Letters to the Editor
417
Recurrent JAK1 and JAK3 somatic mutations in T-cell prolymphocytic leukemia
T-cell prolymphocytic leukemia (T-PLL) is a rare hematological disease in elderly people characterized by a mature T-cell phenotype, a large tumor burden and a dismal prognosis (for review see1). Highly recurrent genetic alterations have been identified, including chromosomal rearrangements between TCR genes and MTCP1/TCL1 paralogs, ATM inactivation and haploinsufficiency of the CDKN1B gene in about 50% of the cases. However, T-PLL genome profiling revealed large number of recurrent gains and losses,2 suggesting that many actors of the T-PLL malignant transformation are yet to be identified. Among the key actors of the malignant process in the hematopoietic lineage are the Janus kinase (JAK) family genes (for a recent review3). The JAK family members (JAK1, JAK2, JAK3 and TYK2) are non-receptor tyrosine kinases that bind to cytokine receptors and subsequently phosphorylate and activate STAT (Signal Transducers and Activators of Transcription) latent transcription factors. The demonstration of the direct involvement of JAK in human malignancies came from the identification of the TEL–JAK2 fusion gene in rare cases of leukemia. A major milestone was the discovery of the recurrent activating mutation p.V617F in the pseudokinase (JH2) domain of JAK2 in myeloproliferative neoplasms (MPNs). Subsequently, a number of other mutations or in-frame rearrangements of JAK2 were identified essentially within JH2 domain in MPNs, a fraction of myelodysplasia and B-cell acute lymphoblastic leukemia. Furthermore, mutations in JAK1 and JAK3 have also been observed in various lymphoid malignancies of T-cell and B-cell lineages as well in some acute myeloblastic leukemia. In contrast, TYK2 mutations are extremely rare in malignancies. As both MTCP1 and TEL–JAK2 transgenic models develop similar clonally mature T-cell malignancies,4,5 we thought to experimentally test oncogenic cooperation between these two oncogenes. MTCP1 transgenic mice develop clonal T-cell expansion after 18 months of age, which closely resembles human T-PLL. MTCP1 transgenics were crossed to a TEL–JAK2 transgenic line (line #41), which expresses TEL-JAK2 at a lower level as compared with our published line,4 and is characterized by poorly penetrant and late leukemia onset (Figure 1). All progenies of this cross were closely surveyed for leukemia onset. Mice were killed when manifesting leukemic pathologic symptoms (see Figure 1 legend) or at 7.5 months of age. Survival curves of each of the four progeny genotypes, wild-type (WT; n ¼ 35), MTCP1 transgenics (MTCP1; n ¼ 33), TEL–JAK2 transgenics (TEL–JAK2; n ¼ 42) and MTCP1/TEL–JAK2 double transgenics (MTCP1 TEL– JAK2; n ¼ 50) are shown in Figure 1. MTCP1 TEL–JAK2 double transgenic mice died significantly faster than the three other genotypes (Po10 9). At 269 days, surviving mice represented the following: 100.0% for WT, 91.0% for MTCP1, 90.4% for TEL–JAK2 and 6.2% for MTCP1 TEL–JAK2, demonstrating strong leukemia penetrance in double transgenic mice (Figure 1). A macroscopic examination of diseased mice revealed typical symptoms of lymphoid malignancy, including a pronounced thymus enlargement and/or splenomegaly with a prominent lymph node involvement, which were confirmed to be of T-cell origin by flow cytometry analyses (not shown). Taken together, these data indicated the oncogenic cooperation between MTCP1 and the activation of the JAK–STAT pathway.
In order to explore whether this oncogenic cooperation in animal models reflects the human disease, we searched for evidence of JAK/STAT activation in human T-PLL. We explored the STAT phosphorylation status on archived blood samples from 24 patients with this rare disease. No sample was available from the proliferative tissues such as lymphoid tissues or bone marrow. Phosphorylation of STAT5 could not be detected in any samples, whereas concomitant phosphorylation of STAT1 and STAT3 were observed in five T-PLLs (Supplementary Figure S1). We therefore screened for JAK1, JAK2, JAK3 and TYK2 mutations in seven T-PLL cases by sequencing the whole coding transcripts for these four genes. No JAK2 or TYK2 mutation was identified, whereas missense mutations or in-frame deletions in JAK1 (n ¼ 3) or JAK3 (n ¼ 5) were found in the seven tumors, one sample (TP22) carrying mutations in both genes. Thus, an additional series of 38 T-PLLs was explored at genomic the level for JAK1, JAK2 and JAK3 hotspot mutations (COSMIC, http://www.sanger.ac.uk/genetics/ CGP/cosmic as of mars 2013) or extended gene regions depending on available biological materials (Supplementary Tables S1 and S2). JAK1 and JAK3 mutations were found in half T-PLL cases (49%, 22/45), whereas no mutation was found in the JAK2 gene (Figure 2a and Supplementary Tables S2 and S3). JAK3 mutations were found in 19 out of 45 T-PLL cases (42%) with missense mutations (n ¼ 19) and one in-frame deletion (n ¼ 1). Three different single-nucleotide changes leading to the same JAK3M511I mutant protein (c.1533G4A, c.1533G4T or c.1533G4C/p.Met511Ile) were found in 10 out of 45 cases (22%; Figure 2b). Another minor hotspot mutation, JAK3A573V, was found in 3 out of 45 cases (7%). Two mutations were complex: a nine-base in-frame deletion and a Q501H/Q503H double mutant (confirmed to be in cis by subcloning and sequencing; Figure 2c). A complex JAK3 rearrangement was evidenced in case TP35, which could not be fully elucidated. Survival Curve of MTCP1*TEL-JAK2 mice 1 0.8
WT MTCP1
Survival
Leukemia (2014) 28, 417–419; doi:10.1038/leu.2013.271
TEL-JAK2
0.6
PA
c.1533G>C
c.1533G>T
AGATGACAT
JAK3 M511I
JAK3 K563_C565del
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CACAGGGATATTTC
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c.1886_1891del
CACATTTC
WT
TTCAGCCCCAATCCC
JAK3 TTCASCCCCAWTCCC
c.1503G>C c.1509A>T
TTCACCCCCATTCCC
Figure 2. JAK1 and JAK3 mutations in T-PLL. (a) Localization of affected residues with missense (full circle) and deletion (full triangle) mutations in JAK1 and JAK3 relative to the domain organization of JAK proteins: FERM (protein 4.1, ezrin, radixin, moesin), SH2 (SH2-like domain), JH1 (JAK homology 1 kinase domain), JH2 (JAK homology 2 pseudokinase domain) and L (linker). (b) Chromatograms of the three mutations c.1533 G4A/C/T leading to the hotspot JAK3M511I mutant kinase. (c) Chromatograms of complex JAK1 and JAK3 mutations (left panel) elucidated after subcloning and sequencing (right panel). Nucleotides are numbered relative to the start codon.
Four non-synonymous mutations of JAK1 were identified in this series of 45 T-PLLs (9%) all in the pseudokinase domain: c.1884_1889 and c.1886_1891 deletions leading to the same JAK1R629_D630del mutant protein and the previously reported c.1957C4T/p.Leu653Phe and c.1972G4T/p.Val658Phe mutations (Supplementary Table S3). The JAK1 c.1886_1891del in TP01 was detected at a low level in the chromatogram and confirmed by subcloning, contrasting with the almost pure leukemic population from which the sequence was obtained (Figure 1c). JAK mutations may thus be acquired during the course of the disease. Most identified mutations (11 out of 16) are already known to induce proliferation in in vitro models (Supplementary Table S3). It is noteworthy that the JAK3M511I hotspot mutation was shown by Yamahita et al.6 to lead to the most efficient oncokinase with highest transforming properties. Accordingly, analyzed cases with evidence of STAT1/3 phosphorylation were all JAK1/3-mutated samples (Supplementary Figure S1). Some patients carried more than one JAK mutation. Patient TP22 carried both JAK1L653F and JAK3M511I mutations; patient TP02 carried both JAK3M511I and JAK3A572V mutants and patient TP35 both a JAK3M511I mutation and a complex rearrangement of JAK3. This suggests that either these mutations have additional or complementary effects or independent JAK mutations occurred in different subclones of these leukemias. There is growing evidence of a role of JAK1- and JAK3-activating mutations in human lymphoid leukemia. We demonstrated here that T-PLL is by far associated with the highest rate of JAK1/3 mutations. JAK1 mutations were described in T-acute lymphoblastic leukemia (ALL), and rare B-cell ALL. JAK3 mutations have been detected in adult T-cell Leukemia (2014) 404 – 463
leukemia/lymphoma, in cutaneous T-cell lymphoma and in extranodal nasal-type natural killer cell lymphoma.7–11 JAK1/3 mutations have been recently associated with early T-cell precursor ALL (ETP-ALL), and, strikingly, T-PLL and ETP-ALL share the same JAK3M511I and JAK3A573V hotspot mutations.11 The preferential mutations for one or another JAK paralogs may be influenced by the interaction with a specific receptor and/or the critical growth factor signaling pathway inducing a given disease. In this regard, IL7R-activating mutations are almost exclusive from JAK1 and JAK3 mutations in T-ALL. However, no T-PLL case presented in this study showed STAT activation independently of JAK1/3 mutations, suggesting that the additional JAK-STATactivating mechanism is unlikely to be frequent. Another important point is the downstream consequences of JAKactivating mutations. Whereas STAT5 is generally considered as the major oncogenic transducer, we found no evidence of its activation in T-PLL. Conversely, STAT1 and STAT3 were found frequently in a phosphorylated state in this disease. This is also reminiscent of STAT3-bearing activating mutations in T-cell large granular lymphocytic leukemia, as well as various lymphoid malignancies, with so far no evidence of such mutations in T-PLL.12,13 By its high rate of JAK1/3 mutations and its dismal prognosis, T-PLL appears as a model disease to evaluate JAK inhibitors to suppress uncontrolled growth of cancer cells with deregulated JAK. CONFLICT OF INTEREST The authors declare no conflict of interest.
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Letters to the Editor
419 ACKNOWLEDGEMENTS We are indebted to the French hematologists who provided patient samples: B Cazin (Lille); R Garand (Nantes); MJ Grange, V Leblond, F Nguyen Khac, H Merle-Beral, F Valensi, B Varet, I Radford-Weiss, O Hermine, R Delarue, V Levy, JC Brouet, P Rousselot (Paris); G Damaj (Creteil); X Troussard (Caen); S Daliphard, P Cornillet (Reims); D Lusina (Aulnay); K Ghomari (Beauvais); C Bertout (Brest); O Tournilhac (Clermond-Ferrand); M Maynadie (Dijon); V Izydirczyk (Le Havre); E Callet-Bauchu, B Coffier (Lyon); F Lellouche (Quimper). This work was supported by grants from the Institut National du Cancer (INCa), the Institut National de la Sante´ et de la Recherche Me´dicale (INSERM) and the Institut Curie, Section de Recherche. This work is a part of the ‘Cance´ropole Ile-de-France–Mouse models of human cancer’ program coordinated by M Giovannini. VJ is a recipient of grants from the Association pour la Recherche sur le Cancer (ARC).
D Bellanger1,2, V Jacquemin1,2, M Chopin3, G Pierron1,2, OA Bernard4,5, J Ghysdael6,7,8 and M-H Stern1,2 1 Institut Curie, Centre de Recherche, Paris, France; 2 INSERM U830, Paris, France; 3 INSERM U940, Institut Universitaire d’He´matologie, Saint-Louis Hospital, Paris, France; 4 INSERM U985, Institut Gustave Roussy, Villejuif, France; 5 Universite´ Paris-Sud, Orsay, France; 6 Institut Curie, Centre Universitaire, Orsay, France; 7 CNRS UMR 3306, Orsay, France and 8 INSERM U1005, Orsay, France E-mail:
[email protected] REFERENCES 1 Dungarwalla M, Matutes E, Dearden CE. Prolymphocytic leukaemia of B- and T-cell subtype: a state-of-the-art paper. Eur J Haematol 2008; 80: 469–476.
2 Soulier J, Pierron G, Vecchione D, Garand R, Brizard F, Sigaux F et al. A complex pattern of recurrent chromosomal losses and gains in T-cell prolymphocytic leukemia. Genes Chromosomes Cancer 2001; 31: 248–254. 3 Vainchenker W, Constantinescu SN. JAK/STAT signaling in hematological malignancies. Oncogene 2013; 32: 2601–2613. 4 Carron C, Cormier F, Janin A, Lacronique V, Giovannini M, Daniel MT et al. TELJAK2 transgenic mice develop T-cell leukemia. Blood 2000; 95: 3891–3899. 5 Gritti C, Dastot H, Soulier J, Janin A, Daniel MT, Madani A et al. Transgenic mice for MTCP1 develop T-cell prolymphocytic leukemia. Blood 1998; 92: 368–373. 6 Yamashita Y, Yuan J, Suetake I, Suzuki H, Ishikawa Y, Choi YL et al. Array-based genomic resequencing of human leukemia. Oncogene 2010; 29: 3723–3731. 7 Elliott NE, Cleveland SM, Grann V, Janik J, Waldmann TA, Dave UP. FERM domain mutations induce gain of function in JAK3 in adult T-cell leukemia/lymphoma. Blood 2011; 118: 3911–3921. 8 Flex E, Petrangeli V, Stella L, Chiaretti S, Hornakova T, Knoops L et al. Somatically acquired JAK1 mutations in adult acute lymphoblastic leukemia. J Exp Med 2008; 205: 751–758. 9 Kameda T, Shide K, Shimoda HK, Hidaka T, Kubuki Y, Katayose K et al. Absence of gain-of-function JAK1 and JAK3 mutations in adult T cell leukemia/lymphoma. Int J Hematol 2010; 92: 320–325. 10 Bouchekioua A, Scourzic L, de Wever O, Zhang Y, Cervera P, Aline-Fardinet A et al. JAK3 deregulation by activating mutations confers invasive growth advantage in extranodal nasal-type natural killer cell lymphoma. Leukemia 2014; 28: 338–348. 11 Zhang J, Ding L, Holmfeldt L, Wu G, Heatley SL, Payne-Turner D et al. The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature 2012; 481: 157–163. 12 Koskela HLM, Eldfors S, Ellonen P, van Adrichem AJ, Kuusanma¨ki H, Andersson EI et al. Somatic STAT3 mutations in large granular lymphocytic leukemia. N Engl J Med 2012; 366: 1905–1913. 13 Couronne´ L, Scourzic L, Pilati C, Della Valle V, Duffourd Y, Solary E et al. STAT3 mutations identified in human hematological neoplasms induce myeloid malignancies in a mouse bone marrow transplantation model. Haematologica 2013; 98: 1748–1752.
Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)
Molecular monitoring in NUP214-ABL-positive T-acute lymphoblastic leukemia reveals clonal diversity and helps to guide targeted therapy Leukemia (2014) 28, 419–422; doi:10.1038/leu.2013.272
A 20-year-old male patient presented to our department because of cervical lymphadenopathy, which progressed over a month. No acute EBV or CMV titers were detected. B symptoms (fever, night sweats and weight loss of 4 kg in 8 weeks) were accompanied by axillary and inguinal lymphadenopathy. LDH was found to be increased to 900 U/l (reference o240 U/l). In addition, CT scanning revealed both hilar and mediastinal lymphadenopathy. The spleen was enlarged to 7.6 13.3 16.8 cm. Immunophenotyping of the bone marrow (BM) revealed dense infiltration by T-lymphoblasts expressing cyCD3, CD5, CD7 and CD34, but lacking CD1a, CD2, sCD3, CD4 and CD8, although coexpressing CD79a and CD33. CD52 was not expressed on the blasts. Cytogenetic analysis at diagnosis showed three different clones harboring a complex aberrant karyotype, which were derived from a common ancestor, comprising 70% (14 of 20) metaphases, whereas 30% of metaphases depicted a normal karyotype. Fluorescence in situ hybridization (FISH) analysis failed to show
any BCR–ABL translocated clones but did reveal deletion of 9q34 in 6% (12 of 200) and an ABL amplification in 66% (132 of 200) interphase nuclei (Figure 1). The amplified ABL region showed an extrachromosomal localization pattern. These results, demonstrating the deletion of the ABL gene combined with extrachromosomal amplification of ABL, were suggestive of NUP214-ABL-positive T-acute lymphoblastic leukemia (T-ALL). In fact, molecular genetic analysis with a conventional six-primer multiplex PCR demonstrated the presence of a NUP214-ABL fusion transcript (fusion of NUP214 exon 31 and ABL exon 2) (Figure 2).1 Quantitative real time (qRT)-PCR analysis was established for the transcript using PCR primers NUP31 and ENR561 together with the hydrolysis probe ENP541 and GUSB as the control gene.1–3 The relative expression level was calculated using the D(ct) method. In addition, two clonally rearranged T-cell receptor (TCR) genes were sequenced (TCRB Db1-Jb2.2 and TCRG Vg11-Jg1/2), and clone-specific RQ-PCR assays were established for DNA-based quantification of minimal residual disease (MRD). Polychemotherapy according to the German ALL study group protocol 07/2003 (www.clinicaltrials.gov: NCT00198991) was initiated after a written informed consent was obtained from the patient. BM aspiration on day 11 and day 26 showed persistence
Accepted article preview online 19 September 2013; advance online publication, 4 October 2013
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Leukemia (2014) 404 – 463
