Janus Kinase 3–Activating Mutations Identified in Natural Killer/T-Cell

Published OnlineFirst June 15, 2012; DOI: 10.1158/2159-8290.CD-12-0028

RESEARCH BRIEF Janus Kinase 3–Activating Mutations Identiﬁ ed in Natural Killer/T-cell Lymphoma

Ghee Chong Koo1, Soo Yong Tan5,10, Tiffany Tang1,3, Song Ling Poon1, George E. Allen1, Leonard Tan5, Soo Ching Chong1, Whee Sze Ong2, Kevin Tay3, Miriam Tao3, Richard Quek3, Susan Loong4, Kheng-Wei Yeoh4, Swee Peng Yap4, Kuo Ann Lee4, Lay Cheng Lim6, Daryl Tan6, Christopher Goh7, Ioana Cutcutache8, Willie Yu1, Cedric Chuan Young Ng1, Vikneswari Rajasegaran1, Hong Lee Heng1, Anna Gan1, Choon Kiat Ong1, Steve Rozen8, Patrick Tan9,11,12, Bin Tean Teh1,9, and Soon Thye Lim3,10

ABSTRACT The molecular pathogenesis of natural killer/T-cell lymphoma (NKTCL) is not well understood. We conducted whole-exome sequencing and identiﬁ ed Janus kinase 3 (JAK3) somatic–activating mutations (A572V and A573V) in 2 of 4 patients with NKTCLs. Further validation of the prevalence of JAK3 mutations was determined by Sanger sequencing and high-resolution melt (HRM) analysis in an additional 61 cases. In total, 23 of 65 (35.4%) cases harbored JAK3 mutations. Functional characterization of the JAK3 mutations support its involvement in cytokine-independent JAK/ STAT constitutive activation leading to increased cell growth. Moreover, treatment of both JAK3-mutant and wild-type NKTCL cell lines with a novel pan-JAK inhibitor, CP-690550, resulted in dose-dependent reduction of phosphorylated STAT5, reduced cell viability, and increased apoptosis. Hence, targeting the deregulated JAK/STAT pathway could be a promising therapy for patients with NKTCLs.

SIGNIFICANCE: Gene mutations causing NKTCL have not been fully identifi ed. Through exome sequencing, we identifi ed activating mutations of JAK3 that may play a signifi cant role in the pathogenesis of NKTCLs. Our fi ndings have important implications for the management of patients with NKTCLs. Cancer Discov; 2(7); 1–7. ©2012 AACR.

INTRODUCTION However, compared with the more common B-cell lymphomas, very little is known about its molecular characteristics Natural killer/T-cell lymphoma (NKTCL) is particularly and pathogenesis. There has been little progress in basic sci- prevalent in Asian countries and some parts of Latin America. ence and clinical research in this subtype of lymphoma, which It accounts for up to half of all mature TCL cases in Asia (1). continues to constitute a major challenge in managing these

Authors’ Afﬁ liations: 1NCCS-VARI Translational Research Laboratory, Corresponding Authors: Bin Tean Teh, NCCS-VARI Translational Research Department of Medical Sciences, 2Division of Clinical Trials and Epidemiolog- Laboratory, Department of Medical Sciences, National Cancer Centre ical Sciences, Departments of 3Medical Oncology and 4Radiation Oncology, Singapore, 11 Hospital Drive, Singapore 169610, Singapore. Phone: 65- National Cancer Centre Singapore; Departments of 5Pathology, 6Hematol- 64368309; Fax: 65-63720161; E-mail: [email protected]; ogy, and 7Ear, Nose and Throat, Singapore General Hospital; 8Neuroscience and [email protected]; and Patrick Tan, Cancer and Stem Cell Biology and Behavioral Disorders, 9Cancer and Stem Cell Biology Program, 10Duke- Program, Duke-NUS Graduate Medical School of Singapore, Duke-NUS NUS Graduate Medical School Singapore, 11Cancer Science Institute of Graduate Medical School, 8 College Road, Singapore 169857, Singapore. Singapore, NUS, and 12Genome Institute of Singapore, Singapore E-mail: [email protected]

Note: Supplementary data for this article are available at Cancer Discovery doi: 10.1158/2159-8290.CD-12-0028 Online (http://cancerdiscovery.aacrjournals.org/). ©2012 American Association for Cancer Research.

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RESEARCH BRIEF Koo et al. patients as there is currently no accepted standard fi rst-line heterozygous JAK3A572V, 2 homozygous JAK3A572V, 2 het- treatment for NKTCLs. Despite multiagent chemotherapy and erozygous JAK3A573V, 1 homozygous JAK3A573V, and 1 het- involved-fi eld radiotherapy, the 5-year overall survival rate is erozygous with both JAK3A572V and JAK3A573V mutations. approximately 9% for non-nasal NKTCLs and 42% for nasal These results were further confi rmed by high-resolution NKTCLs (2, 3). There is thus an urgent need to identify melt (HRM) analysis (Supplementary Fig. S1). The presence novel genetic aberrations and potential treatment targets in of nonmalignant stroma (our samples contained at least NKTCLs. 50% tumor content) or tumor subclones makes it diffi cult to In this study, we conducted whole-exome sequencing assess whether these “heterozygous” tumors might actually to identify somatic mutations in protein-coding genes of represent a mixture of JAK3 homozygous–mutated cancer NKTCL tumors to shed light on their pathogenesis and to cells admixed with normal tissue. As such, it is possible that uncover potential new therapeutic targets, which are urgently the number of homozygous tumors reported is actually an needed. underestimate and that this value should be regarded as a lower limit. We also conducted Epstein-Barr virus–encoded RNA RESULTS (EBER) testing on all cases. Apart from 4 older cases that can- Identifi cation and Validation of JAK3 Mutations not be interpreted, all but one case were positive for EBER, Whole-exome sequencing was successfully conducted on regardless of JAK3 mutation status. The single EBER-negative case was a cutaneous deposit taken from a patient with fresh-frozen NKTCLs and paired blood samples from 4 + different patients. The average coverage of each base in EBER nasal NKTCLs (Supplementary Table S3). In parallel, the targeted regions was 111-fold; 84% of the bases were 50% of extra-nasal cases possessed JAK3 mutations and 31.7% represented at least 20 times (Supplementary Table S1) of nasal cases had JAK3 mutations. This latter difference was and a total of 208 somatic mutations were identifi ed in 201 not statistically signifi cant (Supplementary Table S3). genes (Supplementary Table S2). Known somatic mutations A572V in NKTCLs, such as TP53, KRAS, and NRAS (4), identi- JAK3 -Activating Mutations Confer fi ed by exome sequencing were further validated by Sanger Cytokine-Independent Growth sequencing in the same tumors (Supplementary Table S2). Interleukin (IL)-2 is an essential cytokine required for the Interestingly, several somatic heterozygous Janus kinase proliferation and activation of NK cells (6). JAK1 and JAK3 (JAK) mutations were found in 2 separate samples. One mediate IL-2 receptor signaling through phosphorylation of tumor harbored both JAK1Y652D and JAK3A572V mutations, STAT transcription factors (7). In line with the functional whereas the other tumor harbored a JAK3A573V mutation. importance of the activating JAK3 mutations identifi ed, we The JAK3A572V and JAK3A573V mutations were located at exon tested whether JAK3 mutations could confer IL-2–independ- 12, in the Janus homology domain 2 (JH2) that negatively ent growth to the NKTCL cell line (NK-S1) that harbors a regulates the Janus homology domain 1 (JH1) kinase activ- homozygous JAK3A572V mutation. JAK-mutant (NK-S1) cells ity (Fig. 1). The JAK1Y652D mutation was located in the JH2 showed IL-2–independent growth (Fig. 2A) and constitutive domain as well. All 3 missense mutations were predicted by phosphorylation of both JAK3 and STAT5 (Fig. 2B). In con- PolyPhen to be damaging (5). trast, JAK3 wild-type KHYG-1 cells were clearly IL-2–dependent To determine the prevalence of JAK1 and JAK3 muta- (Fig. 2C and D). Importantly, NK-S1 cells treated with JAK3 tions in NKTCLs, we Sanger sequenced an additional 61 siRNAs exhibited a signifi cant reduction in cell prolifera- NKTCL formalin-fi xed, paraffi n-embedded (FFPE) cases. In tion and also decreased autophosphorylation of JAK3 and total, we found mutations in JAK3 in 23 of 65 (35.4%) cases STAT5, compared with cells treated with control siRNAs (Supplementary Table S3) and for JAK1, besides the case (Fig. 3A). Reciprocally, KHYG-1 cells transiently overex- with concomitant JAK1Y652D and JAK3A572V mutations pressing a mutated JAK3 (JAK3A572V) cDNA showed IL-2– described above, no additional mutations were identifi ed. independent proliferation and autophosphorylation of Among the patients with JAK3 mutations, there were 17 JAK3 and STAT5 (Fig. 3B). These results indicate that the

Sample ID: #31 #10 #7 Figure 1. Identiﬁ cation and charac- WT c.1715 C>T c.1718 C>T terization of JAK3-activating mutations. Domain structure of JAK3 (bottom) and the positions of JAK3A572V (c.1715C>T) and JAK3A573V (c.1718C>T; top) identi- ﬁ ed through Sanger sequencing of NKTCL GGAA CCA G GGA AGC/AA GGGGGGCCAAA C/A samples. Glu Ala Ala Glu Ala/Val Ala Glu Ala Ala/Val

A572V A573V

N JH7 JH6 JH4JH5 JH3 JH2 JH1 C

Domain: FERM SH2 Pseudokinase Kinase

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AC Figure 2. IL-2–independent growth and A572V KHYG-1 (JAK3 WT) 1.4 NK-S1 (JAK3 ) 1.4 constitutive JAK3 and STAT5 phosphoryla- IL-2 (0 IU/mL) A572V IL-2 (0 IU/mL) tion in a JAK3 -mutant NKTCL cell line. 1.2 1.2 IL-2 (50 IU/mL) NK-S1 (JAK3A572V homozygous mutant) IL-2 (50 IU/mL) 1 IL-2 (100 IU/mL) and KHYG-1 (wild-type JAK3) cells were 1 IL-2 (100 IU/mL) cultured with or without recombinant IL-2 (200 IU/mL) 0.8 IL-2 (200 IU/mL) 0.8 human IL-2 up to 7 days and followed by MTS assay. A, the NK-S1 cells harboring 0.6 0.6 JAK3A572V were able to grow in an IL-2– independent manner. B, NK-S1 cell lysates 0.4 0.4 were harvested for Western blotting, and 0.2 0.2

the results showed that phosphorylation Absorbance (490–650 nm) Absorbance (490–650 nm) of JAK3 and STAT5 are IL-2–independent. 0 0 C, KHYG-1 cells carrying wild-type JAK3 012345 67 01234567 showed IL-2–dependent proliferation. Time (d) Time (d) D, phosphorylation of JAK3 and STAT5 in KHYG-1 cells were IL-2–dependent. BD Experiments were repeated at least NK-S1 (JAK3A572V) KHYG-1 (JAK3 WT) 3 times. IL-2 – + IL-2 –+

p-JAK3 p-JAK3

JAK3 JAK3

p-STAT5 p-STAT5

STAT5 STAT5

JAK3-activating mutations are gain-of-function alleles and Besides hematologic neoplasia, nonhematologic can- contribute to the constitutive activity of the JAK/STAT path- cers, including breast, stomach, and lung cancer, have also way in an IL-2–independent manner. been found to harbor JAK3 mutations (17, 18). To date, transforming ability of the activating mutations of JAK3 Effects of CP-690550 on NKTCL Cell Lines (such as P132T, L156P, R172Q, E183G, Q501H, M511I, To further confi rm the involvement of JAK/STAT signaling A572V, A573V, R657Q, and V722I) has been previously in the survival of NKTCLs, we next evaluated the effect of a validated in Ba/F3 cells (8, 12, 19, 20). In line with these pan-JAK inhibitor, CP-690550, in NK-S1, KHYG-1, and K562 observations, we identifi ed the presence of activating JAK3 cells. As expected, both the NK-S1 and KHYG-1 cells showed mutations in 35% of NKTCL tumors. The JAK3A572V and a reduction of phosphorylated STAT5 (Fig. 4A) and cell JAK3A573V mutations found in our samples were located viability in a dose-dependent fashion (Fig. 4B). Furthermore, at the JH2 pseudokinase domain that is known to have Annexin V staining revealed that the reduction of NK-S1 an autoinhibitory effect on the JH1 kinase domain. Cel- viability was due to an increase in cellular apoptosis (Fig. 4C). lular studies revealed that the NK-S1 cells harboring the However, this phenomenon was not observed in the K562 homozygous JAK3A572V mutation are able to proliferate cells in which STAT5 phosphorylation is dependent on BCR/ in cell culture without IL-2 stimulation, with constitu- ABL1 (8) and not JAK3 (Fig. 4A and B). tive expression of both phosphorylated JAK3 and STAT5. Cornejo and colleagues (21) showed that when JAK3A572V retroviral–transduced bone marrow cells were transplanted DISCUSSION into C57BL/6 and BALB/c mice, there was a constitutive The JAK/STAT pathway is a key component in normal activation of JAK/STAT signaling which led to the develop- hematopoiesis. The JAK family of tyrosine kinases comprises ment of fatal polyclonal T-cell lymphoproliferative disorder. 4 members: JAK1, JAK2, JAK3, and TYK2. Among these In accordance, transiently overexpressing a JAK3A572V in 4 members, JAK3 signaling is specifi cally related to T-cell a JAK3 wild-type NKTCL cell line (KHYG-1) resulted in development and proliferation (8) with loss-of-function IL-2–independent cell proliferation and the activation of mutations resulting in severe combined immunodefi ciency JAK/STAT signaling pathways. Thus, it is conceivable that characterized by the lack of T and NK cells (9). Recent data the JAK3 mutation may play an important role in the suggest that mutations resulting in persistent activation of pathogenesis of NKTCLs. JAK/STAT signaling are involved in the pathogenesis of T-cell CP-690550, a novel pan-JAK inhibitor, has recently been acute lymphoblastic lymphoma/leukemia, cutaneous TCL, shown to inhibit adult TCL/leukemia (ATLL) cells (22) mantle cell lymphoma, acute megakaryoblastic leukemia, and and ATLL xenograft tumors and is currently in phase III myeloproliferative diseases (8, 10–16). trials for the treatment of rheumatoid arthritis (23).

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A A572V si-Ctrl si-JAK3 NK-S1 (JAK3 ) Figure 3. JAK3A572V mutation causes constitutive JAK3 activity and IL-2–inde- 160 si-Ctrl p-JAK3 pendent proliferation of NKTCL cells. 140 si-JAK3 A, NK-S1 cells were treated with 100 nmol/L JAK3 siRNA (si-JAK3) or control 120 p-STAT5 siRNA (si-Ctrl) for 24 hours before being subjected to proliferation assays up to 100 72 hours (right). In parallel, these cells were harvested, and protein extracts 80 JAK3 were subjected to Western blotting 60 with antibodies against phosphorylated JAK3 (p-JAK3), phosphorylated STAT5

(% vs. si-Ctrl) (% vs. * 40 β STAT5 (p-STAT5), JAK3, STAT5, or -actin as * a normalization control. B, KHYG-1 20

Relative cell proliferation cells were transiently transfected with 0 wild-type JAK3 (JAK3 WT) or mutated β-Actin A572V 0 h 24 h 48 h 72 h JAK3 expression vectors (i.e., JAK3 ). The relative p-JAK3, p-STAT5, JAK3, and STAT5 levels in these cells were detected B KHYG-1 (JAK3 WT) by Western blotting (top), and proliferation assays using these cells were A572V A572V Vehicle JAK3JAK3 Vehicle JAK3 JAK3 conducted for 48 hours with or without IL-2 (bottom). All results are expressed p-JAK3 p-JAK3 as mean ± SEM of 3 independent experiments. *, P < 0.05 compared with vehicle control (vehicle). p-STAT5 p-STAT5

JAK3 JAK3

STAT5 STAT5

β-Actin β-Actin

–IL-2 +IL-2

–IL-2 250 +IL-2

200 *

150 * *

100 (% vs. vehicle) (% vs. 50 Relative cell proliferation

icle WT icle WT A572V A572V Veh Veh JAK3 JAK3 JAK3 JAK3

Consistent with the high frequency of JAK3 mutations potentially effective therapeutic approach that warrants fur- (35%) in NKTCLs, use of CP-690550 in the JAK3-mutant ther investigation. NKTCL cell line showed inhibition in the phosphorylation of STAT5 along with reduced cell viability. These data are compelling and suggest a potential target for this otherwise METHODS fatal disease. Tissue Samples In summary, our studies identiﬁ ed, for the ﬁ rst time, Matched fresh-frozen tissue and peripheral blood samples were frequent JAK3 mutations in NKTCLs. They also indicated obtained from 4 consented patients with NKTCLs for whole-exome that targeting the JAK/STAT pathway in this disease is a sequencing. The JAK3, JAK1, JAK2, and TYK2 mutation status in these

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A NK-S1 (–IL-2) KHYG-1 (+IL-2) K562 CP-690550 (μmol/L) 0 0.5 1.0 2.0 0 0.5 1.0 2.0 0 0.5 1.0 2.0

p-STAT5

STAT5

BC K562 NK-S1 KHYG-1 70 K562 NK-S1 *** 100 *** * * 60 * * *** 80 50 ** * 60 40 * * 30 40 * *** *** 20 *** * * * 20 *** *** ***

Viability (% of control) 10

*** V–positive cells (%) Annexin 0 *** *** *** 0 012345678910 Vehicle 0.5 1.0 2.0 5.0 10.0 Concentration of JAK inhibitor (μmol/L) Concentration of JAK inhibitor (μmol/L)

Figure 4. Effects of CP-690550 on NKTCL cell lines. A, NK-S1, KHYG-1, and K562 cells were treated with CP-690550 for 48 hours, and the effect on STAT5 phosphorylation was evaluated by Western blotting. B, cell viability was analyzed by MTS assay after the cells were treated with their respective treatment for 72 hours. C, drug-induced apoptosis was evaluated by Annexin V-FITC (fl uorescein isothiocyanate) staining, followed by fl ow cytometric analysis. Both NK-S1 and KHYG-1 cells showed a dose-dependent reduction in STAT5 phosphorylation. Treatment with CP-690550 resulted in reduced cell viability of NK-S1 and KHYG-1 cells but not in K562 cells. Experiments were repeated at least 3 times. Data were analyzed by paired t test, and values signifi cantly different from control are shown as *, P < 0.05; **, P < 0.005; ***, P < 0.001.

samples was confi rmed by Sanger sequencing of all coding exons. uses a GATK Unifi ed Genotyper that does the consensus calling FFPE tissue blocks from 61 patients with NKTCLs were procured for to identify SNVs. Only well-mapped reads (mapping quality ≥30, mutation analysis. The diagnosis of NKTCL was made according to number of mismatches within a 40-bp window ≤3) were used as the 2008 World Health Organization classifi cation of tumors of the input to the genotyper. We retained SNVs that passed additional hematopoietic and lymphoid tissues (24). All samples were centrally quality fi lters (a quality by depth ≥5, a variant depth ≥5, a normal reviewed by our hematopathologists. This study was approved by depth ≥5) and discarded any SNV close to a micro-indel or to sev- the SingHealth Centralized Institutional Review Board (CIRB), study eral other SNVs. We compared our variants against the common / / number 2004 407 B. polymorphisms present in dbSNP 131 and in the 1000 genomes databases to discard any common SNPs. All variants retained fol- Preparation of Genomic DNA lowing this step were considered to be novel. Several gene transcript DNA of frozen tissue and paired blood samples was isolated annotation databases (CCDS, RefSeq, Ensembl, and UCSC) were used for transcript identifi cation and for determining the amino using a DNeasy blood and tissue mini kit and a QIAmp DNA blood acid change. Only SNVs in exons or in canonical splice sites were midi kit (Qiagen), respectively, according to the manufacturer’s further analyzed. Amino acid changes corresponding to SNVs were instruction. For FFPE samples, genomic DNA was extracted from annotated according to the largest transcript of the gene. one or two 10-μm slices from each sample by removal of paraffi n followed by proteinase K digestion according to standard proce- Mutation Validation by HRM and Sanger Sequencing dures. DNA was then extracted using a DNeasy blood and tissue Sanger sequencing and HRM (25, 26) were used to confi rm the mini kit (Qiagen). JAK3 and JAK1 mutations identifi ed and validate their prevalence in our NKTCL patient population. The primer sequences used for vali- Whole-Exome Sequencing and Identifi cation dation are listed in Supplementary Table S4. For Sanger sequencing, of Candidate Mutations PCR was carried out with Platinum Taq Polymerase (Life Technolo- A total of 3 μg of genomic DNA extracted from each sample gies, catalog number 10966-083) and cycled at 95°C for 10 minutes; was used for exome sequencing. Captured DNA libraries were 39 cycles of 95°C for 30 seconds; 60°C for 30 seconds, 72°C for sequenced with the Illumina GAIIx Genome Analyzer, yielding 1 minute, and a fi nal extension of 72°C for 10 minutes. Sequencing 150 (2 × 75) base pairs from the fi nal library fragments. We used PCR was carried out with ABI BigDye Terminator v3.1 (Life Tech- Burrows Wheeler Aligner to align the sequence reads to the human nologies, catalog number 4337457) and cycled at 96°C for 1 minute; reference genome NCBI built 37.1 (hg19) and then we ran SamTools 29 cycles of 96°C for 10 seconds; 50°C for 5 seconds, and 60°C to remove PCR duplicates. To detect single-nucleotide variants for 4 minutes. The resulting products were run on an ABI 3730 (SNV), we used a discovery pipeline based on the Genome Analyzer DNA analyzer. For HRM analysis, SsoFast EvaGreen Supermix Toolkit (GATK). Our pipeline fi rst recalibrates the base qualities (Bio-Rad, catalog number 172-5200) was used in the amplifi cation and realigns the sequence reads around micro-indels. The next step of the sample amplicons using the JAK3 HRM primers at a fi nal

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RESEARCH BRIEF Koo et al. concentration of 600 nmol/L on a Bio-Rad CFX96 Real-Time PCR S.C. Chong, K. Tay, M. Tao, R. Quek, S. Loong, K.-W. Yeoh, S.P. detection system in replicates. The cycling and melting conditions Yap, K.A. Lee, L.C. Lim, D. Tan, C. Goh, C.C.Y. Ng, V. Rajasegaran, were as follows: 1 cycle of 98°C for 2 minutes; 39 cycles of 98°C for H.L. Heng, A. Gan, S.T. Lim 5 minutes; 58°C for 10 minutes; 1 cycle of 95°C for 30 minutes; and Analysis and interpretation of data (e.g., statistical analysis, a melt from 72°C to 95°C increasing at 0.2°C/s. The HRM curves biostatistics, computational analysis): G.C. Koo, S.Y. Tan, S.L. Poon, were analyzed with the Bio-Rad Precision Melt Analysis Software. G.E. Allen, W.S. Ong, K.-W. Yeoh, K.A. Lee, I. Cutcutache, W. Yu, HRM difference curves deviating from the wild-type curve were C.C.Y. Ng, V. Rajasegaran, H.L. Heng, A. Gan, C.K. Ong, S. Rozen, P. Tan, considered to be harboring a mutation. B.T. Teh, S.T. Lim Writing, review, and/or revision of the manuscript: G.C. Koo, Cell Lines S.Y. Tan, T. Tang, S.L. Poon, W.S. Ong, K. Tay, R. Quek, K.-W. Yeoh, NK-S1, established from a previously described NKTCL xenograft L.C. Lim, C. Goh, W. Yu, C.C.Y. Ng, C.K. Ong, P. Tan, B.T. Teh, S.T. Lim (27), was cultured in Dulbecco’s Modifi ed Eagle’s Medium (DMEM; Administrative, technical, or material support (i.e., reporting Life Technologies) supplemented with 10% heat-inactivated FBS or organizing data, constructing databases): S.L. Poon, K. Tay, and 10% equine serum. KHYG-1 was obtained from the Japanese C.C.Y. Ng, S.T. Lim Collection of Research BioResources (28) and cultured in RPMI Study supervision: S. Rozen, B.T. Teh, S.T. Lim medium (Life Technologies) supplemented with heat-inactivated Supplied pathologic diagnosis for case series: L. Tan / FBS (10%), equine serum (10%), and 200 IU mL of recombinant Acknowledgments human IL-2 (Novartis). K562 (CCL-234) was purchased from Amer- A572V ican Type Culture Collection and cultured in DMEM supplemented The wild-type JAK3 and JAK3 expression vectors were kindly with 10% heat-inactivated FBS and 10% equine serum. The cod- provided by Dr. Brian Druker. The authors also thank the Lee ing exons of JAK3 were fully sequenced in these 3 cell lines, and Foundation for its support, Huang Dachuan and Waraporn we confi rmed that only NK-S1 harbored a homozygous JAK3A572V Chan-on for proofreading the manuscript, and Sabrina Noyes for mutation. assistance in manuscript submission. This study is dedicated to Dr. Han Mo Koo. Cell Line Transfections Grant Support JAK3 siRNA or control siRNA (Dharmacon) were transfected into This study was funded by the National Medical Research Council NK-S1 cell line using RNAiMAX (Invitrogen) according to the manu- of Singapore (NMRC/PPG/NCC/2011) as well as a grant from HSBC facturer’s protocols. MIGR1 expression vectors containing full-length Trustee (Singapore) Limited as trustees of the Major John Long Trust wild-type JAK3 or JAK3A572V mutant were generously provided by Dr. Fund and the Chew Woon Poh Trust Fund. Brian Druker (Howard Hughes Medical Institute, Chevy Chase, MD; ref. 8). Transient overexpression of these 2 constructs in KHGY-1 cells was then generated using Effectene Transfection Reagent (Qiagen Inc.). Received January 27, 2012; revised May 7, 2012; accepted May 8, 2012; published OnlineFirst June 15, 2012. Cell Viability and Apoptosis Assays Cells were seeded at 2 × 104 cells/100 μL/well in 96-well plates and REFERENCES treated with or without CP-690550 (Selleck Chemical, S5001) at vari- 1. Kwong YL, Anderson BO, Advani R, Kim WS, Levine AM, Lim ST. ous concentrations before being subjected to MTS assay (Promega). 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JAK3 Mutations Identiﬁ ed in NKTCLs RESEARCH BRIEF

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JULY 2012CANCER DISCOVERY | OF7

Janus Kinase 3−Activating Mutations Identified in Natural Killer/T-cell Lymphoma

Ghee Chong Koo, Soo Yong Tan, Tiffany Tang, et al.

Cancer Discovery Published OnlineFirst June 15, 2012.

Updated version Access the most recent version of this article at: doi:10.1158/2159-8290.CD-12-0028

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