Mass Cytometry Analysis of Dysregulated Cytokine Production and Intracellular Signaling in Myelofibrosis

Abstract Myeloproliferative neoplasms including myelofibrosis (MF) are characterized by a malignant clone containing JAK2 V617F or other mutations leading to unregulated JAK2 kinase activity. MF is characterized by anemia, splenomegaly, bone marrow fibrosis, inflammatory cytokine production, and a propensity for transformation to secondary acute myeloid leukemia. Inhibition of JAK2 with ruxolitinib improves constitutional symptoms and splenomegaly and lowers circulating plasma cytokine levels. However, improvements in anemia, fibrosis, and malignant clonal burden are infrequent. These observations illustrate the need for improved therapy for MF. The objective of this study is to better understand the relationship between dysregulated cytokines and downstream signaling in MF, with the goal of determining how these pathways can be more effectively manipulated for therapeutic benefit. To interrogate altered signaling in MF, we have employed mass cytometry (CyTOF), a method which enables the quantitative analysis of signaling throughout hematopoiesis. In our survey of signaling in MF, hyperactivation of the NFκB signaling pathway was found to be widespread. This finding was corroborated by gene set enrichment analysis (GSEA) of a published gene expression dataset of CD34+ cells from MF versus normal controls (Norfo et al. 2014 Blood). Evidence for NFκB hyperactivation, both by mass cytometry and GSEA, was strongest in JAK2V617F-mutant MF patients. This supports a hypothesis that pronounced NFκB signaling hyperactivation is a consequence of mutant JAK2. In MF, hyperactivated NFκB signaling was widespread among hematopoietic cell populations, including T cells. This distribution suggests that NFκB activation may be partly driven by non-cell-autonomous mechanisms. Consistent with previous studies, plasma TNFα levels were found to be elevated in these patients, suggesting that excessive production of TNFα could result in downstream activation of NFκB across multiple cell populations in a non-cell-autonomous fashion. To further elucidate the etiology of systemic NFκB hyperactivation and understand the interplay of inflammatory cytokines and downstream signaling, we extended our mass cytometry approach to study the cellular distribution of cytokine production in MF. A panel of 22 surface marker antibodies and 12 cytokine antibodies was developed for these experiments. Examination of peripheral blood from two JAK2V617F-mutant MF patients revealed that intracellular levels of several cytokines were constitutively elevated in both MF patients compared to healthy controls. Monocytes produced the highest levels of TNFα among hematopoietic populations, and these were higher in MF versus control monocytes. Supranormal cytokine expression was accentuated by stimulation with PMA/ionomycin or TLR ligands R848 or PAM3CSK4. Incubation with TNFα led to supranormal levels of the cytokines MIP1β and IL-6, in monocytes from one or both patients. Therefore, abnormal production of TNFα by MF patient monocytes could result in overproduction of IL-6, and MIP1β in the same cells. PMA/ionomycin led to above normal production of TNFα from MF Lin-CD34+ cells and CD33+CD34- immature myeloid cells, suggestive that these cells could be hypersensitive to pathophysiologic signaling stimulations in a manner resulting in elevated cytokine production. MF patient T cells also showed hypersensitivity to PMA/ionomycin stimulation, compared with controls, in their production of IFNγ and MIP1β. These cytokines, along with the MF monocyte-overexpressed cytokines TNFα, IL-6, and MIP1β, were found to be elevated in MF patient plasma, consistent with prior studies. These findings imply that multiple cell populations in JAK2 V617F-mutant MF patients overexpress inflammatory cytokines and are hypersensitive to inflammatory insults. The upregulation of cytokines is likely to underlie the systemic hyperactivation of NFκB signaling observed in MF, and could generate non-cell-autonomous effects on the malignant myeloid clone. While NFκB phosphorylation responses to TNFα appear strongest in Lin-CD34+ cells, other cytokines may mediate signaling abnormalities across a variety of cell types. Future experiments will attempt to identify signaling effects of multiple elevated cytokines, which may underlie features of MF that persist despite JAK2 inhibitor therapy. Disclosures Oh: CTI: Research Funding; Janssen: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding.