Deciphering Neutrophil Heterogeneity and Thrombosis-Related Alterations in JAK2V617F-Mutated Myeloproliferative Neoplasms through Multi-Omics and Single-Cell Transcriptome Analysis

Background:

Neutrophils, the most abundant leukocytes, are increasingly recognized as crucial pathogenic mediators linking pro-inflammatory signatures to pro-thrombotic risk in myeloproliferative neoplasms (MPNs). However, the underlying mechanisms linking these changes to metabolic reprogramming of neutrophils and thrombosis in JAK2V617F-mutated MPN patients remain unknown.

Methods:

We studied the clinical data of 621 treatment-naïve MPN patients and 35 healthy donors (HDs) in our hospital. Integrated analysis of scRNA-seq of neutrophils and plasma cell-free RNA sequencing were performed to explore the underlying mechanism. The GSE54644 dataset, contains 49 JAK2V617F-mutated essential thrombocythemia (ET)/polycythemia vera (PV) and 11 HDs were used to validate our findings. scRNA-seq in cells dissociated from atherosclerosis model in Jak2^VFLdlr^-/- mice was utilized to uncover changes in neutrophil profiles.

Results:

Our findings indicated that the JAK2V617F mutation results in the highest neutrophil counts and proportions in both ET (n=203, p<0.0001) and PV patients (n=264, p<0.0001), when compared with other driver mutations (CALR, MPL, triple-negative) and HDs. Correlation analysis was conducted to determine the relationship between neutrophil counts and the allelic burden of the JAK2V617F mutation, and it revealed a significant positive correlation in both ET (r=0.43, p=1.4e-08) and PV (r=0.53, p<2.2e-16) patients. Additionally, elevated neutrophils count was associated with worse thrombosis-free survival in JAK2V617F-mutated ET (p=0.0098) and PV patients (p=0.019).

Next, plasma cell-free RNA from 23 ET patients and 28 HDs was profiled, and cell-type-specific RNA fractions was deconvolved using the Cibersort algorithm. CD4 naïve T cells, neutrophils, and naïve B cells comprised the majority of observed signal. Moreover, we observed distinct proportions of cell types of origin between ET patients and HDs, with neutrophil proportions was significantly higher in ET patients than HDs (p<0.001). Regarding the proportion of neutrophils, it was highest in patients with JAK2V617F mutation compared with other driver gene mutations, and in patients with thrombosis compared with those without thrombosis.

Through scRNA-seq analysis of neutrophils, we provided a comprehensive characterization of the neutrophils landscape, including 61364 cells from JAK2V617F-mutated MPN patients and 32305 cells from HDs. Four distinct neutrophil subclusters (Neu-1, 2, 3, 4) were identified. Using monocle2 and cytoTRACE algorithms, we determined that the Neu-3 phenotype was the least mature, and was most represented in JAK2V617F-mutated MPNs when compared to HDs. Neu-3 exhibited the most significant roles in ROS response, inflammatory, glycolysis, and lipid metabolism. Flow cytometry confirmed that the mean fluorescence intensity (MFI) of neutrophil ROS was significantly increased in JAK2V617F-mutated MPN than HDs. Furthermore, pathways including JAK-STAT signaling, fluid shear stress atherosclerosis, neutrophil extracellular traps formation, and mitogen-activated protein kinase signaling were significantly upregulated in JAK2V617F-mutated MPN than HDs, especially in Neu-3 phenotype. These results were corroborated by neutrophils transcriptome from GSE54644 dataset. Neutrophils in an atherosclerosis model of Jak2^VFLdlr^-/- mice were also enriched in glycolysis, ferroptosis, lipid metabolism and atherosclerosis pathways when compared to the control.

Conclusions:

Our findings reveal the pivotal roles of neutrophils in metabolic reprogramming and thrombosis-related pathways in JAK2V617F-mutated MPN patients. Neutrophils also demonstrated significant clinical relevance for atherosclerosis in precursor disease models of MPN.