Proteomic Profiling and Cytokine Analysis Reveals Molecular Signatures and Inflammatory Factors Associated with Thrombosis in JAK2-V617F Positive Myeloproliferative Neoplasm (MPN) Cohorts

Background: Myeloproliferative neoplasms (MPNs), a hematological malignancy commonly associated with the JAK2-V617F mutation, exhibit a considerable burden of comorbidities resulting from inflammation-mediated processes. The JAK2-V617F malignant clones instigate systemic inflammation by upregulating various pro-inflammatory cytokines. This leads to dysregulated immune and inflammatory responses, thereby driving a process called thrombo-inflammation. Neutrophils play a crucial role as inflammatory mediators and effectors of pro-thrombotic activity in MPNs. We, therefore, hypothesize that perturbations in the neutrophil proteome may alter the molecular communication networks within neutrophils, thereby potentially contributing to the pathological thrombus formation in veins and arteries.

Aim: The study was aimed to elucidate (i) the pathological significance of dysregulated protein expression profile of neutrophils carrying the JAK2-V617F mutation, and (ii) the impact of pro-inflammatory cytokines on neutrophils during thrombo-inflammation.

Methods: In this study, which was conducted at the University Hospital Magdeburg, Germany, 11 patients carrying the JAK2-V617F mutation (6 with thrombosis; 5 without thrombosis) and 9 healthy volunteers were enrolled. Neutrophils were isolated from freshly collected whole blood using a negative selection kit. The isolated neutrophils were then lysed, and protein extraction was performed. High-resolution liquid chromatography - mass spectrometry (LC-MS/MS) was employed to investigate the protein inventory of neutrophils. Furthermore, platelet-poor plasma was isolated from the same cohort to analyse plasma cytokine/chemokine levels using a bead-based multiplex assay.

Results: LC-MS/MS identified 2,006 proteins in neutrophils, which were then used for quantitative comparative analysis. Amongst these, 28 and 26 proteins exhibited significant up-regulation in the JAK2-V617F positive patients ‘with thrombosis’ and ‘without thrombosis’, respectively. Notably, ATP2A3 and ROCK1 were significantly upregulated in the JAK2-V617F thrombotic group, ranking among the top 10-upregulated proteins. Of note, ROCK1 activation has previously been implicated in cell proliferation, survival and cell cycle control. Functional annotation analysis using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that upregulated proteins in the JAK2-V617F cohorts were significantly enriched for proteins involved in the calcium and TGF-ß signaling pathways.

Furthermore, we assessed the levels of 60 cytokines/chemokines and soluble adhesion molecules in the same cohort, wherein significantly increased levels of ALCAM, ICAM-2 and VCAM-1 were observed in the JAK2-V617F thrombotic group. These molecules are known to play a critical role in vasculature-related inflammation and endothelial cell activation. Additionally, the JAK2-V617F thrombotic group exhibited significant upregulation of free active TGF-ß and pro-inflammatory cytokines such as IL-6 and IL-8 compared to both healthy subjects and JAK2-V617F non-thrombotic patients, indicating their contribution to the thrombo-inflammatory response. Moreover, the thrombotic cohort displayed elevated plasma levels of CXC chemokines and myeloperoxidase (MPO), a key enzyme linked to NETosis for neutrophil extracellular trap (NET) formation.

Conclusions: The proteomic profile of neutrophils in MPN patients exhibit significant alterations, particularly between JAK2-V617F patients with and without thrombosis.

The observed elevated expression of markers related to NETosis and calcium signaling pathways point towards the underlying mechanisms that could potentially contribute to the differential behavior of neutrophils, thereby facilitating the development of a thrombotic state in MPN patients.