Expression of JAK2-V617F Kinase Profoundly Dysregulates the Functional Properties of β1 Integrins on Myeloid Cells

Introduction: Deregulated signal transduction pathways driven by the constitutively active JAK2-V617F kinase, play a central role in pathogenesis of the classical Philadelphia negative myeloproliferative neoplasms (MPNs). Polycythemia vera, essential thrombocythemia and primary myelofibrosis are characterized by clonal proliferation of myeloid cells and development of inflammatory syndrome (CRP elevation, fever, high proinflammatory cytokine plasma levels) particularly, in advanced phases of the disease. Integrins are heterodimeric cell surface receptors that mediate a wide range of anchorage dependent events fundamentally essential in adhesion and mobility of leukocytes during inflammatory states. An intricate network of intracellular signaling pathways triggers the activation of intergins by allosteric conformational changes and/or by altering lateral mobility to increase receptor clustering.

Methods: We investigated the role of JAK2-V617F in the regulation of β1 integrin activation, function and expression in myeloid cells using in vitro cell culture models, in vivo JAK2-V617F knock-in model, murine bone marrow transplantation model and finally, clinical patient samples (collected after informed consent obtained in accordance with the Declaration of Helsinki). Integrin activation was studied using flow cytomery based soluble ligand binding assay and by analyzing the expression of 9EG7 activation epitope. The functional static adhesion assay was performed on ligand coated plates and data was acquired using a fluorescence microplate reader. Surface expression of integrins was investigated by flow cytometry.

Results: To identify a potential biological impact of JAK2-V617F on β1 integrins, we generated a 32D murine myeloid progenitor cell line transfected with EpoR and either wildtype JAK2 or JAK2-V617F. Under serum starved conditions, the presence of the oncogenic JAK2-V617F mutation strongly induced activation of β1 integrins as revealed by a 6-fold rise in binding to its soluble ligand, VCAM-1-Fc and enhanced exposure of the 9EG7 high-affinity conformation-specific epitope. This also translated into increased adhesion on VCAM-1 in comparison to wildtype cells. Importantly, inhibition of the constitutive JAK2-V617F kinase activity by JAK inhibitor treatment, significantly downregulated β1 integrin activation as well as cellular adhesion. Furthermore, shRNA based knockdown of the endogenous JAK2-V617F expression in HEL (Human Erythroleukemia) cells, reduced adhesion on VCAM-1, thereby underscoring the functional role of the JAK2-V617F kinase in the regulation of β1 integrins. Interestingly, the cell surface expression of β1 integrins remains unaltered in the presence of JAK2-V617F or inhibitor treatment in the 32D cells as well as, following genetic inhibition in HEL cells. Additionally, in an in vivo retroviral bone marrow transplantation model in Balb/c mice, the overexpression of wildtype or JAK2-V617F, showed similar β1 integrin expression in granulocytes. Currently, in vivo studies in a JAK2-V617F knock-in murine model are under way. We then extended our study to investigate the role of constitutively active JAK2-V617F in integrin function in MPN patients. Again, no significant difference was found in β1 integrin expression on primary granulocytes from JAK2-V617F positive MPN patients and age-matched healthy donors. However, granulocytes from JAK2-V617F-positive patients showed higher activation of β1 integrins as exhibited by a 6-fold increase in binding to soluble VCAM-1-Fc when compared to healthy donors. In addition, preliminary analysis suggests stronger adhesion of JAK2-V617F positive granulocytes on VCAM-1. In an attempt to dissect the underlying molecular mechanism, we uncovered a previously undescribed role of JAK2 in the inside-out signaling of integrin activation in 32D myeloid cells. Overexpression of wildype JAK2 or V617F resulted in activation of the small GTPase Rap1, a key integrin regulator, and was subsequently downregulated upon treatment with JAK inhibitor.

Conclusions: Taken together, our study highlights a unique and important role of JAK2-V617F in regulation of β1 integrin activation in myeloid cells. This may have functional consequences in pathophysiology of the disease, particularly, in dysregulation of leukocyte adhesion during thrombosis and in induction of inflammation.