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1843 and PEST domains, respectively (data not shown). Overall, we found 17 mutations in 16 of 33 childhood T-ALL cases (48.5%), recapitulating the previous observation.3 It is intriguing to consider g-secretase inhibitors as antileukemia agents, with Notch signaling being a new therapeutic target, since their efficacy is predicted and there are ongoing clinical trials of g-secretase inhibitors as anti-Alzheimer drugs. However, at least two issues must be considered further. First, according to the previous report, many Notch1-mutated T-ALL cell lines are not likely to respond to g-secretase inhibitors, although some are definitely sensitive to these agents. Indeed, we found that g-secretase inhibitors that induce apoptosis in some T-ALL cell lines did not affect many Notch1-mutated T-ALL cell lines despite the fact that these g-secretase inhibitors unambiguously blocked the activation of Notch1 (data not shown). These findings indicate that Notch1 activation is not always required for the growth of T-ALL cell lines even if they have mutations. This may be due to additional mutations during establishment of the cell line or presense of Notch-independent cell growth machinery in T-ALL cells from patients. To see whether Notch signaling is a good therapeutic target, it is important to examine fresh T-ALL cells for frequency of responsiveness to g-secretase inhibitors. Second, with the development of g-secretase inhibitors for the treatment of Alzheimer’s disease, major effort has been made to find compounds that have less effect on Notch signaling. Indeed, it has been clearly shown that the administration of large amounts of Ly411575, a compound with a strong g-secretase inhibiting activity, to mice induces severe abnormalities in the immune system and digestive tract.8 Therefore, it is unlikely that we can divert a g-secretase inhibitor that has been developed for treatment of Alzheimer’s disease to an anti-T-ALL drug. We need a careful strategy to find g-secretase inhibitors or other Notch inhibitors that could be used for T-ALL and potentially other malignancies, with acceptable side effects due to the inhibition of Notch signaling, which is required for cell life physiologically.
Acknowledgements This work was supported in part by Grant-in-Aid for Scientific Research on Priority Areas, KAKENHI-17013022 (to SO) and
KAKENHI-17390274 (to SC) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by Japan Health Sciences Foundation (to SC).
S-Y Lee1 K Kumano1,2 S Masuda1,2 A Hangaishi2 J Takita4 K Nakazaki2 M Kurokawa2 Y Hayashi5 S Ogawa2,3 S Chiba1,2
1
Department of Cell Therapy and Transplantation Medicine, University of Tokyo, Tokyo, Japan; 2 Department of Hematology and Oncology, University of Tokyo, Tokyo, Japan; 3 Department of Regeneration Medicine for Hematopoiesis, University of Tokyo, Tokyo, Japan 4 Department of Pediatrics, University of Tokyo, Tokyo, Japan; and 5 Department of Hematology and Oncology, Gunma Children’s Medical Center, Gunma, Japan
References 1 Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science 1999; 284: 770–776. 2 Radtke F, Raj K. The role of Notch in tumorigenesis: oncogene or tumour suppressor? Nat Rev Cancer 2003; 3: 756–767. 3 Weng AP, Ferrando AA, Lee W, Morris IV JP, Silverman LB, Sanchez-Irizarry C et al. Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 2004; 306: 269–271. 4 Wolfe MS. Therapeutic strategies for Alzheimer’s disease. Nat Rev Drug Discov 2002; 1: 859–866. 5 Hoelzer D, Gokbuget N. New approaches to acute lymphoblastic leukemia in adults: where do we go? Semin Oncol 2000; 27: 540–559. 6 Aplan PD. Adults are not simply big children. Blood 2004; 103: 2437–2438. 7 Ogawa S, Hangaishi A, Miyawaki S, Hirosawa S, Miura Y, Takeyama K et al. Loss of the cyclin-dependent kinase 4-inhibitor (p16; MTS1) gene is frequent in and highly specific to lymphoid tumors in primary human hematopoietic malignancies. Blood 1995; 86: 1548–1556. 8 Wong GT, Manfra D, Poulet FM, Zhang Q, Josien H, Bara T et al. Chronic treatment with the gamma-secretase inhibitor LY-411,575 inhibits beta-amyloid peptide production and alters lymphopoiesis and intestinal cell differentiation. J Biol Chem 2004; 279: 12876–12882.
JAK2 Val617Phe activating tyrosine kinase mutation in juvenile myelomonocytic leukemia Leukemia (2005) 19, 1843–1844. doi:10.1038/sj.leu.2403903; published online 4 August 2005 TO THE EDITOR
Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative/myelodysplastic disorder of early childhood.1 Genetic abnormalities of the three genes RAS (15–20%), neurofibromatosis type I (NF1) (25%), and protein-tyrosine phosphatase, nonreceptor type 11 (PTPN11) (34%), all of which are positioned in the GM-CSF/Ras signal transduction pathway, Correspondence: Dr E Ito, Department of Pediatrics, Hirosaki University School of Medicine, 53 Honcho, Hirosaki, Aomori, 0368563, Japan; Fax: 81 172 39 5071; E-mail:
[email protected] Received 13 June 2005; accepted 5 July 2005; published online 4 August 2005
have been implicated in the pathogenesis of JMML.2–4 One of these genetic abnormalities is observed in 75% of JMML patients, leaving 25% of the reported cases in which a specific mutation has yet to be detected. Recent reports described an acquired mutation of the tyrosine kinase JAK2 gene that has been found in human myeloproliferative disorders, and the single-point mutation (Val617Phe) in exon 12 was identified.5–7 Several data demonstrate that JAK2 is physically associated with the GM-CSFRb chain, becoming activated upon challenge of myeloid cells with GM-CSF.8 To clarify the involvement of JAK2 in the pathogenesis of JMML, we searched for mutations in the JAK2 gene in five JMML patients. The diagnosis of JMML for each patient was confirmed according to diagnostic criteria agreed upon by the International JMML Working Group.1 Genomic DNA was extracted from patients’ bone marrow cells, and the human JAK2 exon 12 was Leukemia
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1844 identical sibling 8 months later. He has been in complete remission for more than 3 years. We report that the JAK2 mutation Val617Phe was found in one of five cases of JMML that were analyzed. Our results suggest that identification of the Val167Phe JAK2 mutation holds promise for new approaches, not only in myeloproliferative disorders of adults but also in JMML. Our finding warrants further investigation in a larger series of JMML patients.
Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research, Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Technology.
Figure 1 Restriction enzyme-based assessment of the JAK2 genotype in JMML patients. (a) Exon 12 in the human JAK2 gene was amplified by PCR. The resulting 460-bp amplified fragment was digested with BsaXI. The mutant allele remained undigested (JMML3), whereas the wild-type allele was digested into fragments that were 241, 189, and 30 bp. (b) DNA sequencing was performed directly on PCR products using cycle sequencing. The fragment that was not digested by BsaXI was sequenced, revealing a G to T mutation at nucleotide 1849 of JAK2 that encoded a V617F substitution.
amplified by polymerase chain reaction as described previously.5 The resulting 460-bp amplified fragment was digested with BsaXI. The mutant allele remained undigested, whereas the wild-type allele was digested into 241, 189, and 30 bp fragments (Figure 1a). The mutant fragment was purified and its DNA sequence was determined. The single-point mutation (Val617Phe) was detected in one patient (Figure 1b). Here, we describe the case with the JAK2 mutation. A 4-year-old boy was admitted to our hospital with complaints of pallor, a bleeding tendency, and abdominal distention. Physical examination revealed marked hepatosplenomegaly. Peripheral blood analysis showed severe anemia, thrombocytopenia, and hyperleukocytosis with the appearance of blasts: red blood cell count, 2.24 1012/l; hemoglobin, 51 g/l; platelet count, 3 109/l; and white blood cell count, 62.7 109/l (neutrophils, 19%; lymphocytes, 58%; monocytes, 8%; atypical lymphocytes, 4%; metamyelocytes, 1%; myelocytes, 4%; blasts, 5%). The fetal hemoglobin level was 1.1%. Bone marrow aspiration revealed hypercellularity with 12.5% of blasts, and chromosomal analysis revealed 45 XY, 7 (20/20 cells). Spontaneous in vitro colony formation was also found. The mutation PTPN11 was not detected. The patient was diagnosed as JMML, and oral 6-MP and low-dose cytarabine were given. He received a bone marrow transplantation from his HLA-
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C Tono1 G Xu1,2 T Toki1 Y Takahashi1 S Sasaki1 K Terui1 E Ito1
1
Department of Pediatrics, Hirosaki University School of Medicine, Hirosaki, Japan; and Department of Pediatrics, The second Affiliated Hospital of China Medical University, Sheyang, China
2
References 1 Niemeyer CM, Arico M, Basso G, Biondi A, Cantu Rajnoldi A, Creutzig U et al. Chronic myelomonocytic leukemia in childhood: a retrospective analysis of 110 cases. Blood 1997; 89: 3534–3543. 2 Miyauchi J, Asada M, Sasaki M, Tsunematsu Y, Kojima S, Mizutani S. Mutations of the N-ras gene in juvenile chronic myelogenous leukemia. Blood 1994; 83: 2248–2254. 3 Side LE, Emanuel PD, Taylor B, Franklin J, Thompson P, Castleberry RP et al. Mutations of the NF1 gene in children with juvenile myelomonocytic leukemia without clinical evidence of neurofibromatosis, type 1. Blood 1998; 92: 267–272. 4 Tartaglia M, Niemeyer CM, Fragale A, Song X, Buechner J, Jung A et al. Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet 2003; 34: 148–150. 5 Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005; 365: 1054–1061. 6 Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005; 352: 1779–1790. 7 James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C et al. A unique clonal JAK2 mutation leading to constitutive signaling causes polycythaemia vera. Nature 2005; 434: 1144–1148. 8 Quelle FW, Sato N, Witthuhn BA, Inhorn RC, Eder M, Miyajima A et al. JAK2 associates with the b c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region. Mol Cell Biol 1994; 14: 4335–4341.
