Novel synthetic bisindolylmaleimide alkaloids inhibit STAT3 activation by binding to the SH2 domain and suppress breast xenograft tumor growth

Article abstract of DOI:10.1038/s41388-017-0076-0

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in malignant tumors and plays important roles in multiple aspects of cancer aggressiveness. Thus, targeting STAT3 promises to be an attractive strategy for the treatment of advanced metastatic tumors. Bisindolylmaleimide alkaloid (BMA) has been shown to have anti-cancer activities and was thought to suppress tumor cell growth by inhibiting protein kinase C. In this study, we show that a newly synthesized BMA analog, BMA097, is effective in suppressing tumor cell and xenograft growth and in inducing spontaneous apoptosis. We also provide evidence that BMA097 binds directly to the SH2 domain of STAT3 and inhibits STAT3 phosphorylation and activation, leading to reduced expression of STAT3 downstream target genes. Structure activity relationship analysis revealed that the hydroxymethyl group in the 2,5-dihydropyrrole-2,5-dione prohibits STAT3 inhibitory activity of BMA analogs. Altogether, we conclude that the synthetic BMA analogs may be developed as anti-cancer drugs by targeting and binding to the SH2 domain of STAT3 and inhibiting the STAT3 signaling pathway.

Full text of DOI:10.1038/s41388-017-0076-0

Oncogene
https://doi.org/10.1038/s41388-017-0076-0
ARTICLE
Novel synthetic bisindolylmaleimide alkaloids inhibit STAT3
activation by binding to the SH2 domain and suppress breast
xenograft tumor growth
Xia Li1,2 Hongguang Ma3 Lin Li2 Yifan Chen1 Xiao Sun2 Zizheng Dong1 Jing-Yuan Liu1,4 Weiming Zhu3
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Jian-Ting Zhang
Received: 27 August 2017 / Accepted: 19 November 2017
© Macmillan Publishers Limited, part of Springer Nature 2018
Abstract
Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in malignant tumors and
plays important roles in multiple aspects of cancer aggressiveness. Thus, targeting STAT3 promises to be an attractive
strategy for the treatment of advanced metastatic tumors. Bisindolylmaleimide alkaloid (BMA) has been shown to have anti-
cancer activities and was thought to suppress tumor cell growth by inhibiting protein kinase C. In this study, we show
that a newly synthesized BMA analog, BMA097, is effective in suppressing tumor cell and xenograft growth and in
inducing spontaneous apoptosis. We also provide evidence that BMA097 binds directly to the SH2 domain of STAT3
and inhibits STAT3 phosphorylation and activation, leading to reduced expression of STAT3 downstream target
genes. Structure activity relationship analysis revealed that the hydroxymethyl group in the 2,5-dihydropyrrole-2,5-dione
prohibits STAT3 inhibitory activity of BMA analogs. Altogether, we conclude that the synthetic BMA analogs may be
developed as anti-cancer drugs by targeting and binding to the SH2 domain of STAT3 and inhibiting the STAT3 signaling
pathway.
Introduction
Electronic supplementary material The online version of this article
STAT3, signal transducers and activators of transcription 3,
(https://doi.org/10.1038/s41388-017-0076-0) contains supplementary
is a member of transcription factors in the Janus kinase
(JAK)/STAT signaling pathway and can be activated
by multiple stimuli [1]. Activation of STAT3 involves
phosphorylation of its conserved Tyr705, dimerization,
and translocation into nucleus, where it binds to STAT3-
specific DNA elements and induces transcription of
downstream target genes including survivin, Bcl-xl, and
VEGF [2]. Constitutive activation of STAT3 has been
detected in a number of human malignancies including
breast and lung cancers [3–7]. Activated STAT3 is essential
for cancer cell survival and inhibiting STAT3 signaling
pathway causes apoptosis [8]. Over-expression of a con-
stitutively activated STAT3c molecule transformed human
mammary epithelial cells [9] and subcutaneously injected
cells expressing STAT3c formed xenograft tumors [10].
Transgenic over-expression of STAT3c in alveolar type II
epithelial cells of mice led to lung inflammation and con-
sequently spontaneous lung bronchoalveolar adenocarci-
noma [11].
material, which is available to authorized users.
* Xia Li
xiali@sdu.edu.cn
* Jing-Yuan Liu
jliu2@iu.edu
* Weiming Zhu
weimingzhu@ouc.edu.cn
* Jian-Ting Zhang
jianzhan@iu.edu
1
Department of Pharmacology and Toxicology, Indiana University
School of Medicine, Indianapolis, IN, USA
2
School of Ocean, Shandong University, Weihai, China
3
Key Laboratory of Marine Drugs, Ministry of Education of China,
School of Medicine and Pharmacy, Ocean University of China,
Qingdao, China
4
Department of Computer and Information Science, Indiana
University-Purdue University, Indianapolis, IN, USA
5
IU Simon Cancer Center, Indiana University School of Medicine,
Indianapolis, IN, USA

X. Li et al.
Fig. 1 Synthesis of BMA
analogs and their effects on
STAT3-dependent luciferase
activity. a Synthesis of BMA
analogs. a NaH, CH3CH2I, THF:
1 95% yield; b NaH, Br(CH2)
nCN, MeCN: 2a 94% yield, 3a
57% yield, 4a 83% yield; c (i)
(COCl)2, CH2Cl2, (ii) (CH3CH2)
3N, CH2Cl2: 2b 23% yield, 3b
45% yield, 4b 51% yield, 5b
44% yield, 6b 32% yield, 7b
39% yield; d HMDS, MeOH,
DMF: BMA016 95% yield,
BMA019 98% yield, 4c 95%
yield, BMA097 91% yield,
BMA100 90% yield, 7c 90%
yield; e 37% HCHO, NaHCO3,
85 °C: BMA088 96% yield,:
BMA127 95% yield; f NaH, Br
(CH2)nCN, THF: 5a 30% yield,
6a 83% yiled; g NaH,
C6H5CH2Br, DMF: 7a 99%
yiled; h O2, DMSO, t-BuOK,
THF: 7d 89% yiled. b Effect of
BMA analogs on STAT3-
dependent luciferase activity.
Stable MDA-MB-231 cells
expressing STAT3-dependent
luciferase reporter were treated
with synthetic BMA analogs or
DMSO control followed by
determination of luciferase
activity. (*p < 0.05; **p < 0.01)
Bioactive compounds of microbial origin such as bisin-
dolylmaleimide alkaloid (BMA) have been shown to display
a wide spectrum of structural diversity with versatile biolo-
gical activities including anti-inflammatory, antibacterial,
antiparasitic, antifungal, and anti-tumor activities [12]. For
example, staurosporine, a structurally and biosynthesis-
related natural product of BMA, has been shown to possess
various strong biological activities such as anti-proliferation
possibly via inhibiting protein kinase C [13]. However, it
has also been shown that BMA induces apoptosis by inhi-
biting other pathways such as Wnt/β-catenin signaling in
addition to inhibiting protein kinase C [14].
In this study, we tested a class of novel BMA derivatives,
synthesized based on a 3,4-diindolyl-2, 5-dihydropyrrole-2,
5-dione nucleus, for their potential activity in inhibiting
STAT3 signaling pathway and inducing cancer cell
apoptosis. We found that the synthetic BMA analogs
are effective in suppressing tumor cell proliferation,
inducing spontaneous apoptosis, and inhibiting xenograft
tumor growth, possibly by binding directly to the SH2
domain of STAT3 and inhibiting STAT3 phosphorylation
and activation. Thus, synthetic BMA analogs may be
developed as anti-cancer drugs by targeting STAT3 sig-
naling pathway.

Synthetic BMA analogs as novel STAT3 inhibitors
Fig. 2 BMA097 inhibition of STAT3 activity and expression of
STAT3 target genes. a, b Dose response and time course of BMA097
inhibition of STAT3-dependent luciferase expression. MDA-MB-231
cells with stable expression of STAT3-dependent luciferase were
incubated with BMA097 at different concentrations (a) or at 2 µM for
different times (b) followed by determination of luciferase activity. c
BMA097 inhibition of STAT3 downstream target gene expression.
MDA-MB-231 cells were treated without or with different con-
centrations of BMA097 followed by real-time RT–PCR analysis of
mRNAs encoding STAT3, cyclin D1 and Bcl-xL. GAPDH was used
as an internal control. (n = 3; *p < 0.05; **p < 0.01)
Results
in STAT3 inhibition. Furthermore, alteration in length or in
end group of the two hydrocarbon chains on the indole may
also affect the STAT3 inhibitory activity, although they do
not appear to be very critical with possibly optimal length of
the hydrocarbon chain in BMA097.
Synthesis of BMA derivatives
As shown in Fig. 1a, 2-(1H-indol-3-yl)acetic acid reacted
with ethyl iodide and bromonitriles in the presence of
sodium hydride (NaH) to yield compounds 1, 5a and 6a,
respectively. Indole derivatives 2a−4a and 7a were syn-
thesized by reaction of indole with bromonitriles and benzyl
bromide, respectively, in the presence of NaH. Compounds
2a−4a and 7a reacted with oxalyl chloride, leading to
acidylated intermediates that were used to react with com-
pounds 1, 5a and 6a to afford bisindolylmaleic anhydrides
2b−7b. The anhydrides were subjected to aminolysis with
hexamethyldisilazane (HMDS) and MeOH in DMF to
produce the corresponding bisindolymaleimides BMA016,
BMA019, 4c, BMA097, BMA100 and 7c. BMA088 was
prepared from the nucleophilic addition of 4c with HCHO
under basic condition. Compound 7c was subjected to
deprotection of benzyl in the presence of t-BuOK, O2 and
DMSO, followed by the additive reaction with HCHO and
NaHCO3 to give BMA127 (Fig. 1a).
Because BMA097 appears to be most effective (Fig. 1b),
it was selected for further evaluation. First, re-synthesized
BMA097 was tested to verify its activity. Figure 2a, b
shows that the re-synthesized BMA097 inhibits STAT3-
dependent luciferase expression in a dose- and time-
dependent manner with an IC50 of ~1.8 µM and t1/2 of
~16 h. Furthermore, BMA097 inhibited the expression of
STAT3 target genes, cyclin D1 and Bcl-xL, as determined
using both real-time RT–PCR for mRNAs (Fig. 2c) and
western blot for proteins (Fig. 3a), indicating that BMA097
inhibits the transcriptional activation of cyclin D1 and Bcl-
xL gene possibly by inhibiting STAT3 activity.
BMA097 suppresses Tyr705 phosphorylation and
activation of STAT3
To determine the mechanism of BMA097 inhibition of
STAT3 activity, we first examined the phosphorylation sta-
tus and activation of STAT3 following BMA097 treatment.
Figure 3a, b shows that BMA097 inhibits phosphorylation of
the Tyr705 in dose-dependent and time-dependent manners
but not the phosphorylation of Ser727. It also had little effect
on the level of total STAT3. Furthermore, BMA097 treat-
ment reduced IL-6-induced phosphorylation and activation
of STAT3 in serum-starved MDA-MB-231 cells (Fig. 3c).
Thus, BMA097 likely inhibits STAT3 activity by inhibiting
Tyr705 phosphorylation and activation of STAT3.
Inhibition of STAT3 activity by BMA derivatives
To determine whether BMA are potential STAT3 inhibitors,
we tested six synthetic BMA derivatives for their effects on
STAT3-dependent luciferase expression in a cell-based
assay. As shown in Fig. 1b, 5 BMA derivatives (BMA016,
019, 097, 100, and 127) significantly inhibited STAT3-
dependent luciferase expression in MDA-MB-231 cells
with BMA127 having the least inhibition and BMA088
having no inhibition (Fig. 2).
We next analyzed subcellular localization of STAT3
using both immunofluorescence staining and separation of
nuclear and cytoplasmic fractions. As shown in Fig. 3d, e,
The above findings also suggest that the hydroxymethyl
group on the nitrogen in 2, 5-dihydropyrrole-2,5-dione in
BMA088 and BMA127 may be inhibitory to BMA activity
the nucleus-localized STAT3, representing the Tyr705
-

X. Li et al.
Fig. 3 Effect of BMA097 on STAT3 phosphorylation and activation.
a, b BMA097 inhibition of constitutive STAT3 phosphorylation.
MDA-MB-231 cells were treated with different concentrations of
BMA097 for 24 h (a) or at 2 μM for different times (b) followed by
Western blot analysis of total and phosphorylated STAT3. STAT3
downstream target proteins Bcl-xL and Cyclin D1 were also tested.
Actin was used as a loading control for Western blot. c BMA097
inhibition of IL-6-induced STAT3 phosphorylation. Serum-starved
MDA-MB-231 cells were pre-treated with BMA097 and then with IL-
6 followed by Western blot analysis of total and Tyr705-phosphorylated
STAT3. d, e BMA097 effect on pSTAT3 subcellular localization.
MDA-MB-231 cells were treated without or with BMA097 for 24 h
followed by immunofluorescence staining or fractionation and Wes-
tern blot analysis of Tyr705-phosphorylated STAT3. DAPI was used to
counterstain nuclei in d. Histone and actin were used as markers for
nuclear and cytoplasmic fractions, respectively, in e. f, g Effect of
BMA097 on STAT3 dimerization. MDA-MB-231 cells were treated
with BMA097 as described in panel a followed by separation using
non-denaturing PAGE and Western blot analysis of STAT3. h Effect
of BMA097 on JAK2 activation. MDA-MB-231 cells were treated
with BMA097 followed by Western blot analysis of total and Tyr1007/
1008-phosphorylated JAK2
phosphorylated and activated STAT3, disappeared follow-
ing BMA097 treatment, confirming that BMA097 inhibits
Tyr705 phosphorylation and activation and, thus, nuclear
localization of STAT3.
upstream kinase JAK2, we performed Western blot analysis
of JAK2 Tyr1007/1008 phosphorylation and activation. Figure
3h shows that BMA097 treatment has no effect on the level
of total JAK2 and does not inhibit JAK2 phosphorylation/
activation. Thus, BMA097 inhibition of STAT3 phosphor-
ylation and activation is unlikely by inhibiting its upstream
kinase JAK2.
It is well known that STAT3 activation following Tyr705
phosphorylation results in homo-dimerization before relo-
calization into nucleus [15]. To further determine if
BMA097 inhibition of Tyr705 phosphorylation would con-
sequently reduce STAT3 dimerization and activation, we
performed non-denaturing PAGE analysis of dimeric
STAT3 following BMA097 treatment. As shown in Figs.
3f, g, BMA097 inhibited STAT3 dimerization in dose-
dependent and time-dependent manners. Taken together, we
conclude that BMA097 treatment likely inhibits STAT3
activation (phosphorylation and dimerization) and translo-
cation into nucleus.
BMA097 binds directly to STAT3 at the SH2 domain
Because BMA097 does not inhibit STAT3 upstream kinase
JAK2, we hypothesized that BMA097 might bind directly
to STAT3 at its SH2 domain and inhibit phosphorylation of
Tyr705 and activation by JAK. To test this hypothesis, we
first performed structural analysis of STAT3 (Pdb code:
1BG1) and docked BMA097 to the phospho-Tyr-binding
pocket in the SH2 domain Next, we performed 20-ns
molecular dynamics (MD) simulation on the docked
To determine whether BMA097 inhibits STAT3 phos-
phorylation and activation possibly by inhibiting its

Synthetic BMA analogs as novel STAT3 inhibitors
Fig. 4 Binding of BMA097 to the SH2 domain of STAT3. a, b The
predicted BMA097-binding pocket (a) and binding mode (b) in the
SH2 domain of STAT3. Molecular surface in a is colored by dodger
blue for the most hydrophilic to orange red for the most hydrophobic.
The green lines in b indicate H-bonds. c Pulldown assay. Total lysate
from MDA-MB-231 cells were subjected to pulldown assay by CNBr-
immobilized BMA097, an irrelevant compound (IC) or vehicle control
followed by western blot analysis of pulldown materials using STAT3
BMA097 (d) or elution of STAT3 bound to the immobilized BMA097
(e) by excess free BMA097, DMSO vehicle control, or an irrelevant
negative control compound (IC). f Schematic domain structures of
STAT3 and of recombinant proteins. NTD amino terminal domain,
CCD coiled coil domain, DBD DNA-binding domain, LD linker
domain, SH2 Src homology 2 domain, TAD transactivation domain. g
Pulldown assay of purified recombinant STAT3 proteins with different
deletions by immobilized BMA097
antibody. d,
e Competition of STAT3-binding to immobilized
STAT3-BMA097 complex and calculated the total binding
free energies (ΔGbind) of BMA097. The difference of
enthalpy (ΔHbind) is calculated to be −15.21 kcal/mol using
Generalized Born method. The difference of entropy
(ΔSbind) is estimated to be −10.71 kcal/mol by normal mode
analyses. By solving the equation ΔGbind = ΔHbind - T
ΔSbind, the total binding free energy is estimated to be
−4.50 kcal/mol, indicating a favorable binding of BMA097
to the SH2 domain of STAT3.
The frame with lowest total potential energy of the
simulation was further examined. As shown in Fig. 4a,
BMA097 (pink) docked in the phospho-Tyr-binding pocket
in the SH2 domain (green). It appears that BMA097 inter-
acts with the SH2 domain of STAT3 via 3 strong hydrogen
bonds (green lines in Fig. 4b). First, the nitrogen atom on
the dioxo-pyrrole ring of BMA097 interacts with the
oxygen of Gln635 of STAT3 via a hydrogen bond. Second,
the oxygen atom on the dioxo-pyrrole ring of BMA097
hydrogen bonds with the main chain nitrogen of Ser636 of
STAT3. Finally, the nitrogen atom on the indole ring of
BMA097 and the side chain oxygen of Ser636 of STAT3
forms the third hydrogen bond. In addition, residues Arg609
,
Glu612 and Glu638 interact with BMA097 via electrostatic
interactions and residues Thr620, Val637 and Pro639 con-
tribute to the binding of BMA097 via hydrophobic
interactions.
To validate the above computational analysis and to
determine whether BMA097 binds directly to STAT3, we
took advantage of the imino group of BMA097 and con-
jugated it to CNBr-activated Sepharose for pulldown assay.
As shown in Fig. 4c, BMA097-conjugated Sepharose suc-
cessfully pulled down STAT3 from lysate of MDA-MB-

X. Li et al.
Fig. 5 BMA097 inhibits proliferation and induces apoptosis in breast
cancer cells. a Survival assay. Human breast cancer cells were treated
with BMA097 at different concentrations for various times followed
by MTT assay. b–d Apoptosis assay. Human breast cancer cells were
treated with BMA097 at different concentrations (b, d) or at 2 µM for
different times (c) followed by Annexin V/PI (b, c) or JC-1 staining
(d) and FACS analysis of apoptosis and mitochondrial membrane
potential (Δψm). (*p < 0.05; **p < 0.01)
231 cells while control Sepharose and Sepharose-
conjugated with an irrelevant compound did not. Pre-
treatment of cell lysate with excess BMA097 but not
vehicle nor the irrelevant compound as competitors com-
pletely inhibited STAT3 pulldown by BMA097-conjugated
Sepharose (Fig. 4d). Furthermore, only excess BMA097 but
not vehicle nor the irrelevant compound can elute STAT3
from BMA097-bound STAT3 (Fig. 4e). These findings
together suggest that STAT3 can bind to BMA097 via non-
covalent interactions.
of STAT3, we performed a pulldown assay using purified
recombinant STAT3 followed by SDS-PAGE analysis with
silver staining. Figure 4f shows the schematic domain
structures of different recombinant STAT3 proteins for
pulldown assays. Figure 4g shows that the recombinant
STAT3 lacking the TAD domain (ΔTAD-C) can be suc-
cessfully pulled down by immobilized BMA097. However,
further deletion of the SH2 domain (ΔSH2-C) abolished the
binding by BMA097, suggesting that the BMA097-binding
site may be located in the SH2 domain of STAT3. To
confirm this finding, we engineered another construct for
production of recombinant STAT3 with only the carboxyl
domains containing the SH2 domain. As shown in Fig. 4g,
this protein was successfully pulled down by immobilized
In above studies, total cell lysate was used and, thus, the
interaction between STAT3 and BMA097 could be indirect
via another protein. To rule out this possibility and to
demonstrate that BMA097 binds directly to the SH2 domain

Synthetic BMA analogs as novel STAT3 inhibitors
Fig. 6 BMA097 inhibits
xenograft breast tumor growth
and STAT3 phosphorylation and
induces cancer cell apoptosis
in vivo. a Volume of xenograft
tumors and body weight of mice
following treatments. b, c Wet
weight and gross anatomy of
final dissected xenograft tumor
masses. (n = 5; *p < 0.05; **p
< 0.01). d IHC staining of
xenograft tumors using
antibodies against total or
Tyr705-phosphorylated STAT3
and cleaved caspase 3
BMA097. Altogether, these findings suggest that BMA097
may bind directly to STAT3 at its SH2 domain as predicted
by docking analysis.
treatment with 0, 2, and 4 μM BMA097 for 24 h, respec-
tively. The population of annexin V-positive MDA-MB-
231 cells also increases with time of treatment using 2 μM
BMA097 (Fig. 5c).
BMA097 inhibits survival and induces apoptosis of
breast cancer cells
We also examined the effects of BMA097 treatment on
mitochondrial membrane potential (Δψm) in both MDA-
MB-231 and MCF7 cells as another indicator of apoptosis.
As shown in Fig. 5d, BMA097 treatment induced Δψm loss
in a dose-dependent manner for both cell lines. Together,
the above findings suggest that BMA097 suppresses breast
cancer cell survival likely by causing dose and time-
dependent apoptosis.
Previously it has been reported that STAT3 signaling is
required for survival of breast cancer cells [16]. Thus, we
next tested if BMA097 suppresses survival of breast cancer
cells and has any anti-cancer activity using MTT assay. As
shown in Fig. 5a, BMA097 inhibited survival of breast
cancer cell lines MDA-MB-231, MDA-MB-468, and MCF7
in dose-dependent and time-dependent manners. The IC50
for 48-hr treatments were estimated to be 3.6, 4.0, 6.4 µM for
MDA-MB-231, MDA-MB-468, and MCF7 cells, respec-
tively. These values at 72 h of treatments are reduced to 0.9,
1.8, and 3.9 μM, respectively. In contrast, the cytotoxicity of
BMA097 is relatively low to non-cancerous human mam-
mary epithelial cell line MCF10A with an IC50 of 10.6 at 48
h and 6.5 µM at 72 h of treatments (Supplementary Fig. S1).
Thus, there may be a therapeutic window for BMA097.
Next, we examined the effect of BMA097 on apoptosis
first using annexin V-FITC and PI staining and flow cyto-
metry in MDA-MB-231 and MCF7 cells. Figures 5b shows
that the proportion of cells stained with annexin V and PI
increases following BMA097 treatment in a dose-dependent
manner in both cell lines. The total annexin V-positive cells
represent 13.4, 51.9 and 63.8% of the population for MDA-
MB-231 and 7.8, 34.2 and 48.1% for MCF7 cells following
BMA097 suppresses growth of xenograft tumors
and STAT3 activation in vivo
We next evaluated the in vivo anti-tumor activity of
BMA097 in nude mice bearing MDA-MB-231 xenografts.
As shown in Fig. 6a–c, the growth and final weight of
MDA-MB-231 xenograft tumors were effectively and sig-
nificantly reduced by BMA097 treatment with doses at 10
and 40 mg/kg, similar to the vincristine control treatment
group. Although the body weight of mice in all groups
dropped slightly at the conclusion of the experiment (Fig.
6a), possibly due to disease burden, there are no significant
differences in body weight reduction between the control
and treatment groups. Immuohistochemistry staining of the
xenograft tumors shows that the tumors in the BMA097-
treated group have reduced level of phosphorylated STAT3
compared to vehicle control treatment group (Fig. 6d).

X. Li et al.
BMA097 treatment also induced caspase 3 cleavage in the
xenograft tumors (Fig. 6d). These observations are con-
sistent with our findings that BMA097 inhibits STAT3
phosphorylation and activation and induces apoptosis.
Thus, BMA097 likely inhibits xenograft tumor growth by
inhibiting STAT3 activation in vivo.
Although BMA097 has a potential therapeutic window
with IC50 of 0.9–3.9 µM for breast cancer cells and 6.2 μM
for non-cancerous mammary epithelial cells, developing it
into cancer therapeutics by targeting STAT3 needs to be
cautiously performed. STAT3 is known to play important
roles in hematopoiesis and innate immunity [18, 19],
chronic use of STAT3 inhibitors may cause adverse effect
on bone marrow and innate immunity. However, lack of
weight loss and apparent phenotypic changes in the mouse
model suggest that 40 mg/kg BMA097 in mice may not
cause sever adverse effect and, thus, may warrant further
development.
Discussion
Using rational screening of synthetic BMA analogs, we
identified BMA097 as an effective inhibitor of STAT3 and
a potential therapeutics for breast cancer treatment. It
appears that BMA097 binds directly to the pTyr-binding
pocket in the SH2 domain and, thus, effectively inhibits
Tyr705 phosphorylation and activation by JAK2. The inhi-
bition of STAT3 phosphorylation at Tyr705 and BMA097
occupation in the pTyr-binding pocket of the SH2 domain
led to inhibition of STAT3 dimerization and translocation
into nucleus for activation of its downstream target genes
and, thus, inhibition of proliferation and induction of
apoptosis. Thus, BMA097, as a synthetic derivative of
BMA, may be developed as a potential therapeutics for
breast cancer treatment by targeting STAT3.
Although BMA097 binds to and inhibits STAT3 acti-
vation, we cannot rule out the possibility that its effect on
cancer cell survival and apoptosis may be compounded by
its potential inhibition of PKC because bisindolylmaleimide
are known to also inhibit PKC. However, BMA097 did not
affect phosphorylation of Ser727 of STAT3, which is known
to be phosphorylated by PKC [17]. Thus, the specific syn-
thetic analog of bisindolymaleimide, BMA097, may not
inhibit PKC. Indeed, it remains to be determined whether
BMA097 possibly inhibits other pathways such as Wnt/β-
catenin signaling [14], which then contributes to BMA097-
induced cell death.
Materials and methods
Materials
MTT [3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bro-
mide], DAPI [4′,6-diamidino-2-phenylindole], and JC-1
(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl
benzimidazolo-
carbocyanine iodide) were purchased from Sigma (St. Louis,
MO). All electrophoresis reagents and polyvinylidene
difluoride (PVDF) membranes were purchased from Bio-
Rad (Hercules, CA). The antibodies against pJAK2 (3776 s),
pSTAT3 (9145 s, 9134p), JAK2 (3230 s), STAT3 (9139 s),
and cleaved caspase 3 (9661 s) were obtained from Cell
Signaling Technology (Danvers, MA). Antibodies against
cyclin D1 (SC-753) and Bcl-xL (SC-8392) were from Santa
Cruz Biotechnology (Dallas, TX). DMEM and Ham’s F-12
were purchased from Media Tech. (Herndon, CA). All other
chemicals were of molecular biology grade from Sigma or
Fisher Scientific (Chicago, IL).
Cell lines and culture
Domain mapping of the binding site using purified
recombinant STAT3 protein and pulldown assay indicate
that BMA097 likely binds directly to the SH2 domain as
predicted using computational modeling. The predicted
interaction between BMA097 and STAT3 also provides
theoretical evidence that explains why attaching a hydro-
xymethyl group to the nitrogen in 2,5-dihydropyrrole-2,5-
dione would result in reduction in activity. It is possible that
the hydroxymethyl group forms an internal hydrogen bond
with one of the oxygen in the dioxo-pyrrole ring and, thus,
eliminates formation of two hydrogen bonding with
STAT3, which would effectively reduce the binding of
BMA088 and BMA127 to the SH2 domain of STAT3.
However, exactly how and if BMA097 binds to the pocket
in the SH2 domain of STAT3 requires further investigation
using experimental approaches such as generating co-
crystal structures.
Human breast cancer cell lines MCF7, MDA-MB-231, and
MDA-MB-468 were from ATCC and cultured in DMEM
supplemented with 10% fetal bovine serum (Invitrogen,
USA) in a humidified atmosphere with 5% CO2 at 37 °C.
The non-cancerous mammary epithelial cell line MCF10A
cells, also from ATCC, were cultured in DMEM/F12 (50/
50) with 10% equine serum, 10 ug/mL insulin, 25 ng/mL
EGF, 500 ng/mL hydrocortisone, and 100 ng/mL cholera
toxin. All media contained 100 µg/ml penicillin and 100 µg/
ml streptomycin. All cell lines were authenticated by short
tandem repeat analysis on 21 January 2013 and then on 3
August 2016.
Synthesis and analysis of BMA analogs
BMA analogs were synthesized (Fig. 1a) and their struc-
tures were unequivocally determined using NMR and MS

Synthetic BMA analogs as novel STAT3 inhibitors
as we previously described [20]. To synthesize compound
1, 2-(1H-indol-3-yl)acetic acid (3.5 g, 20 mmol) was added
to a stirred suspension of NaH (4 g, 100 mmol, mixture of
60% NaH in mineral oil) in THF (80 mL) at 0°C. The
resulting mixture was stirred for 30 min followed by drop-
wise addition of ethyl iodide (5 mL, 60 mmol) in THF (30
mL), allowed to warm to room temperature (RT), and stir-
red overnight. The reaction mixture was then cooled to 0 °C
and quenched by addition of 10 mL MeOH and 20 mL H2O
followed by extraction with Et2O (2 × 100 mL). The aqu-
eous layer was acidified by addition of 6N hydrochloric acid
(30 mL) and extracted with ethyl acetate (EtOAc, 2 × 100
mL). The combined organic extracts were washed with H2O
(2 × 100 mL) and brine (2 × 100 mL), dried over anhydrous
Na2SO4, filtered and concentrated under vacuum. The
residue was purified by flash chromatography (1:3 EtOAc-
petroleum ether, v/v) to yield 2-(1-ethyl-1H-indol-3-yl)
acetic acid (1) as a colorless solid (3.87 g, 95% yield).
Using the same procedures, 2-[1-(3-cyanopropyl)-1H-
indol-3-yl]acetic acid (5a) was synthesized as a colorless
solid (541 mg, 30% yield) from 2-(1H-indol-3-yl)acetic acid
(1.4 g, 8 mmol), NaH (1.6 g, 40 mmol) and 4-
bromobutanenitrile (2.4 mL, 24 mmol); 2-(1-(4-cyanobu-
tyl)-1H-indol-3-yl)acetic acid (6a) was synthesized as a
colorless solid (1.69 g, 83% yield) from 2-(1H-indol-3-yl)
acetic acid (1.4 g, 8 mmol), NaH (1.6 g, 40 mmol) and 5-
bromobutanenitrile (2 mL, 16 mmol).
To synthesize compound 2a, indole (702 mg, 6.0 mmol)
was added to a stirred suspension of NaH (360 mg, 9.0
mmol, mixture of 60% NaH in mineral oil) in MeCN (80
mL) at –5 °C. The resulting mixture was stirred for 30 min
followed by dropwise addition of 3-bromopropanenitrile
(744 μL, 9.0 mmol), allowed to warm to RT and stirred for
24 h, followed by quenching with addition of saturated
aqueous NH4Cl solution (100 mL) and extraction with
EtOAc (2 × 100 mL). The combined organic layers were
washed with H2O (2 × 100 mL) and brine (2 × 100 mL),
dried over anhydrous Na2SO4, filtered and concentrated
under vacuum. The residue was purified by flash chroma-
tography (1:15 EtOAc-petroleum ether, v/v) to give 3-(1H-
indol-1-yl)propanenitrile (2a) as a colorless oil (949 mg,
94% yield).
indole (585 mg, 5 mmol), NaH (300 mg, 7.5 mmol), and
benzyl bromide (0.89 mL, 7.5 mmol) in DMF (30 mL), and
purified by flash chromatography (1:100 EtOAc-petroleum
ether, v/v).
To synthesize compound 2b, oxalyl chloride (1.11 g,
8.72 mmol) was added to a solution of 2a (988 mg, 5.81
mmol) in CH2Cl2 (25 mL) at –5 °C. The resulting mixture
was stirred for 4 h and allowed to warm to RT and then
concentrated under vacuum and redissolved in 30 mL
CH2Cl2. The resulting solution was added to a mixture of
compound 1 (1.18 g, 5.81 mmol) and triethylamine (Et3N,
1.18 g, 11.64 mmol) in CH2Cl2 (30 mL) and stirred over-
night followed by removing the solvent under vacuum. The
residue was purified by flash chromatography (1:2 EtOAc-
petroleum ether, v/v), leading to 2-(1-ethyl-1H-indol-3-yl)-
3-[1-(2-cyanoethyl)-1H- indol-3-yl]maleic anhydride (2b)
as a red powder (530 mg, 23% yield).
Using the same procedures, 2-(1-ethyl-1H-indol-3-yl)-3-
[1-(3-cyanopropyl)-1H-indol-3-yl]maleic anhydride (3b)
was prepared as a red powder (415 mg, 45% yield) from 3a
(407 mg, 2.21 mmol), oxalyl chloride (421 mg, 3.32 mmol),
1 (449 mg, 2.21 mmol) and Et3N (447 mg, 4.42 mmol), and
purified by flash chromatography (1:3 EtOAc-petroleum
ether, v/v); 2-(1-ethyl-1H-indol-3-yl)-3-[1-(4-cyanobutyl)-
1H-indol-3-yl]maleic anhydride (4b) was synthesized as a
red powder (712 mg, 51% yield) from 4a (627 mg, 3.17
mmol), oxalyl chloride (604 mg, 4.76 mmol), 1 (644 mg,
3.17 mmol) and Et3N (640 mg, 6.34 mmol); 2,3-bis[1-(3-
cyanopropyl)-1H-indol-3-yl]maleic anhydride (5b) was
synthesized as a red powder (500 mg, 44% yield) from 3a
(452 mg, 2.46 mmol), oxalyl chloride (469 mg, 3.69 mmol),
5a (595 mg, 2.46 mmol) and Et3N (497 mg, 4.92 mmol),
and purified by flash chromatography (1:1 EtOAc-
petroleum ether, v/v); 2,3-bis[1-(4-cyanobutyl)-1H-indol-
3-yl] maleic anhydride (6b) was synthesized as a red
powder (900 mg, 32% yield) from 4a (1140 mg, 5.76
mmol), oxalyl chloride (1097 mg, 8.64 mmol), 6a (1470
mg, 5.76 mmol) and Et3N (1163 mg, 11.5 mmol); 2-(1-
ethyl-1H-indol-3-yl)-3-(1-benzyl-1H-indol-3-yl)maleic
anhydride (7b) was synthesized as a red powder (300 mg,
39% yield) from 7a (356 mg, 1.72 mmol), 1 (0.397 g, 1.92
mmol), oxalyl chloride (0.25 ml, 2.88 mmol) and Et3N
(0.531 mL, 3.84 mmol), and purified by flash chromato-
graphy (1:5 EtOAc-petroleum ether, v/v).
Using the same procedures, 4-(1H-indol-1-yl)butaneni-
trile (3a) was synthesized as a colorless oil (1.04 g, 57%
yield) from indole (1.17 g, 10 mmol), NaH (0.6 g, 15 mmol)
and 4-bromobutanenitrile (1.6 mL, 15 mmol), and purified
by flash chromatography (1:20 EtOAc-petroleum ether, v/
v); 5-(1H-indol-1-yl)pentanenitrile (4a) was synthesized as
a colorless oil (0.63 g, 83% yield) from indole (0.59 g, 5
mmol), NaH (0.30 g, 7.5 mmol) and 5-bromopentanenitrile
(880 μL, 7.5 mmol) and purified by flash chromatography
(1:10 EtOAc-petroleum ether, v/v); 1-benzyl-1H-indole (7a)
was synthesized as a colorless solid (1.0 g, 99% yield) from
BMA016 was synthesized by first mixing 2b (230 mg,
0.56 mmol) in DMF (10 mL) with the mixture of HMDS
(12 mL, 57 mmol) and MeOH (1.2 mL, 28.5 mmol) in DMF
(5 mL), which was sealed and stirred at RT overnight fol-
lowed by quenching the reaction with addition of ice-cold
water (25 mL), extraction with EtOAc (2 × 100 mL), and
washing with H2O (3 × 100 mL). The organic layers were
combined and dried over anhydrous Na2SO4, filtered and
evaporated under vacuum. The residue was purified by flash

X. Li et al.
chromatography (4:1 CH2Cl2-petroleum ether, v/v) to give
2-(1-ethyl-1H-indol-3-yl)-3-[1-(2-cyanoethyl)-1H-indol-3-
yl]maleimide (BMA016) as a red powder (219 mg, 95%
yield).
(2 × 100 mL). The organic layer was dried over anhydrous
Na2SO4, filtered and concentrated under vacuum. The
residue was purified by flash chromatography (6:1 CH2Cl2-
ethyl acetate, v/v) to yield 2-(1-ethyl-1H-indol-3-yl)-3-(1H-
indol-3-yl)maleimide (7d) as a red powder (70.5 mg, 89%
yield).
Using the same procedures, 2-(1-ethyl-1H-indol-3-yl)-3-
[1-(3-cyanopropyl)-1H-indol-3-yl]maleimide
(BMA019)
was synthesized as a red powder (203 mg, 98% yield) from
3b (205 mg, 0.49 mmol), HMDS (10.2 mL, 48.5 mmol) and
MeOH (0.97 mL, 24.3 mmol), and purified by flash chro-
matography (1:1 EtOAc-petroleum ether, v/v); 2-(1-ethyl-
1H-indol-3-yl)-3-[1-(4-cyanobutyl)-1H-indol-3-yl]mal-
eimide (4c) was synthesized as a red powder (585 mg, 95%
yield) from 4b (616 mg, 1.14 mmol), HMDS (12 mL, 57
mmol) and MeOH (1.14 mL, 28.5 mmol), and purified by
flash chromatography (1:2 EtOAc-petroleum ether, v/v);
2,3-bis[1-(3-cyanopropyl)-1H-indol-3-yl]maleimide
Analysis and characteristics of all compounds are
described in the Supplementary Method.
Docking and binding free energy estimation
The crystal structure of STAT3 was obtained from the
RCSB protein data bank (ID: 1BG1) with missing residues
modeled using ModLoop [21]. Chain B was removed from
the structure and only chain A was kept and prepared for
docking by the UCSF Chimera Dock Prep module [22].
Since Chain B was removed, the binding pocket in chain A
for pTyr705 in chain B was chosen as the docking site. The
2D structure of BMA097 was converted to 3D format by
ChemBioDraw 13.0, prepared using the UCSF Chimera
Dock Prep module, and docked to the SH2 domain of
STAT3 by DOCK6 suite [23]. The binding free energies
(ΔGbind) of BMA097 were calculated as we previously
described [24]. Briefly, 20-ns product MD simulations were
performed and then 200 snapshots were extracted for
enthalpy calculation using Generalized Born method and 20
frames were used to estimate entropy due to the expen-
siveness of the computation.
(BMA097) was synthesized as a red powder (341 mg, 91%
yield) from 5b (375 mg, 0.81 mmol), HMDS (375 mg, 0.81
mmol) and MeOH (0.66 mL, 16.2 mmol), and purified by
flash chromatography (1:2 EtOAc-petroleum ether, v/v);
2,3-bis[1-(4-cyanobutyl)-1H-indol-3-yl]
maleimide
(BMA100) was synthesized as red powder (426 mg, 90%
yield) from 6b (470 mg, 0.96 mmol), HMDS (8.1 mL, 38.4
mmol) and MeOH (0.77 mL, 19.2 mmol), and purified by
flash chromatography (3:2 EtOAc-petroleum ether, v/v); 2-
(1-ethyl-1H-indol-3-yl)-3-(1-benzyl-1H-indol-3-yl)mal-
eimide (7c) was synthesized as a red powder (426 mg, 90%
yield) from 7b (260 mg, 0.58 mmol), HMDS (2.44 mL,
11.7 mmol) and MeOH (0.23 mL, 5.8 mmol), and purified
by flash chromatography (4:1 CH2Cl2-petroleum ether, v/v).
To synthesize BMA088, a suspension of 4c (6.7 mg,
14.4 μmol) and NaHCO3 (2.4 mg, 28.8 μmol) in 37%
HCHO (5 mL) was stirred for 10 h at 85 °C. The reaction
was stopped by addition of excess ice-cold water and
extracted with EtOAc (2 × 50 mL) and washed twice with
brine (2 × 50 mL). The organic layer was dried over
anhydrous Na2SO4, filtered and concentrate under vacuum.
The residue was purified by flash chromatography (1:4
EtOAc-petroleum ether, v/v) to yield N-hydroxymethyl-2-
(1-ethyl-1H-indol-3-yl)-3-[1-(4-cyanobutyl)-1H-indol-3-yl]
maleimide (BMA088) as a red powder (6.9 mg, 96% yield).
N-hydroxy-2-(1-ethyl-1H-indol-3-yl)-3-(1-hydroxy-1H-
indol-3-yl)maleimide (BMA127) was prepared in a similar
manner as a red powder (111 mg, 95% yield) from 7d (100
mg, 0.28 mmol), NaHCO3 (71 mg, 0.84 mmol) and 37%
HCHO (5 mL), and purified by flash chromatography (2:1
EtOAc-petroleum ether, v/v).
Luciferase reporter assay
MDA-MB-231-STAT3 cells (stably transfected with
STAT3-dependent luciferase reporter) were used for testing
BMA as we previously described [25, 26]. Briefly, the cells
were treated with vehicle DMSO control or BMA com-
pounds at different concentrations for various times fol-
lowed by measurement of luciferase activity using a
luciferase assay kit (Promega).
Western blot and real-time RT–PCR
Western blot and real-time RT–PCR analyses were per-
formed as we previously described [25, 26]. Briefly, fol-
lowing separation by SDS-PAGE and transfer onto PVDF
membranes, the membranes were probed with primary
antibodies and then HRP-conjugated secondary antibodies.
The signals were detected using the Amersham ECL wes-
tern blotting detection reagent (GE Healthcare, Uppsala,
Sweden) and signal recorded using x-ray films.
The real-time RT–PCR was performed using an ABI
Prism@7500 sequence detection system (Applied Biosys-
tems) with SYBR Green diction according to the manu-
facturer’s instruction as we previously described [25, 26].
To synthesize 7d, 1 M t-BuOK/THF (8.4 mL, 8.4 mmol)
solution was mixed with a solution of 7c (100 mg, 0.225
mmol) in DMSO (0.85 mL, 12.0 mmol) and pure O2 was
bubbled into the mixture for 30 min. The reaction was
quenched by addition of saturated aqueous NH4Cl solution,
extracted with EtOAc (2 × 100 mL), and washed with H2O

Synthetic BMA analogs as novel STAT3 inhibitors
Primers used for PCR are 5′-GGCCCCTCGTCAT-
CAAGA-3′ (forward) and 5′-TTTGACCAGCAACCT-
staining with STAT3 or pSTAT3 antibody. The staining
was detected using secondary antibody-conjugated with
Alexa Fluor 555. Nuclei were counter-stained by DAPI
before mounting the cover glasses onto slides for fluores-
cence imaging using a confocal microscope [28].
GACTTTAGT-3′
(reverse)
for
STAT3,
5′-
CTTCCTCTCCAAAATGCCAG-3′ (forward) and 5′-
AGAGATGGAAGGGGGAAAGA-3′ (reverse) for cyclin
D1, 5′-TGCATTGTTCCCATAGAGTTCCA-3′ (forward)
and 5′- CCTGAATGACCACCTAGAGCCTT-3′ (reverse)
for Bcl-xL, and 5′-AAGGACTCATGACCACAGTCCAT-
3′ (forward) and 5′-CCATCACGCCACAGTTTCC-3′
(reverse) for GAPDH. The threshold cycle (Ct) was defined
as the PCR cycle number at which the reporter fluorescence
crosses the threshold reflecting a statistically significant
point above the calculated baseline. The relative level was
calculated using 2ΔΔCt with GAPDH as an internal control.
Pulldown assay
Pulldown assay using immobilized BMA097 was per-
formed as previously described [25, 26]. Briefly, BMA097
was incubated with CNBr-activated Sepharose 4B (CNBr-
S4B) to immobilize BMA097. Sepharose-conjugated
BMA097 was then blocked by 10% milk in the binding
buffer (20 mmol/L Tris-HCl (pH 8.0), 150 mmol/L KCl, 1
mmol/L EDTA, 15% glycerol, 0.5% NP-40, 0.2 mmol/L
PMSF and protease inhibitor cocktail) followed by mixing
with cell lysate or purified STAT3 protein for pulldown
assay. The pulldown materials were then subjected to
separation on SDS-PAGE and Western blot analysis probed
by STAT3 antibody or silver staining. For competition, cell
lysate was pre-incubated with DMSO vehicle, 400 μM
BMA097 or an irrelevant compound [25, 26] for 30 min at
37°C prior to pulldown assay using immobilized BMA097.
For elution, the pulldown materials were incubated in the
presence of vehicle, 400 μM BMA097 or an irrelevant
compound for 1 h followed by centrifugation. The eluent
supernatant was then collected for Western blot analysis.
Survival and apoptosis assays
The cytotoxicity of BMA097 was evaluated using MTT
survival assay as previously described [25, 26]. Briefly,
3–4 × 103 cells/well were seeded in 96-well plates and
treated with BMA097 at different concentrations for various
times, followed by addition of MTT and analysis of viable
cells.
The BD Pharmingen Annexin V-FITC Apoptosis
Detection Kit was used to evaluate BMA097-induced
apoptosis according to manufacturer’s instructions. Briefly,
3 × 105 cells/well were seeded in 6-well plates and treated
with BMA097 at different concentrations for various times
followed by staining with annexin V-FITC and propidium
iodide (PI) and FACS analysis using a flow cytometer
(Becton Dickinson, USA). Both annexin V-positive/PI
negative and double-positive cells were considered apop-
totic cells in this study.
Mitochondrial membrane potential Δψm depolarization
was determined using a fluoresce probe JC-1 according to
manufacturer’s instructions. Briefly, cells were exposed to
BMA097 for 24 h and stained with JC-1 (5 μg/mL) for 30
min at 37 °C. Cells were then washed with PBS and intra-
cellular fluorescence intensity of JC-1 was quantified using
flow cytometry. Data analysis was performed using Cell
Quest software (Becton Dickinson). The Δψm was calcu-
lated as the ratio of red (JC-1 aggregates) to green (JC-1
monomers) fluorescence.
In vivo efficacy
For efficacy study, 1 × 107 MDA-MB-231 cells were first
injected subcutaneously into the flank of a 5-week old Balb/
c athymic (nu+/nu+) female mouse and allowed to pro-
liferate for two weeks. The exuberantly proliferating
xenograft tumor mass was then collected and cut into ~1.5-
mm thick pieces and inoculated subcutaneously on the left
flank of 4–6 weeks old Balb/c athymic (nu+/nu+) female
mice. When tumors reached ~100 mm3, the mice were
randomized into four groups using an online tool (https://
www.randomizer.org/) with 5 in each group and treated
every 3 days for 18 days with vehicle only, 10 mg/kg or 40
mg/kg BMA097, and 0.6 mg/kg vincristine, respectively,
via IP injection. The tumor volume and body weight were
measured every 3 days. The treatments were not but mea-
surements of tumor volume were blinded. Based on our
preliminary experiments and experiences working with
MDA-MB-231 cells as xenografts, five animals in each
group were estimated that would give enough power
without statistical analysis. All animals were included for
final data analyses without any exclusion. The above in vivo
studies were approved by the Institutional Animal Care and
Use Committee.
Immunostaining
Immunofluorescence staining was performed as we pre-
viously described [27]. Briefly, cells were seeded onto 12-
mm round glass cover slips in 24-well plates and treated
with or without BMA097 for 24 h. Cells were washed and
fixed in methanol/acetone (1:1) and permeabilized with
0.1% Triton X-100 before blocking with 3% BSA and

X. Li et al.
Statistical analysis
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of the signal transducers and activators of the transcription 3
pathway in alveolar epithelial cells induces inflammation and
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All experiments except in vivo studies were performed at
least three times. Statistical analysis was performed with an
analysis of variance (ANOVA) followed by the Turkey’s t-
test. P-values of <0.05 were considered statistically sig-
nificant. For every figures, the statistical tests were appro-
priate and met the assumptions of the tests. However, the
variance within and between each treatment group of the
in vivo study is different.
Acknowledgements We thank IU Big Red supercomputers for the
CPU time. This work was supported in part by grants from the Pro-
gram for Changjiang Scholars and Innovative Research Team in
University (PCSIRT, No IRT_17R68), the National Natural Science
Foundation of China (Nos. 81273532 & U1501221) and by National
Institute of Health Grant R01 CA211904.
Compliance with ethical standards
17. Gartsbein M, Alt A, Hashimoto K, Nakajima K, Kuroki T, Ten-
nenbaum T. The role of protein kinase C delta activation and
STAT3 Ser727 phosphorylation in insulin-induced keratinocyte
proliferation. J Cell Sci. 2006;119:470–81.
Conflict of interest The authors declare that they have no conflict of
interests.
18. Welte T, Zhang SS, Wang T, Zhang Z, Hesslein DG, Yin Z, et al.
STAT3 deletion during hematopoiesis causes Crohn’s disease-like
pathogenesis and lethality: a critical role of STAT3 in innate
immunity. Proc Natl Acad Sci USA. 2003;100:1879–84.
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