clinician to detect possible relapse in patients if TK1 levels start
A minor limitation of measuring serum TK1 activity is the
substantial variance between patients. This variation between
individuals prevents direct comparison between patients. For
this reason, we presented the changes in serum TK1 activity for
the ALL and AML patients as a percentage of the maximum
measured for each individual (Figures 3 and 4). Our results show
that the changes in serum TK1 activity are only useful for
prognosis when compared with the individual’s previous
samples. Therefore, a baseline level of serum TK1 activity must
be established for each individual if serum TK1 is to be used as a
prognostic tool in leukemia, similar to the method currently
used in monitoring prostate-specific antigen in men. Variations
in TK1 levels between serial samples from the same individual
are directly reflective of the response to treatment, indicating
that TK1 measurements can accurately predict patient prog-
Our results comparing the mean serum TK1 levels of ALL
significant differences between the two groups. This confirms
the reality of measuring serum TK1 levels as a diagnostic
tool. Statistical significance between serum TK1 levels in ALL
patients of different stages confirms TK1 as a possible prognostic
This study also indicates that the immunoassay, utilizing
the TK1-specific mAb, is an accurate method of measuring
serum TK1 levels. The development of an immunoassay
to measure serum TK1 levels would be more practical and
efficient for use in a clinical setting. Through continued
investigation of TK1, we hope to provide clinicians with a
valuable tool for earlier detection and improved evaluation of
We thank KEM Baillie and JM Bridges for providing the serial
blood samples from the ALL and AML patients. We also thank the
Biological Carcinogenesis Branch for providing blood samples.
KL O’Neill, F Zhang, H Li, DG Fuja and BK Murray
Department of Microbiology and Molecular Biology,
Brigham Young University, Provo, UT, USA
1 Kantarjian HM. Adult acute lymphocytic leukemia: critical review
of current knowledge. Am J Med 1994; 97: 176–184.
2 O’Neill KL, Buckwalter MR, Murray BK. Thymidine kinase:
diagnostic and prognostic potential. Expert Rev Mol Diagn 2001;
3 Hannigan BM, Barnett YA, Armstrong DB, McKelvey-Martin VJ,
McKenna PG. Thymidine kinases: the enzymes and their clinical
usefulness. Cancer Biother 1993; 8: 189–197.
4 Sherley JL, Kelly TJ. Regulation of human thymidine kinase during
the cell cycle. J Biol Chem 1988; 263: 8350–8358.
5 Johansson M, Karlsson A. Cloning of the cDNA and chromosome
localization of the gene for human thymidine kinase 2. J Biol Chem
1997; 272: 8454–8458.
6 Zhang J, Jia Q, Zou S, Zhang P, Zhang X, Skog S et al. Thymidine
kinase 1: a proliferation marker for determining prognosis and
monitoring the surgical outcome of primary bladder carcinoma
patients. Oncol Rep 2006; 15: 455–461.
7 He Q, Zhang P, Zou L, Li H, Wang X, Zhou S et al. Concentration of
thymidine kinase 1 in serum (S-TK1) is a more sensitive proliferation
marker in human solid tumors than its activity. Oncol Rep 2005; 14:
8 Zhang F, Shao X, Li H, Robison JG, Murray BK, O’Neill KL. A
monoclonal antibody specific for human thymidine kinase 1.
Hybridoma 2001; 20: 25–34.
Phosphoproteomic analysis identifies the M0-91 cell line as a cellular model
for the study of TEL-TRKC fusion-associated leukemia
Leukemia (2007) 21, 563–566. doi:10.1038/sj.leu.2404555;
published online 25 January 2007
The TEL-TRKC gene fusion associated with the t(12;15)(p13;q25)
translocation has been implicated in both hematological
(acute myeloid leukemia (AML))1
malignancies (congenital fibrosarcoma, congenital mesoblastic
nephroma and secretory breast carcinoma). In AML, the
TEL-TRKC (TEL-TRKC(L)) derives from the in-frame fusion of
exons 1–4 of TEL to exons 13–18 of TRKC. In contrast, the
TEL-TRKC variant associated with solid tumors (TEL-TRKC(F))
contains exons 1–5 of the TEL gene. Activation of both the
RAS-MAPK and PI3K-AKT pathways by TEL-TRKC contributes
to oncogenic signaling in transfected NIH3T3 cells.2However,
no human cell lines are available to study the TEL-TRKC
In this study, we screened over 40 AML cell lines for
constitutive phosphorylation of STAT5 by Western blot. The
M0-91, an AML-M0-derived cell line3, showed constitutive
tyrosine phosphorylation of STAT5 (Figure 1a). To identify
protein tyrosine kinases responsible for the constitutive phos-
phorylation of STAT5 in M0-91, cell lysates were trypsin-
digested, and phosphopeptides were immunoprecipitated with
phosphotyrosine antibody (pY-100), and analyzed by LC-MS/MS
mass spectrometry.4,5LC-MS/MS mass spectrometry identified
393 phosphotyrosine sites in 265 proteins (Supplementary
Table 1). Among these proteins, over 15 tyrosine kinases
were tyrosine-phosphorylated (Figure 1b). Multiple tyrosine-
phosphorylated peptides corresponded to either TRKB or TRKC,
including three tyrosines in the activation loop. Thus, either
TRKB or TRKC could be aberrantly activated in M0-91
cells. While full-length TRKB/C have a molecular weight of
140–145kDa, we observed a 50kDa by Western blot with a
pan-TRK antibody in M0-91 cells (Figure 1c). In addition, we
observed tyrosine-phosphorylated peptides deriving from the
TEL protein (Supplementary Table 1). TEL is a member of the ETS
family transcription factor and is essential for hematopoiesis. It is
a frequent target of chromosomal translocations in human
cancers, and one of its fusion partners is TRKC.1To determine
whether a chimeric TEL-TRKC transcript was present, we
performed 30rapid amplification of complementary DNA
(cDNA) ends on the sequence encoding the HLH domain of
TEL. Sequence analysis of the resultant product revealed that the
Letters to the Editor
kinase domain of TRKC was fused to TEL gene (Figure 1d). In
agreement with a previous observation in a patient diagnosed
with TEL-TRKC AML,1the M0-91 cell line expressed TEL-TRKC
fusion transcript (Figure 1e), with exon 4 of TEL fused in-frame to
exon 14 of TRKC. It lacks a 42bp alternative spliced exon in
the TRKC moiety (data not shown). The lack of this exon
enhances tyrosine kinase activity.6,7Thus, this result confirmed
the presence of the ‘leukemic’ form of the TEL-TRKC fusion.
No reciprocal TRKC-TEL fusion gene was detected. The M0-91
cell line did express wild type TEL, but not wild type TRKC
To investigate whether the TEL-TRKC fusion protein con-
tributes to the growth and viability of the M0-91 cells, the
expression of the TEL-TRKC fusion was down regulated with an
siRNA designed against the TRKC portion of the fusion protein.
Western-blot analysis revealed that the expression of the
TEL-TRKC fusion was specifically and significantly reduced at
24 and 48h following transfection of the TRKC siRNA into
M0-91 cells. This is accompanied by a decrease in the
effectors involving RAS-MAPK, PI3K-AKT and PLCg pathways
(AKT, ERK, PLCg1 and PLCg2) (Figure 2a). For the first time, we
phosphorylation of STAT5 in the M0-91 cell line. Detection of phospho-STAT5 is shown in the upper panel and total STAT5 is shown below.
(b) Phosphorylated tyrosine kinases identified by mass spectrometry analysis. ‘y’ in peptide indicates phosphorylated tyrosine residue. ‘y’ stands for
published tyrosine phosphorylation sites. (c) Detection of a truncated form of TRKB/C in M0-91 cells by Western blot. ‘c’ indicates full-length TRK
proteins. (d) Schematic representation of TEL, TRKC and TEL-TRKC proteins. The position of the breakpoint is indicated by arrowhead.
(e) Detection of TEL-TRKC and TEL transcript in theM0-91 cell line.
Fusion of TEL to TRKC in the M0-91 cell line. (a) Westernblot analysis of whole-cell lysates revealed constitutive tyrosine
Letters to the Editor
show that both STAT3 and STAT5 are tyrosine phosphorylated
downstream to TEL-TRKC (Figure 2a), in contrasts to a previous
report, where STATs were not activated in TEL-TRKC trans-
formed BaF3 cells.8Thus, activation of STATs by TEL-TRKC
maybe cell-type specific. Down regulation of TRKC inhibits cell
growth (Figure 2b), and treatment with TRKC siRNA also
increases apoptosis of the M0-91 cell line as measured by a
cleaved Caspase-3 flow cytometric assay (Figure 2c). Similar
results were observed when M0-91 cells were treated with a
relatively selective TRKC inhibitor AG879 (data not shown).
These results suggest that the TEL-TRKC fusion is essential for the
growth and survival of M0-91 cells. The siRNA knockdown
experiments also revealed that phosphorylation of Shc, SHP-2
and SHIP1, as well as expression of both c-Myc and cyclin D1,
is regulated by TEL-TRKC fusion (Figure 2d).
In conclusion, we identified M0-91 as the first cell line
expressing the TEL-TRKC fusion gene from its endogenous
promoter, and therefore the first valuable cell culture model for
the screening for TRKC inhibitors and for understanding both
hematological and non-hematological diseases associated with
TEL-TRKC. In addition, our phosphoproteomic analysis provided
the most comprehensive tyrosine phosphorylation signaling
profile reported for TEL-TRKC fusion to date. This information
will be useful to develop novel therapeutic approaches for the
effective treatment of human cancers associated with deregu-
lated TRKC activity.
T-L Gu1, L Popova1, C Reeves1, J Nardone1, J MacNeill1,
J Rush1, SD Nimer2and RD Polakiewicz1
1Cell Signaling Technology, Inc., Danvers, MA, USA and
2Division of Hematologic Oncology, Memorial
Sloan-Kettering Cancer Center, New York, NY, USA
1 Eguchi M, Eguchi-Ishimae M, Tojo A, Morishita K, Suzuki K,
Sato Y et al. Fusion of ETV6 to neurotrophin-3 receptor TRKC in
acute myeloid leukemia with t(12;15)(p13;q25). Blood 1999; 93:
2 Tognon C, Garnett M, Kenward E, Kay R, Morrison K, Sorensen PH.
The chimeric protein tyrosine kinase ETV6-NTRK3 requires both
Ras-Erk1/2 and PI3-kinase-Akt signaling for fibroblast transforma-
tion. Cancer Res 2001; 61: 8909–8916.
3 Okabe M, Kunieda Y, Shoji M, Nakane S, Kurosawa M, Tanaka J
et al. Megakaryocytic differentiation of a leukemic cell line, MC3,
by phorbol ester: induction of glycoprotein IIb/IIIa and effects on
expression of IL-6, IL-6 receptor, mpl and GATA genes. Leuk Res
1995; 19: 933–943.
4 Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ et al.
Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.
Nat Biotechnol 2005; 23: 94–101.
5 Walters DK, Mercher T, Gu TL, O’Hare T, Tyner JW, Loriaux M
et al. Activating alleles of JAK3 in acute megakaryoblastic leukemia.
Cancer Cell 2006; 10: 65–75.
24h after siRNA transfection. Down regulation of TEL-TRKC fusion results in decreased phosphorylation of STAT5, STAT3, AKT, ERK, PLCg1 and
PLCg2. NP and TRKC stand for sicontrol non-targeting siRNA pool and TRKC SMARTpool siRNA, respectively. (b) Electroporation with TRKC
siRNA into M0-91 cells results in a decrease of proliferation and (c) an increase in apoptosis. (d) Inhibition of TEL-TRKC results in decreased
phosphorylation of Shc, SHP-2 and SHIP1, decreased expression of c-Myc and cyclin D1.
Effects of TRKC siRNA on M0-91 cell line. (a) siRNA-induced silencing of TRKC was assessed by immunoblot of whole-cell lysates
Letters to the Editor
6 Lamballe F, Tapley P, Barbacid M. TrkC encodes multiple Download full-text
neurotrophin-3 receptors with distinct biological properties and
substrate specificities. EMBO J 1993; 12: 3083–3094.
7 Tsoulfas P, Stephens RM, Kaplan DR, Parada LF. TrkC isoforms with
inserts in the kinase domain show impaired signaling responses.
J Biol Chem 1996; 271: 5691–5697.
8 Liu Q, Schwaller J, Kutok J, Cain D, Aster JC, Williams IR et al.
Signal transduction and transforming properties of the TEL-TRKC
fusions associated with t(12;15)(p13;q25) in congenital fibro-
sarcoma and acute myelogenous leukemia. EMBO J 2000; 19:
Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)
CD200 as a prognostic factor in acute myeloid leukaemia
Leukemia (2007) 21, 566–568. doi:10.1038/sj.leu.2404559;
published online 25 January 2007
The CD200 gene (aka MOX-2, OX-2) is located on chromosome
3 and encodes a type-1 membrane glycoprotein.1This protein
belongs to the immunoglobulin superfamily and is expressed on
many different cell types including T and B lymphocytes and
dendritic cells.2CD200 binds multiple membrane receptor
isoforms (CD200R) which have a more tissue restricted
expression.3,4The best characterized isoform, CD200R1, has a
longer cytoplasmic tail containing three conserved tyrosine
residues that can be phosphorylated.1These phosphorylated
residues interact with signalling adaptor molecules such as Shc,
suggesting that the CD200R1 can signal upon binding CD200
ligand providing a more localized response than that provided
by cytokines.5Although CD200(?/?)-deficient mice appear
grossly normal and live a normal lifespan, they are susceptible
to tissue-specific autoimmunity, suggesting that the function
of this protein is to induce immune suppression through
the CD200R.6CD200 also appears to have an alternative
role in regulating osteoclast development.7In leukaemia,
CD200 has been shown to be upregulated in B cells from
patients with chronic lymphocytic leukemia.8More recently,
CD200 has been shown to be an independent prognostic
factor for patients with multiple myeloma.9In this latter
study, expression of CD200 was associated with a bad
prognosis. Here we report that in acute myeloid leukaemia
(AML), there is a correlation between CD200 expression and the
presence of the core binding factor (CBF) associated abnorm-
alities, t(8;21) and inv(16). However, at the same time CD200
expression was linked to worse overall survival in other AML
subsets. These data indicate that CD200 is also of prognostic
value in AML.
Using a cohort of 184 AML trial patients (all processed and
diagnosed at Cardiff as part of the Medical Research Council
(MRC) AML Trials), complementary RNA was prepared from
each sample and hybridized to Affymetrix Human 133A
oligonucleotide arrays which allowed the analysis of CD200
gene expression. In approximately 43% of AML patients, CD200
had a ‘present’ Affymetrix call (CD200present) with the remainder
giving an absent call (CD200absent). CD200presentcalls were
highly correlated with abnormalities affecting CBF (24/28 vs
56/156, P¼0.0001). There was no evidence of any association
with age at diagnosis (mean ages: present 49 years vs absent 50
years, P¼0.7), WBC (P¼0.3) or sex (P¼0.6), however, larger
numbers of patients will be required to provide more reliable
evidence and allow for adjustment of clinical and demographic
parameters. Despite the association of CD200 expression with
these good risk subtypes, analyses of survival stratified for CBF
abnormalities showed that CD200 was significantly associated
with worse survival (HR 1.68, 95% CI 1.08–2.62, P¼0.02;
Figure 1a and b).
Meier plot of the overall survival in non-CBF leukaemia patients
expressing CD200 (CD200present) or not (CD200absent). (b) Odds ratio
plots of survival stratified by mutations affecting CBF (t(8;21) and
inv(16)). (c) Box and whisker plots depicting microarray data of the
normalized expression of CD200 in AML patients (n¼184) classified
according to FAB criteria.wM2 patients without a t(8;21).yM2 patients
with a t(8;21).nData outliers.
CD200 expression in patients with AML. (a) Kaplan–
Letters to the Editor