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

Megumi Funakoshi-Tago,Eriko Aizu-Yokota,Tadashi Kasahara,Kenji Tago,Tomoyuki Oshio,Miyuki Abe,Yoshiko Sonoda,Kazuya Sumi

doi:10.1074/jbc.m808879200

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

Megumi Funakoshi-Tago, Eriko Aizu-Yokota + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m808879200

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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.

Highlights

Tein kinase family (ERK, c-Jun NH2-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]
It is expected that an acute lymphoblastic leukemia (ALL) patient-derived L611S mutation would induce the constitutive activation of Janus kinase 2 (JAK2)
In the case of several tyrosine kinase family members, aberrant expression, point mutation, and chromosomal translocation are reported to cause their disorderly activity. Such alterations of tyrosine kinases have been found in various tumors, and these variations of tyrosine kinases have been frequently reported to result in the promotion of cellular transformation and tumorigenesis [4, 5, 23]

Summary

EXPERIMENTAL PROCEDURES

Reagents—Recombinant human Epo (ESPO௡3000) was purchased from Kirin Brewery Co. (Tokyo, Japan). Cell Cultures—BaF3 cells were infected with empty virus (MSCV), wild type JAK2 (WT) c-HA, or mutant JAK2 c-HA (L611S) with erythropoietin receptor c-FLAG and established as described previously [14] These cells were cultured in RPMI 1640 medium (Nissui Seiyaku, Tokyo, Japan) supplemented with 10% fetal bovine serum (BioWest), 100 ␮g/ml L-glutamine (Nacalai Tesque, Tokyo, Japan), 100 ␮g/ml penicillin (Nacalai Tesque), 100 ␮g/ml streptomycin (Nacalai Tesque), and 5 units/ml Epo. BaF3 Cell Growth Assay—Transduced and exponentially growing BaF3 cells were washed twice with PBS and incubated with RPMI 1640 medium supplemented with 1% fetal bovine serum and 100 mg/ml L-glutamine in the presence or absence of Epo (5 units/ml) for the indicated times. Histological Examination—After sacrifice, liver and spleen of each nude mouse were fixed in 4% paraformaldehyde and

RESULTS

DISCUSSION

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