Effect of Integrin α 4β 1 -VCAM-1 Interactions in JAK2V617F Myeloproliferative Neoplasm Pro-Thrombotic Interactions

Abstract

Introduction: The JAK2 V617Fmutation, which occurs in up to 95% of polycythemia vera (PV) myeloproliferative neoplasm (MPN) patients, increases thrombosis risk 6-fold. A recent study in PV mice demonstrated that blocking JAK2 V617F+ leukocyte-mediated β1/β2 integrin interactions with anti-VCAM-1 antibodies decreases thrombosis. Interestingly, hydroxyurea (HU), a common PV treatment, reduces leukocyte and red cell α4β1 integrin expression. Overall, JAK2 V617F+ mouse models show inconsistent thrombosis and bleeding phenotype, hindering pre-clinical study reproducibility; therefore, we have developed an in vitro endothelialized microfluidic model to assess MPN-flow behavior. We hypothesized that in our microfluidic model, disrupting α4β1 integrin expression on JAK2 V617F+ leukocytes would reduce pro-adhesive interactions. We tested this hypothesis by assessing JAK2 V617F+ MPN blood flow in untreated and hydroxyurea treated subjects and through addition of a monoclonal antibody against α4β1 integrin, natalizumab. Methods: Primary human umbilical vein endothelial cells (HUVECs) were exposed to venous shear (15 dynes/cm 2) in microfluidics for a 4-day culture period. On day 4, endothelialized microfluidics were treated +/- 10 ng/mL TNFα for 4 hours. Blood samples were obtained from consented JAK2 V617F MPN individuals aged >18 years with ET, PV, or MF as diagnosed by the WHO 2016 Criteria identified in the M Health Fairview Masonic Cancer Center Clinics. Healthy age, race, and sex-matched blood samples served as controls. Clinical data on patients was collected and stored in REDCAP. Platelets and leukocytes were visualized using Calcein AM. Samples were treated +/- 0.25 μg/mL natalizumab for 30 minutes, then perfused through the microfluidic under a microscope to observe blood-endothelial interactions in real-time. Post-experiment, videos were analyzed to quantify velocity and adhesion of platelets and leukocytes for each treatment condition. Results: When perfused over TNF-α activated endothelium, both controls ( JAK2 negative, n=11) and JAK2 V617F MPN (n=20) had a significant reduction in cell velocity. Furthermore, compared to controls, JAK2 V617F MPN individuals exhibited significantly lower cell velocities. In multi-variate analysis, reduction in cell velocity was independent of white blood cell, hemoglobin, hematocrit or platelet counts. Hydroxyurea is a standard therapy in JAK2 V617F MPN and is known to reduce α4β1 expression. Therefore, we performed subgroup analysis in our JAK2 V617F MPN cohort based on treatment. When perfused over TNF-α activated endothelium, individuals with JAK2 V617F MPN (n=9) not on therapy (newly diagnosed) or on phlebotomy/aspirin only had a significant reduction in cell velocity. In the same conditions, HU-treated JAK2 V617F MPN patients (n=9) had no change in cell velocity. There was no difference in complete blood counts between these groups. In a separate subgroup analysis, compared to untreated JAK2 V617F MPN patients (n=9), interferon-treated JAK2 V617F MPN patients (n=5) had a greater reduction in cell velocity. Last, we specifically targeted α4β1 integrin by adding natalizumab to either control (n=7) or untreated JAK2 V617F MPN (n=3) samples. On inactivated endothelium, treatment of either control or JAK2 V617F MPN blood with natalizumab did not significantly change cell velocity. On a TNF-α activated endothelium, treatment of control (n=7) and JAK2 V617F MPN blood (n=3) with natalizumab led to a recovery in cell velocity. Conclusion: Our data suggests that the interaction between integrin α4β1 on JAK2 V617F cell surface and VCAM-1 on activated endothelium is important for initiation of pro-thrombotic interactions. JAK2 V617F MPN individuals treated with HU exhibit decreased adhesion to activated endothelium compared to control; JAK2 V617F MPN individuals treated with interferon exhibit increased adhesion. In the microfluidic system, based on current findings, blockage of integrin α4β1-VCAM-1 interactions using natalizumab is a potential therapeutic strategy. Further mechanistic studies evaluating interferon and cell populations changed by therapies as they pertain to integrin α4β1 are underway.