The JAK Inhibitor Ruxolitinib Substantially Affects NK Cells in MPN Patients
Authors: Schönberg Kathrin , Rudolph Janna , Vonnahme Maria , Cornez Isabelle , Parampalli Yajnanarayana Sowmya , Verbeek Walter , Koschmieder Steffen , Brümmendorf Tim H. , Brossart Peter , Heine Annkristin , Wolf Dominik 
Published: April 2020

Abstract
Introduction: Ruxolitinib (INCB018424) is the first JAK inhibitor approved for treatment of myelofibrosis (MF). Ruxolitinib-induced reduction of splenomegaly and symptoms control is linked to a substantial suppression of MF-associated circulating pro-inflammatory and pro-angiogenic cytokines. However, an increased rate of infections in ruxolitinib-exposed patients with MF was recently described. Natural killer (NK) cells are innate immune effector cells eliminating malignant or virus-infected cells. Thus, the aim of this project was to define in more detail the impact of JAK inhibition on NK cell biology both in vitro and in vivo.
Methods: 28 patients with myeloproliferative neoplasms (MPN) with or without ruxolitinib therapy and 12 healthy donors were analyzed for NK cell frequency, NK receptor expression and function. Phenotypic and functional NK cell markers (e.g. CD11b, CD27, KIR, NKG2A, NKG2D, NKp46, CD16, granzyme B, and perforin) were analyzed by FACS. NK cell function was evaluated by classical killing assays upon stimulation with MHC class I-deficient target cells K562. Finally, a set of additional in vitro experiments (e.g. analysis of lytic synapse formation by FACS and confocal microscopy) were performed to define in more detail the characteristics and potential mechanisms of ruxolitinib-induced NK cell dysfunction.
Results: In addition to our recent finding that ruxolitinib induces NK cell dysfunction in vitro (e.g. reduced killing, degranulation and IFN-γ production), we here demonstrate that NK cell proliferation and cytokine-induced receptor expression as well as cytokine signalling are drastically impaired by ruxolitinib. Interestingly, reduced killing is at least in part due to a reduced capacity to form a mature lytic synapse with target cells. The significance of the in vitrofindings is underscored by a dramatically reduced proportion and absolute number of NK cells in ruxolitinib-treated MPN patients when compared to treatment-naïve patients or to healthy controls (mean percentage of NK cell frequency: ruxolitinib-naïve MPN patients 12.63% ±1.81; healthy donors 13.51% ±1.44; ruxolitinib-treated patients 5.47% ±1.27). A systematic analysis of NK cell receptor expression revealed that the reduction of NK cells in ruxolitinib-exposed individuals is most likely due to an impaired NK cell differentiation and maturation process, as reflected by a significantly increased ratio of immature to mature NK cells. Finally, the endogenous functional NK cell defect in MPN is further aggravated by intake of the JAK inhibitor ruxolitinib.
Conclusion: We here provide compelling in vitro and in vivo evidence that inhibition of the JAK/STAT-pathway by ruxolitinib exerts substantial effects on the NK cell compartment in MPN patients due to the inhibition of NK cell differentiation and NK cell key functions. Our data may help to better understand the increased rate of severe infections and complement recent reports on ruxolitinib-induced immune dysfunction.

Disclosures
Koschmieder: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel, Accomodation, Expenses Other. Brümmendorf:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Wolf:Novartis: Consultancy, Honoraria, Research Funding, Travel and Accommodation Other.