P008 | JAK2E837K: a novel mutation post-polycythemia vera myelofibrosis

Abstract

Mutations in the non-receptor tyrosine kinase JAK2 play a central role in myeloproliferative disorders. JAK2 associates with various cytokine receptors and activates signaling pathways involved in cellular proliferation and survival, including the STAT and PI3K-AKT pathways. Activating mutations, beyond the well-known JAK2V617F, lead to constitutive pathway activation contributing to the myeloproliferative phenotype. This study aims to characterize a novel JAK2E837K mutation discovered in a patient with post polycythemia vera (PV) myelofibrosis (PPV-MF) and to investigate its potential pathogenic role.
The JAK2E837K mutation was identified by Next-Generation Sequencing (NGS) performed on the NextSeq 550® platform using the Archer VariantPlex® panel. A colony-forming unit (CFU) assay was performed on peripheral blood mononuclear cells (PBMCs) from the JAK2E837K PPV-MF patient, 4 healthy donors (HDs) and 8 JAK2V617F PV patients, cultured with increasing concentrations of erythropoietin (EPO). STAT3/5 and AKT activation was assessed by Western blot. Mitochondrial function and glycolytic activity were analyzed using a Seahorse Bioscience XFe96 analyzer.
According to the NCBI Genome Data Viewer and UniProt, the JAK2E837K mutation is located in exon 19, within the interdomain linker connecting the pseudokinase (JH2) and tyrosine kinase (JH1) domains. This linker determines the spatial arrangement between these domains, with JH2 exerting an inhibitory effect on JH1. Phylogenetic analysis showed that residue E837 lies within a highly conserved region across species, suggesting its functional importance (Figure 1A-B). Colony-forming assays revealed that, compared with HDs, the JAK2E837K PPV-MF patient exhibits hypersensitivity to EPO, similar to JAK2V617F PV patients (Figure 1C). Western blot analysis showed increased STAT5 and AKT phosphorylation in colonies from the JAK2E837K PPV-MF patient compared with an HD, whereas no increase in STAT3 phosphorylation was detected (Figure 1D). Finally, the Seahorse analyzer showed increased mitochondrial respiration and glycolytic activity in the JAK2E837K PPV-MF patient compared with the same patient undergoing JAK2 inhibitor treatment and HDs (Figure 1E-F).
In conclusion, we report a novel noncanonical JAK2 mutation in a PPV-MF patient lacking other known driver mutations. JAK2E837K affects a conserved residue within the interdomain linker between JH2 and JH1, likely altering their regulatory interaction. Functional studies demonstrate that the mutation causes constitutive JAK2 activation with increased STAT5 and AKT phosphorylation suggesting a selective activation of the JAK/STAT and PI3K-AKT pathways. Metabolic analyses reveal that JAK2E837K enhances mitochondrial respiration and glycolysis, similar to the canonical JAK2V617F mutation. Overall, these results support the pathogenic role of the JAK2E837K mutation and the contribution of noncanonical JAK2 variants to the pathogenesis of myeloproliferative neoplasms.