Differential effects of itacitinib, fedratinib, and ruxolitinib in mouse models of hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) comprises a severe hyperinflammatory phenotype driven by the overproduction of cytokines, many of which signal via the JAK/STAT pathway. Indeed, the JAK1/2 inhibitor ruxolitinib has demonstrated efficacy in preclinical studies and early-phase clinical trials in HLH. Nevertheless, concerns remain for ruxolitinib-induced cytopenias, which are postulated to result from the blockade of JAK2-dependent hematopoietic growth factors. To explore the therapeutic effects of selective JAK inhibition in mouse models of HLH, we carried out studies incorporating the JAK1 inhibitor itacitinib, JAK2 inhibitor fedratinib, and JAK1/2 inhibitor ruxolitinib. All 3 drugs were well-tolerated and at the doses tested, they suppressed interferon-gamma (IFN-γ)-induced STAT1 phosphorylation in vitro and in vivo. Itacitinib, but not fedratinib, significantly improved survival and clinical scores in CpG-induced secondary HLH. Conversely, in primary HLH, in which perforin-deficient (Prf1-/-) mice are infected with lymphocytic choriomeningitis virus (LCMV), itacitinib, and fedratinib performed suboptimally. Ruxolitinib demonstrated excellent clinical efficacy in both HLH models. RNA-sequencing of splenocytes from LCMV-infected Prf1-/- mice revealed that itacitinib targeted inflammatory and metabolic pathway genes in CD8 T cells, whereas fedratinib targeted genes regulating cell proliferation and metabolism. In monocytes, neither drug conferred major transcriptional impacts. Consistent with its superior clinical effects, ruxolitinib exerted the greatest transcriptional changes in CD8 T cells and monocytes, targeting more genes across several biologic pathways, most notably JAK-dependent proinflammatory signaling. We conclude that JAK1 inhibition is sufficient to curtail CpG-induced disease, but combined inhibition of JAK1 and JAK2 is needed to best control LCMV-induced immunopathology.

Keenan C ，Albeituni S ，Oak N ，Stroh A ，Tillman HS ，Wang Y ，Freeman BB 3rd ，Alemán-Arteaga S ，Meyer LK ，Woods R ，Verbist KC ，Zhou Y ，Cheng C ，Nichols KE ... -  《-》

JAK/STAT pathway inhibition sensitizes CD8 T cells to dexamethasone-induced apoptosis in hyperinflammation.

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.

Meyer LK ，Verbist KC ，Albeituni S ，Scull BP ，Bassett RC ，Stroh AN ，Tillman H ，Allen CE ，Hermiston ML ，Nichols KE ... -  《-》

Janus kinase inhibition lessens inflammation and ameliorates disease in murine models of hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) comprises an emerging spectrum of inherited and noninherited disorders of the immune system characterized by the excessive production of cytokines, including interferon-γ and interleukins 2, 6, and 10 (IL-2, IL-6, and IL-10). The Janus kinases (JAKs) transduce signals initiated following engagement of specific receptors that bind a broad array of cytokines, including those overproduced in HLH. Based on the central role for cytokines in the pathogenesis of HLH, we sought to examine whether the inhibition of JAK function might lessen inflammation in murine models of the disease. Toward this end, we examined the effects of JAK inhibition using a model of primary (inherited) HLH in which perforin-deficient (Prf1(-∕-)) mice are infected with lymphocytic choriomeningitis virus (LCMV) and secondary (noninherited) HLH in which C57BL/6 mice receive repeated injections of CpG DNA. In both models, treatment with the JAK1/2 inhibitor ruxolitinib significantly lessened the clinical and laboratory manifestations of HLH, including weight loss, organomegaly, anemia, thrombocytopenia, hypercytokinemia, and tissue inflammation. Importantly, ruxolitinib treatment also significantly improved the survival of LCMV-infectedPrf1(-∕-)mice. Mechanistic studies revealed that in vivo exposure to ruxolitinib inhibited signal transducer and activation of transcription 1-dependent gene expression, limited CD8(+)T-cell expansion, and greatly reduced proinflammatory cytokine production, without effecting degranulation and cytotoxic function. Collectively, these findings highlight the JAKs as novel, druggable targets for mitigating the cytokine-driven hyperinflammation that occurs in HLH. These observations also support the incorporation of JAK inhibitors such as ruxolitinib into future clinical trials for patients with these life-threatening disorders.

Das R ，Guan P ，Sprague L ，Verbist K ，Tedrick P ，An QA ，Cheng C ，Kurachi M ，Levine R ，Wherry EJ ，Canna SW ，Behrens EM ，Nichols KE ... -  《-》

Therapeutic effect of JAK1/2 blockade on the manifestations of hemophagocytic lymphohistiocytosis in mice.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome, characterized by severe hyperinflammation and immunopathological manifestations in several tissues. These features result from organ infiltration by overactivated CD8 T-cells and macrophages, which produce high levels of pro-inflammatory cytokines, such as IFN-γ, TNF-α, IL-6, and IL-18. Recently, several Janus kinase 1/2 (JAK1/2) inhibitors, such as ruxolitinib, have been developed as immunosuppressive agents. They have proven beneficial effects in the treatment of myeloproliferative disorders and inflammatory conditions. To determine whether pharmacological inhibition of the JAK1/2 not only prevents the onset of HLH immunopathology but also is effective against existing HLH, cytotoxicity-impaired Prf1(-/-) and Rab27a(-/-) mice with full-blown HLH syndrome were treated with a clinically relevant dose of ruxolitinib. In vivo, ruxolitinib treatment suppressed signal transducer and activator of transcription 1 activation and led to recovery from HLH manifestations in both murine models. In the Prf1(-/-) mice, these beneficial effects were evidenced by a greater survival rate, and in both murine models, they were evidenced by the correction of blood cytopenia and a rapid decrease in serum IL-6 and TNF-α levels. During ruxolitinib treatment, liver tissue damage receded concomitantly with a decrease in the number of infiltrating inflammatory macrophages and an increase in the number of alternatively activated macrophages. In Rab27a(-/-) mice, central nervous system involvement was significantly reduced by ruxolitinib therapy. Our findings demonstrate that clinically relevant doses of the JAK1/2 inhibitor ruxolitinib suppresses the harmful consequences of macrophage overactivation characterizing HLH in 2 murine models. The results could be readily translated into the clinic for the treatment of primary, and perhaps even secondary, forms of HLH.

Maschalidi S ，Sepulveda FE ，Garrigue A ，Fischer A ，de Saint Basile G ... -  《-》

JAK inhibition for murine HLH requires complete blockade of IFN-γ signaling and is limited by toxicity of JAK2 inhibition.

Hemophagocytic lymphohistiocytosis (HLH) is an inflammatory disorder in which numerous cytokines are elevated, though interferon-γ (IFN-γ) is central to disease pathogenesis and a key therapeutic target. Experimental and early clinical reports have shown that ruxolitinib, a small molecule inhibitor of Janus kinases (JAKs), which are essential for cytokine signaling, may be therapeutic in HLH. In contrast, we found that intermittently administered ruxolitinib at various dose levels failed to prevent HLH development or treat established murine HLH. High doses of ruxolitinib blocked IFN-γ signaling only transiently after administration, consistent with human pharmacokinetics, and only continuously administered drug could prevent HLH development or treat established HLH. Continuously administered ruxolitinib was therapeutic in only a narrow dose range and intermittently dosed ruxolitinib worsened survival and decreased bone marrow cellularity of animals concurrently treated with anti-IFN-γ antibody, indicating a narrow therapeutic window and potential toxicity. Because JAK2 is essential for hematopoietic cytokine signaling, we also tested a JAK1-selective inhibitor and observed therapeutic benefit without apparent toxicity, though it did not improve survival when combined with anti-IFN-γ. We conclude that continuous blockade of IFN-γ signaling is necessary for optimal control of HLH and that JAK2 inhibition may be toxic in this disorder.

Chaturvedi V ，Lakes N ，Tran M ，Castillo N ，Jordan MB ... -  《-》