Transcriptional differences between JAK2-V617F and wild-type bone marrow cells in patients with myeloproliferative neoplasms

Abstract

The JAK2-V617F mutation is the most common cause of myeloproliferative neoplasms. Although experiments have revealed that this gain-of-function mutation is associated with myeloid blood cell expansion and increased production of white cells, red cells, and platelets, the transcriptional consequences of the JAK2-V617F mutation in different cellular compartments of the bone marrow have not yet been fully elucidated. To study the direct effects of JAK2-V617F on bone marrow cells in patients with myeloproliferative neoplasms, we performed joint single-cell RNA sequencing and JAK2 genotyping on CD34⁺-enriched cells from eight patients with newly diagnosed essential thrombocythemia or polycythemia vera. We found that the JAK2-V617F mutation increases the expression of interferon-response genes (e.g., HLAs) and the leptin receptor in hematopoietic progenitor cells. Furthermore, we sequenced a population of CD34^- bone marrow monocytes and found that the JAK2 mutation increased expression of intermediate monocyte genes and the fibrocyte-associated surface protein SLAMF7 in these cells.

Figure 1.. JAK2-V617F megakaryocyte and erythroid progenitors have higher expression of pro-inflammatory and antigen presentation genes.

A. UMAP of scRNA-seq data from bone marrow from 8 MPN patients, colored by cell type classifications. B. JAK2-WT (blue) and JAK2-mutant (red) transcripts detected in single cells in MPN patient bone marrow. C. Smoothed JAK2-V617F transcript fraction for all patients combined. D. Detection of mutations associated with JAK2-V617F in patient ET 1. Additional mutations were called using targeted amplification of loci identified from WGS (e.g., UPF1) and by directly identifying somatic mutations in the scRNA-seq data. E-F. Volcano plots showing differential expression analysis results from comparing cells with mutant transcripts to cells with WT transcripts within the MEP, erythroid progenitor, and CD14+ compartments for ET patients (E) and PV patients (F). Ribosomal genes, antigen presentation genes, and proteasomal genes are colored in blue, green, and red, respectively.

Figure 2.. JAK2-V617F induces SLAMF7 expression and an intermediate monocyte phenotype in MPN patients.

A. Marker gene expression UMAPs of the bone marrow monocyte compartment measured by scRNA-seq in 8 MPN patients. B. JAK2-WT (blue) and JAK2-V617F (red) transcripts detected in monocytes. C. UMAP of scRNA-seq data from monocytes colored by transcriptionally defined monocyte subset classifications. D. Fraction of CD14+ cells with a JAK2-WT or JAK2-mutant transcript detected by scRNA-seq that are classical, intermediate, or nonclassical monocytes. The monocyte subset definitions and color scheme are the same as in C. E. Differential expression analysis of monocytes from all 8 MPN patients, comparing cells with at least one mutant transcript detected to cells with a WT transcript detected. Ribosomal genes, antigen presentation genes, and proteasomal genes are colored in blue, green, and red, respectively. F-G. Flow cytometry and SLAMF7 staining of CD14+ cells from three MPN patients and three healthy controls. Gating scheme to identify SLAMF7 is shown in F, and the proportion of CD14+ cells expressing SLAMF7 is shown in G.