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Risk-stratification of acute myeloid leukemia (AML) based on (cyto)genetic aberrations, including hotspot mutations, deletions and point mutations have evolved substantially in recent years. With the development of next-generation sequence technology, more and more novel mutations in the AML were identified. Thus, to unravel roles and mechanism of novel mutations would improve prognostic and predictive abilities. In this study, two novel germline JAK2 His608Tyr (H608Y) and His608Asn (H608N) mutations were identified and the molecular basis of these mutations in the leukemiagenesis of AML was elucidated. Our results indicated that JAK2 H608Y and H608N mutations disrupted the hydrogen bond between Q656 and H608 which reduced the JH2 domain's activity and abolished interactions between JH1 and JH2 domains, forced JAK2 into the active conformation, facilitated the entrance of substrates and thus caused JAK2 hyperactivation. Further studies suggested that JAK2 H608Y and H608N mutations enhanced the cell proliferation and inhibited the differentiation of Ba/F3 and MV4-11 cells via activating the JAK2-STAT5 signaling pathway. Moreover, rescue experiments demonstrated that mutations repaired the hydrogen bond between Q656 and H608 displayed opposite results. Thus, this study revealed the molecular basis of JAK2 H608Y and H608N mutations in the pathology of AML., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Activating mutations in JAK2 have been described in patients with various hematologic malignancies including acute myeloid leukemia (AML) and myeloproliferative neoplasms. However, mechanism of these mutations in JAK2's activity, structural stability and pathology of AML remains poorly understood. The JAK2 T875N somatic mutation has been detected in about 5.2% of AML patients. But the structural basis and mechanism of JAK2 T875N mutation in the pathology of AML is still unclear. Our results suggested that JAK2 T875N mutation disrupted the T875 and D873 interaction which destroyed the compact structure of JH1 domain, forced it into the active conformation, facilitated the entrance of substrate and thus led to JAK2 hyperactivation. Mutations (T875N, T875A, D873A and D873G) disrupted the T875 and D873 interaction enhanced JAK2's activity, decreased its structural stability and JH2 domain's activity which further enhanced JAK2's activity, while mutations (T875R, D873E, T875R/D873E) repaired this interaction displayed opposite results. Moreover, JAK2 T875N mutation enhanced the activity of JAK2-STAT5 pathway, promoted the proliferation and transformation of OCI-AML3 cells. This study provides clues in understanding structural basis of T875N mutation caused JAK2 hyperactivation and its roles in the pathology of AML., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Janus tyrosine kinase 2 (JAK2) mediates downstream signaling of cytokine receptors in all hematological lineages, constitutively active somatic JAK2 mutations were important for the leukemogenesis of acute leukemia (AL). The JAK2 R867Q somatic mutation is detected in a subset of AL patients. However, roles of JAK2 R867Q mutation in the pathogenesis of AL remain unclear. In this study, homology modeling analysis showed that loss of interaction between R867 and Y613 disrupted the JAK2 JH1/JH2 domain's interactions was responsible for its activation. JAK2 R867Q and mutations (R867A and R867G) abolished this interaction caused JAK2 constitutive activation. While, mutations (R867K, Y613E, R867K/Y613E) repairing this interaction reduced JAK2 R867Q mutation's activity. Furthermore, our studies showed that abolished R867 and Y613 interaction disrupted JH1/JH2 domains' interactions and led to JAK2 constitutive activation. More importantly, mutations (R867Q, R867A and R867G) disrupted this interaction enhanced the activity of JAK2-STAT5 pathway and the proliferation of Ba/F3 and MV4-11 cells. Further study showed that JAK2 R867Q mutation promoted the expression of proliferation marker and inhibited the differentiation marker of Ba/F3 and MV4-11 cells. Thus our studies provide clues in understanding the pathogenesis of JAK2 R867Q mutation in AL., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Oncogenic activation of tyrosine kinase signaling pathway is recurrent in human leukemia. The acquired Janus kinase 2 (JAK2) K607N somatic mutation was detected in about 6.8% of acute myeloid leukemia (AML) patients. However, roles of JAK2 K607N mutation in the leukemogenesis of AML remain unclear. In this study, loss of interaction between K607 and E877 was identified as key reasons for JAK2 K607N mutation constitutive activation. JAK2 K607N and mutations (K607A, K607G and E877A) abolished the K607 and E877 interaction caused JAK2 constitutive activation. While, mutations (K607R, E877D) repairing this interaction reduced K607N mutation's activity. Furthermore, our studies showed that disruption of K607 and E877 interaction abolished JH1/JH2 domains' interactions and led to JAK2 constitutive activation. More importantly, JAK2 K607N and mutations disrupted this interaction enhanced JAK2-STAT5 pathway activation and the proliferation of Ba/F3 cells. Thus our studies provide clues in understanding the leukemogenesis of JAK2 K607N mutation in AML., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Although roles of somatic JAK2 mutations in clonally myeloproliferative neoplasms (MPNs) are well established, roles of germline JAK2 mutations in the pathogenesis of MPNs remain unclear. Recently, a novel activating, germline JAK2 F556V mutation was identified and involved in the pathogenesis of MPNs, but, its pathogenesis mechanism was still unknown. In this study, homology models of JAK2 demonstrated that F556 located between two threonine residues which interacted with ATP phosphate groups by hydrogen bonds, Thr555 with the γ-phosphate and Thr557 with the β-phosphate in the active site of JAK2's JH2 domain. Moreover, the hydrogen bond between Thr557 and Arg715 played vital roles in sustaining the structural conformation of JH2's active site and JH1-JH2 domains' interactions. When F556 was replaced by other amino acids except Trp, the hydrogen bond, JH2 domain's structural conformation and JH1-JH2 domains' interactions disrupted for changing the helix between β2 and β3 strands which finally caused JAK2 activation. Mechanistic and functional studies showed that JAK2 F556V mutation disrupted JAK2 JH2 domain's activity, caused JAK2-STAT5 pathway activation and promoted the proliferation of BaF3 cells. Thus, our results herein may provide clues to understand the pathogenesis mechanism of JAK2 F556V mutation in the MPNs., (Copyright © 2018. Published by Elsevier B.V.)

Janus tyrosine kinase 2 (JAK2) mediates downstream signaling of cytokine receptors in all hematological lineages, constitutively active somatic JAK2 mutations play key roles in the pathology of myeloproliferative neoplasms (MPNs). Recently, germline JAK2 mutations are also associated with triple-negative MPNs. A novel germline mutation JAK2 V625F is reported to be involved in a subset of MPNs patients. However, the pathogenesis of this mutation caused MPN is still unclear. In this study, the homology models of JAK2 V625F showed that the newly formed interaction between F625 and Y613 disrupted the JAK2 JH1-JH2 domain interactions was responsible for its activation, when F625 and Y613 interaction was disrupted, its activity significantly decreased. While, when this interaction was repaired whether by forming hydrogen bond or salt bond, it would cause JAK2 activation. Biochemical studies also demonstrated that JAK2 V625F mutation led to JAK2-STAT5 pathway activation and promoted the proliferation of BaF3 cells. Thus, our results herein provide clues to understand the mechanism JAK2 V625F mutation caused MPNs and give information for the development of JAK2 mutation specific inhibitors., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Janus kinase 2 (JAK2) is an important mediator of cytokine receptor signaling and plays key roles in the hematopoietic and immune response. The acquired JAK2 R683S (G) mutations are presumed to be a biomarker for B-cell acute lymphoblastic leukemia (B-ALL). However, how these mutations leading to the B-ALL is still unclear. The crystal structure of JAK2 JH2 domain suggests that the residue R683 locating in the linker between the N and C lobes of JH2 domain is important for keeping the compact structure, activity and structural stability of this domain. Mutations R683S, R683G and R683E significantly increase JAK2 activity and decrease its structural stability. While the R683K and R683H mutations almost have no effects on the JAK2 activity and structural stability. Furthermore, the spectroscopic experiments imply that mutations R683S, R683G and R683E impair the structure of JAK2 JH2 domain, and lead JAK2 to partially unfolded state. It may be this partially unfolded state that caused JAK2 R683S (G) constitutive activation. This study provides clues in understanding the mechanism of JAK2 R683S (G) mutations caused B-ALL., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Janus kinase 2 (JAK2) is an important mediator of cytokine receptor signaling and plays a key role in the hematopoietic and immune responses. The acquired JAK2 C618R somatic mutation is detected in a subset of myeloproliferative disorders (MPDs) patients and presumed to be a biomarker for MPDs. However, how JAK2 C618R mutation causes MPDs is still unclear. Our results indicate that the amino acid residue E543 in JAK2 C618R is indispensable for its constitutive activation. When the glutamic acid at this position was mutated to alanine (E543A) in the JAK2 C618R, its activity significantly decreased. However when the glutamic acid was mutated to the acidic amino acid, aspartic acid, JAK2 C618R activity changed little. These results suggest that there is an interaction between the amino acid residue R618 and E543, and that this interaction is crucial to sustain the constitutive activation of JAK2 C618R. More importantly, the E543 single mutation had no effects on the function of wild type JAK2 (WT JAK2). This study suggests that the amino acid residue E543 might be a potential target for specific inhibitors to treat MPDs caused by the JAK2 C618R mutation., (Copyright © 2012 Elsevier Inc. All rights reserved.)