JAK2 E837K. FIRST CHARACTERIZATION OF A NOVEL JH2-JH1 INTERDOMAIN LINKER MUTATION DRIVING STAT ACTIVATION AND METABOLIC REPROGRAMMING IN POST-POLYCYTHEMIA VERA MYELOFIBROSIS

Background: JAK2 mutations are key drivers of myeloproliferative neoplasms (MPNs), inducing constitutive kinase activation by disrupting JH2-JH1 autoinhibition or stabilizing active dimers, leading to aberrant JAK2-STAT signaling (Glassman et al., 2022; Abraham et al., 2024). Aberrant JAK2-STAT activation is also associated with metabolic reprogramming in MPNs, including coordinated increases in glycolysis and oxidative phosphorylation (Rao et al., 2019; Ma et al., 2025; Sharma et al., 2020). To date, activating mutations have been reported exclusively within functional domains and regulatory interfaces of the protein. Here, we describe E837K, the first pathogenic mutation identified within the JH2-JH1 interdomain linker. Aims: We aimed to characterize the structural, functional, and metabolic consequences of the noncanonical JAK2 E837K mutation identified in post-polycythemia vera myelofibrosis (PPV-MF), localized in the JH2-JH1 linker, to define its pathogenic role. Methods: The JAK2 E837K mutation was identified by NGS performed on the NextSeq 550 platform using the Archer VariantPlex panel. Wild-type and E837K JAK2 structures were modeled using AlphaFold2 via ColabFold-AF2. A colony-forming unit (CFU) assay was performed on peripheral blood mononuclear cells (PBMCs) from the JAK2 E837K PPV-MF patient, 4 healthy donors (HDs) and 8 JAK2 V617F PV patients, cultured with increasing erythropoietin (EPO) concentrations. STAT3/5 and AKT activation were assessed by Western blot. Mitochondrial respiration and glycolytic activity were analyzed using a Seahorse XFe96 analyzer. Results: According to NCBI Genome Data Viewer and UniProt, the E837K mutation lies in exon 19 within the JH2-JH1 interdomain linker, a highly conserved region across species (Fig. 1A-B). Using AlphaFold2, five high-confidence structural models were generated for wild-type and E837K JAK2. Wild-type JAK2 samples both the closed (autoinhibited) and elongated (active) conformations, with preference for the closed state, whereas E837K adopts exclusively the elongated conformation (Fig. 1C-D). The mutation disrupts a salt-bridge network in the JH2-JH1 linker (E837-R839-E845-R847), introducing electrostatic repulsion between K837 and R839 and causing rotation of the K837 side chain. This rearrangement promotes further repulsive interactions with basic residues in the adjacent FERM domain, stabilizing JAK2 in the open conformation, promoting dimerization and constitutive activation (Fig. 1E-F). Functionally, this structural shift is reflected in enhanced cellular responsiveness as indicated by the pronounced hypersensitivity to EPO of JAK2 E837K-derived colonies, comparable to that of JAK2 V617F-derived colonies (Fig. 1G). Consistent with this, JAK2 E837K colonies showed increased STAT5 and AKT phosphorylation, whereas STAT3 activation remained unchanged (1H). Moreover, metabolic profiling revealed that JAK2 E837K-derived colonies possess elevated mitochondrial respiration and glycolytic activity compared to healthy donors (Fig. 1I-L), linking the conformational change directly to both signaling and metabolic rewiring. In conclusion, we provide the first evidence that a mutation located within the JH2-JH1 interdomain linker can act as a driver mutation, as demonstrated by E837K in a PPV-MF patient lacking other known mutations. The E837K mutation locks the protein in the open conformation, inducing EPO hypersensitivity, constitutive STAT5 and AKT activation and metabolic reprogramming, supporting its pathogenic role.