Targeting JAK-STAT in macrophage activation syndrome—no MAS! Free

In this issue of Blood, Ma et al demonstrate that neutrophil activation is mediated via JAK-STAT signaling in macrophage activation syndrome (MAS) developing in patients with adult-onset Still disease (AOSD) and that neutrophils play an important role in disease pathogenesis.¹ These findings represent an important advance in our understanding of MAS. AOSD-MAS is a systemic inflammatory cytokine release syndrome that develops in approximately 20% of patients with AOSD² and manifests clinical findings that overlap with those of hemophagocytic lymphohistiocytosis (HLH).³ MAS is diagnosed using the 2016 American College of Rheumatology/European Alliance of Associations for Rheumatology classification criteria for MAS in the setting of Still disease,⁴ and HLH is diagnosed using HLH-2004 criteria.⁵ Distinguishing MAS and HLH may be challenging, particularly with the numerous syndromes that lead to development of secondary HLH.⁶ 

In the current report, Ma et al studied an initial cohort of 13 patients with AOSD (7 with AOSD-MAS, 6 with AOSD alone), a second cohort of 10 patients with refractory AOSD-MAS, and a third cohort of 195 patients with AOSD, including 41 cases of AOSD-MAS. RNA sequencing of paired neutrophils and peripheral blood mononuclear cells from the 7 patients with AOSD-MAS in cohort 1 were consistent with a distinct role for neutrophils in AOSD-MAS; moreover, in a murine model of MAS, selective neutrophil depletion using an anti-Ly6G antibody led to significant reductions in cytokine levels similar to those seen following monocyte depletion with clodronate liposomes. Comparison of differential gene expression in neutrophils from cohort 1 patients with AOSD-MAS vs neutrophils from patients with AOSD without MAS by gene set enrichment analysis suggested specific upregulation of several biological pathways in MAS neutrophils including interferon-γ (IFN-γ) and IFN-α responses, as well as greater activation of the JAK-STAT signaling pathway. Increased expression of JAK1/JAK2 expression correlated with that of other genes associated with neutrophil activation.

The clinical significance of these studies is supported by the observation that treatment of 10 patients with refractory AOSD-MAS (cohort 2) with the JAK inhibitor ruxolitinib led to complete remission in 8 patients and partial remission in another. These responses were accompanied by resolution of clinical symptoms such as skin rash and sore throat and improvement in multiple measures of disease activity including C-reactive protein, erythrocyte sedimentation rate, and other inflammatory indicators such as soluble interleukin 2 receptor (sIL-2R), free IL-18 and neutrophil-derived cytokines CXCL1, CXCL9, CXCL11, CASP8 and IL-33. Reductions in the systemic score and HScore, used to measure MAS severity, were observed during the first month of ruxolitinib. Patients treated with ruxolitinib also demonstrated lower levels of circulating neutrophil extracellular trap (NET) markers including cell-free DNA, myeloperoxidase-DNA, and neutrophil elastase-DNA; similar reductions were observed in Padi4^−/− mice compared with control mice in a CpG-induced murine model of MAS.

Though several comparatively small studies have shown that ruxolitinib may be effective in some patients with MAS, there is no information available on the effects of ruxolitinib on neutrophil function in these patients. However, Ma et al demonstrate that ruxolitinib blocks phosphorylation of STAT3 and STAT5 in normal donor neutrophils exposed to sera from patients with AOSD-MAS. These investigators also performed whole exome sequencing of DNA from the 10 patients in cohort 2 treated with ruxolitinib to pursue potential HLH/MAS and/or JAK-STAT pathway variant gene sequences. These studies demonstrated 1 variant in NCKAPIL (associated with immunodeficiency and autoinflammation) and 12 additional variants in genes associated with JAK-STAT signaling including PDGFRB, PDGFRA, SOCS4, PIAS4, PDGFRA, LEPR, IL17R, IL27RA, IL12RBI, IL11RA, and CSF2RB (see figure). Expanding these studies to the third cohort of 193 patients, the investigators confirmed a higher prevalence of JAK-STAT variants in the patients with AOSD-MAS than those with AOSD alone, although these differences were not statistically significant. However, of the 13 patients with AOSD-MAS in this cohort treated with ruxolitinib, all 10 with JAK-STAT variants achieved a complete remission, and only 1 of 3 without JAK-STAT variants achieved a partial remission. Though these numbers are small and lack statistical significance, they suggest that additional studies may be warranted to determine the role of JAK-STAT pathway gene sequencing to identify patients with AOSD-MAS who are most likely to benefit from ruxolitinib. The rationale for such an approach is supported by the fact that when JAK-STAT pathway proteins containing the identified variants were expressed in 293T cells, activation of JAK-STAT signaling cascades, either spontaneously or to an excessive degree after exposure to their cognate ligands, was observed.

Taken together, observations from this study provide an important step forward in understanding the pathogenesis of AOSD-MAS by demonstrating a pathogenic role for neutrophil activation and NET release in animal models and humans. The identification and characterization of hyperactive JAK-STAT variants in patients responsive to ruxolitinib suggests the potential for application of precision medicine approaches for management of MAS, though testing this hypothesis will be challenging given the rarity of the disease. These studies also raise several questions. For example, several patients in this study had been previously treated and apparently did not respond to baricitinib, a JAK inhibitor with specificity for JAK 1/3. Does this suggest that JAK2, a primary target of ruxolitinib, may be the critical target in AOSD-MAS? If so, might other JAK2-focused inhibitors such as fedratinib or pacritinib be even more effective than ruxolitinib? In addition, though this study demonstrated a role for ruxolitinib in dampening neutrophil activation and NET release, its effects on other immune cells, including antigen-presenting cells and natural killer/T cells that also play an important role in AOSD-MAS as well as HLH, should be further studied. Finally, the role of JAK/STAT variants in other MAS-related disorders such as HLH requires further consideration. Nevertheless, we look forward to a time when these perplexing cytokine storm disorders are diagnosed through molecular approaches, leading to the advent of effective targeted therapies.

Conflict-of-interest disclosure: The authors declare no competing financial interests.