A small molecule blocking oncogenic protein EWS-FLI1 interaction with RNA helicase A inhibits growth of Ewing's sarcoma.
ABSTRACT Many sarcomas and leukemias carry nonrandom chromosomal translocations encoding tumor-specific mutant fusion transcription factors that are essential to their molecular pathogenesis. Ewing's sarcoma family tumors (ESFTs) contain a characteristic t(11;22) translocation leading to expression of the oncogenic fusion protein EWS-FLI1. EWS-FLI1 is a disordered protein that precludes standard structure-based small-molecule inhibitor design. EWS-FLI1 binding to RNA helicase A (RHA) is important for its oncogenic function. We therefore used surface plasmon resonance screening to identify compounds that bind EWS-FLI1 and might block its interaction with RHA. YK-4-279, a derivative of the lead compound from the screen, blocks RHA binding to EWS-FLI1, induces apoptosis in ESFT cells and reduces the growth of ESFT orthotopic xenografts. These findings provide proof of principle that inhibiting the interaction of mutant cancer-specific transcription factors with the normal cellular binding partners required for their oncogenic activity provides a promising strategy for the development of uniquely effective, tumor-specific anticancer agents.
- SourceAvailable from: Haydar Celik[Show abstract] [Hide abstract]
ABSTRACT: Background: The erythroblastosis virus E26 transforming sequences (ETS) family of transcription factors consists of a highly conserved group of genes that play important roles in cellular proliferation, differentiation, migration and invasion. Chromosomal translocations fusing ETS factors to promoters of androgen responsive genes have been found in prostate cancers, including the most clinically aggressive forms. ERG and ETV1 are the most commonly translocated ETS proteins. Over-expression of these proteins in prostate cancer cells results in a more invasive phenotype. Inhibition of ETS activity by small molecule inhibitors may provide a novel method for the treatment of prostate cancer. Methods and Findings: We recently demonstrated that the small molecule YK-4-279 inhibits biological activity of ETV1 in fusion-positive prostate cancer cells leading to decreased motility and invasion in-vitro. Here, we present data from an in-vivo mouse xenograft model. SCID-beige mice were subcutaneously implanted with fusion-positive LNCaP-luc-M6 and fusion-negative PC-3M-luc-C6 tumors. Animals were treated with YK-4-279, and its effects on primary tumor growth and lung metastasis were evaluated. YK-4-279 treatment resulted in decreased growth of the primary tumor only in LNCaP-luc-M6 cohort. When primary tumors were grown to comparable sizes, YK-4-279 inhibited tumor metastasis to the lungs. Expression of ETV1 target genes MMP7, FKBP10 and GLYATL2 were reduced in YK-4-279 treated animals. ETS fusion-negative PC-3M-luc-C6 xenografts were unresponsive to the compound. Furthermore, YK-4-279 is a chiral molecule that exists as a racemic mixture of R and S enantiomers. We established that (S)-YK-4-279 is the active enantiomer in prostate cancer cells. Conclusion: Our results demonstrate that YK-4-279 is a potent inhibitor of ETV1 and inhibits both the primary tumor growth and metastasis of fusion positive prostate cancer xenografts. Therefore, YK-4-279 or similar compounds may be evaluated as a potential therapeutic tool for treatment of human prostate cancer at different stages.PLoS ONE 12/2014; · 3.53 Impact Factor
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ABSTRACT: Ewing sarcoma is a cancer of bone and soft tissue in children that is characterized by a chromosomal translocation involving EWS and an Ets family transcription factor, most commonly Fli-1. EWS-Fli-1 fusion accounts for 85% of cases. The growth and survival of Ewing sarcoma cells are critically dependent on EWS-Fli-1. A large body of evidence has established that EWS-Fli-1 functions as a DNA-binding transcription factor that regulates the expression of a number of genes important for cell proliferation and transformation. However, little is known about the biochemical properties of the EWS-Fli-1 protein. We undertook a series of proteomic analyses to dissect the EWS-Fli-1 interactome. Employing a proximity-dependent biotinylation technique, BioID, we identified cation-independent mannose 6-phosphate receptor (CIMPR) as a protein located in the vicinity of EWS-Fli-1 within a cell. CIMPR is a cargo that mediates the delivery of lysosomal hydrolases from trans-Golgi network to endosome, which are subsequently transferred to the lysosomes. Further molecular cell biological analyses uncovered a role for lysosomes in the turnover of the EWS-Fli-1 protein. We demonstrate that an mTORC1 active-site inhibitor torin 1, which stimulates the TFEB-lysosome pathway, can induce the degradation of EWS-Fli-1, suggesting a potential therapeutic approach to target EWS-Fli-1 for degradation.Journal of Proteome Research 07/2014; · 5.00 Impact Factor
- Nucleic Acids Research 01/2015; · 8.81 Impact Factor
A small molecule blocking oncogenic protein EWS-FLI1
interaction with RNA helicase A inhibits growth of
Hayriye V Erkizan1, Yali Kong1, Melinda Merchant2, Silke Schlottmann1, Julie S Barber-Rotenberg1,
Linshan Yuan1, Ogan D Abaan1, Tsu-hang Chou2, Sivanesan Dakshanamurthy1, Milton L Brown1,
Aykut U¨ren1& Jeffrey A Toretsky1,3,4
Many sarcomas and leukemias carry nonrandom chromosomal translocations encoding tumor-specific mutant fusion transcription
factors that are essential to their molecular pathogenesis. Ewing’s sarcoma family tumors (ESFTs) contain a characteristic t(11;22)
translocation leading to expression of the oncogenic fusion protein EWS-FLI1. EWS-FLI1 is a disordered protein that precludes
standard structure-based small-molecule inhibitor design. EWS-FLI1 binding to RNA helicase A (RHA) is important for its
oncogenic function. We therefore used surface plasmon resonance screening to identify compounds that bind EWS-FLI1 and might
block its interaction with RHA. YK-4-279, a derivative of the lead compound from the screen, blocks RHA binding to EWS-FLI1,
induces apoptosis in ESFT cells and reduces the growth of ESFT orthotopic xenografts. These findings provide proof of principle
that inhibiting the interaction of mutant cancer-specific transcription factors with the normal cellular binding partners required for
their oncogenic activity provides a promising strategy for the development of uniquely effective, tumor-specific anticancer agents.
There is a considerable need for new cancer therapies that enhance
efficacy and reduce long-term morbidity. Protein products of tumor-
specific chromosomal translocations provide unique targets for anti-
tumor therapies1. These translocations span a broad range of malig-
nancies, including carcinomas, hematopoietic malignancies and
sarcomas2–4. In many cancers, these translocations lead to new fusion
proteins that both initiate and maintain oncogenesis. Although some
of these translocations, such as breakpoint cluster region–Abelson
oncoprotein (BCR-ABL)5, lead to constitutively activated kinases, the
majority lead to fusion proteins that function as transcription factors
and lack intrinsic enzymatic activity. These translocation-generated
transcription factor fusion proteins are ideal targets of anticancer
therapies, yet no specific pharmaceuticals have been developed to date.
The ESFTs are undifferentiated tumors that can occur anywhere in
the body, most often in the second and third decades of life. ESFTs
often respond well to initial chemotherapy, yet 40% of patients will
develop lethal recurrent disease. Seventy-five to eighty percent of
people who present with metastatic ESFTs will die within 5 years,
despite high-dose chemotherapy6. ESFTs contain a well-characterized
chromosomal translocation that fuses the amino half of EWS to the
carboxy half of an ets (erythroblastosis virus E26 transforming
sequence gene) family DNA binding protein7. The most common
fusion protein is the oncogenic transcription factor EWS-FLI1.
Elimination of EWS-FLI1 through antisense and small interfering
RNA approaches results in the prolonged survival of ESFT
xenograft–bearing mice8, but this approach currently lacks translation
to clinical therapy9,10. As EWS-FLI1 lacks intrinsic enzymatic activity,
small-molecule targeting would be directed toward the disruption of
EWS-FLI1 from established transcriptional complexes. The EWS-FLI1
transcriptional complex includes: RNA polymerase II, cyclic AMP
response element–binding protein and RHA11–13. Our previous inves-
tigations showed that RHA augments EWS-FLI1–modulated onco-
genesis, suggesting that this protein-protein complex is particularly
essential for tumor maintenance13. Small-molecule inhibitors that
block RHA interaction by targeting the oncogenic fusion protein
EWS-FLI1 would be the first in a new class of antitumor therapy
directed at these proteins.
RHA has a crucial role in embryogenesis and thus might be a
reasonable option as a partner for an oncoprotein in undifferentiated
tumors and is indispensable for ectoderm survival in gastrulation
of mammals14. RHA is also required beyond embryogenesis because
RHA-null mouse fibroblast cells are not viable (C.-G. Lee (University
of Medicine and Dentistry of New Jersey), personal communication).
However, transient reduction of RHA protein expression in COS cells
did not affect their viability15. RHA provides a transcriptional coacti-
vator role in models of tumorigenesis, and in the nuclear factor-kB
(NF-kB)16and signal transducer and activator of transcription-6
(ref. 17) transcriptomes. RHA binds DNA in a sequence specific
manner within the promoters of the genes encoding cyclin-dependent
kinase inhibitor 2A (ref. 18) and multidrug resistance protein-1
Received 2 March; accepted 8 May; published online 5 July 2009; doi:10.1038/nm.1983
1Georgetown University, Lombardi Comprehensive Cancer Center, Department of Oncology, Washington, DC, USA.2Memorial Sloan-Kettering Cancer Center, New York,
New York, USA.3Georgetown University, Department of Pediatrics, Washington, DC, USA.4Correspondence should be addressed to J.A.T. (firstname.lastname@example.org).
750VOLUME 15 [ NUMBER 7 [ JULY 2009 NATURE MEDICINE
© 2009 Nature America, Inc. All rights reserved.
(ref. 19). The amino-terminal region of RHA is most often the site for
protein-protein interactions. cAMP-binding protein binds amino
acids 1–250 of RHA20, RNA polymerase II and breast cancer
protein-1 (ref. 21), and RNA-induced silencing complex compo-
nents22bind in the amino-terminal region. EWS-FLI1 binds RHA in
a unique region that is not occupied by other transcriptional or RNA
metabolism proteins13, thus increasing the attractiveness of this
protein-protein interaction target.
Disruption of protein-protein interactions by small molecules is a
rapidly evolving field. Proteins with more flexible structures, in some
cases disordered proteins, have a greater potential for small-molecule
binding than rigid proteins because of higher induced-fit sampling
probabilities23. A disordered protein is defined, in part, by increased
intrinsic movement and the inability to form rigid three-dimensional
structures (reviewed in ref. 24). EWS-FLI1 is a disordered protein and
requires the disorder for maximal transactivation of transcription25,26.
On the basis of these observations, EWS-FLI1, along with its binding
to RHA, may provide a unique drug target even without structural
information of the EWS-FLI1 protein.
RHA is a validated target in ESFTs
A region of RHA that binds EWS-FLI1 was identified based upon
phage-display epitope screening13(Fig. 1a). To validate RHA as
essential for the survival of ESFT cells, we lowered RHA levels
with short hairpin RNA (shRNA), and ESFT cell viability was reduced
by 90% (Fig. 1b,c). We stably transfected PANC1 cells, a pancreatic
cell line that does not express EWS-FLI1, with the same shRNA
vectors, yielding a similar reduction in RHA abundance (Supplemen-
tary Fig. 1a) but with no decrease in cell viability (Supplementary
Fig. 1b). We further validated the RHA and EWS-FLI1 interaction
with site-directed mutagenesis in the GST-RHA647–1075fragment to
identify mutants that don’t interact with EWS-FLI1. We expressed
GST-RHA647–1075mutants and immunoprecipitated them with full
length recombinant EWS-FLI1. Mutants P824A and D827A showed a
significant decrease in binding to EWS-FLI1 compared to wild-type
control RHA (P ¼ 0.0129 and P¼ 0.0034, respectively; Fig. 1d).
The full-length RHA mutant D827A maintained wild-type ATPase
activity (Supplementary Fig. 2); therefore, we chose the D827A
mutant to test whether RHA binding to EWS-FLI1 is required for
We stably transfected mouse embryonic fibroblasts (W cells) that
express low levels of endogenous RHA13with EWS-FLI1 (WEF1 cells)
and either full-length wild-type RHA or full-length RHA D827A. We
observed a greater than additive effect of RHA and EWS-FLI1 expres-
sion on anchorage independent growth when comparing the colony
numbers from W cells expressing RHA (227 ± 66 colonies) and WEF1
cells (115 ± 8 colonies) to those of WEF1 cells expressing RHA (582 ±
30 colonies) (Fig. 1e,f). The RHA D827A–expressing cells showed
a 60% reduction in anchorage-independent growth (P ¼ 0.0028)
compared to cells expressing wild-type RHA (Fig. 1e,f). We quantified
EWS-FLI1 expression by densitometry of the immunoblot (Fig. 1g,h).
The markedly lower colony formation by the RHA D827A–expressing
cells suggests a crucial role of RHA for transforming activity of
EWS-FLI1 that is abrogated by RHA not binding to EWS-FLI1.
E9R peptide disrupts binding and inhibits growth
As RHA is necessary for optimal EWS-FLI1 activity, we developed
reagents to block RHA binding to EWS-FLI1. The E9R peptide
corresponds to amino acids 823–832 of RHA. With the immunopre-
cipitation assay, we assessed binding between bacterially expressed
GST-RHA647–1075and full-length purified recombinant EWS-FLI1
(Fig. 2a). Titration of E9R showed a dose-dependent reduction in
the binding of GST-RHA647–1075and full-length EWS-FLI1 with a
decreased association to 50% with 0.1 mM E9R (Fig. 2a). We thus
sought to determine whether disrupted EWS-FLI1–RHA binding
inhibits cell growth.
Peptide delivery to growing cells is greatly facilitated by cell-
permeable peptides (CPP)27. We fused the CPP antennapedia to the
amino terminus of E9R or with the D827A mutation (E9R-P and
469 181 250 411767
RGG Box and NTS
EWS-FLI1 b inding (%)
RHA 5RHA 7
RHA 5 RHA 7
WEF1 + RHA
W + RHA
WEF1 + RHA
W + RHA
Figure 1 RHA is necessary for optimal transformation by EWS-FLI1. (a) A schematic representation of RHA, including the
region that binds EWS-FLI1. The E9R peptide corresponds to amino acids 823–832, located just proximal to the HA2 region
of RHA. dsRBD, double-stranded RNA–binding domain, RGG box, arginine glycine glycine box; NTS, nuclear transport signal; aa, amino acid residue.
(b) An shRNA expression vector was transfected into TC71 (ESFT) cells to reduce RHA levels. (c) TC71 viability after RHA knockdown, as measured
by cell proliferation reagent water-soluble terazolium salt (WST) reduction. (d) Alanine mutagenesis within E9R sequence was followed by in vitro
immunoprecipitation with EWS-FLI1. The density of the GST-RHA band was measured, and this graph is the average of three experiments. RHA P824A
and D827A mutants have significantly lower binding to EWS-FLI1 (*P ¼ 0.0129 and **P ¼ 0.0034, respectively). (e) Mouse fibroblasts were placed in
soft agar for anchorage-independent growth assays (empty vector (W), EWS-FLI1 alone (WEF1)). (f) The graph enumerates the colonies counted in three
separate experiments. (g) Protein expression of fibroblasts, detected with antibody to Flag (top) or antibody to FLI1 (bottom). (h) Densitometry of the
EWS-FLI1 blot, performed with MultiGauge software.
NATURE MEDICINE VOLUME 15 [ NUMBER 7 [ JULY 2009751
© 2009 Nature America, Inc. All rights reserved.
E9R(D5A)-P, respectively; Supplementary Table 1). We treated
monolayer cultures of the EWS-FLI1–positive ESFT cell line, TC32,
or a control EWS-FLI1–negative cell line, SKNAS (neuroblastoma),
with fluorescein-conjugated peptides. Only the EWS-FLI1–containing
TC32 cells showed reduced growth with E9R peptide, and the SKNAS
cells showed mild stimulation from the E9R peptide via an unknown
mechanism (Fig. 2b). Confocal microscopy showed uptake through-
out the cell, including nuclei (as evidenced by DAPI overlay, Fig. 2c).
E9R-P significantly reduced ESFT cell growth (P ¼ 0.048), while
neither the D5A mutant control nor antennapedia peptides alone
reduced ESFT cell growth (Fig. 2d). Neuroblastoma cells treated with
the same peptides did not have a statistically significant alteration in
growth, although we observed a slight increase with E9R(D5A)-P–
treated cells (P ¼ 0.175; Fig. 2d). To determine the effect of E9R on
sequence into an EGFP-expressing plasmid (pGE9R). We also
expressed EGFP-E9R peptide only in cytoplasm by adding a nuclear
export signal sequence (LQLPPLERLTL) to the plasmid28. We stably
transfected EGFP-E9R plasmid into TC71 (ESFT) or SKNAS (neuro-
blastoma) cells. Transfected cells showed E9R peptide expression either
throughout the cell or excluded from the nucleus, as predicted on the
basis of the intended targeting (Fig. 2e). TC71 colony formation was
95% lower owing to the expression of E9R, except when the peptide
was excluded from the nucleus (P ¼ 0.0012; Fig. 2e,f). The anchorage-
independent growth of SKNAS was not affected by the E9R peptide
(Fig. 2e,f). Further supporting the specificity of the E9R peptide, a
second small round blue cell tumor, embryonal rhabdomyosarcoma,
expressing pGE9R did not show reduced anchorage-independent
growth (data not shown). Only expression of EGFP-E9R in TC71
reduced anchorage-independent growth (Fig. 2e,f).
Optimized small molecule binds to EWS-FLI1
We screened a library of 3,000 small molecules (National Cancer
Institute Drug Targeting Program) for EWS-FLI1 binding by using
surface plasmon resonance (SPR). We selected compounds that bind
monomeric EWS-FLI1. We evaluated the binding state of the com-
pound to EWS-FLI1 by the ratio of actual binding resonance units
(RUactual) to the theoretical binding resonance units (RUtheor). A ratio
below 1.0 indicated monomeric binding of compound to EWS-FLI1.
NSC635437 had an RUactualto RUtheorratio of 0.9, signifying mono-
meric binding to EWS-FLI1. NSC635437 had a greater potential to
chemical derivatization with favorable drug-like properties29. We
synthesized 1.0 g of NSC635437 to complete our studies and for use
as a standard during compound optimization (Fig. 3a).
In a cell-free assay, NSC635437 reduced the direct binding of GST-
RHA647–1075to full-length recombinant EWS-FLI1 (Fig. 3b). We used
an aromatic optimization strategy to design analogs to improve the
inhibition of RHA binding to EWS-FLI1 by NSC635437. One of these
compounds (YK-4-279), substituted with a methoxy group at the para
position (p-methoxy) of the aromatic ring (Fig. 3a), markedly reduced
the protein-protein interaction of EWS-FLI1 with GST-RHA647–1075
in vitro (Fig. 3b). We calculated a KDof 9.48 mM for the affinity of
YK-4-279 with EWS-FLI1 by SPR (Fig. 3c). To support a model of
YK-4-279 as having similar interaction qualities to E9R, we used an
SPR displacement assay to show that 10 mM YK-4-279 reduces the
binding of 64 mM E9R from 17 RU to 7 RU, and 32 mM E9R reduces
the binding from 13 RU to 5 RU (Fig. 3d). YK-4-279 at 30 mM
completely displaced E9R from EWS-FLI1 binding, as measured by
fluorescence polarization assay (Fig. 3e).
YK-4-279 functionally inhibits EWS-FLI1 and ESFT cells
ESFT cells treated with YK-4-279 showed a dissociation of EWS-FLI1
from RHA by 10 mM, consistent with the KDvalue (Fig. 4a). YK-4-279
did not directly affect EWS-FLI1 or RHA levels (Fig. 4a and Supple-
mentary Fig. 3). To further support YK-4-279 as a functional
inhibitor of EWS-FLI1, we transfected COS7 cells with EWS-FLI1
and NR0B1 reporter-luciferase plasmid (containing EWS-FLI1 regu-
latory GGAA elements30). The EWS-FLI1–transfected cells showed a
dose-dependent decrease in promoter activity when treated for 18 h
with 3 mM and 10 mM YK-4-279 (Fig. 4b,c). As an additional control
for nonspecific promoter effects, we transfected an NF-kB–responsive
reporter into COS7 cells and activated it with phorbol 12-myristate
13-acetate. YK-4-279 did not affect the NF-kB–responsive promoter
(Supplementary Fig. 4a). In a recent publication, EWS-FLI1 was
Cell growth (%)
10 101 0.1 0.01
Cell growth (%)
Figure 2 E9R peptide prevents EWS-FLI1 binding to RHA with
specific detrimental effects upon ESFT growth and transformation.
(a) Immunoprecipitation of GST-RHA647–1075using recombinant full-length
EWS-FLI1 bound to a FLI1-specific antibody. (b) Growth reduction upon
E9R-P (antennapedia-E9R) treatment (10 mM) in TC32 cells but not SKNAS
cells. (c) E9R-P peptide uptake, tracked with FITC label (top images).
DAPI nuclear counterstain (middle images) and merged (bottom images)
are shown. Scale bar, 20 mm. (d) Graph showing growth response of
TC32 and SKNAS cells to E9R–P, Antennapedia alone (Antp) or E9R-D5A-P.
(e) TC71 and SKNAS cells expressing EGFP empty vector (pG), EGFP-E9R
(pGE9R), EGFP with nuclear export sequence (pGC) or EGFP-E9R
with nuclear export sequence (pGCE9R). (f) Average colony numbers
of three experiments in TC71 cells expressing E9R throughout the cell.
Scale bar, 20 mm.
752VOLUME 15 [ NUMBER 7 [ JULY 2009 NATURE MEDICINE
© 2009 Nature America, Inc. All rights reserved.
shown to modulate cyclin D1 protein abundance by altering a cyclin
D1 splice site31. Blocking the interaction of EWS-FLI1 with RHAusing
YK-4-279 nearly eliminated cyclin D1 in TC32 cells treated for 14 h
(Fig. 4d) but did not affect cyclin D1 expression in four non–EWS-
FLI1–containing cell lines (Supplementary Fig. 4b,c).
We found that NSC635437 has a half-maximal inhibitory
concentration (IC50) of 20 mM for TC32 cells growing in monolayer;
however, the derivative, YK-4-279, reduced the IC50to 900 nM
(Fig. 5a). YK-4-279 was relatively specific for ESFT cells as compared
to the nontransformed HEK293 cells, showing a tenfold difference in
IC50(Fig. 5b). Primary cell lines, ES925 and GUES1, established
from individuals with ESFTs with recurrent tumors showed sensitivity
to YK-4-279 with antiproliferative IC50values of 1 mM and 8 mM,
respectively (Fig. 5c). A panel of ESFT cell lines showed IC50
values between 0.5 mM and 2 mM for YK-4-279, whereas cell lines
that lack EWS-FLI1 had IC50values in excess of 25 mM (Fig. 5d).
An additional panel of nontransformed human foreskin keratinocytes
(HFK cells) and human ectocervical cells (HEC cells) treated
for 3 d with 30 mM YK-4-279 showed an IC50that exceeded 30 mM
(Supplementary Fig. 5a).
As an apoptotic indicator, caspase-3 activity32rose in a dose-
dependent fashion in TC32 cells treated with YK-4-279 for 24 h
(Supplementary Fig. 5b). Caspase-3 activation in response to YK-4-
279 was similar to that induced by doxorubicin, a standard agent in
the treatment of patients with ESFT6. We evaluated additional
malignant and nonmalignant cell lines for caspase-3 activation in
response to YK-4-279. YK-4-279 induced caspase-3 activity in four
ESFT cell lines (TC32, A4573, TC71 and ES925 cells), however none
of the five non–EWS-FLI1 cancer cell lines nor any of the three
nontransformed cell lines (HFK, HEC and HEK293 cells) treated with
YK-4-279 underwent apoptosis (Fig. 5e). Treatment of TC32,
HEK293, HFK and HEC cells with short-term (6-h) high-dose
(50 mM) YK-4-279 resulted in substantial apoptosis of the ESFT
cells but no death of the nontransformed cells (Fig. 5f). Together,
these results support the specific toxicity of YK-4-279 in tumor cell
lines containing EWS-FLI1 compared with other tumor and non-
To further support for the target specificity of YK-4-279 toxicity in
ESFT cells, we reduced the levels of EWS-FLI1 and RHA proteins by
using shRNA in A673 cells33. Cells with knocked down RHAR showed
a YK-4-279 IC50of 410 mM, whereas cells treated with the control
shRNA (targeting luciferase) had a YK-4-279 IC50of less than 1 mM
(Supplementary Fig. 5c). When we lowered EWS-FLI1 expression
with shRNA, the IC50increased tenfold from 0.5 mM to approximately
5 mM (Supplementary Fig. 5d,e).
ESFT xenograft growth is inhibited by YK-4-279
We established ESFT (orthotopic) with CHP-100 and TC71 or
prostate cancer with PC3 cell xenograft tumors in severe combined
YK-4-279 concentration (µM)
10.0 15.0 20.0 25.0
KD = 9.48 µM
YK-4-279 (10 µM)
NSC635473 (10 µM)
64 µM E9R-P alone
64 µM E9R-P + YK-4-279
32 µM E9R-P alone
32 µM E9R-P + YK-4-279
Figure 3 Small molecule binds EWS-FLI1 and
displaces E9R from EWS-FLI1. (a) NSC635437,
indol-2-one, was synthesized with 100% yield.
Aromatic optimization produced YK-4-279 a
para-methoxy derivative of NSC635437.
(b) EWS-FLI1 was incubated with NSC635437
(left) or YK-4-279 (right) followed by the
addition of GST-RHA647–1075. EWS-FLI1 and
GST-RHA647–1075were precipitated from the
solution with an FLI1-specific antibody.
(c) YK-4-279 steady-state kinetics for binding to
recombinant EWS-FLI1 that was immobilized on
a CM5 Biacore chip. (d) SPR displacement assay of 64 mM E9R alone and with addition of YK-4-279; 32 mM E9R alone and with addition of YK-4-279.
(e) Graph showing YK-4-279 displacement of E9R-P from EWS-FLI1, as measured by fluorescent polarization.
YK-4-279 (µM) 0
3 1003 10
Total cell lysate
Figure 4 YK-4-279 reduces EWS-FLI1 functional activity. (a) TC32
cells were treated with YK-4-279, and resolved protein lysates were
immunoblotted for co-precipitated RHA (top), EWS-FLI1 (middle) or total
RHA (bottom). IP, immunoprecipitate; IB, immunoblot. (b) Luciferase
reporter assay of the EWS-FLI1–responsive NR0B1 promoter upon dose-
dependent (18-h) YK-4-279 treatment in COS7 cells. (c) Protein lysates
from transfected cells showing expression of EWS-FLI1. (d) YK-4-279–
treated TC32 cell lysates (treated for 14 h) were blotted for cyclin D1
NATURE MEDICINE VOLUME 15 [ NUMBER 7 [ JULY 2009753
© 2009 Nature America, Inc. All rights reserved.
immunodeficient–beige mice. The tumor growth rate of YK-4-279–
treated mice bearing CHP-100 (Fig. 6a) was lower than that in mice
having PC3 prostate tumors (Fig. 6b). The cumulative data from five
independent experiments with the ESFT xenografts (TC71 and
CHP-100) show a marked overall tumor reduction (P o 0.0001) in
the YK-4-279–treated mice (Fig. 6c). Pathological analysis of mice
treated with YK-4-279 did not show any signs of toxicity, except for
sterile inflammatory lesions in the abdominal cavities of mice, where
at 72 h (%)
YK-4-279 (log µM)
at 72 h (%)
0.1 110 100
at 72 h (%)
Fold caspase-3 activity
Figure 5 YK-4-279 is a potent and specific inhibitor of ESFTs. (a) TC32 cells were treated with a dose range of YK-4-279 and NSC635437. Cell growth,
as measured by 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) or WST reduction after 7 d in culture. (b) TC32 and HEK293
(nontransformed, lacking EWS-FLI1) cells were treated similarly to those in a. (c) Primary ESFT explant cell lines GUES1 and ES925 were treated for 3 d
with YK-4-279. (d) Cell lines expressing EWS-FLI1 were compared to non–EWS-FLI1 malignant cell lines after 3 d in culture to establish the IC50using
WST assay. (e) Caspase-3 activity of a panel of ESFT (TC32, TC71, A4573 and ES925), malignant non–EWS-FLI1–expressing (MCF-7, MDA-MB-231, PC3,
ASPC1 and COLO-PL) and nontransformed (HEK-293, HFK and HEC) cells. Graph shows fluorescence in treated lysate divided by that of untreated lysate.
(f) Arrows indicate apoptotic nuclear fragmentation after 50 mM YK-4-279 treatment of ESFT (TC32) cells and nontransformed cells (HEK-293, HFK and
HEC). Scale bar, 200 mm.
Time (d) after tumor cell injection
0 7 14 2128 3542
Tumor volume fold increase
lower extremity volume (cm3)
lower extremity volume (cm3)
Time (d) after tumor cell injection
TC71 treated with DMSO
CH100 treated with DMSO
TC71 treated with YK-4-279
CHP100 treated with YK-4-279
Figure 6 YK-4-279 inhibits the growth of ESFT xenograft
tumors. Xenografts were established with injection of
either ESFT (CHP-100 or TC71) or prostate cancer (PC3)
cells. (a) CHP-100 intramuscular xenografts (arrow
indicates when tumors were palpable) received DMSO
(n ¼ 4) or 1.5 mg YK-4-279 (n ¼ 5) (P ¼ 0.016, by t
test comparison). The single-experiment growth curves
depicted are representative of five independent
experiments. (b) PC3 subcutaneous xenografts (arrow
indicates when tumors were palpable) were treated
as the CHP-100 cells were in a (n ¼ 5 per group,
representative of three independent experiments). (c) Overall response of ESFT xenografts (TC71 and CHP-100) to YK-4-279 (1.5 mg per dose). Tumor
volumes at day 14 after treatment initiation compared across five experiments are shown (DMSO, n ¼ 19; YK-4-279, n ¼ 25; P o 0.0001, by Mann-
Whitney test). (d) Tumors from the mice in a were analyzed by immunohistochemistry for activation of caspase-3 activity. (e) Caspase-3–positive cells were
counted (n 4 500 in three high-power fields) in four separately stained slides for each group (P ¼ 0.041).
754VOLUME 15 [ NUMBER 7 [ JULY 2009 NATURE MEDICINE
© 2009 Nature America, Inc. All rights reserved.
intraperitoneal injections were applied. Tumors from mice treated
with YK-4-279 were compared with those after DMSO treatment by
immunohistochemistry to identify caspase-3 activity (Fig. 6d). The
CHP-100 xenograft tumors from treated mice had a threefold increase
in caspase-3 activity compared to control mice (Fig. 6e). These results
show inhibition of tumor growth and concomitant increased apop-
tosis after YK-4-279 treatment in two models of ESFT.
EWS-FLI1 is a unique, cancer-specific molecule that is a potential
therapeutic target in ESFT cells. RHA is essential for the function of
EWS-FLI1. We showed that an E9R peptide that blocks RHA binding
to EWS-FLI1 (E9R) specifically reduced the transformation activity of
EWS-FLI1. We also identified a small-molecule lead compound that
binds EWS-FLI1. The lead compound derivative, YK-4-279, along
with E9R peptide, shows that the EWS-FLI1–RHA interaction can be
blocked with a detrimental effect on ESFT cells both in vitro and
in vivo. These findings validate a highly specific cancer target, the
interaction of EWS-FLI1 with RHA.
These are to our knowledge the first experiments that evaluate a
small-molecule inhibitor of EWS-FLI1 function. A series of xenograft
experiments show that 60–75 mg per kg body weight YK-4-279 substan-
tially decreases tumor growth. The small molecule not only inhibits
RHA binding to EWS-FLI1 but also decreases EWS-FLI1 modulated
transcription, on the basis of reporter assays. An additional putative
function of EWS-FLI1 is splice-site modification34, which was recently
supported by the EWS-FLI1–altered splicing of cyclin D1 (ref. 31).
Treatment of ESFT cells with YK-4-279 led to decreased cyclin D1
levels. Additional investigations of the splicing complex are necessary to
determine whether this effect is due to the disruption of an EWS-FLI1–
RHA complex or allosteric interference with EWS-FLI1. Small-molecule
inhibitors of protein-protein interactions have great therapeutic potential
and will be immediately useful as functional probes.
EWS-FLI1 was recognized as a potential therapeutic target over
15 years ago, almost immediately after the protein was identified as a
product of the breakpoint region t(11;22)35. We hypothesized that
RHA is a functionally crucial partner of EWS-FLI1. We developed
small-molecule protein-protein interaction inhibitors against EWS-
FLI1 and RHA without benefit of a fixed structure of EWS-FLI1. The
exact nature of the requirement of RHA by EWS-FLI1 is currently
under investigation; however, we speculate that RHA could be
involved in EWS-FLI1 function, synthesis or stability. Our data
support multiple mechanisms and therefore require further enzymatic
and structural studies of EWS-FLI1–bound RHA for resolution. The
fact that YK-4-279 is still toxic to A673 cells with low EWS-FLI1
expression could be due to residual EWS-FLI1 or suggest broader
action of the compound. In addition, although our data suggest that
YK-4-279 has ESFT cell-specific toxic effects, we recognize that as
additional cell and tumor models are tested, other protein interactions
of YK-4-279 may be revealed.
Inhibitory peptides offer a higher likelihood of specificity than
small molecules to validate protein-protein interaction targets and to
evaluate protein-complex disruption; however, peptides are proble-
matic for clinical development. Although small peptides are currently
being developed as therapeutic agents36,37, 10–20–amino acid peptides
present formidable pharmacokinetic stability and delivery challenges.
Our investigations use peptides to compare the effects of disrupting
protein-protein interactions with our small molecules. The E9R
peptide may compete with full-length RHA binding to EWS-FLI1,
and our data support a functional displacement of RHA by E9R.
Small molecule YK-4-279 can ‘displace’ E9R peptide from EWS-FLI1,
as shown by SPR and fluorescence polarization. Although our results
support E9R and YK-4-279 binding to the same site on EWS-FLI1,
allosteric interference cannot be excluded. Therefore, a structural
model of EWS-FLI1 is required to fully prove both this interaction
and the YK-4-279 binding site but is yet unavailable owing to the
challenges of disordered proteins23.
The interaction of RHA with EWS-FLI1 presents an ideal oppor-
tunity for the development of small-molecule protein-protein inter-
action inhibitors. Both the evidence and the prevailing opinion
support disordered proteins as potential targets of small molecule
therapeutics38. Our data also support EWS-FLI1 protein interaction
targeting to modulate oncogene function and potentially lead to new
therapeutics. Additional experiments to evaluate multispecies specifi-
city, toxicity and absorption, distribution, metabolism and excretion
are required to advance a further optimized derivative of YK-4-279
into clinical trials. Small molecules that disable EWS-FLI1 function
with minimal toxicity, in particular sparing hematopoetic stem cells,
could potentially provide a valuable adjuvant therapy for patients with
ESFT. In addition, this paradigm for drug discovery could be applied
to many related sarcomas that share similar oncogenic fusion proteins.
Methods and any associated references are available in the online
version of the paper at http://www.nature.com/naturemedicine/.
Note: Supplementary information is available on the Nature Medicine website.
This work was generously supported by the Children’s Cancer Foundation of
Baltimore (J.T. and A.U¨.), Go4theGoal Foundation (J.T.), Dani’s Foundation
of Denver (J.T.), the Liddy Shriver Sarcoma Initiative (J.T.), the Amschwand
Sarcoma Cancer Foundation (J.T.), the Burroughs-Wellcome Clinical Scientist
Award in Translational Research (J.T.), US National Institutes of Health grants
R01CA138212 (J.T.) and R01CA133662 (J.T.), and the Georgetown University
Medical Center Drug Discovery Program. US National Institutes of Health
support is through the Cancer Center Support Grant P30 CA051008 for use of
Flow Cytometry and Cell Sorting, Biacore Molecular Interaction, Tissue Culture
and microscopy core facilities and grant P01 CA47179 (M.M.). We would like to
thank S. Metallo for training in fluorescence polarization. Also, T. Cripe and
L. Whitesell provided critical review of our manuscript. We also thank S. Lessnick
from Hunstamn Cancer Institute, for providing NROB1 reporter plasmid,
J.V. Frangioni from Beth Israel Deaconess Medical Center for providing pG, pGN
and pGC vectors, O. Delattre from INSERM France for providing the A673
shEWS-FLI1 cell line, and R. Schlegel, Lombardi Comprehensive Cancer Center,
for providing HFK and HEC cell lines. We thank the Developmental Therapeutics
Program of the US National Cancer Institute for providing the Diversity set of
compounds for screening. This article is dedicated to our patients who have
fought but succumbed to ESFT.
H.V.E., J.S.B.-R., M.M., L.Y., O.D.A., S.S., T.-h.C., A.U¨. and J.A.T. designed
and carried out experiments. Y.K., S.D. and M.L.B. designed and synthesized
chemical compounds. H.V.E. and J.A.T. wrote the manuscript. All authors
reviewed, critiqued and offered comments to the text.
COMPETING INTERESTS STATEMENT
The authors declare competing financial interests: details accompany the full-text
HTML version of the paper at http://www.nature.com/naturemedicine/.
Published online at http://www.nature.com/naturemedicine/
Reprints and permissions information is available online at http://npg.nature.com/
1. Mitelman, F., Johansson, B. & Mertens, F. The impact of translocations and gene
fusions on cancer causation. Nat. Rev. Cancer 7, 233–245 (2007).
2. French, C.A. et al. Midline carcinoma of children and young adults with NUT
rearrangement. J. Clin. Oncol. 22, 4135–4139 (2004).
3. Helman, L.J. & Meltzer, P. Mechanisms of sarcoma development. Nat. Rev. Cancer 3,
NATURE MEDICINE VOLUME 15 [ NUMBER 7 [ JULY 2009755
© 2009 Nature America, Inc. All rights reserved.
4. Poppe, B. et al. Expression analyses identify MLL as a prominent target of 11q23
amplification and support an etiologic role for MLL gain of function in myeloid
malignancies. Blood 103, 229–235 (2004).
5. Carroll, M. et al. CGP 57148, a tyrosine kinase inhibitor, inhibits the growth of
cells expressing BCR-ABL, TEL-ABL, and TEL-PDGFR fusion proteins. Blood 90,
6. Grier, H.E. et al. Addition of ifosfamide and etoposide to standard chemotherapy for
Ewing’s sarcoma and primitive neuroectodermal tumor of bone. N. Engl. J. Med. 348,
7. Delattre, O. et al. The Ewing family of tumors—a subgroup of small-round-cell
tumors defined by specific chimeric transcripts. N. Engl. J. Med. 331, 294–299
8. Hu-Lieskovan, S., Heidel, J.D., Bartlett, D.W., Davis, M.E. & Triche, T.J. Sequence-
specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA
inhibits tumor growth in a murine model of metastatic Ewing’s sarcoma. Cancer Res.
65, 8984–8992 (2005).
9. Kovar, H., Ban, J. & Pospisilova, S. Potentials for RNAi in sarcoma research
and therapy: Ewing’s sarcoma as a model. Semin. Cancer Biol. 13, 275–281
10. Tanaka, K., Iwakuma, T., Harimaya, K., Sato, H. & Iwamoto, Y. EWS-Fli1 antisense
oligodeoxynucleotide inhibits proliferation of human Ewing’s sarcoma and primitive
neuroectodermal tumor cells. J. Clin. Invest. 99, 239–247 (1997).
11. Petermann, R. et al. Oncogenic EWS-Fli1 interacts with hsRPB7, a subunit of human
RNA polymerase II. Oncogene 17, 603–610 (1998).
12. Nakatani, F. et al. Identification of p21WAF1/CIP1 as a direct target of EWS-Fli1
oncogenic fusion protein. J. Biol. Chem. 278, 15105–15115 (2003).
13. Toretsky, J.A. et al. Oncoprotein EWS-FLI1 activity is enhanced by RNA helicase A.
Cancer Res. 66, 5574–5581 (2006).
14. Lee, C.G. et al. RNA helicase A is essential for normal gastrulation. Proc. Natl. Acad.
Sci. USA 95, 13709–13713 (1998).
15. Hartman, T.R. et al. RNA helicase A is necessary for translation of selected messenger
RNAs. Nat. Struct. Mol. Biol. 13, 509–516 (2006).
16. Tetsuka, T. et al. RNA helicase A interacts with nuclear factor kB p65 and functions as
a transcriptional coactivator. Eur. J. Biochem. 271, 3741–3751 (2004).
17. Va ¨lineva, T., Yang, J. & Silvennoinen, O. Characterization of RNA helicase A as
component of STAT6-dependent enhanceosome. Nucleic Acids Res. 34, 3938–
18. Myo ¨ha ¨nen, S. & Baylin, S.B. Sequence-specific DNA binding activity of RNA helicase
A to the p16INK4a promoter. J. Biol. Chem. 276, 1634–1642 (2001).
19. Zhong, X. & Safa, A.R. RNA helicase A in the MEF1 transcription factor complex up-
regulates the MDR1 gene in multidrug-resistant cancer cells. J. Biol. Chem. 279,
20. Nakajima, T. et al. RNA helicase A mediates association of CBP with RNA polymerase
II. Cell 90, 1107–1112 (1997).
21. Anderson, S.F., Schlegel, B.P., Nakajima, T., Wolpin, E.S. & Parvin, J.D. BRCA1
protein is linked to the RNA polymerase II holoenzyme complex via RNA helicase A.
Nat. Genet. 19, 254–256 (1998).
22. Robb, G.B. & Rana, T.M. RNA helicase A interacts with RISC in human cells and
functions in RISC loading. Mol. Cell 26, 523–537 (2007).
23. Bhalla, J., Storchan, G.B., MacCarthy, C.M., Uversky, V.N. & Tcherkasskaya, O. Local
flexibility in molecular function paradigm. Mol. Cell. Proteomics 5, 1212–1223
24. Xie, H. et al. Functional anthology of intrinsic disorder. 1. Biological processes and
functions of proteins with long disordered regions. J. Proteome Res. 6, 1882–1898
25. Ng, K.P. et al. Multiple aromatic side chains within a disordered structure are critical
for transcription and transforming activity of EWS family oncoproteins. Proc. Natl.
Acad. Sci. USA 104, 479–484 (2007).
26. U¨ren, A., Tcherkasskaya, O. & Toretsky, J.A. Recombinant EWS-FLI1 oncoprotein
activates transcription. Biochemistry 43, 13579–13589 (2004).
27. Terrone, D., Sang, S.L., Roudaia, L. & Silvius, J.R. Penetratin and related cell-
penetrating cationic peptides can translocate across lipid bilayers in the presence of
a transbilayer potential. Biochemistry 42, 13787–13799 (2003).
28. Voss, S.D., DeGrand, A.M., Romeo, G.R., Cantley, L.C. & Frangioni, J.V. An integrated
vector system for cellular studies of phage display-derived peptides. Anal. Biochem.
308, 364–372 (2002).
29. Leeson, P.D. & Springthorpe, B. The influence of drug-like concepts on decision-
making in medicinal chemistry. Nat. Rev. Drug Discov. 6, 881–890 (2007).
30. Gangwal, K. et al. Microsatellites as EWS/FLI response elements in Ewing’s sarcoma.
Proc. Natl. Acad. Sci. USA 105, 10149–10154 (2008).
31. Sanchez, G. et al. Alteration of cyclin D1 transcript elongation by a mutated
transcription factor up-regulates the oncogenic D1b splice isoform in cancer. Proc.
Natl. Acad. Sci. USA 105, 6004–6009 (2008).
32. Li, F. et al. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396,
33. Knoop, L.L. & Baker, S.J. EWS/FLI alters 5¢-splice site selection. J. Biol. Chem. 276,
34. Tirode, F. et al. Mesenchymal stem cell features of Ewing tumors. Cancer Cell 11,
35. Delattre, O. et al. Gene fusion with an ETS DNA-binding domain caused by chromo-
some translocation in human tumours. Nature 359, 162–165 (1992).
36. Plescia, J. et al. Rational design of shepherdin, a novel anticancer agent. Cancer Cell 7,
37. Palermo, C.M., Bennett, C.A., Winters, A.C. & Hemenway, C.S. The AF4-mimetic
peptide, PFWT, induces necrotic cell death in MV4–11 leukemia cells. Leuk. Res. 32,
38. Cheng, Y. et al. Rational drug design via intrinsically disordered protein. Trends
Biotechnol. 24, 435–442 (2006).
756VOLUME 15 [ NUMBER 7 [ JULY 2009 NATURE MEDICINE
© 2009 Nature America, Inc. All rights reserved.
Materials. We obtained E9R peptide from Bio-synthesis. We obtained protein
G beads (Invitrogen), antibody to GST, antibody to FLI1, antibody to cyclin D1
(all from Santa Cruz), fluorogenic caspase-3 substrate N-acetyl-Asp-Glu-Val-
Asp-AMC (7-amino-4- methylcoumarin) (Ac-DEVD-AMC), caspase-3 fluoro-
genic substrate (BD Biosciences Pharmingen) and antibody to cleaved caspase 3
(Asp175) (Cell Signaling) from commercial sources.
Site-directed mutagenesis. We changed every nonalanine amino acid in the
amino acids 823–832 region of RHA to alanine by site-directed mutagenesis
with QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene) according
to the manufacturer’s protocol.
Cell cultures. We maintained established TC32, TC71, A4573, CHP-100 and
primary ES925 and GUES1 ESFT cell lines in RPMI (Invitrogen) medium
supplemented with 10% FBS (Gemini Bioproducts). HEC and HFK cell lines,
kind gifts from R. Schlegel, are previously described39. We tested subclones of
these cells stably expressing EWS-FLI1 tested in an anchorage-independent
growth assay as previously described13.
Protein immunoprecipitation assays. We made protein lysates and performed
immunoprecipitations as previously published13. We prepared recombinant
GST-RHA647–1075from crude bacterial extracts without further purification.
Small molecule library screening and selection of lead compound. We
established an SPR assay using the Biacore T100 with EWS-FLI1, prepared in
our laboratory as previously published26. We used DNA oligonucleotides to
quality-control the proper conformation of EWS-FLI1 on the surface of a CM5
chip. We prioritized small molecules from the Developmental Therapeutics
Program of the National Cancer Institute, US National Institutes of Health on
the basis of their molecular weight and solubility. We performed an initial
screening of molecules at 1 mM or 10 mM compound, based on solubility. We
used a model that compares the actual binding maximum (actual RU) with the
theoretical binding maximum (RUtheor). If the RUactualto RUtheorratio is 0.9–
1.0, this suggests a binding, and such a compound is considered a ‘hit’. A team
of medicinal chemists then reviewed hits, and those with structural potentials
were selected for further study. We tested selected molecules in vitro in a
solution co-immunoprecipitation assay with recombinant EWS-FLI1 and GST-
Synthesis and analysis of small molecule compounds. Details provided in the
Fluorescence polarization assay. We added increasing concentrations of FITC-
E9R to a fixed concentration of EWS-FLI1 (4.8 mM) to obtain a saturated
binding curve. We performed the assay in 20 mM Tris, 500 mM NaCl and
0.67M imidazole, pH 7.4. We analyzed the fluorescence polarization in a
QuantaMaster fluorimeter (Photon Technology International) equipped with
polymer sheet polarizers at an excitation wavelength of 495 nm and emission
wavelength of 517 nm. We added increasing concentrations of YK-4-279 to a
fixed concentration of EWS-FLI1 and FITC-E9R (3.2 mM, as determined from
saturated binding curve) with the same buffer and instrumental settings as
Plasmids and reporter assay. We prepared EGFP-E9R fusion constructs as
previously published40. We transiently transfected the NR0B1 (ref. 31) luciferase
reporter and full-length EWS-FLI1 into COS-7 cells with Fugene-6 (Roche) and
performed the luciferase assay per the manufacturer’s protocol (Dual Luciferase
Kit, Promega). Six hours after transfection, we treated cells with either 3 mM or
10 mM YK-4-279. We standardized cell lysate luciferase activity to Renilla
activity from a nonaffected promoter and plotted as relative luciferase activity
Caspase-3 activity measurement and nuclear fragmentation. We treated cells
for 24 h with 10 mM YK-4-279. We incubated the Caspase-3 substrate DEVD-
AMC with equal amounts of protein lysate and measured the fluorescence from
cleaved substrate in a fluorimeter. We treated TC32 cells and nontransformed
HEK-293, HFK and HEC cells for 6 h with high-dose (50 mM) YK-4-279. We
photographed DAPI-stained cells at 600? magnification on an inverted
Mouse strains and in vivo small-molecule testing. We orthotopically injected
1 million TC71 or CHP-100 cells in 100 ml HBSS into the gastrocnemius muscle
of 4- to 8-week-old severe combined immunodeficient–beige mice (Taconic).
We established prostate cancer xenografts by subcutaneous injection of 5
million PC3 cells into the flanks of 4- to 8-week-old nude mice (Taconic).
We randomized mice to treatment groups receiving thrice weekly intraperito-
neal injections of DMSO or YK-4-279 at 1.5 mg per dose when tumors were
palpable. We began each of the mouse experiments with ten mice that were
randomized into treatment and control groups when the tumors reached
palpable size. In the control groups, some tumors exceeded the Institutional
Animal Care and Use Committee maximal size (2 cm in any dimension) and
were euthanized before day 14 and thus not included in the day 14 analysis
(Fig. 6c). We measured tumor length and width every 2–4 d and calculated
volume with the formula volume ¼ D ? d2? p/6, where D is the longest
diameter and d is the shorter diameter. Xenograft studies were approved by the
Memorial Sloan-Kettering Cancer Center Institutional Animal Care and Use
Statistical analyses. We performed statistical analyses with GraphPad Prism.
39. Uren, A. et al. Activation of the canonical Wnt pathway during genital keratinocyte
transformation: a model for cervical cancer progression. Cancer Res. 65, 6199–6206
40. Frangioni, J.V. & Neel, B.G. Use of a general purpose mammalian expression
vector for studying intracellular protein targeting: identification of critical residues
in the nuclear lamin A/C nuclear localization signal. J. Cell Sci. 105, 481–488
© 2009 Nature America, Inc. All rights reserved.