice lacking 2 out of 4 α globin genes [HbA1 knockout *α2/*α2]. Compared to wild type (wt) controls, the thoracodorsal artery (TDA, a resistance arteriole) from mutant animals exhibited abnormal morphology with thinned internal elastic lamina, excessive dilation, and reduced contractility after treatment with the vasoconstrictor phenylephrine (EC50 8.2e-6M-1 n=6 in wt vs 2.3e-5 M-1 n=6 in mutant; p Wild type TDAs expressed αHb (Fig. 1A), but not βHb mRNA and protein. However, free αHb is unstable and not expected to exist as an isolated monomer. Alpha hemoglobin stabilizing protein (AHSP) is a molecular chaperone that binds free αHb, stabilizes its structure and facilitates assembly of HbA tetramers (α2β2) in red blood cells. Immunohistochemistry showed that AHSP and αHb co-localize in endothelial cells lining the TDA. In the TDA of Ahsp knockout (KO) mice, αHb immunostaining was disorganized and reduced in intensity (Fig. 1A), and Western blotting showed reduced αHb protein compared to wt controls. Moreover, TDAs from Ahsp KO mice exhibited abnormal thinning of the internal elastic lamina, excessive dilation (lumen diameter 7500 μM2 in Ahsp KO vs 4100 μM2 in controls; n=5 mice, p=0.0037) and reduced constriction after phenylephrine treatment (EC50 9.6e-6 M-1 in wt n=7 vs 5.6e-6 M-1 in mutant n=6; p To examine physical and functional interactions between AHSP, αHb and eNOS, we expressed fluorescent-tagged proteins in cultured human coronary ECs. αHb-GFP alone was expressed at relatively low level (mean fluorescent intensity (MFI) = 631) (Fig. 1D). Coexpressed mCherry-AHSP colocalized with αHb-GFP and increased its expression level (MFI 5312) (Fig. 1D). Similarly, coexpressed mCherry-eNOS colocalized with αHb-GFP and enhanced its expression (MFI 1519) (Fig. 1D). Purified αHb co-immunoprecipitated with AHSP or eNOS, but not both, suggesting mutually exclusive interactions. Overall, our studies provide genetic evidence that αHb expressed in arteriolar endothelial cells regulates blood vessel tone in vivo. Moreover, biochemical studies show that AHSP stabilizes endothelial-expressed αHb and facilitates its assembly with eNOS. Thus, AHSP acts as a molecular chaperone for αHb in erythrocytes and endothelial cells to promote the formation of HbA (α2b2) for O2 transport, and αHb-eNOS for NO degradation. We hypothesize that the αHb-AHSP-eNOS axis functions to fine-tune systemic blood pressure and/or regional blood flow to specific vascular beds. Defining the magnitude of these effects in mice and humans during specific circulatory stresses and in α thalassemia is an important and interesting topic for future investigation. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.