U.S. patent application number 15/647054 was filed with the patent office on 2018-02-01 for novel compositions and methods for cancer treatment.
This patent application is currently assigned to Boston Biomedical, Inc.. The applicant listed for this patent is Boston Biomedical, Inc.. Invention is credited to Chiang Jia Li, Youzhi Li, Keith Mikule.
Application Number | 20180030021 15/647054 |
Document ID | / |
Family ID | 40452461 |
Filed Date | 2018-02-01 |
United States Patent
Application |
20180030021 |
Kind Code |
A1 |
Li; Chiang Jia ; et
al. |
February 1, 2018 |
NOVEL COMPOSITIONS AND METHODS FOR CANCER TREATMENT
Abstract
The present invention relates to the composition and methods of
use of Stat3 pathway inhibitors or cancer stem cell inhibitors in
combination treatment of cancer.
Inventors: |
Li; Chiang Jia; (Cambridge,
MA) ; Mikule; Keith; (Norwood, MA) ; Li;
Youzhi; (Westwood, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Boston Biomedical, Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
Boston Biomedical, Inc.
Cambridge
MA
|
Family ID: |
40452461 |
Appl. No.: |
15/647054 |
Filed: |
July 11, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12677516 |
Aug 12, 2010 |
9732055 |
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PCT/US08/75906 |
Sep 10, 2008 |
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15647054 |
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60971144 |
Sep 10, 2007 |
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61013372 |
Dec 13, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/02 20180101;
A61P 25/00 20180101; A61K 31/343 20130101; A61P 19/02 20180101;
A61P 25/02 20180101; A61P 17/06 20180101; A61P 35/04 20180101; A61P
35/00 20180101; A61P 37/00 20180101; C07D 307/92 20130101; A61P
1/00 20180101; A61P 9/00 20180101; A61P 37/08 20180101; A61P 37/06
20180101; A61P 29/00 20180101; A61P 17/02 20180101; A61K 31/38
20130101; A61P 35/02 20180101; A61P 9/10 20180101; A61P 17/12
20180101; C07D 333/74 20130101; A61P 11/06 20180101; A61P 43/00
20180101; A61P 1/04 20180101; A61P 31/00 20180101; Y02A 50/30
20180101; A61P 7/00 20180101; A61P 25/28 20180101 |
International
Class: |
C07D 307/92 20060101
C07D307/92; C07D 333/74 20060101 C07D333/74; A61K 31/38 20060101
A61K031/38 |
Claims
1.-111. (canceled)
112. A method for treating a non-cancer disorder in a subject in
need thereof, comprising administering: a) a composition comprising
a therapeutically effective amount of at least one first agent
chosen from 2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,
2-acetyl-7-chloronaphtho[2,3-b]furan-4,9-dione,
2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,
2-acetylnaphtho[2,3-b]furan-4,9-dione,
2-ethyl-naphtho[2,3-b]furan-4,9-dione, and pharmaceutically
acceptable salts thereof; and b) a composition comprising a
therapeutically effective amount of at least one second agent.
113. The method according to claim 112, wherein the non-cancer
disorder is chosen from autoimmune diseases, inflammatory diseases,
inflammatory bowel diseases, arthritis, asthma, systemic lupus
erythematosus, autoimmune demyelination disorders, Alzheimer's
disease, stroke, ischemia reperfusion injuries, and multiple
sclerosis.
114. The method according to claim 112, wherein the at least one
second agent is chosen from cytotoxic agents, targeted agents,
radiotherapy agents, biologic agents, hormonal agents, HDAC
inhibitors, retinoid agents, checkpoint activators, proteasome
inhibitors, adjuvant agents, and adjunctive agents.
115. The method according to claim 112, wherein the non-cancer
disorder is refractory to chemotherapy or radiotherapy.
116. A method for treating a cancer disorder in a subject in need
thereof, comprising administering: a) a composition comprising a
therapeutically effective amount of at least one first agent chosen
from 2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,
2-acetyl-7-chloronaphtho[2,3-b]furan-4,9-dione,
2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,
2-acetylnaphtho[2,3-b]furan-4,9-dione,
2-ethyl-naphtho[2,3-b]furan-4,9-dione, and pharmaceutically
acceptable salts thereof; and b) a composition comprising a
therapeutically effective amount of at least one second agent
chosen from growth factor-targeting agents, kinase-targeting
agents, angiogenesis inhibitors, DNA-damaging agents, antimitotic
agents, and antimetabolite agents, wherein the cancer disorder is
selected from head and neck cancer, brain cancer, breast cancer,
lung cancer, liver cancer, pancreatic cancer, ovarian cancer,
cervical cancer, uterine cancer, gastrointestinal cancer, kidney
cancer, bladder cancer, vulvar cancer, cancer of the peritoneum,
prostate cancer, thyroid cancer, sarcomas, squamous cell cancer,
melanoma, leukemia, lymphoma, and myeloma, and wherein said cancer
expresses activated Stat3.
117. The method according to claim 116, wherein the cancer disorder
is refractory to chemotherapy or radiotherapy.
118. A pharmaceutical composition comprising: a) a therapeutically
effective amount of at least one first agent chosen from
2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,
2-acetyl-7-chloronaphtho[2,3-b]furan-4,9-dione,
2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,
2-acetylnaphtho[2,3-b]furan-4,9-dione,
2-ethyl-naphtho[2,3-b]furan-4,9-dione, and pharmaceutically
acceptable salts thereof; and b) a therapeutically effective amount
of at least one second agent chosen from cytotoxic agents, targeted
agents, radiotherapy agents, biologic agents, hormonal agents, HDAC
inhibitors, retinoid agents, checkpoint activators, proteasome
inhibitors, adjuvant agents, adjunctive agents, and
pharmaceutically acceptable salts thereof.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of U.S.
provisional patent application Nos. 60/971,144 and 61/013,372,
respectively filed on Sep. 10, 2007 and Dec. 13, 2007, the entire
contents of which applications are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the composition and methods
of use of Stat3 pathway inhibitors and cancer stem cell inhibitors
in combinatorial treatment of cancers and other disorders.
SEQUENCE LISTING
[0003] This application contains a Sequence Listing which has been
submitted in electronic format. The Sequence Listing file was
created on Sep. 26, 2017, is named "sequencelisting.txt," and its
size is 4.00 KB (4,096 bytes). The Entire contents of the Sequence
Listing in the sequencelisting.txt file are incorporated herein by
this reference.
BACKGROUND OF THE INVENTION
[0004] Cancer Stem cells (CSCs).
[0005] In recent years, a new model of tumorigenesis has gained
wide acceptance, where it is hypothesized that only a small
fraction of the entire tumor mass are responsible for the
tumorigenic activities within the tumor, whereas the old or clonal
genetic model posits that all the mutated tumor cells contribute
equally to such tumorigenic activities. This small fraction of
tumorigenic cells, according to the new model, are transformed
cells with stem-cell-like qualities and are called "cancer stem
cells" (CSCs). Bonnet and Dick first demonstrated, in vivo, the
presence of CSCs in acute myeloid leukemia (AML) during the 1990s.
Their data showed that only a small subpopulation of human AML
cells had the ability to transfer AML, when transplanted into
immunodeficient mice while other AML cells were incapable of
inducing leukemia. Later, these CSCs were shown to have the same
cellular markers, CD34.sup.+/CD38.sup.-, as primitive hematopoietic
stem cells [1]. Since then, researchers have found CSCs
conclusively in various types of tumors including those of the
brain, breast, skin, prostate, and so on.
[0006] The CSC model of tumorigenesis would explain why tens or
hundreds of thousands of tumor cells need to be injected into an
experimental animal in order to establish a tumor transplant. In
human AML, the frequency of these cells is less than 1 in 10,000
[2]. Even though rare within a given tumor cell population, there
is mounting evidence that such cells exist in almost all tumor
types. However, as cancer cell lines are selected from a
sub-population of cancer cells that are specifically adapted to
grow in tissue cultures, the biological and functional properties
of cancer cell lines can undergo dramatic changes. Therefore, not
all cancer cell lines contain CSCs.
[0007] Cancer stem cells share many similar traits with normal stem
cells. For example, CSCs have self-renewal capacity, namely, the
ability to give rise to additional tumorigenic cancer stem cells,
typically at a slower rate than other dividing tumor cells, as
opposed to a limited number of divisions. CSCs also have the
ability to differentiate into multiple cell types, which would
explain histological evidences that not only many tumors contain
multiple cell types native to the host organ, but also that
heterogeneity is commonly retained in tumor metastases. CSCs have
been demonstrated to be fundamentally responsible for
tumorigenesis, cancer metastasis, and cancer reoccurrence. CSCs are
also called tumor initiating cells, cancer stem-like cells,
stem-like cancer cells, highly tumorigenic cells, tumor stem cells,
solid tumor stem cells, or super malignant cells.
[0008] The existence of cancer stem cells has fundamental
implications for future cancer treatments and therapies. The
efficacy of current cancer treatments are, in the initial stages of
testing, often measured by the size of the tumor shrinkage, i.e.,
the amount of tumor mass that is killed off. As CSCs would form a
very small proportion of the tumor and have markedly different
biologic characteristics than their more differentiated progenies,
the measurement of tumor mass may not necessarily select for drugs
that act specifically on the stem cells. In fact, cancer stem cells
appear to be resistant to radiotherapy (XRT) and are also
refractory to chemotherapeutic and targeted drugs [3-5]. Normal
somatic stem cells are naturally resistant to chemotherapeutic
agents--they have various pumps (such as MDR) that pump out drugs,
and efficient DNA repair mechanisms. Further, they also have a slow
rate of cell turnover while chemotherapeutic agents target rapidly
replicating cells. Cancer stem cells, being the mutated
counterparts of normal stem cells, may also have similar mechanisms
that allow them to survive drug therapies and radiation treatment.
In other words, conventional chemotherapies and radiotherapies kill
differentiated or differentiating cells, which form the bulk of the
tumor that are unable to regenerate tumors. The population of
cancer stem cells that gave rise to the differentiated and
differentiating cells, on the other hand, could remain untouched
and cause a relapse of the disease. A further danger for the
conventional anti-cancer therapy is the possibility that the
treatment of, for instance, chemotherapy, leaves only
chemotherapy-resistant cancer stem cells, and the ensuing recurrent
tumor will likely also be resistant to chemotherapy.
[0009] Since the surviving cancer stem cells can repopulate the
tumor and cause relapse, it is imperative that anti-cancer
therapies include strategies against CSCs (see FIG. 1). This has
been likened to the need for eliminating dandelion roots in order
to prevent weed regrowth [6]. By selectively targeting cancer stem
cells, it becomes possible to treat patients with aggressive,
non-resectable tumors, and refractory or recurrent cancers, as well
as preventing tumor metastasis and recurrence. Development of
specific therapies targeting cancer stem cells therefore holds hope
for survival and improved quality of life of cancer patients,
especially for those with metastatic cancers. The key to unlocking
this untapped potential is the identification and validation of
pathways that are selectively important for cancer stem cell
self-renewal and survival. Though multiple pathways underlying
tumorigenesis in cancer and in embryonic stem cells or adult stem
cells have been elucidated in the past, no pathways have been
identified and validated for cancer stem cell self-renewal and
survival.
[0010] There has also been a lot of research into the
identification and isolation of cancer stem cells. Methods used
mainly exploit the ability of CSCs to efflux drugs, or are based on
the expression of surface markers associated with cancer stem
cells.
[0011] For example, since CSCs are resistant to many
chemotherapeutic agents, it is not surprising that CSCs almost
ubiquitously overexpress drug efflux pumps such as ABCG2 (BCRP-1)
[7-11], and other ATP binding cassette (ABC) superfamily members
[12, 13]. Accordingly, the side population (SP) technique,
originally used to enrich hematopoetic and leukemic stem cells, was
also employed to identify and isolate CSCs [14]. This technique,
first described by Goodell et al., takes advantage of differential
ABC transporter-dependent efflux of fluorescent dyes such as
Hoechst 33342 to define and isolate a cell population enriched in
CSCs [10, 15]. Specifically, the SP is revealed by blocking drug
efflux with verapamil, at which point the dyes can no longer be
pumped out of the SP.
[0012] Researchers have also focused on finding specific markers
that distinguish cancer stem cells from the bulk of the tumor. Most
commonly expressed CSC surface markers include CD44, CD133, and
CD166 [16-24]. Sorting tumor cells based primarily upon the
differential expression of these surface marker (s) have accounted
for the majority of the highly tumorigenic CSCs described to date.
Therefore, these surface markers are well validated for the
identification and isolation of cancer stem cells from cancer cell
lines and from bulk tumor tissues.
Stat3 Pathway.
[0013] There are many different genetic defects in mammalian or
human cancer cells, and many have been studied in the quest to cure
cancer. For example, the p53 tumor suppressor has been found to be
defective or altogether absent in more than half of the human
cancers. The STAT (Signal Transducers and Activator of
Transcription) protein family are latent transcription factors
activated in response to cytokines/growth factors to promote
proliferation, survival, and other biological processes. Among
them, Stat3 is activated by phosphorylation of a critical tyrosine
residue mediated by growth factor receptor tyrosine kinases, Janus
kinases, or the Src family kinases, etc. These kinases include but
are not limited to EGFR, JAKs, AbI, KDR, c-Met, Src, and Her2 [25].
Upon tyrosine phosphorylation, Stat3 forms homo-dimers,
translocates to the nucleus, binds to specific DNA-response
elements in the promoter regions of the target genes, and induces
gene expression [26] (see FIG. 2).
[0014] In normal cells, Stat3 activation is transient and tightly
regulated, lasting from 30 minutes to several hours. However, Stat3
is found to be aberrantly active in a wide variety of human
cancers, including all the major carcinomas as well as some
hematologic tumors. Stat3 plays multiple roles in cancer
progression. As a potent transcription regulator, it targets genes
involved in many important cellular functions, such as Bcl-xl,
c-Myc, cyclin D1, Vegf, MMP-2, and survivin [27-32]. It is also a
key negative regulator of tumor immune surveillance and immune cell
recruitment [33-35].
[0015] Ablating Stat3 signaling by antisense, siRNA, a
dominant-negative form of Stat3, and/or blockade of tyrosine
kinases inhibits certain cancer cell lines or tumors in vitro
and/or in vivo [26, 28, 36, 37]. But no clear link between Stat3
and cancer stem cell functionality have ever been empirically made.
Nor have researchers found an effective Stat3 pathway inhibitor to
explore potential therapeutic uses with regard to cancers that have
been found to contain cancer stem cells. As described earlier,
cancer stem cells (CSCs) have been recently demonstrated to be
fundamentally responsible for tumorigenesis, cancer metastasis and
cancer reoccurrence, and should be taken into consideration in
designing any curative therapy that targets a tumor known to have
these cells no matter how small a fraction of the tumor mass they
may constitute.
[0016] In diseases other than cancer, over-activation of Stat3 by
various cytokines, such as interleukin 6 (IL6), has been
demonstrated in a number of autoimmune and inflammatory diseases
[38], Recently, it has been revealed that the Stat3 pathway also
promotes pathologic immune responses through its essential role in
generating TH17 T cell responses [39], In addition, IL6-Stat3
pathway mediated inflammation has been found to be the common
causative origin for atherosclerosis, peripheral vascular disease,
coronary artery disease, hypertension, osteoporosis, type 2
diabetes, and dementia.
SUMMARY
[0017] The present invention is predicated, in part, on empirical
evidence provided herein that Stat3 plays a key role in both the
survival and self-renewal capacity of cancer stem cells (CSCs)
across a broad spectrum of cancers. The present invention also
provides data that confirms certain compounds act as Stat3 pathway
inhibitors and that they effectively inhibit CSCs both in vitro and
in vivo.
[0018] Accordingly, a first aspect of the invention provides a
method of treating a subject with a disorder that is associated
with aberrant Stat3 pathway activity, the method comprising the
steps of: (a) administering to the subject a first amount of a
first agent to inhibit at least some of the aberrant Stat3 pathway
activity; and (b) administering to the subject a second amount of a
second agent comprising a signal transduction inhibitor.
[0019] The first agent may inhibit Stat3 pathway activity through
at least one of the following actions: substantially inhibiting
phosphorylation of the Stat3 protein, substantially inhibiting
dimerization of the Stat3 protein, substantially inhibiting nuclear
translocation of the Stat3 protein, substantially inhibiting
DNA-binding activity of the Stat3 protein, and substantially
inhibiting transcription activities of the Stat3 protein.
[0020] In one embodiment, the first agent is selected from the
group consisting of
2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,
2-Acetyl-7-Chloro-naphtho[2,3-b]furan-4,9-dione,
2-Acetyl-7-Fluoro-naphtho[2,3-b]furan-4,9-dione,
2-acetylnaphtho[2,3-b]furan-4,9-dione,
2-ethyl-naphtho[2,3-b]furan-4,9-dione, an enantiomer, diastereomer,
tautomer, and a salt or solvate thereof (hereafter referred to as
the "Compound of the Invention").
[0021] Non-cancer disorders that can be treated by methods of the
first aspect of the invention include but are not limited to:
autoimmune diseases, inflammatory diseases, inflammatory bowel
diseases, arthritis, asthma, and systemic lupus erythematosus,
autoimmune demyelination disorder, Alzheimer's disease, stroke,
ischemia reperfusion injury and multiple sclerosis. Cancers that
can be treated by the methods include but are not limited to:
breast cancer, head and neck cancer, lung cancer, ovarian cancer,
pancreatic cancer, colorectal carcinoma, prostate cancer, renal
cell carcinoma, melanoma, hepatocellular carcinomas, cervical
cancer, sarcomas, brain tumors, gastric cancers, multiple myeloma,
leukemia, and lymphomas. These non-cancer and cancerous disorders
are known to be associated with aberrant Stat3 pathway
activities.
[0022] In one feature, the second agent is a targeted agent, which
can be a growth factor receptor-targeting agent, a kinase-targeting
agent, or an angiogenesis inhibitor.
[0023] In a second aspect, the present invention provides a method
of treating a subject of a cancer that is associated with aberrant
Stat3 pathway activity, the method comprising the steps of: (a)
administering to the subject a first amount of a first agent to
inhibit at least some of the aberrant Stat3 pathway activity; and
(b) administering to the subject a second amount of a second
anti-cancer agent.
[0024] While features regarding the first agent can be similar to
those described with regard to the first aspect of the invention,
the second anti-cancer agent can be a cytotoxic agent, or a
chemotherapeutic agent. In one embodiment, the second agent is a
standard first-line treatment for at least one cancer.
[0025] In one feature, the anti-cancer agent is a DNA-damaging
agent, an antimitotic agent, and/or an antimetabolite agent. For
example, the DNA-damaging agent can be an alkylating agent, a
topoisomerase inhibitor, or a DNA intercalator. In one embodiment,
the second agent is one of carboplatin, doxorubicin, gemcitabine,
docetaxel, or etoposide
[0026] Cancers that can be treated by methods of the second aspect
of the invention include those known to be associated with aberrant
Stat3 pathway activities, which are listed above, and not repeated
here.
[0027] Since cancer stem cells are generally resistant to
radiotherapy and conventional chemotherapies, a drug that targets
cancer stem cells should have synergistic effect when used in
combination with other anti-cancer therapies. Therefore, according
to a third aspect in the present invention, a method of treating
cancer in a subject includes the steps of: (a) administering to the
subject a first amount of a first anti-cancer agent to inhibit a
cancer stem cell (CSC) population; and (b) administering to the
subject a second amount of a second anti-cancer agent to inhibit a
plurality of normal cancer cells.
[0028] In various embodiments, step (a) of this method inhibits at
least one CSC from self-renewal, and/or kills at least one CSC. In
an embodiment, the first amount of the first anti-cancer agent also
kills a plurality of normal cancer cells. In an embodiment, step
(a) inhibits at least some Stat3 pathway activity in cancer stem
cells. The first anti-cancer agent shares the same features and
characteristics as the first agent in methods according to the
first aspect of the invention, since this invention has provided
evidence that Stat3 pathway inhibitors can effectively inhibit
CSCs. The shared features include, for example, various steps of
the Stat3 pathway that the first anti-cancer agent recited here can
target. In various embodiments, the first anti-cancer agent can be
a small molecule Stat3 inhibitor, an RNAi agent against Stat3, an
antisense agent against Stat3, a peptidomimetic Stat3 inhibitor, or
a G-quartet oligodeoxynucleotides Stat3 inhibitor.
[0029] Cancers that can be treated by this method preferably are
those known or confirmed to contain CSCs, which include, but are
not limited to: breast cancer, head and neck cancer, lung cancer,
ovarian cancer, pancreatic cancer, multiple myeloma, colorectal
carcinoma, prostate cancer, melanoma, kaposi sarcoma, ewing's
sarcoma, liver cancer, gastric cancer, medulloblastoraa, brain
tumors, and leukemia.
[0030] The "second anti-cancer agent" in methods according to the
third aspect of the invention can be the same "second anti-cancer
agent" in methods according to the second aspect of the invention,
and all the shared features are not repeated here.
[0031] In one embodiment, the second agent is a standard first-line
treatment for at least one cancer. The second agent can be a
cytotoxic agent. In one feature, the anti-cancer agent is a
DNA-damaging agent, an anti-mitotic agent, and/or an
anti-metabolite agent. For example, the DNA-damaging agent can be
an alkylating agent, a topoisomerase inhibitor, or a DNA
intercalator. In one embodiment, the second agent is one of
carboplatin, doxorubicin, gemcitabine, docetaxel, or etoposide.
[0032] According to a fourth aspect of the invention, a method is
provided for treating cancer in a subject, comprising the steps of:
(a) administering to the subject a first amount of a first cancer
stem cell inhibitor to inhibit Stat3 pathway activities; and (b)
administering to the subject a second amount of a second cancer
stem cell inhibitor to inhibit activities of a different
pathway.
[0033] In an embodiment, the second cancer stem cell inhibitor is
lapatinib. In some embodiments, the second amount of the second
anti-cancer agent is not therapeutically effective against the
cancer stem cell population by itself. Cancers that can be treated
by this method preferably are those known or confirmed to contain
CSCs and some examples are listed above. In various embodiments,
the cancer is metastatic, refractory to a standard first-line
cancer treatment, or relapsed.
[0034] According to a fifth aspect of the invention, a method is
provided for treating cancer in a subject, comprising the steps of:
(a) administering to the subject a therapeutically effective amount
of a first anti-cancer agent selected from the group consisting of
2-(1-hydroxyethyl)-naphtho [2,3-b] furan-4,9-dione,
2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,
2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,
2-acetylnaphtho[2,3-b]furan-4,9-dione,
2-ethyl-naphtho[2,3-b]furan-4,9-dione, and a pharmaceutically
acceptable salt or solvate thereof; and (b) administering a second
anti-cancer agent that is not selected from the same group.
[0035] The second anti-cancer agent can be any of the agents
described in other aspects of the invention, including any of the
cytotoxic or chemotherapeutic agents, and any of the targeted
agents.
[0036] In a sixth aspect, the present invention provides a
pharmaceutical composition comprising a therapeutically effective
amount of a first anti-cancer agent selected from the group
consisting of 2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,
2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,
2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,
2-acetylnaphtho[2,3-b]furan-4,9-dione,
2-ethyl-naphtho[2,3-b]furan-4,9-dione, and a pharmaceutically
acceptable salt or solvate thereof; and a second anti-cancer
therapy selected from the group consisting of a cytotoxic agent, a
targeted agent, a radiotherapy agent, a biologic agent, a hormonal
agent, a HDAC inhibitor, a retinoid agent, a checkpoint activator,
a proteasome inhibitor, an adjuvant agent, or an adjunctive
agent.
[0037] In an embodiment, the composition further includes a
pharmaceutically-acceptable excipient, carrier, or diluent.
[0038] Other aspects including all compositions and kits related to
methods described herein, and embodiments of the present invention
are set forth or will be readily apparent from the following
detailed description of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0039] FIG. 1 illustrates the differences between
cancer-stem-cell-specific and conventional cancer therapies.
[0040] FIG. 2 shows the Stat3 pathway in cancer.
[0041] FIG. 3A shows that Stat3 is constitutively active in Hoechst
Side Population cells.
[0042] FIG. 3B shows that Stat3 is constitutively active in
CD133.sup.+ cells.
[0043] FIG. 4A and FIG. 4B show that Stat3 knockdown in cancer stem
cells induces apoptosis.
[0044] FIG. 5 shows that Stat3 knockdown in cancer stem cells
inhibits cancer stem cell spherogenesis.
[0045] FIG. 6 shows that compound 401 inhibits Stat3 transcription
activation activity.
[0046] FIG. 7A shows that compound 401 inhibits Stat3 DNA-binding
activity in nuclear extract.
[0047] FIG. 7B shows that compounds 401, 416 and 418 inhibit Stat3
DNA-binding activity in nuclear extract.
[0048] FIG. 8A shows that compound 401 inhibits Stat3 DNA-binding
activity in xenograft tumor tissues.
[0049] FIG. 8B shows that compound 401 inhibits the expression
level of the Stat3 downstream effectors in xenograft tumor
tissues.
[0050] FIG. 9A shows the sorting and analysis of the Hoechst Side
Population.
[0051] FIG. 9B shows that Hoechst Side Population is as sensitive
as non-side population to compound 401.
[0052] FIG. 10A shows that compound 401 is apoptotic to Hoechst
Side Population cells.
[0053] FIG. 10B shows that compound 401 is apoptotic to CD133.sup.+
cells.
[0054] FIG. 11A and FIG. 11B show that compound 401 blocks
CD44.sup.high sphere formation.
[0055] FIG. 12 shows that in vivo compound 401 treatment decreases
the spherogenesis of the xenografted tumor cells.
[0056] FIG. 13 shows that compound 401 inhibits metastasis in ISMS
model.
[0057] FIG. 14 shows that compound 401 has a synergistic effect
with sorafenib in A549 human lung cancer cells.
[0058] FIG. 15 shows that compound 401 has a synergistic effect
with erlotinib in A549 human lung cancer cells.
[0059] FIG. 16 shows that compound 401 has a synergistic effect
with lapatinib in A549 human lung cancer cells.
[0060] FIG. 17 shows that compound 401 has a synergistic effect
with sutent in A549 human lung cancer cells.
[0061] FIG. 18 shows that compound 401 has a synergistic effect
with gemcitabine in Paca-2 human pancreatic xenograft model.
DETAILED DESCRIPTION
[0062] As used herein, the singular form "a", "an", and "the"
include plural references unless the context clearly dictate
otherwise. For example, the term "a cell" includes a plurality of
cells including mixtures thereof.
[0063] The terms "isolated" or "purified" as used herein refer to a
material that is substantially or essentially free from components
that normally accompany it in its native state. Purity and
homogeneity are typically determined using analytical chemistry
techniques such as polyacrylamide gel electrophoresis or high
performance liquid chromatography.
[0064] As used herein, the terms "cancer stem cell(s)" and "CSC(s)"
are interchangeable. CSCs are mammalian, and in preferred
embodiments, these CSCs are of human origin, but they are not
intended to be limited thereto. Cancer stem cells are defined and
functionally characterized as a population of cells originating
from a solid tumor that: (1) have extensive proliferative capacity;
(2) are capable of asymmetric cell division to generate one or more
kinds of differentiated progeny with reduced proliferative or
developmental potential; and (3) are capable of symmetric cell
divisions for self-renewal or self-maintenance. Other common
approaches to characterize CSCs involve morphology and examination
of cell surface markers, transcriptional profile, and drug
response. CSCs are also called in the research literature
tumor/cancer initiating cells, cancer stem-like cells, stem-like
cancer cells, highly tumorigenic cells, tumor stem cells, solid
tumor stem cells, drug survival cells (DSC), drug resistant cells
(DRCs) or super malignant cells.
[0065] As used herein, the term "self-renewal" refers to cancer
stem cells' ability to give rise to new tumorigenic cancer stem
cells to replenish or increase their number.
[0066] As used herein, the terms "cancer" and "cancerous" refer to
or describe the physiological condition in mammals in which a
population of cells are characterized by unregulated cell growth.
"Cancer cells" and "tumor cells" as used herein refer to the total
population of cells derived from a tumor including both
non-tumorigenic cells, which comprise the bulk of the tumor cell
population, and tumorigenic stem cells (cancer stem cells).
Examples of cancer include, but are not limited to, carcinoma,
lymphoma, blastoma, sarcoma, and leukemia. More particular examples
of such cancers include squamous cell cancer, small-cell lung
cancer, non-small cell lung cancer, adenocarcinoma of the lung,
squamous carcinoma of the lung, cancer of the peritoneum,
hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,
glioblastoma, cervical cancer, ovarian cancer, liver cancer,
bladder cancer, hepatoma, breast cancer, colon cancer, colorectal
cancer, endometrial or uterine carcinoma, salivary gland carcinoma,
kidney cancer, liver cancer, prostate cancer, vulval cancer,
thyroid cancer, hepatic carcinoma and various types of head and
neck cancer.
[0067] "Tumor" as used herein refers to any mass of tissue that
result from excessive cell growth or proliferation, either benign
(noncancerous) or malignant (cancerous) including pre-cancerous
lesions.
[0068] "Metastasis" as used herein refers to the process by which a
cancer spreads or transfers from the site of origin to other
regions of the body with the development of a similar cancerous
lesion at the new location. A "metastatic" or "metastasizing" cell
is one that loses adhesive contacts with neighboring cells and
migrates via the bloodstream or lymph from the primary site of
disease to invade neighboring body structures.
[0069] As used herein, the term "subject" refers to any animal
(e.g., a mammal), including, but not limited to humans, non-human
primates, rodents, and the like, which is to be the recipient of a
particular treatment. Typically, the terms "subject" and "patient"
are used interchangeably herein in reference to a human
subject.
[0070] Terms such as "treating" or "treatment" or "to treat" or
"alleviating" or "to alleviate" as used herein refer to both 1)
therapeutic measures that cure, slow down, lessen symptoms of,
and/or halt progression of a diagnosed pathologic condition or
disorder and 2) prophylactic or preventative measures that prevent
or slow the development of a targeted pathologic condition or
disorder. Thus those in need of treatment include those already
with the disorder; those prone to have the disorder; and those in
whom the disorder is to be prevented. A subject is successfully
"treated" according to the methods of the present invention if the
patient shows one or more of the following: a reduction in the
number of or complete absence of cancer cells; a reduction in the
tumor size; inhibition of, or an absence of cancer cell
infiltration into peripheral organs including the spread of cancer
into soft tissue and bone; inhibition of, or an absence of tumor
metastasis; inhibition or an absence of tumor growth; relief of one
or more symptoms associated with the specific cancer; reduced
morbidity and mortality; and improvement in quality of life.
[0071] As used herein, the term "inhibiting", "to inhibit" and
their grammatical equivalents, when used in the context of a
bioactivity, refer to a down-regulation of the bioactivity, which
may reduce or eliminate the targeted function, such as the
production of a protein or the phosphorylation of a molecule. In
particular embodiments, inhibition may refers to a reduction of
about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the targeted
activity. When used in the context of a disorder or disease, the
terms refer to success at preventing the onset of symptoms,
alleviating symptoms, or eliminating the disease, condition or
disorder.
[0072] "Normal cancer cells," as used herein either in singular or
plural form, refers to cancer cells that are not cancer stem
cells.
[0073] "Combination" or "combinatorial" therapy or treatment, as
used herein means the administration of at least two different
therapeutics to treat a disorder, condition or symptom, e.g., a
cancer condition. Such combination therapy may involve the
administration of one therapeutic before, during, and/or after the
administration of the other therapeutic. The administrations of the
therapeutics may be separated in time by up to several weeks, but
more commonly within 48 hours, and most commonly within 24
hours.
[0074] The term "pharmaceutically-acceptable excipient, carrier, or
diluent" as used herein means a pharmaceutically-acceptable
material, composition or vehicle, such as a liquid or solid filler,
diluent, excipient, solvent or encapsulating material, involved in
carrying or transporting the subject pharmaceutical agent from one
organ, or portion of the body, to another organ, or portion of the
body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
injurious to the patient. Some examples of materials which can
serve as pharmaceutically-acceptable carriers include: sugars, such
as lactose, glucose and sucrose; starches, such as corn starch and
potato starch; cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer
solutions; and other non-toxic compatible substances employed in
pharmaceutical formulations. Wetting agents, emulsifiers and
lubricants, such as sodium lauryl sulfate, magnesium stearate, and
polyethylene oxide-polypropylene oxide copolymer as well as
coloring agents, release agents, coating agents, sweetening,
flavoring and perfuming agents, preservatives and antioxidants can
also be present in the compositions.
[0075] The compounds of the present invention may form salts which
are also within the scope of this invention. Reference to a
compound of the present invention herein is understood to include
reference to salts thereof, unless otherwise indicated. The term
"salt(s)", as employed herein, denotes acidic and/or basic salts
formed with inorganic and/or organic acids and bases. In addition,
when a compound of the present invention contains both a basic
moiety, such as but not limited to a pyridine or imidazole, and an
acidic moiety such as but not limited to a carboxylic acid,
zwitterions ("inner salts") may be formed and are included within
the term "salt(s)" as used herein. Pharmaceutically acceptable
(i.e., non-toxic, physiologically acceptable) salts are preferred,
although other salts are also useful, e.g., in isolation or
purification steps which may be employed during preparation. Salts
of the compounds of the present invention may be formed, for
example, by reacting a compound I, II or III with an amount of acid
or base, such as an equivalent amount, in a medium such as one in
which the salt precipitates or in an aqueous medium followed by
lyophilization.
[0076] Solvates of the compounds of the invention are also
contemplated herein. Solvates of the compounds of the present
invention include, for example, hydrates.
Targeting Stat3 Pathway
[0077] The present invention provides compounds that are effective
inhibitors of Stat3 pathway activities (Example 2). As Stat3
pathway is a latent transcription factor activated to promote
proliferation, survival and many other biological processes, it is
implicated in a wide variety of human cancers as well as non-cancer
disorders including a number of autoimmune and inflammatory
diseases (Table 1). Accordingly, the present invention provides, in
a first aspect, combination treatment of disorders associated with
aberrant, e.g., over-expressed, Stat3 pathway activity.
Specifically, the patient subject is administered a first amount of
a first agent to inhibit at least some of the aberrant Stat3
pathway activity and also a second amount of a second agent which
includes a signal transduction inhibitor. In various embodiments,
some (e.g., 20%, 30%, 40%), most (more than about 50%), or
substantially all (e.g., 60%, 70%, 80%, 90%, 95% or 100%), of the
aberrant Stat3 pathway activity is inhibited. One or both of the
first amount and the second amount can be a therapeutically
effective amount of the respective agent before combination use,
i.e., when used by itself against the disorder, or less than that
amount because of outstanding synergistic effects from the
combination. The first agent may target one or multiple steps in
the Stat3 pathway. In one embodiment, the first agent is the
Compound of the Invention.
TABLE-US-00001 TABLE 1 Activation of STAT3 PATHWAY in human
diseases DISEASES REF. ONCOLOGY Solid Tumors Breast Cancer [40]
DISEASES Head and Neck Cancer (SCCHN) [41] Lung Cancer [42] Ovarian
Cancer [43] Pancreatic Cancer [44] Colorectal carcinoma [45]
Prostate Cancer [46] Renal Cell carcinoma [47] Melanoma [48]
Hepatocellular carcinomas [36] Cervical Cancer [49] Endometrial
Cancer [49] Sarcomas [50, 51] Brain Tumors [52] Gastric Cancers
[29] Hematologic Multiple Myeloma [53] Tumors Leukemia
HTLV-1-dependent Leukemia [54] Chronic Myelogenous Leukemia [47[
Acute Myelogenous Leukemia [55] Large Granular Lymphocyte [56]
Leukemia Lymphomas EBV-related/Burkitt's [57] Mycosis Fungoides
[47] HSV Saimiri-dependent (T-cell) [47] Cutaneous T-cell Lymphoma
[58] Hodgkin's Disease [47] Anaplastic Large-cell Lymphoma [59]
IMMUNE Inflammatory Inflammatory Bowel Diseases [60] DISEASES
Diseases Inflammatory Arthritis [61-63] Crohn's Diseases [64]
Chronic inflammatory conditions [65] Autoimmune Reumatoid Arthritis
[61, 62, 66-68] Systemic lupus erythematosus [69] Asthma [70]
Allergy [71] Infections [72] PROLIFERATIVE Psoriasis [73] DISORDERS
Keloids [74] Warts [75] Myelodysplastic syndrome [76] Polycythemia
vera [77] CNS DISEASES Alzhemer's [38, 78, 79] Multiple sclerosis
(MS) [38, 78, 80]
[0078] The second agent, i.e., a signal transduction inhibitor, can
be used to target a different pathway, a related pathway or a
different step in the same Stat3 pathway from the one inhibited by
the first agent. Normally, when the two agents in the combination
therapy target the same pathway, albeit at different steps, the
expected amount of synergism is limited. However, data provided
below in Example 5 show surprisingly high amounts of synergism
between the Compound of the Invention and a second agent that
presumably targets other steps in the same pathway, e.g., tyrosine
kinases and GFR-targeting agents, suggesting unexpected mechanism
of inhibition at work.
[0079] Specifically, Stat3 is activated by phosphorylation of a
critical tyrosine residue mediated by growth factor receptor
tyrosine kinases, Janus kinases, or the Src family kinases, etc;
upon tyrosine phosphorylation, Stat3 forms homo-dimers,
translocates to the nucleus, binds to specific DNA-response
elements in the promoter regions of the target genes, and induces
gene expression. Example 2 of the present invention shows that in
the Stat3 pathway, the inhibitory effect by the Compound of the
Invention is evident by the step of DNA-binding. Further, because
such effect is found on constitutively activated Stat3, it is
likely, that the Compound of the Invention inhibits dimerization
and/or nuclear translocation of the Stat3 protein. Therefore, when
combined with tyrosine kinases (TKI) and GFR-targeting agents that
also target the same Stat3 pathway, the amount of synergism
observed was surprisingly high. For example, 100% inhibition of
cells from a pancreatic cancer cell line was achieved when Compound
401 was combined with TKI Sorafenib, whereas both Compound 401 and
Sorafenib could only respectively achieve 66% inhibition of the
same cell line when administered individually--pancreatic cancer is
known to implicate over-expression of Stat3 [44]. In fact, all four
TKIs tested in combination with Compound 401 showed marked
synergism. In preferred embodiments of the invention, the
combination treatment achieves over about 50%, or 70%, or 90%
inhibition of the cancer cells.
[0080] Methods according to this first aspect of the invention can
be applied to treatment of cancers or non-cancer disorders,
preferably those known to be associated with aberrant Stat3 pathway
activities. Examples of non-cancer disorders associated with
aberrant Stat3 pathway activities include but are not limited to:
autoimmune diseases, inflammatory diseases, inflammatory bowel
diseases, arthritis, asthma, and systemic lupus erythematosus,
autoimmune demyelination disorder, Alzheimer's disease, stroke,
ischemia reperfusion injury and multiple sclerosis. Examples of
cancers associated with aberrant Stat3 pathway activities include
but are not limited to: breast cancer, head and neck cancer, lung
cancer, ovarian cancer, pancreatic cancer, colorectal carcinoma,
prostate cancer, renal cell carcinoma, melanoma, hepatocellular
carcinomas, cervical cancer, sarcomas, brain tumors, gastric
cancers, multiple myeloma, leukemia, and lymphomas.
[0081] The second agent according to this first aspect of the
invention can be a targeted agent, e.g., a growth factor
receptor-targeting agent (see erlotinib (tarceva) data in Example
5), a kinase-targeting agent (see lapatinib, erlotinib, sunitinib
and sorafenib data in Example 5) or an angiogenesis inhibitor (see
sunitinib and sorafenib data in Example 5).
[0082] In one embodiment, the second agent is a growth factor
receptor-targeting agent, for example, an antibody targeting a
growth factor receptor associated with a kinase, such as the
Epidermal Growth Factor Receptor (EGFR) or the Vascular Endothelial
Growth Factor Receptor (VEGFR). For example, the target agent can
be gefitinib (Iressa), erlotinib (tarceva), PD153035, cetuximab
(erbitux), avastin, panitumumab, trastuzumab, and anti-c-Met
antibody.
[0083] In one embodiment, the second agent is a kinase-targeting
agent, which can be a kinase inhibitor such as a tyrosine kinase
inhibitor (TKI). For example, the TKI can be erlotinib (Tarceva),
sutent (sunitinib), lapatinib, sorafenib (nexavar), vandetanib,
axitinib, bosutinib, cedivanib, dasatinib (sprycel), gefitinib
(irressa), imatinib (gleevac), lestaurtinib, and/or ARQ197.
[0084] In various embodiments, the kinase-targeting agent is one of
the following: gefitinib (iressa), ZD6474 (AZD6474), EMD-72000
(matuzumab), panitumab (ABX-EGF), ICR-62, CI-1033 (PD183805),
lapatinib (tykerb), AEE788 (pyrrolo-pyrimidine), EKB-569, EXEL
7647/EXEL 0999, erlotinib (tarceva), imatinib (gleevec), sorafinib
(nexavar), sunitinib (sutent), dasatinib (sprycel), vandetinib
(ZACTIMA), temsirolimus (torisel), PTK787 (vatalanib), pazopanib,
AZD2171, everolimus, seliciclib, AMG 706, axitinib, PD0325901,
PKC-412, CEP701, XL880, bosutinib, BIBF1120, BIBF1120, nilotinib,
AZD6244, HKI-272, MS-275, BI2536, GX15-070, AZD0530, enzastaurin,
MLN-518, and ARQ197.
[0085] In one embodiment, the second agent is an angiogenesis
inhibitor which can be one of the following: CM101, IFN-.alpha.,
IL-12, platelet factor-4, suramin, SU5416, thrombospondin, VEGFR
antagonists, angiostatic steroids plus heparin, Cartilage-Derived
Angiogenesis Inhibitory Factor, matrix metalloproteinase
inhibitors, batimastat, marimastat, angiostatin, endostatin,
2-methoxyestradiol, tecogalan, thrombospondin, .alpha.V.beta.3
inhibitors, linomide, and ADH-1.
[0086] In a related second aspect, the present invention provides a
method of treating a subject of a cancer that is associated with
aberrant Stat3 pathway activity, the method comprising the steps
of: (a) administering to the subject a first amount of a first
agent to inhibit at least some of the aberrant Stat3 pathway
activity; and (b) administering to the subject a second amount of a
second anti-cancer agent. Cancers that can be treated with this
method include those known to be associated with, e.g., caused at
least partly by, aberrant Stat3 pathway activities, a list of which
is provided above with regard to the first aspect of the
invention.
[0087] While features regarding the first agent can be similar to
those described with regard to the first aspect of the invention,
the second anti-cancer agent can be a cytotoxic agent, or, a
chemotherapeutic agent. In one embodiment, the second agent is a
standard first-line treatment for at least one cancer. The amount
of the first agent and second agent used in the method can be a
therapeutically effective amount of the respective agent before
combination use or less.
[0088] In one feature, the anti-cancer agent is a DNA-damaging
agent, an antimitotic agent, and/or an antimetabolite agent. The
DNA-damaging agent can be an alkylating agent, a topoisomerase
inhibitor, and/or a DNA intercalator. As shown in Example 5,
Compound 401 of the present invention was added to Paca2 pancreatic
cancer cells along with each of the following: carboplatin (a
DNA-alkylating agent), etoposide (inhibitor of topoisomerase II),
doxorubicin (a DNA intercalator), docetaxel (an anti-mitotic
agent), and Gemzar/gemcitabine (an anti-metabolite agent).
Significant amount of synergism was found in each combination. For
example, 96% inhibition of the pancreatic cancer cells was achieved
when Compound 401 was combined with Gemzar/gemcitabine whereas
Compound 401 and Gemzar could only respectively achieve 66% and 36%
inhibition of the same cell line when administered
individually.
[0089] The alkylating agent can be one of the following:
chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine,
melphalan, uracil mustard, thiotepa, busulfan, carmustine,
lomustine, streptozocin, carboplatin, cisplatin, satraplatin,
oxaliplatin, altretamine, ET-743, XL119 (becatecarin), dacarbazine,
chlormethine, bendamustine, trofosfamide, uramustine, fotemustine,
nimustine, prednimustine, ranimustine, semustine, nedaplatin,
triplatin tetranitrate, mannosulfan, treosulfan, temozolomide,
carboquone, triaziquone, triethylenemelamine, and procarbazin.
[0090] The topoisomerase inhibitor can be one of the following:
doxorubicin (doxil), daunorubicin, epirubicin, idarubicin,
anthracenedione (novantrone), mitoxantrone, mitomycin C, bleomycin,
dactinomycin, plicatomycin, irinotecan (camptosar), camptothecin,
rubitecan, belotecan, etoposide, teniposide, and topotecan
(hycamptin).
[0091] The DNA intercalator can be proflavine, doxorubicin
(adriamycin), daunorubicin, dactinomycin, and thalidomide.
[0092] The antimitotic agent can be one of the following:
paclitaxel (abraxane)/taxol, docetaxel (taxotere), BMS-275183,
xyotax, tocosal, vinorlebine, vincristine, vinblastine, vindesine,
vinzolidine, etoposide (VP-16), teniposide (VM-26), ixabepilone,
larotaxel, ortataxel, tesetaxel, and ispinesib.
[0093] The antimetabolite agent can be one of the following:
fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate, xeloda,
arranon, leucovorin, hydroxyurea, thioguanine (6-TG),
mercaptopurine (6-MP), cytarabine, pentostatin, fludarabine
phosphate, cladribine (2-CDA), asparaginase, gemcitabine,
pemetrexed, bortezomib, aminopterin, raltitrexed, clofarabine,
enocitabine, sapacitabine, and azacitidine.
[0094] In one embodiment, the second anti-cancer agent is one of
the following: carboplatin, doxorubicin, gemcitabine, docetaxel,
and etoposide. As this method inhibits Stat3 pathway, proven herein
to be critical for both the self-renewal and survival of CSCs (see
data in Example 1), and CSCs have been found to be fundamentally
responsible for drug resistance, tumor relapse and metastasis, in
preferred embodiments, this method is used to treat or prevent a
refractory cancer, relapsed cancer and/or metastatic cancer.
[0095] Further discussions of anticancer chemotherapy and biologic
therapy, and examples of suitable therapeutic protocols, maybe
found in such books as Cancer Chemotherapy and Biotherapy:
Principles and Practice, 3rd ed. (2001), Chabner and Longo, eds.,
and Handbook of Cancer Chemotherapy, 6th ed. (2003), Skeet, ed.,
both from Lippincott Williams & Wilkins, Philadelphia, Pa.,
U.S.A.; and regimens for anticancer therapies, especially
chemotherapies, may be found on Web sites such as those maintained
by the National Cancer Institute (www.cancer.gov), the American
Society for Clinical Oncology (www.asco.org), and the National
Comprehensive Cancer Network (www.nccn.org).
Targeting Cancer Stem Cells
[0096] The present invention also provides both in vitro and in
vivo data that the Compound of the Invention inhibits CSCs'
self-renewal and is apoptotic to CSCs (Example 3). Moreover, the
present invention empirically confirms the Compound of the
Invention's efficacy against metastatic cancer (Example 4).
[0097] The purpose of cancer therapy (anticancer therapy) is to
prevent cancer cells from multiplying, invading, metastasizing, and
ultimately killing their host organism, e.g. a human or other
mammals. Because cell multiplication is a characteristic of many
normal cells as well as cancer cells, most of the existing
anticancer therapies also have toxic effects on normal cells,
particularly those with a rapid rate of turnover, such as bone
marrow and mucous membrane cells. Therefore, an effective cancer
therapy needs to have a marked growth inhibitory or controlling
effect on the cancer cells while exerting a minimal toxic effect on
the host's normal cells.
[0098] Since the first effective anticancer compounds were brought
into clinical trials in the 1940's, cancer relapse and drug
resistance have remained some of the biggest problems in cancer
treatment. Often, regressions of symptoms can be obtained, but
responses are frequently partial and only of short duration, and
relapsed cancers tend to be resistant to the original drug. This
can now be explained by the existence of cancer stem cells (CSCs).
As described above, this tiny population of cells within the entire
cancer mass can evade drugs and radiotherapies that are effective
on the rest of the cancer cells because CSCs probably share the
same kind of biological mechanisms with normal somatic stem cells,
which are naturally resistant to most, if not all, of the
chemotherapeutic agents. Being the true root of tumorigenic
activities in cancer masses, CSCs can re-espouse cancer regrowth or
cause metastasis if left untreated. Since the initial treatment
leaves only drug-resistant cancer stem cells, chances for the
entire regrown or metastatic tumor to become resistant to the
initially "effective" therapy have dramatically increased.
[0099] Presently, anticancer therapies are used in combination for
several reasons. First, treatment with two or more
non-cross-resistant therapies may prevent the formation of
resistant clones in the tumor. Resistance to one anticancer drug,
e.g. a platinum anticancer compound such as cisplatin, is often
associated with cross-resistance to other drugs of the same class,
e.g. other platinum compounds. Further, there is also multiple drag
resistance, also called pleiotropic drug resistance, where
treatment with one drug confers resistance not only to that drug
and others of its class but also to unrelated agents. Second, the
combination of two or more therapies that are active against cells
in different phases of growth may kill cells that are dividing
slowly as well as those that are dividing actively and/or recruit
cells into a more actively dividing state, making them more
sensitive to multiple anticancer therapies. And third, the
combination may create a biochemical enhancement effect by
affecting different pathways or different steps in a single
biochemical pathway.
[0100] These rationales for combinational anti-cancer treatment
have not taken into account recent advances in confirming and
characterizing cancer stem cells. The failure to incorporate a
CSC-specific therapy in the combination therapy could explain why
current combination therapies cannot cure common cancers such as
metastatic colon cancer and prostate cancer. With data provided
herein that confirms the efficacy of the Compound of the Invention
against CSCs (Example 3), the present invention is able to devise a
cancer treatment method that combines a CSC-targeting agent and
another agent targeting normal cancer cells. Further, while not
wishing to be bound by a particular theory, the present invention
provides a drug regimen that preempts the scenario, which is now
supported by some preliminary data, where some normal cancer cells,
left untreated or insufficiently treated, revert back or give rise
to CSCs as the original CSCs get depleted by a single-drug therapy
that targets CSCs only.
[0101] Since cancer stem cells are generally resistant to
radiotherapy and conventional chemotherapies, a drug that targets
cancer stem cells should have synergistic effect when used in
combination with other anti-cancer therapies. Therefore, the
present invention provides a method of treating cancer in a
subject, comprising the steps of: (a) administering to the subject
a first amount of a first anti-cancer agent to inhibit a cancer
stem cell population; and (b) administering to the subject a second
amount of a second anti-cancer agent to inhibit a plurality of
normal cancer cells.
[0102] In various embodiments, some (e.g., 20%, 30%, 40%), most
(more than about 50%), or substantially all (e.g., 60%, 70%, 80%,
90%, 95% or 100%), of the CSCs are inhibited. One or both of the
first amount and the second amount can be a therapeutically
effective amount of the respective agent before combination use,
i.e., when used by itself against the cancer, or less than that
amount because of outstanding synergistic effects from the
combination. In one embodiment, the first agent is the Compound of
the Invention, namely,
2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,
2-Acetyl-7-Chloro-naphtho [2,3-b]furan-4,9-dione,
2-Acetyl-7-Fluoro-naphtho[2,3-b]furan-4,9-dione,
2-acetylnaphtho[2,3-b]furan-4,9-dione,
2-ethyl-naphtho[2,3-b]furan-4,9-dione, and a pharmaceutically
acceptable salt or solvate thereof.
[0103] The "second anti-cancer agent" here can be the same "second
anti-cancer agent" in methods described above, and all the shared
features are not repeated here. In one feature, the second
anti-cancer agent is a DNA-damaging agent, an antimitotic agent,
and/or an antimetabolite agent. For example, the DNA-damaging agent
can be an alkylating agent, a topoisomerase inhibitor, or a DNA
intercalator. Lists of suitable alkylating agents, topoisomerase
inhibitors, DNA intercalators, antimitotic agents, and
antimetabolite agents are listed above and are not repeated here.
Significant synergism was observed in cancer-inhibition experiments
where the Compound of the Invention is used in combination with
each of the above classes of chemotherapeutic agents (see Example
5). In one embodiment, the second agent is one of carboplatin,
doxorubicin, gemcitabine, docetaxel, and etoposide.
[0104] In another feature, the second anti-cancer agent is targeted
agent, e.g., a growth factor receptor-targeting agent (see
erlotinib (tarceva) data in Example 5), a kinase-targeting agent
(see lapatinib, erlotinib, sunitinib and sorafenib data in Example
5) or an angiogenesis inhibitor (see sunitinib and sorafenib data
in Example 5). Significant synergism was observed in
cancer-inhibition experiments where the Compound of the Invention
is used in combination with each of the above classes of targeted
agents. Lists of suitable growth factor receptor-targeting agents,
kinase-targeting agents (especially TKI), and angiogenesis
inhibitors are listed above and not repeated here.
[0105] As this method employs a therapeutic agent specifically
targeting CSC cells in the tumor, which are fundamentally
responsible for drug resistance, tumor relapse and metastasis, in
preferred embodiments, this method is used to treat or prevent a
refractory cancer, relapsed cancer and/or metastatic cancer.
[0106] In targeting CSCs with a combination treatment, one strategy
should aim at targeting more than one pathway that are implicated
in CSCs critical biological functions such as self-renewal and
survival. To that end, the present invention provides a method of
treating cancer in a subject that includes the steps of: (a)
administering to the subject a first amount of a first cancer stem
cell inhibitor to inhibit Stat3 pathway activities; and (b)
administering to the subject a second amount of a second cancer
stem cell inhibitor to inhibit activities of a different pathway.
In an embodiment, the amount of the first and/or second anti-cancer
agent in this method is not therapeutically effective against the
cancer stem cell population by itself--but due to significant
synergism achieved through the combination, lower amount is able to
be used in this method in order to elicit response from the
patient.
[0107] In one embodiment, the second anti-cancer stem cell agent is
lapatinib (INN) or lapatinib ditosylate (USAN), which was approved
by the FDA in 2007 for use in patients with advanced metastatic
breast cancer. Lapatinib is an ATP-competitive epidermal growth
factor receptor (EGFR) and HER2/neu (ErbB-2) dual tyrosine kinase
inhibitor. It inhibits receptor autophosphorylation and activation
by binding to the ATP-binding pocket of the EGFR/HER2 protein
kinase domain. Data presented in Example 5 below shows that marked
synergism was achieved against Paca2 pancreatic cancer cells:
pre-combination rate of inhibition for Compound 401 and lapatinib
were respectively 32% and 27% while the rate of inhibition after
combination shot up to 74%, higher than the sum of the two rates.
As this method pays additional attention to CSCs, which are
fundamentally responsible for drug resistance, tumor relapse and
metastasis, in preferred embodiments, this method is used to treat
or prevent a refractory cancer, relapsed cancer and/or metastatic
cancer.
[0108] Formulations of the present invention include those suitable
for oral, nasal, topical (including buccal and sublingual), rectal,
vaginal and/or parenteral administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any methods well known in the art of pharmacy. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form will vary depending upon the mammal
being treated and the particular mode of administration. The amount
of active ingredient, which can be combined with a carrier material
to produce a single dosage form, will generally be that amount of
the compound which produces a therapeutic effect. Generally, out of
100%, this amount will range, for example, from about 1% to about
99% of active ingredient, from about 5% to about 70%, from about
10% to about 30%.
Materials and Methods
Biological Assays
[0109] Compounds of the present invention can be tested according
to the protocol described above. Table 2 shows the list of
compounds described in the protocol.
TABLE-US-00002 TABLE 2 Compound Name Compound Code
2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione 301
2-Acetyl-7-Chloro-naphtho[2,3-b]furan-4,9-dione 416
2-Acetyl-7-Fluoro-naphtho[2,3-b]furan-4,9-dione 418
2-acetylnaphtho[2,3-b]furan-4,9-dione 401
2-ethyl-naphtho[2,3-b]furan-4,9-dione 101
[0110] Cell Culture: HeLa, DU145, H1299, DLD1, SW480, A549, MCF7,
LN18, HCT116, HepG2, Paca2, Pancl, LNcap, FaDu, HT29, and PC3 cells
(ATCC, Manassas, VA) were maintained in Dulbecco's Modified Eagle
Medium (DMEM) (Invitrogen, Carlsbad, Calif.) supplemented with 10%
fetal bovine serum (FBS) (Gemini Bio-Products, West Sacramento,
Calif.) and 5% penicillin/streptomycin/amphotercin B
(Invitrogen).
[0111] Hoechst Side Population: To identify and isolate side
population (SP) and non-SP fractions, SW480 cells were removed from
the culture dish with trypsin and EDTA, pelleted by centrifugation,
washed with phosphate-buffered saline (PBS), and resuspended at
37.degree. C. in Dulbecco's modified Eagle's medium (DMEM)
containing 2% FBS and 1 mM HEPES. The cells were then labeled with
Hoechst 33342 (Invitrogen) at a concentration of 5 .mu.g/mL. The
labeled cells were incubated for 120 minutes at 37.degree. C.,
either alone or with 50 .mu.tM verapamil (Sigma-Aldrich, St.
Louis). After staining, the cells were suspended in Hanks' balanced
saline solution (HBSS; Invitrogen) containing 2% FBS and 1 mM
HEPES, passed a through 40 .mu.m mesh filter, and maintained at
4.degree. C. until flow cytometry analysis. The Hoechst dye was
excited at 350 nm, and its fluorescence was measured at two
wavelengths using a 450 DF10 (450/20 nm band-pass filter) and a
675LP (675 nm long-pass edge filter) optical filter. The gating on
forward and side scatter was not stringent, and only debris was
excluded [15].
[0112] CSC isolation with surface markers: Sorting tumor cells
based primarily upon the differential expression of the surface
marker(s), such as CD44 or CD133, have accounted for the majority
of the highly tumorigenic CSCs described to date. CD133 isolation
is based upon the method of Ricci-Vitiani et al. [21], with slight
modification. CD133.sup.- cells were isolated by either
fluorescence activated cell sorting (FACS) or magnetic
nanoparticle-based separation. Briefly, 10' cells/mL were labeled
with CD133/1 (AC133)-PE for FACS-based cell sorting; or with
CD133/1 (AC133)-biotin (Miltenyi Biotec, Auburn, Calif.) for
magnetic field-based separation using the EasySep.RTM. biotin
selection kit (Miltenyi Biotec) according to the manufacturer's
recommendations. Non-specific labeling was blocked with the
supplied FcR blocking reagent and antibody incubations (1:11) were
carried out on ice for 15 minutes in PBS with 2% FBS and 1 mM EDTA.
Five washes were done for EasySep.RTM. isolation, whereas cells
were pelleted at 400.times.g for 5 minutes and resuspended at
2.times.10 mL, before sorting by FACS.
[0113] CD44.sup.high cells were isolated by FACS according to the
methods described in Ponti et al, with slight modification [81].
Briefly, after trypsinization and recovery of cells for 30 minutes
at 37.degree. C. in growth media, cells were pelleted at
400.times.g and were resuspended in PBS with 2% FBS and 1 mM EDTA
at 1.times.10.sup.6 cells/mL. Cells were then incubated on ice with
a 1:100 dilution of CD44-FITC (BD Biosicences, San Diego, Calif.)
for 15 minutes. Alternatively, CD24-PE (BD Bioscences, San Diego,
Calif.) (1:100) was utilized for negative selection. After washing
three times, cells were resuspended at 2.times.10.sup.6/mL and
passed through a 40 .mu.M mesh before sorting
[0114] Sphere assay: A reliable method of measuring the
self-renewal capacity of cell population if the ability to be
cultured as spheres in the absence of serum or attachment.
CD4.sup.high or Hoechst side population cancer stem cells were
cultured in ultra low attachment plates in cancer stem cell media
(DMEM/F12, B27 Neurobasal supplement, 20 ng/ml EGF, 10 ng/ml FGF, 4
.mu.g/ml insulin, and 0.4% BSA) to allow spheres formation.
Typically, sphere formation was evaluated by microscopy after 10-14
days in culture and spheres with >50 cells were scored.
[0115] Luciferase Reporter Assay: HeLa Cells were co-transfected
with Stat3-luciferase (Stat3-Luc) reporter vector (Panomics,
Fremont, Calif.) and Renilla luciferase (Promega, Madison, Wis.)
using Lipofectamine 2000 as described by the manufacturer
(Invitrogen). Following transfection, cells were maintained in
medium containing 0.5% FBS for 24 hours. Cells were then treated
with the indicated compound for 30 minutes prior to the addition of
25 ng/ml oncostatin M (OSM) (R&D Systems, Minneapolis, Minn.)
to the medium. 6 hours following OSM addition, cells were harvested
and levels of firefly and renilla luciferase were measured using
the Dual-Glo Luciferase Assay System as described by the
manufacturer (Promega).
[0116] Analysis of Apoptosis: Cells treated with or without
compound were harvested at 5 hours post treatment for Annexin-V
staining. Collected cells were washed with PBS and resuspended in
Annexin-V-FITC containing buffer and stained according to
manufactures directions (Roche). Annexin-V positive cells were
determined by Flow cytometry.
[0117] STAT3 DNA Binding Assay: Electrophoretic mobility shift
assay (EMSA) was performed as described by the manufacturer (Li-Cor
Biosciences, Lincoln, Nebr.). Briefly, nuclear extracts were made
from HeLa cells using the NucBuster Protein Extraction Kit as
described by the manufacturer (EMD Biosciences, San Diego, Calif.).
5 .mu.g of nuclear extract was pre-incubated with the indicated
dose of indicated compound for 30 minutes prior to a 15-minute
incubation with the IR700-labeled consensus Stat3 oligonucleotide.
Samples were then electrophoresed on a polyacrylamide gel and
directly scanned using the Odyssey infrared imaging system (Li-Cor
Biosciences). For the enzyme-linked immunosorbent assay (ELISA), 5
.mu.g of nuclear extract was preincubated with indicated
concentration of indicated compound for 30 minutes prior to the
addition of biotinylated oligo
(5'-Biotin-GATCCTTCTGGGAATTCCTAGATC-3' SEQ ID NO. 1). Stat3-DNA
complexes were then captured on streptavidin coated 96 well plates
(Pierce, Rockford, Ill.). Bound complexes were then incubated with
Stat3 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz,
Calif.) followed by anti-rabbit HRP conjugated secondary antibody
(GE Healthcare, Pittsburgh, Pa.). Bound antibody was then
visualized by addition of TMB substrate (Pierce) and absorbance
measured at 450 nm.
[0118] Cell Viability Determination: For 3-(4,5
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) (Sigma-Aldrich,
St. Louis, Mo.) analysis, cells were plated in 96 well plates at
10,000 cells per well. 24 hours after plating, compound was added
to cells at indicated doses. 22 hours following compound addition,
MTT was added to each well (0.5 mg/ml, final concentration) and
plates were incubated for an additional 2 hours at 37.degree. C.
Medium was then aspirated and the formazan product was solubilized
in 100 .mu.l of isopropyl alcohol. The absorbance of each well was
measured at 570 nm using a microplate reader.
[0119] Immunofluorescence: Cells treated with indicated compound
for an indicated time were either fixed in 4% formaldehyde or cold
methanol for the detection of Annexin V, cleaved caspase 3, or
stat3, respectively. Coverslips were air dried and rehydrated in
PBS at room temperature for 10 min. Samples were then incubated in
blocking buffer (PBS, 5% FBS) for 10 min at room temperature in a
humid chamber. Cells were incubated overnight at 4.degree. C. with
primary antibodies. After washing, the cells were incubated for 1
hour at room temperature with a 1:500 dilution of FITC conjugated
anti-rabbit antibody. Images were captured with a Nikon TE200
microscope equipped with epifluorescence and a SPOT mosaic CCD
camerapolyclonal Anti-cleaved caspase 3 antibody (1:100) was
obtained from Cell Signaling Technology, Danvers, MA.
Annexin-V-FITC was obtained from Roche, Penzberg, Germany.
Polyclonal anti-Stat3 antibody was obtained from Santa Cruz.
[0120] Gene knockdown by TPIV.RTM. technology: The (Therapeutic
Pathway Identification and Validation) technology (Boston
Biomedical Inc., Norwood, Mass., USA) provides plasmids that can be
used to first transfect bacteria that are in turn taken up by a
mammalian subject. After bacterial lysis, dsRNA encoded by the
TPIV.RTM. plasmids and processed by the bacteria get released into
the mammalian cell cytoplasm and effect targeted gene knockdown.
The TPIV.RTM. technology is described in co-owned PCT patent
application no. PCT/US08/68866 filed on Jun. 30, 2008, the entire
content of which is incorporated herein by reference. Specifically,
a TPIV.RTM. plasmid that encodes effective siRNA sequences against
Stat3 was constructed by PCR-cloning of a Stat3 plasmid purchased
from Origene Technologies (Rockville, Md., USA) using the following
primers:
TABLE-US-00003 TPIV-Stat3 (300 bp insert) Primers: Stat3 TPW For
(SEQ ID NO. 2) 5'-GGATCTAGAATCAGCTACAGCAGC Stat3 TPIV Rev (SEQ ID
NO. 3) 5'-TCCTCTAGAGGGCAATCTCCATTG
[0121] The control plasmid is constructed using a pGL2 plasmid
purchased from Promega (Madison, Wis., USA).
TABLE-US-00004 TPIV-GL2 (300 bp insert) Primers: GL2 TPIV For (SEQ
ID NO. 4) 5'-CCCTCTAGATGGTTCCTGGAAC GL2 TPIV Rev (SEQ ID NO. 5)
5'-GCTCTAGAAACCCCTTTTTGG
[0122] Chemically competent E. coli BL21 (DE3) pLYSe bacteria
(50-100 .mu.l) were transformed with control or 100 ng of
Stat3-targeting TPIV.RTM. plasmid according to the manufacturer
instructions (Stratagene). A single colony was then inoculated into
BHI medium containing 100 .mu.g/ml ampicillin, and grown overnight
at 37.degree. C. The next day, 5 ml of each overnight culture was
diluted 1:40 into fresh BHI medium containing 100 .mu.g/ml
ampicillin and grown for a further 2-4 hours (until the
OD.sub.600=0.5). Each culture was then treated with IPTG (1 mM
final concentration) for 2-4 hours to induce transcription of the
long double strand RNAs which would be processed into a cocktail
siRNAs by the bacteria. After IPTG induction, the total number of
bacteria in each culture was calculated by measuring the OD.sub.600
value (8.times.10.sup.8 bacteria/ml culture has an OD.sub.600=1).
The number of bacteria for cell treatment was then calculated
according to cell confluency and the needed multiplicity of
infection (MOI; try ranges of 20:1 to 2000:1, bacteria to cells) in
an appropriate reaction volume. As a rule of thumb, the reaction
volume should be chosen to result in 3.times.10.sup.8/ml for a
1000:1 MOI. The required volume of bacteria culture was then
centrifuged at 2500 g for 10 mins at 4.degree. C. and the pellet
was washed once with serum-free culture medium that was used for
the cells being bactofectioned plus 100 .mu.g/ml ampicillin and 1
mM of IPTG, and resuspended in the same medium at the required
density for bacterial infection (bactofection).
[0123] At the same time, cancer cells or cancer stem cells were
isolated. 30 minutes before bactofection, the cell culture medium
was replaced with 2 ml of fresh serum-free medium containing 100
.mu.g/ml of ampicillin and 1 mM IPTG. Bacteria prepared above were
then added to the cells at the desired MOI for 2 hours at
37.degree. C.
[0124] After the infection period, the cells were washed 3 times
using serum-free cell culture medium. The cells were then incubated
with 2 ml of fresh complete cell culture medium containing 100
.mu.g/ml of ampicillin and 150 .mu.g/ml of gentamycin for 2 hours
to kill any remaining extracellular bacteria. After treatment with
ampicillin and gentamycin, the cells were incubated with 3 ml of
fresh complete RPMI 1640 medium containing 10 .mu.g/ml of ofloxacin
to kill any intracellular bacteria. The cells were then harvested
or analysis at various time points in order to assess the extent of
target gene silencing and the resulting phenotypes.
[0125] In Life Evaluations: Daily examinations into the health
status of each animal were also conducted. Body weights were
checked every three days. Food and water was supplied daily
according to the animal husbandry procedures of the facility.
Treatment producing >20% lethality and or >20% net body
weight loss were considered toxic. Results are expressed as mean
tumor volume (mm.sup.3).+-.SE. P Values <0.05 are considered to
be statistically relevant.
[0126] Animal Husbandry: Male or female athymic nude mice 4-5 weeks
(Charles River Laboratories, Wilmington, Mass.), were acclimated to
the animal housing facility for at least 1 week before study
initiation. All of the experimental procedures utilized were
consistent with the guidelines outlined by the American Physiology
Society and the Guide for the Care and Use of Laboratory Animals
and were also approved by the Institutional Animal Care and Use
Committee of Boston Biomedical Inc. The animals were housed in
groups of four in wood chip bedded cages in a room having
controlled temperature (68.degree. F.-72.degree. F.), light (12-h
light-dark cycle), and humidity (45-55%). The animals were allowed
free access to water and food during the experiment.
[0127] Intrasplenic-nude mouse model system (ISMS model): The
female nude mice were anesthetized and under aseptic conditions, an
incision was made in the left flank to expose the spleen. One
million human colon cancer HT29 cells in 0.1 ml PBS were injected
under the spleen capsule using a 27-gauge needle. The spleen was
replaced in the peritoneal cavity and the incision was closed.
Treatment started the next day after the implantation till the
examination day. The regimen of the treatments is 5qd/wk via i.p.
The mice were sacrificed when moribund or 30 days after the
injection. The spleen and liver were removed and examined, and the
number of tumor lesions was recorded.
EXAMPLE 1
Identification of Stat3 as an Anti-Cancer Stem Cell Target
[0128] Stat3 knockdown in CSCs induces apoptosis. To determine
whether cancer stem cells expressed Stat3 and whether Stat3 was
constitutively active, we performed immunofluorence microscopy,
which allows not only the analysis of rare cell populations, but
also provides additional information on protein localization and
the ability to correlate staining with phenotype (i.e. apoptosis).
Following immunofluorescent detection of p-Stat3 and Stat3 in NSP
and SP cells isolated by FACS from SW480 colon cancer cells, we
determined that Stat3 was indeed present in SP cells and that it
was modestly enriched in the nucleus (FIG. 3A). In addition, we
also observed increased p-Stat3 staining in SP cells over NSP
cells, suggesting that SP cells may rely more heavily on Stat3 for
survival.
[0129] The status of Stat3 was also evaluated in CD133.sup.+ cells
isolated from FaDu human head and neck cancer cells and LN18 human
glioblastoma cells. As shown in FIG. 3B, Stat3 are also
constitutively active in these cells. Taken together, these data
suggest Stat3 as a target that is particularly important for cancer
stem cells.
[0130] We next tested the effect of Stat3 knockdown in CSCs using
TPIV.RTM.. Immunofluorescence analysis revealed that significant
depletion of Stat3 could be achieved within 24 hours of infection
(FIG. 4A) on freshly islolated CSCs (SP) and found that the
majority of cells treated with Stat3-targeting TPIV.RTM. plasmids
underwent apoptosis within 24 hours of infection, whereas control
TPIV.RTM. plasmids did not induce apoptosis to levels above
control, uninfected cells (FIG. 4B). These data demonstrate that
cancer stem cells depend upon Stat3 for survival.
[0131] Knock down Stat3 in CSCs inhibits CSC spherogenesis.
CD44.sup.high/CD24.sup.low FaDu or Hoeschst side population cancer
stem cells were isolated by FACS, and cultured in ultra low
attachment plates in cancer stem cell media (DMEM/F12, B27
Neurobasal supplement, 20 ng/mL EGF, 10 ng/mL FGF, 4 .mu.g/mL
insulin, and 0.4% BSA) to allow sphere formation. Primary spheres
were collected, disaggregated with trypsin, and distributed to
96-well ultra low attachment plated prior to TPIV.RTM. treatment.
Bacteria were administered at an MOI of 1000 for two hours before
addition of anti-biotic cocktail (penstrep, gentamycin, oflaxacin).
Sphere formation was assessed after 10-14 days in culture.
Representative sphere images were captured before (FIG. 5, left
upper panels) or after the addition of trypan blue to identify dead
cells (FIG. 5, left bottom panel). Relative spherogenesis was shown
in the right panel of FIG. 5. The data clearly showed that Stat3
knockdown in cancer stem cells inhibits sphereogenesis,
demonstrating that Stat3 is a key self-renewal factor of cancer
stem cells.
EXAMPLE 2
[0132] Identification of Compounds that Inhibit Stat3 Pathway
Activity
[0133] Inhibition of Stat3 transcription activity. Compounds were
tested for their ability to inhibit Stat3 transcription activation
activity in cells using a Stat3-luciferase (Stat3-luc) reporter
construct. Cells transfected with Stat3-luc were cultured in
reduced serum medium prior to addition of indicated compound for 30
minutes. Cells were then stimulated with 25 ng/ml oncostatin M
(OSM) for 6 hours followed by detection of Stat3-luc reporter
activity. Incubation of cells with compound 401 inhibited
OSM-stimulated Stat3 reporter activity (FIG. 6, left panel). AG490,
a known inhibitor of the Jak-Stat pathway, was included as a
positive control for Stat3 inhibition. Etoposide, included as a
control for genotoxic activity, showed little or no Stat3
inhibition. Compound 1001, which is naphthalene instead of
naphthoquinone as the compounds in this invention, did not inhibit
OSM-stimulated Stat3 reporter activity even at a much higher
concentration (FIG. 6, right panel).
[0134] Additional compounds were tested in the Stat3 luciferase
reporter assays and the results are summarized in Table 3.
TABLE-US-00005 TABLE 3 Compound # IC.sub.50 in Stat3-Luc assays 401
~0.25 .mu.M 416 ~0.75 .mu.M 418 ~0.75 .mu.M 301 ~2 .mu.M
[0135] Inhibition of Stat3 DNA-binding activity. Nuclear extracts
from HeLa cells, which contain constitutively activated Stat3 as
detected by phosphorylation at the tyrosine 705 residue, were used
to perform Stat3 EMSAs to monitor Stat3 DNA binding activity.
Nuclear extracts were incubated with indicated compound prior to
incubation with IR700-labeled Stat3 consensus oligonucleotide.
Binding of Stat3 to the oligonucleotide was monitored by gel
electrophoresis and detection using a LiCor Odyssey infrared
scanner. The Stat3 retarded band was identified and confirmed by
supershift with the anti-Stat3 antibody (FIG. 7A, left panel) and
dose-dependent inhibition with the Stat3 peptide (FIG. 7A, middle
panel). Dose dependent inhibition of Stat3 DNA binding was observed
following incubation of the labeled probe with compound 401 (FIG. 7
A, right panel).
[0136] Additional compounds were tested in the EMSA assays. As
shown in FIG. 7B, compounds 401, 416 and 418 can inhibit Stat3's
DNA binding activity.
[0137] Inhibition of Stat3 downstream effectors in xenograft tumor
tissues. Extracts were prepared from xenografted Paca2 tumors that
were treated with compound 401 or vehicle control 4 hours prior to
harvest. The samples were analyzed by western blots and EMSA to
evaluate the Stat3 downstream effector expression level and Stat3
DNA binding activity. Compound 401 treated sample (T) showed a
decrease in Stat3 DNA binding activity over the control (V) (FIG.
8A). In addition, compound 401 treatment resulted in a decrease in
the expression level of Stat3's downstream effectors cyclin D1 and
survivin (FIG. 8B).
EXAMPLE 3
[0138] Identification of Compounds that Target Cancer Stem
Cells
[0139] Identification of compounds that are apoptotic to cancer
stem cells. Since cancer stem cells have been demonstrated to
actively efflux Hoechst, SW480 cells were stained with Hoechst and
the side population (shown in FIG. 9A, left panel gated area) was
sorted out to enrich the cancer stem cells. To confirm that this
side population is enriched with cancer stem cells, a control set
of SW480 cells were first treated with Verapamil, an inhibitor of
ABC transporters, before stained with Hoechst. As shown in the
right panel of FIG. 9A, Verapamil treatment results in the loss of
the side population.
[0140] The IC.sub.50 of compound 401 against the Hoechst side
population was accessed in MTT assays and was compared to the
IC.sub.50 against the non-side population. The results show that
the side population is as sensitive as the non-side population to
compound 401 (FIG. 9B, right panels). However, the side population
is much more resistant than the non-side population to Doxorubicin
(FIG. 9B, left panels), which is consistent with previous
publications [7, 82], These data suggest that compound 401 kills
cancer stem cells.
[0141] The Hoechst side population cells were treated with compound
401 and the mode of cell death was accessed by Annexin V (an early
marker for apoptosis) staining. The results show that the dying
cells are Annexin V positive (FIG. 10A), demonstrating that
compound 401 is apoptotic to cancer stem cells.
[0142] Alternatively, we performed CD133 (one of the common cancer
stem cell surface markers) antibody magnetic beads pull down to
enrich cancer stem cells. The CD133.sup.+ cells were then treated
with compound 401 followed by staining with antibody against
cleaved-Caspase 3 (a hallmark of apoptosis). As shown in FIG. 10B,
many of the CD133.sup.+ cells become cleaved-Caspase 3 positive
after compound 401 treatment, corroborating that compound 401 is
apoptotic to cancer stem cells.
[0143] Identification of compounds that inhibit CSC spherogenesis
in vitro. One of the hallmarks of cancer stem cells is their
ability to self-renew. A reliable method of measuring the
self-renewal capacity of cell populations is the ability to be
cultured as spheres in the absence of serum or attachment. To
compare the ability of compound 401 to other targeted and
chemotherapeutic agents, FACS-isolated CD44.sup.high CSCs were
grown as spheres for 72 hours before being challenged with a panel
of therapeutic agents. Of the agents tested, only compound 401 was
effective at preventing sphere proliferation (FIGS. 11A and 11B).
Note that spheres were resistant to doxorubicin and docetaxel
despite being applied at approximately ten times their IC.sub.50
concentrations for cell death in similar assays. Tarceva, Sutent,
and Gleevec were added at approximately three times their reported
therapeutic concentrations. This demonstrates that while cancer
stem cells are resistant to conventional chemotherapeutic and
targeted agents, compound 401 is highly effective at inhibiting
their growth.
[0144] Identification of compounds that inhibit CSC spherogenesis
in vivo. Six-week-old female athymic nu/nu mice were obtained from
Charles River Labs (Wilmington, Mass.). Mice were injected
subcutaneously on the flank with 6.times.10.sup.6 FaDu or Paca2
cancer cells in 0.2 mL of serum-free DMEM. After xenografts reached
.about.200 mm.sup.3 in size, animals bearing Paca2 xenograft tumors
were administered with either vehicle, gemcitabine (120 mg/kg,
twice a week), or compound 401 (20 mg/kg) by ip for one week and
animals bearing FaDu xenograft tumors were administered daily with
either vehicle, carboplatin (30 mg/kg), or compound 401 (20 mg/kg)
via ip for two weeks before sacrifice. Tumors were then collected
for Paca2 and FaDu cells, respectively. Single cell suspensions
were obtained following animal sacrifice, and sterile removal of
tumors. Briefly, tumors were minced with sterile scalpels into 0.1
mm.sup.3 pieces before being digested in 1 mg/mL collagenase/HBSS
for 15-30 minutes with constant agitation. Following passage
through a 40 .mu.m mesh filter, RBCs, dead cells, and cell debris
were removed by layering the cell suspension onto 1 mL of
Histopaque and collecting the interface layer after centrifugation
at 1440.times.g for 30 minutes. Live cells were then counted and
used to measure their ability to form spheres. Cells were
distributed to ultra low attachment 96 well plates at a density of
100 cells per well in cancer stem cell media (DMEM/F12, B27
Neurobasal supplement, 20 ng/mL EGF, 10 ng/mL FGF, 4 .mu.g/mL
insulin, and 0.4% BSA). Fresh media was added every three days, and
sphere formation was determined after 10-14 days in culture.
Spheres with >50 cells were scored. At conclusion of experiment,
trypan blue was added to identify dead cells. As shown in FIG. 12,
standard chemotherapies gemcitabine (upper panel) and carboplatin
(bottom panel) enriched cancer stem cells evidenced by the
increased spherogenesis. In contrast, compound 401 treatments
decreased cancer stem cells as is evident by the decreased
spherogenesis.
EXAMPLE 4
[0145] Anti-Metastasis efficacy
[0146] Compound 401 was also tested for its capability to inhibit
metastasis in ISMS model. The intrasplenic-nude mouse model system
(ISMS model) is appropriate for studies of the malignant behavior
of colorectal carcinomas, as this technique can produce
experimental metastases in the liver. In this model, one million
HT29 cells in 0.1 ml PBS were injected under the spleen capsule of
the nude mice. The spleen was replaced in the peritoneal cavity and
the incision was closed. Mice were sacrificed when moribund or 30
days after the injection. The spleen and liver were removed and
examined, and the number of tumor lesions was recorded. Mice were
divided into 2 groups, a control group given vehicle (n=4) and the
other group receiving 20 mg/kg compound 401 (n=4). Drug was
administered via ip. 5 days/week starting from day 2 to day 30
after i.s. injection. The numbers of primary tumors and metastatic
liver tumors were estimated microscopically. Representative
pictures are shown in FIG. 13. In the vehicle control group, there
was heavy burden of primary tumors at spleen (FIG. 13, upper left
panel). Massive spontaneous liver metastases were also observed
(FIG. 13, upper right panel). Compound 401 treatments significantly
reduced the number of primary tumor foci and the spontaneous liver
metastasis (FIG. 13, lower panels).
EXAMPLE 5
Combinatorial Activity
[0147] Paca2 human pancreatic cancer cells, A549 human lung cancer
cells, and HepG2 human liver cancer cells (American Type Culture
Collection) were cultured in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum, 100 units/mL penicillin, 100
.mu.g/mL streptomycin, and 2 mM L-glutamine. Compound 401 and
Sutent were synthesized by Boston Biomedical, Inc. Carboplatin,
doxorubicin, docetaxel, etoposide were obtained from Sigma (St.
Louis, Mo.) and dissolved in water or DMSO at 10 mM. Erlotinib was
from American Custom Chemicals (San Diego, Calif.). Gemcitabine was
from Eli Lilly (Indianapolis, Ind.) in an aqueous 20 mM stock
solution. Sorafenih was purchased from LKT (St. Paul, Minn.).
Lapatinib was from LC Laboratories (Wobum, Mass.). Unless otherwise
noted all compounds were solubilized in DMSO at 10 mM and aliquoted
at -20.degree. C. Exponentially growing Paca2 pancreatic cancer
cells were seeded at 1,000 cells per well in 6-well plates and
allowed to attach for 24 hours. Increasing concentrations of
individual drugs and those in combination were then added to the
media for another 24 hours. After 24 hours exposure, the drug was
removed and fresh media was added for the next 10-14 days, allowing
for colony formation. Cells were fixed and stained with GIEMSA
(Gibco BRL). Colonies of greater than 50 cells were scored as
survivors and percentage of cell survival was normalized to
untreated controls. Results are an average of duplicate
experiments. Alternatively, MTT assays were performed 72 hours
post-treatment in A549 and HepG2 cells.
[0148] Our data demonstrate that compound 401 has beneficial
effects when combined with all the drugs tested. Among them,
combination with tyrosine kinase inhibitors (TKI) showed most
remarkable results. For examples, as shown in FIG. 14, compound 401
has a synergistic effect in combination with sorafenib in human
lung A549 cells at 72 hours. Similarly, FIGS. 15 to 17 show that
compound 401 also has synergistic effects in combination with
erlotinib, lapatinib, and sunitinib (Sutent.RTM.), respectively, in
human lung A549 cells at 72 hours. The rest of the data are
summarized in Table 4 and demonstrate that compound 401 showed
beneficial effects when combined with all the drugs tested.
TABLE-US-00006 TABLE 4 Compound Compound 401 X 401 + X Combo drug
Exemplary (% Inhibition) (% Inhibition) (% Inhibition) (X) function
7 day colony formation PACA2 cells 32 [66 nM] -- -- -- 66 [133 nM]
-- -- -- 32 43 [16 .mu.M] 80 Carboplatin DNA alkylating agent, DNA
damaging 32 89 [16 .mu.M] 100 Doxorubicin DNA- intercalator,
antibiotic, DNA damaging 32 48 [0.33 nM] 62 Docetaxel Anti-mitotic
32 58 [660 nM] 81 Etoposide topoisomerase II inhibitor. DNA
damaging 32 27 [6.75 .mu.M] 74 Lapatinib TKI 32 26 [16 .mu.M] 40
Erlotinib TKI 32 52 [12 .mu.M] 73 Sunitinib TKI 66 36 [33 nM] 96
Gemcitabine Anti-metabolite 66 66 [2 .mu.M] 100 Sorafenib TKI 72
hour MTT A549 cells 35 [500 nM] 43 [2.5 .mu.M] 88 Erlotinib TKI 35
[250 nM] 29 [12.5 nM] 54 Doxorubicin DNA- intercalator, antibiotic,
DNA damaging 35 [500 nM] 75 [2.5 .mu.M] 81 Sunitinib TKI 35 [500
nM] 40 [2.5 .mu.M] 74 Sorafenib TKI 35 [500 nM] 66 [2.5 .mu.M] 85
Lapatinib TKI 72 hour MTT HepG2 cells 43 [250 nM] 23 [12.5 nM] 72
Doxorubicin DNA- intercalator, antibiotic, DNA damaging 43 51 [2.5
.mu.M] 68 Sorafenib TKI 7 [125 nM] 34 [625 nM] 42 Sorafenib TKI
[0149] Furthermore, we tested the combo effect of compound 401 with
gemcitabine in human pancreatic cancer xenograft model. Briefly,
athymic female nude mice (Ncr) were inoculated subcutaneously with
8.times.10.sup.6 MIA PaCa-2 human pancreatic cancer cells, and the
tumors were allowed to grow to approximately 150 mm.sup.3 in size.
The animals were randomized into four groups of six animals per
group, and were treated with vehicle control, compound 401 at 100
mg/kg in the clinical formulation (20% Gelucire) orally daily,
genicitabine (Gemzar.RTM.) at 120 mg/kg (in PBS) intraperitoneally
every three days, or both. The mice received a total of two-week
treatments, and the mean volumes of the tumors were analyzed.
[0150] As shown in FIG. 18, treatment with either compound 401 (100
mg/kg) or gemcitabine (120 mg/kg) alone retarded tumor growth to a
similar extent during the treatment. Animals treated with compound
401 (100 mg/kg) in combination with gemcitabine (120 mg/kg) showed
a synergistic effect on tumor growth. No significant toxicity was
noted for any of the treatment regimens. Our data suggest that
compound 401 in combination with gemcitabine is clinically
beneficial in treating pancreatic cancer
[0151] All references cited herein are incorporated herein by
reference in their entirety to the extent allowed by applicable
laws and for all purposes to the same extent as if each individual
publication or patent or patent application is specifically and
individually indicated to be incorporated by reference in its
entirety for all purposes. To the extent publications and patents
or patent applications incorporated by reference contradict the
disclosure contained in the specification, the specification is
intended to supersede and/or take precedence over any such
contradictory material.
[0152] All numbers expressing quantities of ingredients, reaction
conditions, analytical results and so forth used in the
specification and claims are to be understood as being modified in
all instances by the term "about." Accordingly, unless indicated to
the contrary, the numerical parameters set forth in the
specification and attached claims are approximations that may vary
depending upon the desired properties sought to be obtained by the
present invention. At the very least, and not as an attempt to
limit the application of the doctrine of equivalents to the scope
of the claims, each numerical parameter should be construed in
light of the number of significant digits and ordinary rounding
approaches.
[0153] Modifications and variations of this invention can be made
without departing from its spirit and scope, as will be apparent to
those skilled in the art. The specific embodiments described herein
are offered by way of example only and are not meant to be limiting
in any way. It is intended that the specification and examples be
considered as exemplary only, with a true scope and spirit of the
invention being indicated by the following claims.
REFERENCES
[0154] 1. Bonnet, D., Normal and leukaemic stem cells. Br J
Haematol, 2005. 130(4): p. 469-79. [0155] 2. Bonnet, D. and J. E.
Dick, Human acute myeloid leukemia is organized as a hierarchy that
originates from a primitive hematopoietic cell. Nat Med, 1997.
3(7): p. 730-7. [0156] 3. Baumann, M., M. Krause, and R. Hill,
Exploring the role of cancer stem cells in radioresistance. Nat Rev
Cancer, 2008. 8(7): p. 545-54. [0157] 4. Hambardzumyan, D., M.
Squatrito, and E. C. Holland, Radiation resistance and stem-like
cells in brain tumors. Cancer Cell, 2006. 10(6): p. 454-6. [0158]
5. Dean, M., T. Fojo, and S. Bates, Tumour stem cells and drug
resistance. Nat Rev Cancer, 2005. 5(4): p. 275-84. [0159] 6. Jones,
R. J., W. H. Matsui, and B. D. Smith, Cancer stem cells: are we
missing the target? J Natl Cancer Inst, 2004. 96(8): p. 583-5.
[0160] 7. Ho, M. M., et al., Side population in human lung cancer
cell lines and tumors is enriched with stem-like cancer cells.
Cancer Res, 2007. 67(10): p. 4827-33. [0161] 8. Wang, J., et al.,
Identification of cancer stem cell-like side population cells in
human nasopharyngeal carcinoma cell line. Cancer Res, 2007. 67(8):
p. 3716-24. [0162] 9. Haraguchi, N., et al., Characterization of a
side population of cancer cells from human gastrointestinal system.
Stem Cells, 2006. 24(3): p. 506-13. [0163] 10. Doyle, L. A. and D.
D. Ross, Multidrug resistance mediated by the breast cancer
resistance protein BCRP (ABCG2). Oncogene, 2003. 22(47): p.
7340-58. [0164] 11. Alvi, A. J., et al., Functional and molecular
characterisation of mammary side population cells. Breast Cancer
Res, 2003. 5(1): p. R1-8. [0165] 12. Frank, N. Y., et al.,
ABCB5-mediated doxorubicin transport and chemoresistance in human
malignant melanoma. Cancer Res, 2005. 65(10): p. 4320-33. [0166]
13. Schatton, T., et al., Identification of cells initiating human
melanomas. Nature, 2008. 451(7176): p. 345-9. [0167] 14. Kondo, T.,
T. Setoguchi, and T. Taga, Persistence of a small subpopulation of
cancer stem-like cells in the C6 glioma cell line. Proc Natl Acad
Sci USA, 2004. 101(3): p. 781-6. [0168] 15. Goodell, M. A., et al.,
Isolation and functional properties of murine hematopoietic stem
cells that are replicating in vivo. J Exp Med, 1996. 183(4): p.
1797-806. [0169] 16. Al-Hajj, M., et al., Prospective
identification of tumorigenic breast cancer cells. Proc Natl Acad
Sci U S A, 2003. 100(7): p. 3983-8. [0170] 17. Collins, A. T., et
al., Prospective identification of tumorigenic prostate cancer stem
cells. Cancer Res, 2005. 65(23): p. 10946-51. [0171] 18. Li, C., et
al., Identification of pancreatic cancer stem cells. Cancer Res,
2007. 67(3): p. 1030-7. [0172] 19. Ma, S., et al., Identification
and characterization of tumorigenic liver cancer stem/progenitor
cells. Gastroenterology, 2007. 132(7): p. 2542-56. [0173] 20.
Prince, M. E., et al., Identification of a subpopulation of cells
with cancer stem cell properties in head and neck squamous cell
carcinoma. Proc Natl Acad Sci U S A, 2007. 104(3): p. 973-8. [0174]
21. Ricci-Vitiani, L., et al., Identification and expansion of
human colon-cancer-initiating cells. Nature, 2007. 445(7123): p.
111-5. [0175] 22. Singh, S. K., et al., Identification of a cancer
stem cell in human brain tumors. Cancer Res, 2003. 63(18): p.
5821-8. [0176] 23. Dalerba, P., et al., Phenotypic characterization
of human colorectal cancer stem cells. Proc Natl Acad Sci U S A,
2007. 104(24): p. 10158-63. [0177] 24. Klein, W. M., et al.,
Increased expression of stem cell markers in malignant melanoma.
Mod Pathol, 2007. 20(1): p. 102-7. [0178] 25. Yu, H. Stat3: Linking
oncogenesis with tumor immune evasion, in AACR 2008 Annual Meeting.
2008. San Diego, Calif. [0179] 26. Pedranzini, L., A. Leitch, and
J. Bromberg, Stat3 is required for the development of skin cancer.
J Clin Invest, 2004. 114(5): p. 619-22. [0180] 27. Catlett-Falcone,
R., et al., Constitutive activation of Stat3 signaling confers
resistance to apoptosis in human U266 myeloma cells. Immunity,
1999. 10(1): p. 105-15. [0181] 28. Bromberg, J. F., et al., Stat3
as an oncogene. Cell, 1999. 98(3): p. 295-303. [0182] 29. Kanda,
N., et al., STAT3 is constitutively activated and supports cell
survival in association with survivin expression in gastric cancer
cells. Oncogene, 2004. 23(28): p. 4921-9. [0183] 30. Schlette, E.
J., et al., Survivin expression predicts poorer prognosis in
anaplastic large-cell lymphoma. J Clin Oncol, 2004. 22(9): p.
1682-8. [0184] 31. Niu, G., et al., Constitutive Stat3 activity
up-regulates VEGF expression and tumor angiogenesis. Oncogene,
2002. 21(13): p. 2000-8. [0185] 32. Xie, T. X., et al., Stat3
activation regulates the expression of matrix metalloproteinase-2
and tumor invasion and metastasis. Oncogene, 2004. 23(20): p.
3550-60. [0186] 33. Kortylewski, M., et al., Inhibiting Stat3
signaling in the hematopoietic system elicits multicomponent
antitumor immunity. Nat Med, 2005. 11(12): p. 1314-21. [0187] 34.
Burdelya, L., et al., Stat3 activity in melanoma cells affects
migration of immune effector cells and nitric oxide-mediated
antitumor effects. J Immunol, 2005. 174(7): p. 3925-31. [0188] 35.
Wang, T., et al., Regulation of the innate and adaptive immune
responses by Stat-3 signaling in tumor cells. Nat Med, 2004. 10(1):
p. 48-54. [0189] 36. Darnell, J. E., Validating Stat3 in cancer
therapy. Nat Med, 2005. 11(6): p. 595-6. [0190] 37. Zhang, L., et
al., Intratumoral delivery and suppression of prostate tumor growth
by attenuated Salmonella enterica serovar typhimurium carrying
plasmid-based small interfering RNAs. Cancer Res, 2007. 67(12): p.
5859-64. [0191] 38. Campbell, I. L., Cytokine-mediated
inflammation, tumorigenesis, and disease-associated JAK/STAT/SOCS
signaling circuits in the CNS. Brain Res Brain Res Rev, 2005.
48(2): p. 166-77. [0192] 39. Harris, T. J., et al., Cutting edge:
An in vivo requirement for STAT3 signaling in TH17 development and
TH17-dependent autoimmunity. J Immunol, 2007. 179(7): p. 4313-7.
[0193] 40. Watson, C. J. and W. R. Miller, Elevated levels of
members of the STAT family of transcription factors in breast
carcinoma nuclear extracts. Br J Cancer, 1995. 71(4): p. 840-4.
[0194] 41. Song, J. I. and J. R. Grandis, STAT signaling in head
and neck cancer. Oncogene, 2000. 19(21): p. 2489-95. [0195] 42.
Song, L., et al., Activation of Stat 3 by receptor tyrosine kinases
and cytokines regulates survival in human non-small cell carcinoma
cells. Oncogene, 2003. 22(27): p. 4150-65. [0196] 43. Savarese, T.
M., et al., Coexpression of oncostatin Mand its receptors and
evidence for STAT3 activation in human ovarian carcinomas.
Cytokine, 2002. 17(6): p. 324-34. [0197] 44. Toyonaga, T., et al.,
Blockade of constitutively activated Janus kinase/signal transducer
and activator of transcription-3 pathway inhibits growth of human
pancreatic, cancer. Cancer Lett, 2003. 201(1): p. 107-16. [0198]
45. Corvinus, F. M., et al., Persistent STAT3 activation in colon
cancer is associated with enhanced cell proliferation and tumor
growth. Neoplasia, 2005. 7(6): p. 545-55. [0199] 46. Gao, B., et
al., Constitutive activation of JAK-STAT3 signaling by BRCA1 in
human prostate cancer cells. FEBS Lett, 2001. 488(3): p. 179-84.
[0200] 47. Buettner, R., L. B. Mora, and R. Jove, Activated STAT
signaling in human tumors provides novel molecular targets for
therapeutic intervention. Clin Cancer Res, 2002. 8(4): p. 945-54.
[0201] 48. Carson, W. E., Interferon-alpha-induced activation of
signal transducer and activator of transcription proteins in
malignant melanoma. Clin Cancer Res, 1998. 4(9): p. 2219-28. [0202]
49. Chen, C. L., et al., Stat3 activation in human endometrial and
cervical cancers. Br J Cancer, 2007. 96(4): p. 591-9. [0203] 50.
Lai, R., et al., STAT3 is activated in a subset of the Ewing
sarcoma family of tumours. J Pathol, 2006. 208(5): p. 624-32.
[0204] 51. Punjabi, A. S., et al., Persistent activation of STAT3
by latent Kaposi's sarcoma-associated herpesvirus infection of
endothelial cells. J Virol, 2007. 81(5): p. 2449-58. [0205] 52.
Schaefer, L. K., et al., Constitutive activation of Stat3alpha in
brain tumors: localization to tumor endothelial cells and
activation by the endothelial tyrosine kinase receptor (VEGFR-2).
Oncogene, 2002. 21(13): p. 2058-65. [0206] 53. Puthier, D., R.
Bataille, and M. Amiot, IL-6 up-regulates mcl-1 in human myeloma
cells through JAK/STAT rather than ras/MAP kinase pathway. Eur J
Immunol, 1999. 29(12): p. 3945-50. [0207] 54. Migone, T. S., et
al., Constitutively activated Jak-STAT pathway in T cells
transformed with HTLV-I. Science, 1995. 269(5220): p. 79-81. [0208]
55. Spiekermann, K., et al., Constitutive activation of STAT
transcription factors in acute myelogenous leukemia. Eur J
Haematol, 2001. 67(2): p. 63-71. [0209] 56. Epling-Burnette, P. K.,
et al., Inhibition of STAT3 signaling leads to apoptosis of
leukemic large granular lymphocytes and decreased Mcl-1 expression.
J Clin Invest, 2001. 107(3): p. 351-62. [0210] 57. Weber-Nordt, R.
M., et al., Constitutive activation of STAT proteins in primary
lymphoid and myeloid leukemia cells and in Epstein-Barr virus
(EBV)-related lymphoma cell lines. Blood, 1996. 88(3): p. 809-16.
[0211] 58. Sommer, V. H., et al., In vivo activation of STAT3 in
cutaneous T-cell lymphoma. Evidence for an antiapoptotic function
of STAT3. Leukemia, 2004. 18(7): p. 1288-95. [0212] 59. Lai, R., et
al., Signal transducer and activator of transcription-3 activation
contributes to high tissue inhibitor of metalloproteinase-1
expression in anaplastic lymphoma kinase-positive anaplastic large
cell lymphoma. Am J Pathol, 2004. 164(6): p. 2251-8. [0213] 60. Fu,
X. Y., STAT3 in immune responses and inflammatory bowel diseases.
Cell Res, 2006. 16(2): p. 214-9. [0214] 61. Feldmann, M, F. M.
Brennan, and R. N. Maini, Role of cytokines in rheumatoid
arthritis. Annu Rev Immunol, 1996. 14: p. 397-440. [0215] 62.
Krause, A., et al., Rheumatoid arthritis synoviocyte survival is
dependent on Stat3. J Immunol, 2002. 169(11): p. 6610-6. [0216] 63.
Pfitzner, E., et al., The role of STATs in inflammation and
inflammatory diseases. Curr Pharm Des, 2004. 10(23): p. 2839-50.
[0217] 64. Lovato, P., et al., Constitutive STAT3 activation in
intestinal T cells from patients with Crohn's disease. J Biol Chem,
2003. 278(19): p. 16777-81. [0218] 65. Ishihara, K. and T. Hirano,
IL-6 in autoimmune disease and chronic inflammatory proliferative
disease. Cytokine Growth Factor Rev, 2002. 13(4-5): p. 357-68.
[0219] 66. Ivashkiv, L. B. and I. Tassiulas, Can SOCS make
arthritis better? J Clin Invest, 2003. 111(6): p. 795-7. [0220] 67.
Sengupta, T. K., et al., Activation of monocyte effector genes and
STAT family transcription factors by inflammatory synovial fluid is
independent of interferon gamma. J Exp Med, 1995, 181(3): p.
1015-25. [0221] 68. Shouda, T., et al., Induction of the cytokine
signal regulator SOCS3/CIS3 as a therapeutic strategy for treating
inflammatory arthritis. J Clin Invest, 2001. 108(12): p. 1781-8.
[0222] 69. Harada, T., et al., Increased expression of STAT3 in SLE
T cells contributes to enhanced chemokine-mediated cell migration.
Autoimmunity, 2007. 40(1): p. 1-8. [0223] 70. Simeone-Penney, M.
C., et al., Airway epithelial STAT3 is required for allergic
inflammation in a murine model of asthma. J Immunol, 2007. 178(10):
p. 6191-9. [0224] 71. Hagler, M., Smith-Norowitz, T., Chice, S.,
Wallner, S., Viterbo, D., Mueller, C, Groos, R., Nowakowski, M.,
Schulze, R., Zenilman, M., Sophorolipids decrease IgE production in
U266 cells by downregulation of BSAP (Pax5), TLR-2, STAT3 and IL-6.
Journal of Allergy and Clinical Immunology, 2007. 119(S1): p.
S263-S263. [0225] 72. Benkhart, E. M., et al., Role of Stat 3 in
lipopolysaccharide-induced IL-10 gene expression. J Immunol, 2000.
165(3): p. 1612-7. [0226] 73. Sano, S., et al., Stat3 links
activated keratinocytes and immunocytes required for development of
psoriasis in a novel transgenic mouse model. Nat Med, 2005. 11(1):
p. 43-9. [0227] 74. Lim, C. P., et al., Stat3 contributes to keloid
pathogenesis via promoting collagen production, cell proliferation
and migration. Oncogene, 2006. 25(39): p. 5416-25. [0228] 75.
Arany, L, et al., Correlation between pretreatment levels of
interferon response genes and clinical responses to an immune
response modifier (Imiquimod) in genital warts. Antimicrob Agents
Chemother, 2000. 44(7): p. 1869-73. [0229] 76. Tefferi, A.,
Classification, diagnosis and management of myeloproliferative
disorders in the JAK2V617F era. Hematology Am Soc Hematol Educ
Program, 2006: p. 240-5. [0230] 77. Roder, S., et al., STAT3 is
constitutively active in some patients with Polycythemia rubra
vera. Exp Hematol, 2001. 29(6): p. 694-702. [0231] 78. Kim, O. S.,
et al., JAK-STAT signaling mediates gangliosides-induced
inflammatory responses in brain microglial cells. J Biol Chem,
2002. 277(43): p. 40594-601. [0232] 79. Wyss-Coray, T.,
Inflammation in Alzheimer disease: driving force, bystander or
beneficial response? Nat Med, 2006. 12(9): p. 1005-15. [0233] 80.
Stelmasiak, Z., et al, Interleukin-6 concentration in serum and
cerebrospinal fluid in multiple sclerosis patients. Med Sci Monit,
2000. 6(6): p. 1104-8. [0234] 81. Ponti, D., et al., Isolation and
in vitro propagation of tumorigenic breast cancer cells with
stem/progenitor cell properties. Cancer Res, 2005. 65(13): p.
5506-11. [0235] 82. Szotek, P. P., et al., Ovarian cancer side
population defines cells with stem cell-like characteristics and
Mullerian Inhibiting Substance responsiveness. Proc Natl Acad Sci
USA, 2006. 103(30): p. 11154-9.
Sequence CWU 1
1
5124DNAArtificial SequenceA syntehtic Oligonucleotide
sequencemisc_feature(1)..(1)5'-biotin 1gatccttctg ggaattccta gatc
24224DNAArtificial SequenceA syntehtic Oligonucleotide sequence
2ggatctagaa tcagctacag cagc 24324DNAArtificial SequenceA syntehtic
Oligonucleotide sequence 3tcctctagag ggcaatctcc attg
24422DNAArtificial SequenceA syntehtic Oligonucleotide sequence
4ccctctagat ggttcctgga ac 22521DNAArtificial SequenceA syntehtic
Oligonucleotide sequence 5gctctagaaa cccctttttg g 21
* * * * *