Effect of Integrin α ₄β ₁ -VCAM-1 Interactions in JAK2^V617F Myeloproliferative Neoplasm Pro-Thrombotic Interactions

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.