GLG Pharma STAT3 Inhibitors, Mechanism of Action Summary

(28 Dec2011)

GLG Pharma STAT3 Inhibitors

Mechanism of Action Summary

28 December 2011

Michael W Lovell, PhD

John J Whalen, MD
Hector J Gomez MD, PhD

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 GLG Pharma STAT3 Inhibitors, Mechanism of Action Summary
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ITEM Page

TABLE OF CONTENTS 2
Introduction to STAT3 Pathways and Inhibitors in Cancer and 3
Polycystic Kidney Disease

A. Studies Demonstrating the Mechanisms of Action of GLG-302 6

on the STAT3 Pathway

1) In Vitro Studies on V-Src fibroblasts, Breast Cancer Cell Lines 7

and isolated STAT3 proteins, and In Vivo Studies on Breast
Cancer Xenografts (Siddiquee et al. PNAS 2007)

2) Hepatocellular carcinoma (Lin et al. 2009) 11

3) Pancreatic cancer (Jaganathan et al. 2010 ) 12

4) Glioblastoma Multiforme (GBM) (Sherry et al. 2009.) 14

B. Role of STAT3 in Energy Metabolism in Epithelial Tumor Cell 17

Lines (Demaria et al. 2010)

C. Polycystic kidney disease (PKD) (Takakura et al. 2011) 19

D. Studies Demonstrating the Mechanisms of Action of GLG- 202 20

(Siddiquee et al. ACS Chem Biol, 2007)

E. Studies Demonstrating the Mechanisms of Action of GLG-401 24

(Turkson et al. 2005)

REFERENCES 25

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Introduction to STAT3 Pathways and Inhibitors in Cancer and Polycystic Kidney Disease

To understand the mechanism of action of agents such as GLG-302, that inhibit the functions/actions of the
“STAT3 pathway”, it is necessary to be familiar with the major components of the STAT3 pathway, the
actions that occur at different steps along the activated pathway, and the structural domains of the STAT3
molecule that participate in the pathway.

The [Surface Receptor (Growth Hormone, Cytokine)/JAK; Non-Receptor (Src)], STAT3 Pathway.

Figure A below provides a condensed view of the major components/actions of the STAT3 pathway.

Figure A: Major STAT3 Signaling Components

____________________________________________________________________________________________
Devarazan A, Curr Mol Med, 2009; 9: 626

Figure A outlines that major components of the STAT3 Signaling pathway, with specific mention of
downstream functional proteins that participate in cellular proliferation and metastasis. Key steps in this
pathway are:

Cell surface receptors for growth factors and cytokines are activated by exposure to their respective ligands,
leading to their activation of a JAK kinase.

The activated JAK kinase then phosphorylates tyrosine residues on the inactive STAT3 molecules present in
the cytoplasm, converting it to a phosphorylated or activated “p-STAT3”.

Once phosphorylated at the 705 tyrosine residue, two p-STAT3 molecules spontaneously dimerize by
binding between the SH2 domains and the phosphorylated tyrosines on each molecules. The dimers then
spontaneously translocate to the nucleus.

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In the nucleus, activated STAT3 dimers bind to specific promoter elements of target genes and activate the transcription
of genes regulating key biological functions, including cell proliferation, survival, angiogenesis, tumor invasion, and
metastasis.

Figure 1, below provide a further depiction of steps in the STAT3 pathway and the various pro oncogenic
actions that it mediates.
_______________________________________________________________________________________
Figure 1: Steps in the STAT3 Pathway and its downstream oncogenic proteins/actions

_______________________________________________________________________________________
Siddiquee K and Turkson J, Cell Research, 2008; 18:254-67

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Specific Domains on the STAT3 molecule participate in the STAT3 Pathway. (From Siddiquee, Turkson,
Cell Res, 2008, 18: 254-67).

1. The C Terminal Transactivation Domain contains tyrosine residue 705: When phosphorylated on a first
STAT3 molecule, this tyrosine binds to the SH2 domain on a second STAT3 molecule to form a STAT3
dimer. (Reciprocal binding occurs between the other tyrosine and the other SH2 region on each
molecule).

2. The SH2 domain: Contains a receptor cavity which binds with a phosphorylated tyrosine on another
STAT3 molecule.

3. The DNA binding domain: Following dimerization, the DNA binding domain, binds to the promoter
regions of various genes present in nuclear DNA and initiates their transcription.

4. The C Terminal Transactivation Domain also contains serine residue 727: The additional
phosphorylation of this serine residue maximizes the transcriptional activity of a STAT3 molecule with
tyrosine 705 phosphorylation.

GLG PHARMA (GLGP) COMPOUNDS THAT INHIBIT ONE OR MORE STEPS IN THE
CYTOPLASMIC/NUCLEAR STAT3 PATHWAY IN CANCER OR PKD; OR AFFECT ENERGY
METABOLISM IN CANCER

 GLG-302 (previously named S3I-201, NSC 74859)

 GLG- 202 (previously named S3I-M2001 )
 GLG-401 (previously named, S3I- 295, NSC 295558 )

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A. Studies Demonstrating the Mechanisms of Action of GLG-302 on the STAT3 Pathway

Different investigators have published their findings on the actions of GLG-302 (previously named S3I-201,
NSC 74859) on various cell lines and tissue samples in different types of cancer and non-cancer conditions
using both in vitro evaluations, in vivo mouse xenografts of human cancer cell lines, as well as isolated
proteins, using Western Blot, Elisa, and EMSA techniques; and polycystic kidney disease. These studies
include evaluations in:

1. In Vitro Studies on V-Src fibroblasts, Breast Cancer Cell Lines and isolated STAT3 proteins, and In
Vivo Studies on Breast Cancer Xenografts (Siddiquee et al, PNAS 2007)
2. Hepatocellular carcinoma (Lin et. al)
3. Pancreatic cancer (Jaganathan et al)
4. Glioblastoma Multiforme (GBM) (Sherry et al)
5. The role of STAT3 in Energy Metabolism in Epithelial Tumor Cell Lines (Demaria et al)
6. Polycystic kidney disease (PKD) (Takakura et al)

In each study the researchers confirmed the STAT3 inhibitory activity of GLG-302 at one or more steps
along the STAT3 Pathway. This document summarizes the published work of these researchers.

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1. In Vitro Studies on V-Src fibroblasts, Breast Cancer Cell Lines and isolated STAT3 proteins, and
In Vivo Studies on Breast Cancer Xenografts (Siddiquee et al. PNAS 2007)

Mechanisms of Action of GLG-302 Supported by in vitro studies

 Inhibits or reverses the activation (phosphorylation) of STAT3 (See following Fig 2E, and Fig 2F)

 Inhibits Stat3:Stat3 dimer formation by binding to the SH2 Domain.( See following Fig 2C, and excerpt
below from Siddiquee 2007, pp 7391-2)

GLG-302 was identified as a chemical probe inhibitor of Stat3 activity from the National Cancer
Institute chemical libraries by using structure-based virtual screening with a computer model of the Stat3
SH2 domain bound to its Stat3 phosphotyrosine peptide derived from the x-ray crystal structure of the
Stat3β homodimer. GLG-302 received a high score of -11.7 compared to the highest score of -11.9 for a
native phosphopeptide sequence APpYLK. Typically compounds receiving highly favorable (negative)
scores have structural features that include sulfonyl, carboxyl, and hydroxyl functional groups. GLG-302
has all three groups.

 Inhibits Stat3 Dimer - DNA-binding (See following Fig 2A)

 Inhibits transcriptional activities/gene expression of genes encoding cyclin D1, Bcl-xL and Survivin,
thereby reducing the formation of downstream proteins that promote cancer cell proliferation and which
protect cancer cells against apoptosis. (See excerpt below from Siddiquee 2007, p. 7394, left column).

V-Src-transformed mouse fibroblasts (NIH 3T3/v-Src) and the human breast carcinoma MDA-MB-231
cell line, which harbor constitutively active Stat3 were incubated with S3I-201, and evaluated with
immunoblot analysis of whole-cell lysates. They showed significant reduction in expression of cyclin
D1, Bcl-xL, and Survivin proteins in response to S3I-201 treatment indicating that it represses induction
of the cell cycle and anti-apoptotic regulatory genes in malignant cells.

 S3I-201 Blocks Cell Growth only in Cells Where Stat3 is Persistently Activated. (See excerpt below
from Siddiquee et al. 2007, p. 7393-4).

The human breast carcinoma (MDA-MB-231, MDA-MB-435, and MDA-MB-468) cell lines and the v-
Src-transformed mouse fibroblasts (NIH 3T3/v-Src), with constitutively active Stat3 and the human
breast carcinoma MDA-MB-453 cell line and normal mouse fibroblasts (NIH 3T3), which do not harbor
aberrant Stat3 activity, were treated with S3I-201, and analyzed for viable cell number by trypan blue
exclusion and microscopy. S3I-201 significantly reduced viable cell numbers and inhibited growth of
transformed mouse fibroblasts and breast carcinoma cell lines with activated STAT3. By contrast,
growth and viability of normal mouse fibroblasts and the breast carcinoma cell line (MDA-MB-453)
without aberrant Stat3 activity were not significantly altered

 S3I-201 Preferentially Induces Apoptosis of Malignant Cells Harboring Constitutively Active Stat3. (See
excerpt below from Siddiquee et al. PNAS 2007, p. 7394, right column).

Human breast cancer cell lines MDA-MB-453 and MDA-MB-435, normal mouse fibroblasts (NIH 3T3)
and their v-Srct transformed counterpart (NIH 3T3/v-Src) were untreated (DMSO, control) or treated
with S3I-201 for 48 h and analyzed by annexin V binding. At 30–100 μM, S3I-201 induced significant
apoptosis in the breast cancer cell line MDA-MB-435 and NIH 3T3/v-Src, which harbor constitutively
active Stat3, compared to the MDA-MB- 453 and NIH 3T3 cells, which lack abnormal Stat3 activity and
are less sensitive to S3I-201 at up to 100 M.

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These findings strongly suggest that the antitumor activity of S3I-201 is mediated through inhibition of
aberrant Stat3 activation, as well as inhibition of STAT3 dimer formation, the binding of STAT3 dimers to
DNA, and resultant downstream consequences of these inhibitions.

Further details on several of the above in vitro findings are provided in the following Figure 2 (see Legend
below, and Figure on following page) from the Siddiquee et al., PNAS 2007 publication.

Fig. 2A to 2F Below shows findings for the effects of S3I-201 on STATs, Shc, v-Src transformed mouse
fibroblast, Erks activation, and onLck-SH2 domain phosphopeptide binding.

Fig. 2A Left shows potent inhibition of Stat3:Stat3 Dimer - DNA-binding activity by S3I-201, with an average
IC50 value of 86 ± 33 µM.

Fig. 2A Right S3I-201 preferentially inhibits Stat3 DNA-binding activity over that of Stat1 (IC50 values,
Stat3:Stat3, 86 ± 33 µM; Stat1:Stat3, 160 ± 43 µM; and Stat1:Stat1, ±300 µM) and inhibits that of Stat5
with 1⁄2 the potency (IC50 value, 166 ±17 µM).

Fig. 2B. The unphosphorylated, inactive Stat3 monomer by itself had no significant effect on DNA-binding
activity of activated Stat3 (Fig. 2B, compare lane 2 to lane 1), because inactive Stat3 monomer is incapable
of binding DNA (19). By contrast, the presence of inactive Stat3 monomer diminished the inhibitory effect
of S3I-201 on the activated Stat3 in a dose-dependent manner, resulting in the recovery of the active Stat3
DNA-binding activity (Fig. 2B, lanes 5–7 versus lanes 3 and 4). The Stat3 DNA-binding activity that was
otherwise inhibited (Fig. 2B, lanes 3 and 4) was partially or completely restored in the presence of 4 or 5 μL
of inactive Stat3 lysates, respectively (Fig. 2B, lanes 6 and 7). These findings support the S3I-201–Stat3
nteraction and suggest the interaction is independent of the activation status of Stat3.

Fig. 2C. Western blot analysis probing with anti-FLAG antibody showed no detectable level of FLAG-ST3
protein in the Stat3-YFP immunoprecipitates from S3I-201-treated cells (Fig. 2Ci Upper Left, lane 2 vs. 1),
suggesting the disruption by S3I-201 of the complex formation between Stat3-YFP and FLAGST3
proteins.

Fig 2D. The ELISA show that the copresence of the Lck-SH2-GST protein and its cognate pTyr peptide
results in signal induction (Fig. 2D, bar 4), suggesting an interaction between the two (20). The addition of
30 µM and 100 µM S3I-201 has no effect on the signal induction (Fig. 2D, compare bars 5 and 6 to bar 4),
indicating that S3I-201does not interfere with the binding of the Lck SH2 domain to its cognate pTyr peptide.

Fig 2E. Compared with control (0.05% DMSO-treated cells, lane 1), S3I-201 induced a time-dependent
inhibition of constitutive Stat3 activation in NIH 3T3/v-Src fibroblasts (Fig. 2E, lanes 4–6).

Fig. 2 F. SDS/PAGE and Western blot analysis of whole-cell lysates from NIH 3T3/v-Src fibroblasts show
pTyr-705 Stat3 levels were significantly diminished after 24-h treatment with S3I-201 (Fig. 2F), whereas the
total Stat3 protein level remained unchanged.

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The mechanism of action of GLG-302 is further supported by the following in vivo study findings.

Figure 3. Tumor growth inhibition by S3I-201.

(A) Human breast (MDA-MB-231) tumor-bearing mice were given S3I-201 (5 mg/kg) i.v. every 2 or every 3
days. Tumor sizes were monitored every 2–3 days, converted to tumor volumes, and plotted vs study day.
Data points shown are the mean ± SD of eight tumor-bearing mice each.

(B) Three days after the last S3I-201 injection, animals were killed, tumors from one control animal
(DMSO-treated) or residual tumor tissue from two S3I-201-treated (T1 and T2) mice were extracted, and
lysate preparations with equal total proteins were analyzed for Stat3 activation by incubating with
radiolabeled hSIE probe and subjecting the products to EMSA analysis (lanes 1–3), or lysates from control
tumor tissue of equal total proteins were preincubated with or without increasing concentrations of S3I-201
before incubation with radiolabeled hSIE probe and subjecting the products to EMSA analysis (lanes 1, 4, 5,
and 6). Positions of Stat3_DNA complexes are shown.

This xenograft study provides additional evidence that inhibition of one or more steps in the STAT3 pathway
is the likely mechanism by which GLG-302 (S3I-201) inhibits cell growth in cancer cells with constitutively
activated STAT3.

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2. Hepatocellular Carcinoma (Lin et al)

In a hepatocellular carcinoma (HCC) xenograph study, Lin et. al found that GLG-302 (S3I-201,NSC 74859)
treatment of Huh-7 xenografts in nude mice significantly retarded tumor growth, with an effective dose of
only 5 mg/kg. Moreover, NSC 74859 inhibited tyrosine phosphorylation of STAT3 in HCC cells in vivo
(Lin L. et al. Oncogene 2009; 28: 961–972). Figure 3 summarizes the results of the study.

Figure 3 Inhibiting signal transducer and activator of transcription 3 (STAT3) retards tumor growth.
pY705STAT3 expression was analyzed by immunohistochemistry in untreated and NSC 74859-treated
tumors. Panel a shows the sizes of the tumors over time in days after treatment. Asterisks indicate
significant differences between treated and untreated tumors (*P<0.05). Panel b shows that there is no
difference in mouse weight when comparing the mice treated with NSC 74859 with those that were untreated.
Panels c and e illustrate pY705STAT3 staining of cells in untreated tumors (quantified in (g) from >1200
cells). In contrast, in (d) and (f), cells within NSC74859-treated tumors had few pY705STAT3-positive tumor
cells (quantified in (g) from >1200 cells).

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3. Pancreatic Cancer (Jaganathan S et al JPET 2010; 333:373–381)

The effect of GLG-302 (S3I-201), Das (dasatinib, Sprycel), ZD (gefitinib, Iressa), and inhibitors of Src,
JAK, etc., on DNA binding activity by activated STAT3 homodimers were evaluated using methods
described by Turkson et al, 1998. As shown below in Fig 4 Ai, last row, lanes 13-16, 24 hour exposure
of Panc- cells to S3I-201 showed a dose response relationship among 30, 50 and 100 uM for DNA
binding of STAT3 dimers after to GLG. Activity confirmed for 50 uM for Panc-1 and Colo-357 in Fig
4C. Other agents showed variable effects after 0.5, 1 and 24 hours of exposure.
_______________________________________________________________________________________
Figure 4

_______________________________________________________________________________________
Legend for Figure 4 appears on next page.
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Fig. 4 legend. EMSA and immunoblotting analyses for effects of inhibitors on Stat3 activation. EMSA
analysis of Stat3 DNA-binding activity in Panc-1 (A) or Colo-357 (B) cells treated or untreated with the
pan ErbB inhibitor PD169, ZD, Das, the Jak inhibitor AG490, the ErbB2-selective inhibitor AG879, or
inhibitor combinations for the indicated times, or Panc-1 and Colo-357 cells treated for 24 h with S3I-201
(C), or immunoblotting analysis of whole-cell lysates from Panc-1 cells transfected with EGFR siRNA,
Src siRNA, or scrambled siRNA (control) and probing for pStat3 or Stat3 (D). *, supershift analysis.
Data are consistent with those obtained from three independent experiments.
_______________________________________________________________________________________
Jaganathan et al. also evaluated the effect of Das (dasatinib) 15 mg/Kg, ZD (gefitinib) 75 mg/Kg, and
GLG-302(S3I-201) 5 mg/Kg, in a nude mouse xenograft models implanted with either Panc-1 or Colo-357
cells treated intravenously Q 2-3 days for 2 weeks, then Q 5 of each 7 days for 3 weeks. There were
modest reductions in tumor volume growth with each single agent in both cell lines, and additive effects
for GLG-302 with both agents in the Colo-357 study and with gefitinib only in the Pance-1 Study. No
in vitro assessment of the inhibition of STAT3-DNA binding was done in these studies, so it is not
known with certainty whether the cause for the reduced tumor volume growth in any group was the effect
of these agents on the STAT3 pathway. (Data is shown in the Jaganathan reference Figure 6, page 379)
____________________________________________________________________________________

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4.Glioblastoma Mutliforme (GBM) (Sherry et al, 2009, 27(10): 2383 – 92)

GBM, also called Grade IV astrocytomas, are lethal forms of CNS neoplasms, gliomas, with a median
survival of just 11 to 18 months. STAT3 is activated in 67% of human GBM tumor samples.
(Lo WM, et al). It had previously been shown that knockdown of STAT3 by RNA interference (RNAi)
in differentiated GBM cell lines inhibits the expression of Bcl-xl, and Survivin, and induces apoptosis
(Konnikova). (Note: Thus far, no clinical trials have been conducted with any STAT3 inhibitor in
human patients with GBM.) GBM-SCs are highly tumorigenic in mice and display aberrant
proliferative capacity and gene expression patterns [Gali R, et al].

No one has previously determined the frequency of activated STAT3 in GBM-SCs or their
Responsiveness to STAT3 inhibitors. Sherry et al, have recently addressed these issues. They showed
that activated STAT3 is present in high concentrations in GBM-SCs cell lines (GS6-22 and GS7-2), that
STAT3 effects are reduced by the STAT3 inhibitors [GLG-302 (S3I-201) and STA-21] and that this
reduction is associated with suppression of cell proliferation and neurosphere formation. The mechanism
by which this suppression is believed to occur, is by the STAT3 inhibitor binding to the SH2 domain on
STAT3 and thereby preventing the formation of STAT3 dimers, and the binding of the dimers to nuclear
DNA. This prevents the expression of excessive quantities of pro-oncogenic proteins such as BcL-Xl,
and Survivin. The ability of STAT3 dimers to bind to DNA is evaluated by a bandshift assay using a
32
P-end labeled human sis-inducible element (hSIE) probe.

As shown in Figure 6A top two rows, immunoblotting with phosphospecific antibodies demonstrated that
STAT3 is phosphorylated on both tyrosine-705 and serine-727 in the GS6-22 and GS7-2 GBM-SC lines.
”Untreated” in this figure refers to GBM-SCs that were grown in media lacking serum and remained
undifferentiated. “Treated” refers to cells that were grown in media containing 2% serum, and which
regain differentiation. As noted, concentrations of both tyrosine phosphorylated and serine phosphorylated
STAT3 are present in larger quantities in the “untreated” cells. (Tyrosine phosphorylated STAT3 binds
with the SH3 domain to form dimers. Serine phosphorylation stimulates maximum transcriptional activity).

As shown in Figure 6B, both STA-21 and S3I-201 abolished the ability of STAT3 to bind the hSIE probe
n GS7-2 cells, thus indicating inhibition of STAT3-DNA binding. (Similar results were shown with GS6-22
cells, available online in Supporting Information, Fig 5). This confirms that STA-21 and S3I-201 can inhibit
STAT3 DNA binding in our GBM-SC lysates, which is consistent with published reports of their efficacy
against STAT3 in other cell lines in vitro and in vivo. Figure 7F shows the suppression cell growth in both
cell lines by both STAT3 inhibitors.

Note these findings do not rule out the possibility that other signaling pathways may be involved and could
contribute to the suppression of cell growth. However, neither STA-21 nor S3I-201 affected ERK or AKT
activation, as shown by immunoblotting of the U251 GBM serum line. (Data available online in supporting
information.)

Previously, both STA-21 and S3I-201 have been shown to inhibit STAT3 preferentially as compared with
STAT1 and STAT5. As shown in Figure 8, immunoblots of GS6-22 and GS7-2 GBM-SCs, it is clear that
although these cell lines express STAT5 and STAT1, STAT3 is the major activated STAT. Phosphotyrosine
TAT1 and STAT5 were undetectable under NSC culture conditions. Thus, STAT3 is the principal activated
STAT protein in these cells.

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Figure 6. STAT3 is expressed and activated in GBM-SCs. (A): Immunoblot of STAT3, pSer727 STAT3, and
pTyr705 STAT3 in GBMSCs untreated and differentiated for 7 days. The serum line derived from the GS7-2
parent tumor was also probed. (B): Bandshift assay of GS7-2 neurosphere lysates treated with inhibitors of
STAT3 DNA binding (STA-21, 30 lM; S3I-201, 100 lM), incubated with radiolabeled high-affinity SIE probe,
and separated by PAGE. Abbreviations: DMSO, dimethylsulfoxide; GBM-SC, glioblastoma stem cell; SIE,
sis-inducible element; STAT3, signal transducer and activator of transcription 3.

Figure 7. (F): STAT3 inhibition prevents GBM-SC growth. GS6-22 and GS7-2 cells were dissociated into
single cells and plated in equal numbers. Twenty-four hours after plating, cells were counted and treated
with DMSO, STA-21, and S3I-201. At day 3 and day 6 after drug treatment, cells were counted in triplicate
and plotted as the mean value.

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Figure 8. Previously, both STA-21 and S3I-201 have been shown to inhibit STAT3 preferentially as
compared with STAT1 and STAT5. From immunoblots of GS6-22 and GS7-2 GBM-SCs, it is clear that
although these cell lines express STAT5 and STAT1, STAT3 is the major activated STAT. Phosphotyrosine
STAT1 and STAT5 were undetectable under NSC culture conditions. Thus, STAT3 is the principal activated
STAT protein in these cells.

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B. The Role of STAT3 in Energy Metabolism in Epithelial Tumor Cell Lines (Demaria et al.)

For most of the 20 year history of research on the STAT family of proteins, it was believed that the
primary mechanism by which these molecules exert their effects on cancer cell transformation, invasion
and metastasis, was by acting as a transcription factor. In that function, they convert signals that begin on
the surface of cells into transcription of specific genes in the nucleus that encode for pro-oncogenic
proteins. More recently, it has been discovered that activated STAT3 also affects energy metabolism in
both mouse embryonic fibroblasts and cancerous cells. As with its role in transcription, to be active in
energy metabolism, STAT3 must be activated by tyrosine phosphorylation.

Most normal cells have the capacity to generate energy in the form of adensosine tri-phosphate (ATP) by
two major mechanisms, glycolysis in the cytoplasm, or oxidative respiration in mitochondria. In
glycolysis, glucose molecules are metabolized to pyruvate, which is converted to lactate that is released
from the cell for recycling in the liver. This process generates just 2 ATPs per glucose molecule. In
oxidative respiration, the pyruvate is oxidized to yield CO2, producing 36 ATP molecules from each
glucose. Most normal cells use the oxidative route when they are in a normoxic environment, and convert
to the glycolysis/lactate route when they are exposed to hypoxic conditions. The enzyme “hypoxia
inducible factor-1” (HIF-1) becomes activated in the presence of hypoxia and directs normal cells to
convert from the mitochondrial oxidative respiration to cytoplasmic glycolysis. Pyruvate dehydrogenase
kinase-1 (PDK-1) is a key enzyme in the glycolytic pathway that is turned on by HIF-1α.

In 1924, Otto Warburg discovered the “effect” that bears his name that cancer cells often will switch from
oxidative respiration to glycolysis even in the presence of adequate oxygen concentration. Reasons for
this conversion have been speculated on. One is that rapidly proliferating cancer cells have a greater need
for carbon molecules (from recycled lactate) to be used in the production of all of the proteins and
organelles required to produce new cells, than for energy. Another is that lactate inhibits apoptosis and
senescence, and promotes cancer cell invasion (Vander Heiden). A third is that the switch to the glycolytic
pathway for energy production, is a pre-adaptive change in many solid tumors which are often exposed to
fluctuating oxygen levels (Bertout)

Demaria et al have provided a summary of recent relevant data as well as new information on the role of
STAT3 in energy metabolism in cancer cells. (See Figure 5 on following page.) They assessed the effects
of inhibiting STAT3 on the glycolytic metabolism and mitochondrial activity of three STAT3-dependent
epithelial tumour cell lines, MDA-MB468 (breast), SKBR3 (breast), and DU145 (prostate), all of which
display constitutively active STAT3. In all cell lines, 12 hours S3I (GLG-302, S3I-201, NSC 74859)
treatment dramatically lowered Hif-1α and Pdk-1 expression and decreased lactate production. (See details
in Figure 5 on following page.)

Overall they concluded that in cancer cells with constitutively active STAT3, it acts as a master regulator
of cell metabolism, inducing aerobic glycolysis and down‐regulating mitochondrial activity both in
primary mouse embryonic fibroblasts (MEFs) and in STAT3‐dependent tumour cell lines. As a result,
cells are protected from apoptosis and senescence while becoming highly sensitive to glucose deprivation.
They show that enhanced glycolysis is dependent on HIF‐1α up‐regulation, while reduced mitochondrial
activity is HIF‐1α‐independent and likely caused by STAT3‐mediated down‐regulation of mitochondrial
proteins. The induction of glycolysis is an important component of STAT3 pro‐oncogenic activities, since
inhibition of STAT3 tyrosine phosphorylation in the tumour cell lines down‐regulates glycolysis prior to
leading to growth arrest and cell death, both in vitro and in vivo. (Demaria M et al. AGING, 2010; 2(11):
823 – 842).

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Figure 5. Stat3‐dependent glycolytic metabolism and mitochondrial activity in tumour cell lines.

(A) The STAT3‐dependent MDA‐MB468, SKBR3, DU145 and the STAT3‐independent T47D human
tumour cells were either treated or not with the S3I STAT3 inhibitor for 12 hours followed by the
analysis of total and Y705 phosphorylated STAT3 by Westernblot.

(B) Percentage of Annexin V+ MDA‐MB468 cells treated with the S3I compound for the indicated
times.

Gene expression, lactate production and mitochondrial Ca2+ release were measured in the
SKBR3 (C), DU145 (D) and T47D (E) cell lines. *, p ≤ 0,01 (n=3) for C and D with activated STAT3,
but not E, lacking activated STAT3.

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C. Polycystic Kidney Disease

Polycystic kidney disease (PKD) is not generally classified as a cancer disorder since it lacks the invasive
and metastatic characteristics that are hallmarks of most cancers. It does, however, involve uncontrolled
cellular growth and the destruction of normal kidney structure and function. PKD has also been shown to
possess a frequent occurrence of constitutively activated STAT3; and in an animal models of the condition,
one of the GLG Pharma STAT3 inhibitors, GLG-302 has been shown to reduce cyst volume and serum
creatinine. For this reason we have chosen to include it in this summary of the effects of the GLG Pharma
compounds on proliferative disorders.

Autosomal Dominant Polycystic Kidney Disease (Takakura et al)

Takakura et al showed STAT3 is activated in mouse and human PKD1 disease (Takakura et al.Human
Molecular Genetics, 2011, 20( 21): 4143–4154). To further verify the importance of STAT3 signaling in
PKD, the researchers administered nIKO mice with a chemical inhibitor of STAT3 activity, S3I-201
(GLG-302, NSC 74859; 10 mg/kg) or vehicle beginning at 3 weeks of age for 5 weeks. S3I-201 is known
to inhibit STAT3 homodimer complex formation and STAT3 DNA-binding and transcriptional activities.
In contrast to the rapid widespread cyst formation in the vehicle treated group, the S3I-201-treated group
displayed no body weight loss but had significantly reduced cystogenesis, shown by significantly reduced
kidney weight, kidney/body weight ratio, cyst volume, cyst number and serum creatinine. Pathological
findings seen in H&E-stained kidney sections of vehicle-treated nIKO kidneys including interstitial
expansion, multiple small cysts and dilated tubules aPlround large cysts are significantly reduced in
3I-201-treated nIKO kidneys. Masson’s trichrome staining confirms the striking reduction in collagen
deposition in S3I-201-treated kidneys, compared with vehicle-treated ones. The improvement in renal
histology is accompanied by a reduction in pSTAT3-positive cyst-lining epithelial cells and interstitial
cells in drug-treated mouse kidneys. Moreover, the mRNA expression of STAT3 target genes in S3I-201-
treated nIKO kidneys was significantly reduced, compared with vehicle-treated nIKO kidneys, and was
similar to un-treated wild-type kidneys (Figure 9). S3I-201 is also effective in suppressing STAT3
activation in human ADPKD cells.

Figure 9. (E) Real-time RT–PCR was performed on cDNA from vehicle (Ve)- and S3I-201 (S)-treated Pkd1
IKO kidneys. There was a significant reduction in the expression of STAT3 (P<0.02) and STAT3 target
genes c-Myc (P <0.008), cyclin D1 (P <0.03), cyclin D2 (P<0.04) and bcl-X (P<0.006) in S3I-201-treated
Pkd1 IKO kidneys compared with vehicle-treated ones. Un-treated wild-type kidneys (Ctl) were used to show
the baseline expression level of genes of interest. (F) Immunoblot of pSTAT3 and total STAT3 in ADPKD cells treated with
S3I-201 at the indicated concentrations. (G) Flow-activated cytometry analysis of human ADPKD
cells treated with either vehicle or S3I-201.

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D. Studies Demonstrating Mechanism of Action of GLG-202 (Siddiquee et al.

ACS Chem Biol 2007)
GLG-202 in Src Transformed Mouse Fibroblasts and Human Breast Cancer Cell Lines
GLG-202 (S3I-M2001) is an oxazole-based peptidomimetic of the Stat3 Src homology (SH) 2 domain-
binding phosphotyrosine peptide . It was shown by computational analysis and X-ray crystallography data
to have key structural requirements for hydrophobic hydrogen-bonding, and electrostatic interactions
critical for tight binding with the SH2 domain of the STAT3 molecule. When bound, it selectively disrupts
active Stat3:Stat3 dimers. This prevents the dimers from forming complexes with promoter regions for
survival genes, such as Bcl-xL. Furthermore, Stat3-dependent malignant transformation, survival, and
migration and invasion of mouse and human cancer cells harboring persistently activated Stat3 were
inhibited by S3I-M2001. Finally, S3I-M2001 inhibited growth of human breast tumor xenografts.
Examples of these finding from Siddiquee et al (ACS Chem. Biol., 2007; 2(12):787–798) are shown below.

Figure 10 below, shows examples of the in vitro effects of S3I-M2001 on STAT3:STAT3 dimers in several
cell lines.
 S3I-M2001favorably disrupts Stat3- DNA binding activity in in vitro nuclear extracts containing
activated Stat3 (Figure 10, panel a, left)
 S3I-M2001 preferentially inhibits Stat3;Stat3- DNA binding activity over Stat1:Stat1 dimers by 2-fold
(IC50 values (µM), Stat3:Stat3, 79 ± 09; and Stat1: Stat1, 159 ± 06) (Figure 10 a, middle and right)
 S3I-M2001 inhibited constitutive Stat3 Tyr phosphorylation and activation in the NIH3T3/v-Src and the
human breast cancer MDA-MB-231 cell line that harbor constitutively active Stat3 (Figure 10, panel b,
lanes 1–13)
 S3I-M2001 inhibited constitutive Stat3 Tyr phosphorylation of the NIH3T3/v-Src and the human breast
cancer MDA-MB-435 cell lines that harbor constitutively active Stat3 (Figure 10, panel c, lanes 1–6)
 S3I-M2001 (Figure 2, panel c, lanes 7–12), minimally repressed non-Stat3-related proteins such as
pJak1, pSrc, and pErk1/2 (MAPKs) in NIH3T3/v-Src fibroblasts.

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 GLG Pharma STAT3 Inhibitors, Mechanism of Action Summary
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Figure 10. Effects of S3I-M2001 on STATs activation and on pJak1, pSrc, pErk1/2. a) EMSA analysis of
STATs DNA-binding activities in nuclear extracts containing activated Stat3 (left panel) or Stat1 and Stat3
(middle panel) and pretreated with S3I-M2001 concentrations for 30 min at RT prior to incubation with
radiolabeled hSIE oligonucleotide probe, and IC50 values for the inhibition of STATs DNA-binding activity
in vitro (right panel) derived, as described in Methods. b) EMSA analysis of Stat3 DNA-binding activity in
nuclear extracts from NIH3T3/v-Src or MDA-MB-231 cells, or from recombinant human EGF (rhEGF)-
stimulated NIH3T3/hEGFR mouse fibroblasts treated with or without S3I-M2001. c) SDS-PAGE/Western
blot analysis of whole-cell lysates prepared from S3I-M2001-treated or untreated NIH3T3/v-Src or MDA-
MB-435 cells probing for pTyr705Stat3, Stat3, pJak1, Jak1, pSrc, Src, pErk1/2, Erk1/2, and _-Actin.
Positions of STAT:DNA complex in gel, pTyrStat3 and Stat3 are labeled. Values are the mean and standard
deviation of three replicate experiments. Data are representative of three independent studies. IC 50 values
were determined by quantifying by ImageQuant the bands corresponding to the STAT:DNA complexes;
control lanes represent nuclear extracts untreated with S3I-M2001 or nuclear extract preparations from cells
untreated with the compound.

Figure 11 shows examples of a the in vitro effects of S3I-M2001 in several cell lines, and it’s in vivo effect
in a MDA-MB-231 mouse xenograft.

 inhibition of the migration of NIH3T3/v-Src (76%), MDA-MB-231 (37%), and Panc-1 (21%) and
the invasion of NIH3T3/v-Src (28%), MDA-MB-231 (48%), and Panc-1 (38%) cells harboring
constitutively active (Figure 11, panel a)

 The i.v. injection of S3I-M2001 at 5, 10, and 20 mg per kg, but not vehicle (control) every 2 or 3
days for 26 days, strongly inhibited growth of tumors in xenograft models of human breast (MDA-
MB-231cells that harbor aberrant Stat3) in tumor-bearing mice (Figure 11, panel b).

 DNA binding assay with EMSA analysis and SDS/AGE-Western blotting of lysates from residual

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 GLG Pharma STAT3 Inhibitors, Mechanism of Action Summary
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tumor tissues extracted from control and treated mice showed abrogated Stat3 activity and pTyr
levels in S3I-M2001 treated tumors (T1 and T2) (Figure 11, panels c and d)

Figure 11. S3I-M2001 suppresses malignant cell migration and invasion and inhibits growth of human breast
tumor xenografts: a) Viral Src-transformed mouse fibroblasts (NIH3T3/v-Src), human breast cancer (MDA-
MB-231), and pancreatic cancer (Panc-1) cells were seeded for studies in migration on filters or invasion on
matrigel-coated filters in Bio-Coat chambers and treated with or without S3I-M2001 (100 mM) for 24. Cells
on the other side of filters were photographed (upper panel) and quantified under light microscope
(lower panel; percent inhibition in parentheses); human breast (MDA-MB-231) tumor bearing mice were

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 GLG Pharma STAT3 Inhibitors, Mechanism of Action Summary
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given S3I-M2001 (5, 10, and 20 mg kg_1) i.v. every 2 or 3 days. b) Tumor sizes, measured every 2 or 3
days, were converted to tumor volumes and plotted against treatment days. c) EMSA analysis of Stat3 DNA-
binding activity in lysates from tumor tissues extracted from one control and two residual treated tumors (T1
and T2) 3 days after the last S3I-M2001 (5 mg kg_1) injection. d) SDS-PAGE/Western blot analysis of
whole-cell lysates from control or residual treated (T2) tumor tissue and probing for pTyr705Stat3 and Stat3.
Values are the mean and standard deviation of three independent experiments each in duplicates or replicates
of 12 tumor-bearing mice in each group. Data are representative of two to three independent experiments.
Bands of Stat3:DNA complexes, pStat3, and Stat3 are shown.

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 GLG Pharma STAT3 Inhibitors, Mechanism of Action Summary

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E. Studies Demonstrating the Mechanism of Action of GLG-401 (Turkson et al.)

Previous studies have established constitutive activation of Stat3 protein as one of the molecular
changes that can cause or contribute to tumorigenesis. Turkson et al.(J. Biol. Chem., 2005;
280(38): 32979 -32988) evaluated compounds from the NCI 2000 diversity set for inhibition of
Stat3 DNA-binding activity in vitro to develop novel therapeutics for tumors harboring
constitutively active Stat3. Of these, a novel platinum (IV) compound, GLG-401 (IS3 295, NSC
295558), interacted with Stat3 homodimers and inhibited its binding to specific DNA-response
elements (Figure 12A) that initiate transcriptions of genes whose protein products promote
proliferation. Further analysis suggested noncompetitive-type kinetics for the inhibition of Stat3
binding to DNA.

In human and mouse tumor cell lines with constitutively active Stat3, IS3 295 selectively
attenuated Stat3 signaling, thereby inducing cell growth arrest at G0/G1 phase and apoptosis, and
reducing cell proliferation (Figure 12B). Moreover, in transformed cells, IS3 295 repressed
expression of cyclinD1 and bcl-xL, two of the known Stat3-regulated genes that are overexpressed
in malignant cells, suggesting that IS3 295 mediates anti-tumor cell activity in part by blocking
Stat3-mediated subversion of cell growth and apoptotic signals.

FIGURE 12. Evaluation of the effects of IS3 295 on cellular constitutive Stat3 activation and
cell proliferation. Normal or malignant cells were treated with or without IS3 295. Nuclear extracts
were prepared for Stat3 DNA-binding activity assay with the hSIE probe, or cells were processed
for nuclear Ki67 immunohistochemistry. A, EMSA analysis of Stat3 DNA-binding activity; B,
graphical representation of the quantified nuclear staining of the Ki67 proliferation index. The
positions of the Stat3/Stat3-DNA complexes on the gels are labeled. The Ki67 proliferation index
was calculated as the percentage of positive tumor cells relative to the total number of cells. Ki67
values are representative of three independent assays. DMSO, dimethyl sulfoxide.

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