The JAK Inhibitor Ruxolitinib Impairs Dendritic Cell Migration Via Off-Target Inhibition of Rock

Introduction: Ruxolitinib is a JAK inhibitor that was recently approved for treatment of primary and secondary MF and shows impressive symptom control by suppression of inflammation. The observed effects are irrespective of the JAK mutational status. Ruxolitinib is also a promising drug for treatment of acute and chronic GvHD (Spoerl et al., Blood 2014 and Zeiser et al., Leukemia, in press). We recently demonstrated that the immune-modulatory effects of ruxolitinib might at least in part be due to an inhibitory effect on dendritic cell (DC) biology (Heine et al., Blood 2014). Dendritic cells are important antigen-presenting cells, which mature and migrate from the periphery into draining lymph nodes upon antigen contact to prime T cells. This process follows chemokine gradients and requires cytoskeletal rearrangement to allow cell movement along the gradients. Members of the family of small GTPases are critically involved in cell chemotaxis, migration and formation of the immunological synapse allowing DC-T cell interaction. The aim of this study was to define in detail the impact of the JAK inhibitor ruxolitinib on DC migration.

Methods: CD14⁺ cells isolated from human buffy coats were differentiated for seven days in the presence of GM-CSF and IL-4 to moDCs and finally matured with LPS. Murine bone marrow-derived DCs (bmDCs) were generated by flushing bone marrow from femura and tibiae of mice and subsequent plating of the cells in medium containing GM-CSF followed by final maturation by LPS. Migration of DCs was analyzed in Transwell assays or dynamically by time-lapse microscopy within three dimensional collagen gels towards CCL19 gradients. Adhesion of cells was tested on different substrates and phenotypic DC marker checked by flow cytometry. Signaling events were analyzed by Western Blot to evaluate changes in phosphorylation levels of relevant proteins of the RhoA/ROCK-pathway.

Results: 2D-migration of ruxolitinib-exposed DC on fibronectin is dose-dependently reduced in vitro. By analyzing the migratory phenotype of human moDCs within 3D-collagen gels, ruxolitinib-exposed DCs are still able to sense chemokine gradients as they form lamellipodia at the leading edge of the cell, whereas the retraction of the uropod is clearly inhibited. As a consequence, cell velocity, accumulated and euclidean distance are all significantly reduced by ruxolitinib. These effects are already seen 30 minutes after ruxolitinib exposure. Notably (and in line with the morphological chemokine-sensing by lamellipodia formation), the surface expression of the CCL19-sensing chemokine receptor CCR7 is not altered by ruxolitinib. Moreover, we could not detect any changes of integrin expression or the adhesion of mature DC to fibronectin or collagen as a potential reason for lowered migration due to increased "stickiness" of the DCs. Additional in vivo studies could show that the JAK inhibitor potently reduces homing of bmDCs into draining lymph nodes in mice. Notably, the impaired DC migration is independent of JAK inhibition, as siRNA knockdown experiments revealed that DCs with JAK1, JAK2 or JAK1/2 knockdown migrate appropriately in vitro applying again the 3D-migration system. On a molecular level we could show a reduced phosphorylation of myosin-light chain phosphatase 1 (MYPT1), a direct target of the Rho-associated protein kinase (ROCK) in ruxolitinib-treated moDC upon CCL19 stimulation. Interestingly, the known inhibition of DC activation and maturation seen after treatment with ruxolitinib could not be observed if cells are incubated with the ROCK inhibitor Y-27632. Whereas pharmacological inhibition of ROCK mimicked the migration phenotype seen in moDC exposed to ruxolitinib.

Conclusion: RhoA/ROCK pathway is critical for cell migration, as ROCK is a downstream effector of RhoA and leads to stabilization of the actin cytoskeleton via cofilin and acto-myosin II contraction by the myosin light chain. The observed reduction of ROCK activity may reveal an important novel mechanism of ruxolitinib-induced inhibition of DC migration. Our current efforts focus on the validation of ROCK as off-target JAK1/2-independent target kinase of ruxolitinib as potential mediator of the observed DC inhibitory effects, which may at least in part also explain the potential therapeutic impact of ruxolitinib for treatment of GvHD or autoimmunity.