The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice

Abstract

JAK2 plays important roles in the regulation of a variety of cellular processes including cell migration, proliferation, and protection from apoptosis. Recently the L611S point mutation in JAK2 has been identified in a child with acute lymphoblastic leukemia. Here we analyzed the mechanism by which JAK2 exhibits its oncogenicity. In BaF3 murine hematopoietic cells, L611S mutant increased the expression of antiapoptotic proteins including X chromosome-linked inhibitor of apoptosis protein, inhibitor of apoptosis protein, and Bcl-XL. We also showed that JAK2 L611S mutant protects BaF3 cells from cytokine withdrawal-induced apoptotic cell death and leads to cytokine-independent cell growth. Furthermore BaF3 cells expressing JAK2 L611S mutant gained the ability to induce tumorigenesis in nude mice. The L611S mutant also exhibited malignancy, including prompt invasion and spreading into various organs, leading to rapid lethality of the mice. Finally we showed that a specific JAK2 inhibitor, AG490, potently inhibited cytokine-independent cell growth induced by JAK2 L611S mutant via the induction of apoptotic cell death. In addition, treatment with AG490 significantly inhibited the JAK2 L611S mutant-induced tumorigenesis in nude mice. Thus, our results both in vitro and in vivo strongly suggest that L611S mutant of JAK2 harbors potent oncogenic activity, and this probably requires the antiapoptotic signaling pathway.The tyrosine kinase Janus kinase 2 (JAK2) 2 is the essential component of various cytokine signal transductions. Recent studies showed that JAK2 activates the mitogen-activated protein kinase family (ERK, c-Jun NH 2 -terminal kinase (JNK), and p38), Akt, and the signal transducers and activators of transcription (Stat) family in various tissues and is involved in numerous biological functions such as cell growth, cell survival, and differentiation (1, 2). Therefore, it has been reported that the disruption of the regulation of JAK2 activity is associated with hematopoietic disorders and oncogenesis (3-5).JAK2 contains seven regions with significant sequence homology between the kinases, termed Jak homology (JH) domains (1, 2). The JH1 domain is located within the carboxyl terminus of the protein and contains the tyrosine kinase domain. The adjacent JH2 domain shows close homology to the JH1 domain; however, it lacks critical residues required for tyrosine kinase activity. Under normal conditions, this JH2 domain negatively regulates kinase activity. Theoretical models of JAK2 structure suggest that the JH1 and JH2 domains are facing each other and that the activation loop of JAK2 is buried at this interface (6). Upon activation of JAK2, phosphorylation of the activation loop at Tyr 1007/1008 occurs and is believed to prevent this JH1-JH2 interaction and therefore relieve inhibition (7-9).The JAK2 deletion mutant lacking JH2 domain exhibited receptor-independent constitutive activation (8, 9). Furthermore JH2 domain mutations have been demonstrated to be involved in various myeloproliferative diseases (10 -1...

STAT5 Activation Is Critical for the Transformation Mediated by Myeloproliferative Disorder-associated JAK2 V617F Mutant

“…Recently, JAK2 V617F mutation in JH2 domain has been found as a common genetic abnormality in patients with myeloproliferative disorders (MPDs), and it was reported that this mutation leads to constitutive activation of JAK2 (19 -21). As in previous reports, leukemia-associated L611S mutation in JAK2 also exhibits a similar phenotype to V617F mutation, and these mutations in the JH2 domain of JAK2 may disrupt intramolecular interaction between JH1 and JH2 (26). However, Lu et al (24) demonstrated that constitutive activation of the JAK2 V617F mutant requires coexpression of a cognate homodimeric cytokine receptor, suggesting that the association of V617F mutant with the receptor seems to be essential for exhibition of its disordered proliferative activity.…”

“…Animal Tumorigenesis-To investigate oncogenic potentials in vivo, 1 ϫ 10 7 transduced Ba/F3 cells were injected subcutaneously into female BALB/c nude mice aged 4 weeks (23,26). Twelve or 17 days post-inoculation, the animals were sacrificed, and the weights of the tumor, liver, and spleen were recorded.…”

“…After sacrifice, the liver of each nude mouse was fixed in 4% paraformaldehyde and then dehydrated gradually in alcohol. Tissues were embedded in paraffin and sectioned at a thickness of 4 m. The sections were stained with hematoxylin-eosin and analyzed for the presence of tumor cell infiltration using an OLYMPUS BX50 microscope (Olympus, Tokyo, Japan) with Olympus Micro DP70 software (Olympus) (26).…”

Janus Kinase Deregulation in Leukemia and Lymphoma

“…Increased PI3K-AKT signaling transforms cells by promoting increased cell survival and proliferative index. Inhibition of PI3K activity with pharmacological inhibitors reduces EEC formation from PV progenitors, increases apoptosis of cells expressing MPN-or ALL-associated JAK mutations, and reduces erythroid proliferation (Funakoshi-Tago et al, 2009;Ugo et al, 2004).…”

Combined Inhibition of Janus Kinase 1/2 for the Treatment of JAK2V617F-Driven Neoplasms: Selective Effects on Mutant Cells and Improvements in Measures of Disease Severity

“…In mice, germline or somatic expression of JAK2V617F induces phenotypes similar to human PV, essential thrombocythemia, and even MF (14)(15)(16)(17). Additional activating (18,19) mutations have been identified in JAK2 (e.g., exon 12 mutations), as well as in MPL (e.g., MPLW515 mutations), further supporting that constitutive activation of the JAK2 pathway is important for MPD pathogenesis (20,21). Based on the paradigm shifting impact of imatinib on the treatment of CML (22), it is intriguing to think of the therapeutic potential of JAK inhibitors for the treatment of these diseases.…”