Thrombosis in Myeloproliferative Neoplasms Is Linked to Increased Neutrophil Extracellular Trap (NET) Formation
Authors: Wolach Ofir , Sellar Rob S , Martinod Kimberly , McConkey Marie E. , Silver Alexander J. , Chappell Ryan , Stone Richard M. , Wadleigh Martha , Steensma David P , DeAngelo Daniel J. , Galinsky Ilene , Schneider Rebekka K. , Padera Robert F. , Mullally Ann , Wagner Denisa D. , Ebert Benjamin L. 
Published: November 2019

Abstract

Background: Philadelphia-negative myeloproliferative neoplasms (MPNs) are a group of clonal stem-cell disorders that are associated with an elevated risk for thrombosis. The reason for this association is incompletely understood. While elevated white blood cell (WBC) count is a risk factor for thrombosis in some models, the pathogenic pathways linking WBC abnormalities to thrombosis in MPNs are not clear.
Neutrophil extracellular traps (NETs) are networks of extracellular neutrophil DNA, histones and serine proteases that contain and kill pathogens. There is a growing body of evidence linking NETs to thrombosis in various disease states, including cancer (Demers et al, PNAS 2012). We explored the contribution of NETs to the pro-thrombotic phenotype of MPNs.
Methods: NET formation was assessed in neutrophils isolated from patients with MPNs and age-matched controls by using previously published immunofluorescence (IF) methods. c-KIT enriched marrow cells from Vav-Cre/Jak2V617F Knock-In (KI) mice were grafted into lethally irradiated wild-type (WT) recipients leading to constitutive heterozygous expression of the mutation in hematopoietic cells. Inferior vena cava (IVC) stenosis model was used to assess thrombotic tendency in mice. Mice were treated with ruxolitinib by oral gavage at a dose of 90 mg/kg BID for 3 days prior to thrombosis and NETosis experiments. NET formation was stimulated by ionomycin (4 µM).
Results: Neutrophils from patients with MPNs (n=14) demonstrated significantly higher NET formation as compared to those from age-matched controls (n=10; p=0.003). This was true across specific MPN diagnoses and driver mutations (JAK2, CALR, MPL). Ex-vivo treatment of neutrophils with ruxolitinib (at 300nM for 2.5 hours) resulted in abrogation of NETosis as assessed by IF studies and was comparable to the effect achieved with the addition of compounds known to inhibit NET formation such as specific PAD4 and NADPH inhibitors; no difference was observed in Annexin V-mediated apoptosis between ruxolitinib treated and untreated neutrophils suggesting that ruxolitinib effect was not confounded by alternative apoptosis pathways.
We next evaluated NETosis and its effect on thrombosis in MPN mouse models driven by Jak2 V617F. Neutrophils isolated from Vav-Cre/Jak2V617F demonstrated increased NET production ex-vivo as compared to WT littermate controls. Vav-Cre/Jak2V617F KI mice were grafted into lethally irradiated WT and assessed for their prothrombotic phenotype. VavCre/Jak2V617F KI demonstrated significantly higher thrombus formation at 2 hours using the IVC stenosis model (45% versus 0% for Jak2V617F KI (n=11) and Jak2WT (n=8), respectively; p=0.04 by Fisher's exact test). This was associated with significantly higher levels of free dsDNA in the plasma of KI stenosis mice as compared to WT stenosis controls (p=0.03 by Mann-Whitney). Furthermore, IHC and IF studies of lung tissue demonstrated that Vav-Cre/Jak2V617F KI have widespread thrombosis as compared to controls and IF studies demonstrated higher content of citrullinated histone H3 in the lung suggesting increased NETosis at that site. Treatment of Vav-Cre/Jak2V617F KI mice with ruxolitinib significantly abrogated thrombus formation (45% versus 0% for untreated (n=11) and treated (n=8) mice, respectively; p=0.04 by Fisher's exact test).
We noted that expression of PAD4, an essential driver of NETosis, is elevated in MPN patients (Rampal Blood 2014). We found that PAD4 content was higher in protein lysates of neutrophils isolated from MPN patients as compared to controls. Moreover, abnormal nuclear localization was observed in patients. These observations may support a role for PAD4 in the excess NETosis observed in MPNs.
Conclusion: We found that neutrophils from MPN patients and Jak2 V617F KI mice are sensitized to NET formation that may be mediated by increased PAD4 expression and nuclear localization. In mice, the increased ex-vivo NET formation is associated with an increased thrombosis rate and an abundance of NET related products in thrombus and tissue. Treatment with ruxolitinib abrogates NET formation in humans and significantly reduces thrombus formation in MPN mice models.
These findings provide a pathogenic link between the genetic drivers of MPNs and thrombosis in these diseases. Pharmacologic inhibition of JAK-STAT pathway to reduce NETosis and thrombosis should be further investigated.

Disclosures
DeAngelo: Novartis: Consultancy; Ariad: Consultancy; Celgene: Consultancy; Baxter: Consultancy; Amgen: Consultancy; Incyte: Consultancy; Pfizer: Consultancy.