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Chronic myeloproliferative neoplasms

Cytokine production in myelofibrosis exhibits differential responsiveness to JAK-STAT, MAP kinase, and NFκB signaling

Abstract

The distinct clinical features of myelofibrosis (MF) have been attributed in part to dysregulated inflammatory cytokine production. Circulating cytokine levels are elevated in MF patients; a subset of which have been shown to be poor prognostic indicators. In this study, cytokine overproduction was examined in MF patient plasma and in MF blood cells ex vivo using mass cytometry. Plasma cytokines measured following treatment with ruxolitinib remained markedly abnormal, indicating that aberrant cytokine production persists despite therapeutic JAK2 inhibition. In MF patient samples, 14/15 cytokines measured by mass cytometry were found to be constitutively overproduced, with the principal cellular source for most cytokines being monocytes, implicating a non-cell-autonomous role for monocyte-derived cytokines impacting disease-propagating stem/progenitor cells in MF. The majority of cytokines elevated in MF exhibited ex vivo hypersensitivity to thrombopoietin (TPO), toll-like receptor (TLR) ligands, and/or tumor necrosis factor (TNF). A subset of this group (including TNF, IL-6, IL-8, IL-10) was minimally sensitive to ruxolitinib. All TPO/TLR/TNF-sensitive cytokines, however, were sensitive to pharmacologic inhibition of NFκB and/or MAP kinase signaling. These results indicate that NFκB and MAP kinase signaling maintain cytokine overproduction in MF, and that inhibition of these pathways may provide optimal control of inflammatory pathophysiology in MF.

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Acknowledgements

This work was supported by NIH grants K08HL106576 (Oh), R01HL134952 (Oh), and T32HL007088 (Engle, Fisher). This research was also supported by an American Cancer Society Postdoctoral Fellowship (Fisher). This work was also supported by a Doris Duke-Damon Runyon Clinical Investigator Award (Oh) and a Challenge Grant from the MPN Research Foundation (Oh). Technical support was provided by the Alvin J Siteman Cancer Center Tissue Procurement Core Facility, Flow Cytometry Core, and Immunomonitoring Laboratory, which are supported by NCI Cancer Center Support Grant P30CA91842. The Immunomonitoring Laboratory is also supported by the Andrew M and Jane M Bursky Center for Human Immunology and Immunotherapy Programs. We thank K. Luber for assistance with patient samples, C. Holley and the staff of the Alvin J Siteman Cancer Center Flow Cytometry Core for assistance with cell sorting, D. Bender and C. Wilson for assistance with plasma cytokine assays, R. Lin and R. Betz for assistance with mass cytometry experiments, and M. Fulbright and E. De Togni for providing results of pharmacologic assays not presented here, relevant for determining doses of inhibitors for cell treatments used. We also thank M. Fulbright for laboratory management.

Author contributions

The study was designed by DACF and STO, with contribution from CAM and EKE. DACF and TBC performed intracellular signaling mass cytometry experiments (Fig. 1a, b), with technical support from OM. EKE performed plasma cytokine measurement, and its statistical analysis was done by EKE and DACF (Fig. 1c–f). CAM, EKE, and DACF performed intracellular cytokine mass cytometry experiments (Figs 27), with technical support from OM. Statistical analysis of these experiments was done by HH, CAM, and DACF. GSEA analysis (Fig. 3c, d) was done by EKE. Cell line experiment on signaling effects of pevonedistat (Fig. 5a, b) was done by TBC. Clinical information on study patients was compiled by DACF and STO, with assistance from AZ and MA. All data presented has been examined by DACF and STO. Manuscript was written by DACF and STO, and all authors provided final approval of the submitted manuscript.

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Fisher, D.A.C., Miner, C.A., Engle, E.K. et al. Cytokine production in myelofibrosis exhibits differential responsiveness to JAK-STAT, MAP kinase, and NFκB signaling. Leukemia 33, 1978–1995 (2019). https://doi.org/10.1038/s41375-019-0379-y

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