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Recurrent JAK1 and JAK3 somatic mutations in


T-cell prolymphocytic leukemia
Article in Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
September 2013
DOI: 10.1038/leu.2013.271 Source: PubMed

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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 identied, including chromosomal rearrangements
between TCR genes and MTCP1/TCL1 paralogs, ATM inactivation
and haploinsufciency of the CDKN1B gene in about 50% of the
cases. However, T-PLL genome proling revealed large number of
recurrent gains and losses,2 suggesting that many actors of the
T-PLL malignant transformation are yet to be identied.
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 identication of the TELJAK2
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 identied 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 TELJAK2 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 TELJAK2
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 35), MTCP1
transgenics (MTCP1; n 33), TELJAK2 transgenics (TELJAK2;
n 42) and MTCP1/TELJAK2 double transgenics (MTCP1 TEL
JAK2; n 50) are shown in Figure 1. MTCP1 TELJAK2 double
transgenic mice died signicantly 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 TELJAK2
and 6.2% for MTCP1 TELJAK2, 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 conrmed to be of T-cell origin by ow
cytometry analyses (not shown). Taken together, these data
indicated the oncogenic cooperation between MTCP1 and the
activation of the JAKSTAT pathway.

In order to explore whether this oncogenic cooperation in


animal models reects 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 ve 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 identied,
whereas missense mutations or in-frame deletions in JAK1 (n 3)
or JAK3 (n 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 19) and one in-frame deletion
(n 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
(conrmed 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, 417419; doi:10.1038/leu.2013.271

TEL-JAK2

0.6

P<10-9

MTCP1 TEL-JAK2

0.4
0.2
0
0

50

100

150

200

250

300

Time (days)

Figure 1. The KaplanMeier survival curve of the progeny of MTCP1


 TEL-JAK2 transgenic mice. A total of 160 mice were followed for
7.5 months. Mice were killed at the indicated time points when
showing signs of hunched posture, extreme weakness, splenomegaly and/or severe dyspnea caused by the expansion of leukemic
cells in the thymus. T-cell leukemia was confirmed by flow
cytometry analyses. WT indicates wild-type mice (n 35); MTCP1,
MTCP1 transgenics (n 33); TELJAK2, TELJAK2 transgenics (n 42);
MTCP1 TELJAK2, MTCP1 TELJAK2 double transgenics (n 50).

Accepted article preview online 19 September 2013; advance online publication, 11 October 2013

& 2014 Macmillan Publishers Limited

Leukemia (2014) 404 463

Letters to the Editor

418
L653F V658F

JAK1

FERM

JH1

JH2

SH2

R629_D630del

M511I

Q501H_Q503H

A572V
L857P
A573V V722I
Y824D
V674A

c.1533G>A

c.1533G>C

c.1533G>T

AGATGACAT

JAK3 M511I

JAK3
K563_C565del

WT

CACAGGGATATTTC

JAK1
CACAKKKMYMTKKC

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 identied 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 conrmed 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 identied 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 efcient 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.711 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
inuenced by the interaction with a specic 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 conict of interest.

& 2014 Macmillan Publishers Limited

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 Cofer (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 Medicale (INSERM) and the Institut Curie, Section de Recherche. This
work is a part of the Canceropole Ile-de-FranceMouse 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 dHematologie, 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: marc-henri.stern@curie.fr
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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, 419422; 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 inltration 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 BCRABL translocated clones but did reveal deletion of 9q34


in 6% (12 of 200) and an ABL amplication in 66% (132 of 200)
interphase nuclei (Figure 1). The amplied ABL region showed an
extrachromosomal localization pattern. These results, demonstrating
the deletion of the ABL gene combined with extrachromosomal
amplication 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.13 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-specic RQ-PCR
assays were established for DNA-based quantication 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

& 2014 Macmillan Publishers Limited

Leukemia (2014) 404 463

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