U.S. patent application number 15/735502 was filed with the patent office on 2018-06-21 for cancer treatment and diagnosis.
The applicant listed for this patent is UNIVERSITY OF MIAMI. Invention is credited to Glen N. Barber.
Application Number | 20180169159 15/735502 |
Document ID | / |
Family ID | 57504666 |
Filed Date | 2018-06-21 |
United States Patent
Application |
20180169159 |
Kind Code |
A1 |
Barber; Glen N. |
June 21, 2018 |
CANCER TREATMENT AND DIAGNOSIS
Abstract
The present disclosure provides, in general, a method for
selecting a therapy for treating cancer in a human subject and
subsequently treating cancer in a subject, which includes isolating
a cancer cell from a human subject having cancer, determining the
functional activity of STING or cGAS in the cell; and selecting a
therapy for the cancer based on the functional activity of the
STING or cGAS in the cell. Also provided, if the functional
activity of STING and/or cGAS is determined to be defective in the
cell, the therapy selected is one that is effective at killing
STING-deficient and/or cGAS-deficient cancer cells, e.g., therapy
including administering to the subject an oncolytic virus.
Inventors: |
Barber; Glen N.; (Miami,
FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF MIAMI |
Miami |
FL |
US |
|
|
Family ID: |
57504666 |
Appl. No.: |
15/735502 |
Filed: |
June 13, 2016 |
PCT Filed: |
June 13, 2016 |
PCT NO: |
PCT/US16/37288 |
371 Date: |
December 11, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62174374 |
Jun 11, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 2600/106 20130101; C12Q 2600/154 20130101; C12N 2710/16633
20130101; G01N 33/57492 20130101; A61P 35/00 20180101; A61K 35/763
20130101; C12Q 2600/158 20130101; C12N 2710/16632 20130101; C12Q
2600/156 20130101; G01N 2333/4706 20130101 |
International
Class: |
A61K 35/763 20060101
A61K035/763; A61P 35/00 20060101 A61P035/00; G01N 33/574 20060101
G01N033/574 |
Claims
1. A method for treating cancer in a human subject, the method
comprising the steps of: isolating a sample from a human subject
having cancer; determining the functional activity of STING in the
sample; selecting a therapy for the cancer based on the functional
activity of the STING in the sample; and treating the subject with
the selected therapy.
2. The method of claim 1, wherein the functional activity of STING
is determined to be defective in the cell, and the therapy selected
is one that is effective at killing STING-deficient cancer
cells.
3. The method of claim 1, wherein the subject has failed at least
one chemotherapy regimen.
4. The method of claim 3, wherein the chemotherapy regimen
comprises administering to the subject an agent which causes DNA
mutations.
5. The method of claim 2, wherein the selected therapy comprises
administering to the subject an oncolytic virus.
6. The method of claim 5, wherein the oncolytic virus is derived
from herpes simplex virus, vaccinia virus or Varicella Zoster
virus.
7. The method of claim 1, wherein the step of determining the
functional activity of STING in the sample comprises analyzing the
amount of cGAS in the cell.
8. A method for treating a cancer in a human subject, the method
comprising the steps of: determining the functional activity of
STING in a sample; and if the sample has defective STING activity,
administering an oncolytic virus to the subject.
9. The method of claim 8, wherein the step of determining the
functional activity of STING in the sample comprises analyzing the
amount of cGAS in the cell.
10. A method for treating a cancer in a human subject, the method
comprising the steps of: determining the functional activity of
STING in a sample from the subject having the cancer; and if the
sample does not have defective STING activity, administering a
cancer treatment to the subject that does not cause DNA
mutation.
11. A method for treating cancer in a human subject, the method
comprising the steps of: isolating a sample from a human subject
having cancer; determining the functional activity of cGAS in the
sample; selecting a therapy for the cancer based on the functional
activity of the cGAS in the sample, and administering the selected
therapy to the subject.
12. A method for treating cancer comprising a) administering a
viral oncolytic therapy to a subject, b) determining the level of
STING or cGAS in a sample from the subject, wherein a decrease in
STING or cGAS activity is predictive of a positive outcome of
oncolytic therapy, and c) i) if levels of STING in the subject are
low, continuing oncolytic therapy; or ii) if STING or cGAS levels
are normal or partially active, discontinuing viral oncolytic
therapy and/or administering a second agent that can increase STING
levels in the subject in order to improve the outcome of the viral
oncolytic therapy.
13. The method of claim 12, wherein the viral oncolytic therapy
comprises herpesvirus, Varicella Zoster virus or vaccinia
virus.
14. The method of any one of the preceding claims, wherein the
cancer is selected from the group consisting of colorectal cancer,
colitis-associated cancer and melanoma.
15. The method of any one of the preceding claims, wherein the
sample is a body fluid, cell, tissue sample, biopsy, tissue print,
skin, hair, a soluble fraction of a cell preparation, or media in
which cells were grown.
16. The method of claim 15, wherein the body fluid is blood, urine,
plasma, saliva, or cerebrospinal fluid.
17. The method of any one of the preceding claims wherein an immune
response in the cancer that is lacking STING activity or cGAS
activity is enhanced by administration of an oncolytic virus.
18. The method of claim 18, wherein the immune response includes
modulation of T cell activity, modulation of dendritic cell
activity, or modulation of immune cytokines.
19. The method of any one of the preceding claims, wherein the
therapy results in increased tumor cell death and/or retarded tumor
growth.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
Provisional Patent Application No. 62/174,374, filed Jun. 11, 2015,
herein incorporated by reference in its entirety.
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] Not applicable.
INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0003] This application contains, as a separate part of the
disclosure, a Sequence Listing in computer-readable form which is
incorporated by reference in its entirety and identified as
follows: Filename: 49720_Seqlisting.txt; Size: 2,313 bytes,
created: Jun. 13, 2016.
FIELD OF THE INVENTION
[0004] The present disclosure relates generally to the fields of
molecular biology, immunology, biochemistry, cancer, and medicine.
More particularly, the disclosure relates to methods for diagnosis
and treatment of cancer through the use of a cellular protein.
BACKGROUND
[0005] Cancer is a leading cause of death in the United States of
America and elsewhere. New treatments and diagnostics are needed to
improve outcomes.
[0006] Colorectal cancer (CRC) affects about 1.2 million people in
the United States with approximately 150,000 new cases being
diagnosed every year. Indeed, CRC is the third most common cause of
cancer worldwide, after lung and breast cancer, and the second
leading cause of cancer death in adults (DeSantis et al., 2014).
Intestine-associated malignant disease frequently develops from
colonic epithelial cells that accumulate genetic alterations in key
genes involved in the control of cell growth. Multistep genomic
damage aggravated alterations can be acquired from environmental
factors comprising carcinogens or from genotoxic microbial
pathogens including Helicobacter pylori (Arthur et al., 2014; Kim
and Chang, 2014; Louis et al., 2014). Such genetic amendments
frequently involve activation of cell growth signaling through
mutation of k-ras as well as through mutation or epigenetic
silencing of critical tumor suppressor genes (TSGs) such as p53 and
adenomatous polyposis coli (APC). Mutated TSGs such as APC can also
be inherited, thus increasing the risk of CRC significantly
(Fearon, 2011).
[0007] Orally administered carcinogens such as the DNA-adduct
forming azoxymethane (AOM) induce genomic changes in
gastrointestinal epithelial cells, an event which can trigger the
activation of DNA damage response (DDR) pathways. While these
responses involve repairing DNA breaks and eliminating base
mismatches, they can also include activating the production of
pro-inflammmatory cytokines which alerts the immune surveillance
system to the damaged area and facilitates wound repair. For
example, using murine models, it has been demonstrated that the
administration of AOM followed by inflammatory drug dextran
sulphate sodium (DSS) can cause epithelial cells to produce
IL-1.beta. and IL-18 which becomes processed by the inflammasome, a
multiprotein complex comprising nucleotide-binding
oligomerization-domain protein like receptors (NLRs) such as NLRP3
and NLRP6 as well as apoptotic speck protein containing a CARD
(ASC/PYCARD) and caspase-1, for secretion (Arthur et al., 2012;
Elinav et al., 2011). IL-18, for example, can bind to colonic
dendritic cells and signal through MyD88 to prevent the production
of growth inhibitory IL-22 binding protein (IL-22BP), which enables
unrestricted IL-22 to stimulate tissue repair (Huber et al., 2012;
Salcedo et al., 2010). Thus, mice defective in key
inflammasome-associated molecules such as ASC or caspase-1 are
susceptible to carcinogen induced colitis-associated cancer (CAC).
Similarly, loss of key adaptor molecules such as MyD88, required
for IL1-R signaling are susceptible to AOM/DSS induced CAC.
Plausibly, unrepaired lesions enable the infiltration of microbes
with heightened genotoxic aptitude that can chronically aggravate
inflammatory processes and the production of DNA damaging radical
oxygen species (ROS).
[0008] While the inflammasome has been shown to be important for
processing proinflammatory cytokines such as IL1.beta. and IL-18,
it remained to be fully clarified how such wound repair proteins
become transcriptionally activated in response to actual genomic
damage. However, it has recently been shown that mice lacking the
innate immune regulator STING (stimulator of interferon genes) are
also sensitive to AOM/DSS-induced CAC (Ahn et al., 2015). STING
resides in the endoplasmic reticulum (ER) of hematopoietic cells as
well as endothelial and epithelial cells and controls the induction
of numerous host defense genes, such as type I IFN as well as
pro-inflammatory genes including IL1-.beta. in response to the
detection of cyclic dinucleotides (CDNs) such as cyclic-di-AMP
(c-di-AMP) generated from intracellular bacteria (Ishikawa and
Barber, 2008; Woodward et al., 2010). STING is also the sensor for
CDNs produced from a cellular nucleotidyltransferase referred to as
cGAS (cyclic GMP-AMP synthase, also referred to as Mab-21
Domain-Containing Protein and C6orf150) (Sun et al., 2013).
Cytosolic DNA species which can constitute the genome of invading
pathogens such as HSV-1, or plausibly self-DNA leaked from the
nucleus can bind to cGAS to generate non-canonical cGAMP containing
one 2'-5' phosphodiester linkage and a canonical 3-5' linkage
(c[G(2',5')pA(3',5')p]). The STING pathway may recognize damaged
DNA during early response to intestinal damage and may be essential
for invigorating tissue repair pathways involving IL1.beta. and
IL-18 (Ahn et al., 2015). STING has also been recently reported to
play an essential role in dendritic cell recognition of dying tumor
cells and the priming of anti-tumor cytotoxic T-cell (CTL)
responses (Corrales et al., 2015; Woo et al., 2014). Thus, while
loss of STING may facilitate tumorigenesis through preventing wound
repair and by preventing the production of tumor specific CTLs, the
effectiveness of STING signaling in human tumors remains
unknown.
SUMMARY
[0009] It is reported herein that STING mediated innate immune
signaling is largely impaired in human colon cancers as well as
many other types of human cancers. In many instances, this was
achieved through silencing STING and/or synthase cGAS expression
through epigenetic hypermethylation processes. The findings suggest
that STING pathway may have a major function in suppressing colon
tumorigenesis and that the inhibition of STING function in this
pathway may be selectively suppressed during cancer development.
Additionally, it is discovered that defects in STING signaling
renders cancer cells more susceptible to oncolytic viral infection.
Therefore, the examination of STING activity in cancers may lead to
development of assays that will shed light into the outcome of
select cancer therapies.
[0010] It was discovered that the cellular protein STING, which
controls innate immune responses to cytoplasmic DNA produced by DNA
damaging agents or DNA viruses, is defective in a wide variety of
cancer cells. Defects in STING signaling may help tumor cells evade
purging by the immune system and constitute a common mechanism of
tumorigenesis. Examining STING expression in tumors allows
predicting disease outcome and provides a crucial prognostic marker
in predicting responses to select anti-tumor therapies. Disclosed
herein are experiments showing that mice-deficient in STING (STING
knockout or SKO) are prone to colitis associated cancer (CAC)
induced by DNA-damaging and inflammatory agents. SKO mice harboring
tumors exhibited low levels of tumor suppressive IL22 binding
protein (IL22-BP) compared to normal mice, a cytokine important for
preventing colon-related tumorigenesis. Analysis of human colon
cancer cells and a variety of other cancer cells such as melanoma
indicated widespread defects in STING signaling which frequently
involved complete loss of STING and/or cyclic GMP-AMP synthase
(cGAS), a synthase that generates STING-activating cyclic
dincucleotides (CDN's). Such tumor cells were highly susceptible to
viral oncolytic therapy.
[0011] Disclosed herein are methods for selecting a therapy for
treating cancer in a mammalian (e.g., human) subject, and treating
a subject with the selected therapy. One such method includes the
steps of: isolating a sample from a human subject having cancer;
determining the functional activity of STING and/or cGAS in the
sample; selecting a therapy for the cancer based on the functional
activity of the STING and/or cGAS in the sample, and treating a
subject with the selected therapy. Also contemplated is the
measurement of levels of IL-22BP's suppression of IL-22, as well as
cellular levels of IL-1.beta., IL-18 and IL-22. A decrease in
levels of IL-1.beta., IL-18, IL-22 and IL-22BP may be indicative of
defective STING or cGAS signaling.
[0012] In various embodiments, the sample is a body fluid, cell,
tissue sample, biopsy, tissue print, skin, hair, a soluble fraction
of a cell preparation, or media in which cells were grown. It is
contemplated that the body fluid is blood, urine, plasma, saliva,
or cerebrospinal fluid.
[0013] If the functional activity of STING and/or cGAS is
determined to be defective in the sample, the therapy selected is
one that is effective at killing STING-deficient and/or
cGAS-deficient cancer cells (e.g., therapy including administering
to the subject an oncolytic virus such as one having a dsDNA
genome, including herpes simplex virus (HSV), Varicella Zoster
virus (VZV), or vaccinia virus (VV)). Exemplary virus families that
have dsDNA genomes include, but are not limited to,
Alloherpesviridae, Herpesviridae, Malacoherpesviridae,
Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae,
Ascoviridae, Asfarviridae, Baculoviridae, Bicaudaviridae,
Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae,
Guttaviridae, Hytrosaviridae, Iridoviridae, Marseilleviridae,
Mimiviridae, Nudiviridae, Nimaviridae, Pandoraviridae,
Papillomaviridae, Phycodnaviridae, Plasmaviridae, Polydnaviruses,
Polyomaviridae, Poxviridae, Sphaerolipoviridae, Tectiviridae and
Turriviridae.
[0014] In various embodiments, the examination of STING-signaling
is a useful prognostic marker for whether HSV1 or other viral based
anti-cancer therapies will be efficacious for the treatment of
malignant disease.
[0015] In the methods described herein, the subject can be one that
has failed at least one chemotherapy regimen (e.g., one that
includes administering to the subject an agent which causes DNA
mutations) and the step of determining the functional activity of
STING in the cell can include analyzing the amount of cGAS in the
cell.
[0016] In various embodiments, it is contemplated that the selected
therapy, e.g., an oncolytic virus, is administered in conjunction
with a second therapeutic agent, such as a chemotherapeutic agent.
Exemplary chemotherapeutic agents are described below in the
Detailed Description.
[0017] Also disclosed herein is a method for treating a cancer in a
mammalian (e.g., human) subject that includes the steps of:
determining the functional activity of STING in a cell making up
the cancer; and if the cell does not have defective STING activity,
administering a cancer treatment to the subject that does not cause
DNA mutation.
[0018] Further disclosed herein is a method for treating cancer in
a mammalian (e.g., human) subject which includes the steps of:
isolating a sample from a human subject having cancer; determining
the susceptibility of the cancer to being killed by an oncolytic
virus in vitro; and if the cancer is susceptible to being killed in
this manner, administering an oncolytic virus to the subject. In
various embodiments, the step of determining the functional
activity of STING in the sample comprises analyzing the amount of
cGAS in the cell. Also contemplated is the measurement of levels of
IL-22BP's suppression of IL-22, as well as cellular levels of
IL-1.beta., IL-18 and IL-22. A decrease in levels of IL-1.beta.,
IL-18, IL-22 and IL-22BP may be indicative of defective STING or
cGAS signaling.
[0019] In various embodiments, the cancer is colorectal cancer,
colitis-associated cancer or melanoma. Additional exemplary cancers
contemplated for treatment herein are set out in the Detailed
Description.
[0020] In various embodiments, measurement of the presence or
absence of STING/cGAS expression is predictive of the response of
patients with certain cancers to viral oncolytic therapy. In
various embodiments, measurement of response may be carried out
using fluorescence in situ hybridization, and analysis of STING
and/or cGAS protein or RNA expression, to predict the outcome to
oncolytic viral therapy depending on the presence or absence of
cGAS or STING.
[0021] Provided herein is a method for treating cancer comprising
administering a viral oncolytic therapy to a subject, determining
the level of STING or cGAS in the subject, wherein a decrease in
STING or cGAS activity is predictive of a positive outcome of
oncolytic therapy, and i) if levels of STING or cGAS in the subject
are low, continuing oncolytic therapy; or ii) if STING or cGAS
levels are normal or partially active, discontinuing viral
oncolytic therapy and/or administering a second agent that can
increase STING levels in the subject in order to improve the
outcome of the viral oncolytic therapy.
[0022] In various embodiments, the viral oncolytic therapy
comprises herpesvirus, VZV or vaccinia virus.
[0023] In various embodiments, the determining comprises obtaining
a sample from the subject and measuring levels of STING, cGAS, or
other biomarkers contemplated herein (e.g., IL-18, IL-22, IL-22BP,
IL-1.beta., IFN.beta., type I IFN) in the sample. It is
contemplated that the sample is a body fluid, such as blood, urine,
plasma, saliva, or cerebrospinal fluid; a cell; a tissue; a tissue
print; a fingerprint, skin or hair; and the like; a soluble
fraction of a cell preparation, or media in which cells were grown;
a chromosome, an organelle, or membrane isolated or extracted from
a cell; genomic DNA, RNA, or cDNA, polypeptides, or peptides in
solution or bound to a substrate.
[0024] In various embodiments, an immune response in the cancer
that is lacking STING activity or cGAS activity is enhanced by
administration of an oncolytic virus. In one embodiment, the immune
response includes modulation of T cell activity, modulation of
dendritic cell activity, or modulation of immune cytokines.
[0025] In various embodiments, the therapy results in increased
tumor cell death and/or retarded tumor growth in a subject.
[0026] It is understood that each feature or embodiment, or
combination, described herein is a non-limiting, illustrative
example of any of the aspects of the invention and, as such, is
meant to be combinable with any other feature or embodiment, or
combination, described herein. For example, where features are
described with language such as "one embodiment", "some
embodiments", "certain embodiments", "further embodiment",
"specific exemplary embodiments", and/or "another embodiment", each
of these types of embodiments is a non-limiting example of a
feature that is intended to be combined with any other feature, or
combination of features, described herein without having to list
every possible combination. Such features or combinations of
features apply to any of the aspects of the invention. Where
examples of values falling within ranges are disclosed, any of
these examples are contemplated as possible endpoints of a range,
any and all numeric values between such endpoints are contemplated,
and any and all combinations of upper and lower endpoints are
envisioned.
[0027] Unless otherwise defined, all technical terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Although methods
and materials similar or equivalent to those described herein can
be used in the practice or testing of the present invention,
suitable methods and materials are described below. All
publications, patents, and patent applications mentioned herein are
incorporated by reference in their entirety. In the case of
conflict, the present specification, including definitions will
control. In addition, the particular embodiments discussed below
are illustrative only and not intended to be limiting.
DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1A-1E. Activation of STING-dependent genes by
azoxymethane (AOM) (FIG. 1A) Gene array analysis of Wild type (WT)
and STING deficient (SKO) mouse embryonic fibroblasts (MEFs)
treated with AOM at 0.14 mM for 8 hours (Left) and
1,2-dimethylhydrazine (DMH) at 1 mM for 8 hours (Right). Highest
variable genes are shown. Rows represent individual genes; columns
represent individual samples. Grayscale indicates transcript levels
below, equal to, or above the mean. Scale represents the intensity
of gene expression (log 2 scale ranges between -2.4 and 2.4). (FIG.
1B) qPCR analysis of Cxcl10 and Ifit3 in MEFs treated with AOM and
DMH same as FIG. 1A. (FIG. 1C) qPCR analysis of Cxcl10 in Human
epithelial cell (FHC) treated with AOM and DMH at 1 mM for 24
hours. (FIG. 1D) FHC cells were transfected with STING or control
siRNA for 72 hours followed by AOM and DMH treatment same as FIG.
1C, and were then subjected to Cxcl0 mRNA expression (Left). STING
expression level after siRNA treatment was determined by qPCR
(Right). Data is representative of at least two independent
experiments. Error bars indicate s.d. *; p<0.05, Student's
t-test. (FIG. 1E) STING Immunohistochemistry staining of the colon
tissue from WT and SKO mice (Left) and Human. All images were shown
at original magnification, 200.times..
[0029] FIG. 2A-2E. Loss of STING renders mice susceptible to CAC:
(FIG. 2A) Schematic representation of AOM/DSS induced colitis
model. WT (n=7) and SKO (n=7) mice were intravenously injected with
AOM on Day 1 followed by 7 d administration of dextran sodium
sulfate (DSS) in drinking water for four DSS cycles. Normal
drinking water was used for control group. (FIG. 2B) Representative
photographs of macro-endoscopic colon tumors (Left) and H&E
staining (Right) of WT (n=7) and SKO (n=7) mice either AOM/DSS
treated or normal water treated. Number of polyps (FIG. 2C) and
inflammation score (FIG. 2D, 0: Normal to 3: most severe) from FIG.
2B. (FIG. 2E) Gene array analysis of colon tissue from WT and SKO
mice treated same as FIG. 2A. Highest variable gene lists are shown
(Right table). Rows represent individual genes; columns represent
individual samples. Grayscale indicate transcript levels below,
equal to (black), or above the mean. Scale represents the intensity
of gene expression (log 2 scale ranges between -2.4 and 2.4).
[0030] FIG. 3A-3C. Suppression of IL22BP expression in
STING-deficient mice: (FIG. 3A) Fold changes from gene array
analysis of Il18 in WT and SKO MEFs administrated with 4 ug/ml of
dsDNA90 and IFN.beta. for 8 hours (Left). qPCR analysis of Il18 in
WT and SKO MEFs transfected with 4 .mu.g/ml of dsDNA90 and
cyclic-di-GMP-AMP (cGAMP) for 8 hours (Middle). qPCR analysis of
Il18 in bone marrow derived dendritic cells (BMDCs) from WT and SKO
mice. BMDCs were treated with 1 mM of AOM and 1 mM of DMH for 8
hours (Right). (FIG. 3B) Schematic representation, body weight, and
qPCR analysis of IL18, IL22 bp and IL22 from WT and SKO colon
during one cycle of DSS administration for 5 days followed by 2
days of normal water. (FIG. 3C) qPCR analysis of IL18, IL22 bp and
IL22 in WT and SKO colon tissue from FIG. 2. Data is representative
of at least two independent experiments. Error bars indicate s.d.
*; p<0.05, Student's t-test.
[0031] FIG. 4A-4F. Cytosolic DNA induced innate immune signaling
was mostly defective in human colon cancer cells: (FIG. 4A)
Immunoblot of STING in a series of human colon cancer cell lines of
various type. hTERT and normal human colon epithelial cell line,
FHC, were included as positive controls. 20 .mu.g of total
protein/per lane was loaded and analyzed by rabbit anti STING
polyclonal antibody. .beta.-actin was used as loading control.
(FIG. 4B) ELISA analysis of human Interferon (3 production in the
media of cells, same as in (FIG. 4A) following polyI:C or dsDNA90
transfection at 3 .mu.g/ml for 16 hours. Lipofectamine 2000 alone
was used as mock transfection. (FIG. 4C) Cells, same as in A, were
either mock transfected or transfected with polyI:C or dsDNA90 at 3
.mu.g/ml for 3 hours. Total RNA was extracted and analyzed by qPCR
for IFNB expression. (FIG. 4D) RNA, same as in FIG. 4C, was
analyzed by qPCR for CXCL10 expression. (FIG. 4 G) RNA, same as in
FIG. 4C, was analyzed by qPCR for IL1B expression. (FIG. 4E) Gene
array analysis of normal or colon cancer cells mock transfected or
transfected with 3 .mu.g/ml dsDNA90 for 3 hours. Highest variable
genes are shown. Rows represent individual genes; columns represent
individual samples. Grayscale legend indicates transcript levels
below, equal to, or above the mean. Scale represents the intensity
of gene expression (log 10 scale ranges between -3 and 3). (FIG.
4F) List of highest variable genes shown in FIG. 4E as well as
their fold induction value following dsDNA90 stimulation. Data is
representative of at least two independent experiments. Error bars
indicate s.d.
[0032] FIG. 5A-5F. STING activation and cytosolic DNA pathway in
colon cancer cells were mostly defective: A series of colon cancer
cells as well as normal cell controls were either mock transfected
or transfected with dsDNA90 at 3 .mu.g/ml for 3 hours, and were
analyzed by Immunofluorescence Microscopy for STING translocation
(FIG. 5A), IRF3 translocation (FIG. 5B), and p65 translocation
(FIG. 5C). (FIG. 5D) Cells, same as above, were either mock
transfected or transfected with dsDNA90 at 3 .mu.g/ml for indicated
time periods followed by immunoblot analysis for STING
phosphorylation as well as phosphorylation of TBK1, IRF3 and p65.
.beta.-actin was used as loading control. (FIG. 5E) Cells, same as
above, were analyzed by qPCR for cGAS expression. (FIG. 5F) Cells
that have undetectable level of cGAS in E were treated with 1 .mu.M
5-azacytidine for 7 days, followed by qPCR analysis for cGAS
expression. Data is representative of at least two independent
experiments. Error bars indicate s.d.
[0033] FIG. 6A-6D. HSV1 viral production is more effective in colon
cancer cells that have defected STING innate immune pathway. (FIG.
6A) A series of colon cancer cells as well as normal cell controls
were infected with HSV-luc at M.O.I. 1 or 5 for 24 hours. Cells
were then lysed and analyzed for luciferase activity. Data is
representative of at least two independent experiments. Error bars
indicate s.d. (FIG. 6B) Cells, same as in FIG. 6A, were infected
with HSV-luc at M.O.I. 10 for 6 hours. Total RNA was then
extracted, followed by qPCR analysis of IFNB production. (FIG. 6C)
Same RNA from FIG. 6B was analyzed by qPCR analysis for CXCL10
production. (FIG. 6D) Cells, same as in FIG. 6A, were infected with
HSV1.gamma.34.5 deletion mutant at M.O.I. 1 for 6 hours followed by
qPCR analysis of IFNB production. Data is representative of at
least two independent experiments. Error bars indicate s.d.
[0034] FIG. 7A-7B. Gene expression fold changes of Illumina array
shown in FIG. 1A.
[0035] FIG. 8A-8C. Fluorescence microscopy analysis (related to
FIG. 1) of DAPI staining in WT and SKO MEFs treated with 3 mM of
AOM of 3 mM of DMH (FIG. 8A) and anti-dsDNA staining and the ration
of cytoplasm to nucleus (FIG. 8B) in Human normal colon epithelial
cells (FHC) treated with 3 mM of AOM or 3 mM of DMH for 48 hours.
(FIG. 8C) Immunofluorescence microscopy analysis of FHC treated
with AOM and DMH sane as FIG. 8A for 48 hours using p65 or IRF3
antibody. Images shown at original magnification, 160.times..
[0036] FIG. 9A-9B. FIG. 9A shows the number of polyps and FIG. 9B
shows the inflammation score from FIG. 2A-2E.
[0037] FIG. 10A-10B. Primary MEF cells lacking STING and/or p53
were transduce with retrovirus encoding human H-Ras 12V or human
c-Myc. After drug selection with puromycin and hygromycin, the
cells were cultured in soft agar. After 14 days, colonies were
photographed (FIG. 10A) and colony numbers in one well (n=3) were
counted (FIG. 10B). Error bars indicated standard deviation.
[0038] FIG. 11. IL18 promoter region contains binding sites for
multiple innate immune gene transcription factors. Putative
transcription factor binding sites in the IL18 gene promoter is
listed and highlighted.
[0039] FIG. 12. Shown is a summary of STING signaling pathway in
colon cancer cell lines.
[0040] FIG. 13A-13D. Human colon cancer cells (SW480 and HT116) as
well as hTERT cells were treated with 1 uM 5azacytidine for 7 days,
followed y dsDNA90 transfection at 3 .mu.g/ml for 3 hours. Total
RNA was extracted and analyzed by qPCR for cGAs (FIG. 13A) and IFNB
(FIG. 13B) expression. (FIG. 13C) cGAS production is deregulated in
many colon cancers. cDNA from 5 normal human colon tissues and 43
human colon cancers of various stages were analyzed by qPCR for
cGAS expression. (FIG. 13D) Immunoblot (upper) and qPCR analysis
(lower) of cGAS expression in normal and human colon cancer cells
same as above.
[0041] FIG. 14A-14C. FIG. 14A, Immunoblot of STING in various
transformed or cancer derived human cell lines. HUVEC was a
positive control. FIG. 14B, Northern blot analysis of STING mRNA
expression in cell lines as in FIG. 14A. HUVEC was a positive
control. FIG. 14C, ELISA analysis of IFNB production in the media
of cells transfected with 3 .mu.g/ml polyI:C or dsDNA90 or mock
transfected for 16 hours. PASMC, NHDF-ad and hTERT were included as
positive controls.
[0042] FIG. 15A-15F. cGAS expression is suppressed in many human
colon cancer cell lines and can be partially recapitulated through
DNA demethylation. FIG. 15A, Immunoblot (upper) and qPCR analysis
(lower) of cGAS expression in normal and human colon cancer cells
same as above. FIG. 15B, qPCR analysis of cGAS expression in cGAS
negative colon cell lines mock treated or treated with 1 .mu.M
5-Azacytidine (5AZADC) for 5 days. FIG. 15C, Immunoblot analysis of
STING signal activation in cells (selected from FIG. 15B) mock
treated or treated with 1 .mu.M 5-Azacytidine (5AZADC) for 5 days,
followed by dsDNA90 transfection at 3 .mu.g/ml for indicated time
periods. FIG. 15D, Immunofluorescence Microscopy analysis of IRF3
translocation in SW480 and HT116 cells treated with 5AZADC same as
above followed by dsDNA transfection at 3 .mu.g/ml dsDNA90 for 3
hours. Original magnification, 1260.times.. FIG. 15E, IFNB qPCR
analysis of cells (same as in FIG. 15C) treated with 5AZADC same as
above followed by dsDNA transfection at 3 .mu.g/ml dsDNA90 for 3
hours. FIG. 15F, IL1B qPCR analysis of cells same as FIG. 15E.
Error bars indicate s.d. *, p<0.05; **, p<0.01; ***,
p<0.001; Student's t-test.
[0043] FIG. 16A-16F. STING signal defect leads colon cancer cells
more susceptible to DNA virus infection. FIG. 16A, Cells (same as
in FIG. 4A-4F) were infected with HSV1.gamma.34.5 at M.O.I. 5 for 1
hour and human IFNB induction was analyzed by qPCR 3 hours post
infection. FIG. 16B, normal human hTERT cells and selected human
colon cancer cell lines (cGAS positive: SW1116, HT29; cGAS
negative: SW480, HT116) were infected with HSV1.gamma.34.5 at
indicated M.O.I. for 1 hour, and titration of HSV1.gamma.34.5 was
analyzed by standard plaque assay in Vero cells 24 hours later.
FIG. 16C, Cells (same as in FIG. 16B) were infected with
HSV1.gamma.34.5 at M.O.I. for 1 hour, and cell viability was
analyzed by trypan blue staining 24 hours and 48 hours later. FIG.
16D, Cells (same as in FIG. 16A) were infected with HSV1-Luc at
indicated M.O.I. for 1 hour, and luciferase activity was analyzed
24 hours later. FIG. 16E, Colon Cancer cells were infected with
Vaccinia Virus at M.O.I. 100 and analyzed by qPCR for IFNB
expression 3 hours post infection. FIG. 16F, Cells same as FIG. 16E
were analyzed by qPCR for CXCL10 expression. Error bars indicate
s.d.
[0044] FIG. 17A-17H. RNA in situ hybridization analysis of STING
and cGAS in human colon cancer cell lines and colon cancer tissue
microarray. FIG. 17A, RNA fluorescence in situ hybridization (RNA
FISH) analysis of STING and cGAS expression in normal and human
colon cancer cell lines. Images are shown at 1260.times.. FIG. 17B,
RNA FISH analysis of STING and cGAS expression in SW480 and HT116
mock treated or treated with 1 .mu.M 5 AZADC for 5 days. Images are
shown at 1260.times.. FIG. 17C, Quantitation of STING and cGAS RNA
copy number in FIG. 17A. FIG. 17D, Quantitation of cGAS RNA copy
number in FIG. 17B. FIG. 17E, STING and cGAS expression in
formalin-fixed paraffin-embedded (FFPE) normal and human colon
cancer cell lines were analyzed by Chromogenic RNA in situ
hybridization (RNA CISH). Quantitation of STING and cGAS RNA copy
number are shown in bar graph. Error bars indicate s.d. FIG. 17F,
representative images of STING and cGAS RNA CISH analysis are shown
at 600.times.. FIG. 17G, RNA CISH analysis of STING and cGAS
expression in a FFPE human colon cancer tissue microarray. A total
of 12 normal and 80 cancer tissues were analyzed and number of
tissue that are detected with STING and/or cGAS are summarized in
the table. FIG. 17H, Representative images of RNA CISH in FIG. 17G
are shown at 400.times..
[0045] FIG. 18A-18E. Increased HSV1.gamma.34.5 oncolytic effect was
observed in colon cancer cells with impaired STING signal in vivo.
FIG. 18A, Scheme of HSV1.gamma.34.5 treatment on xenograft tumor in
nude mice. The indicated xenograft tumors (SW116, FIG. 18B; HT29,
FIG. 18C; SW480, FIG. 18D; HT116, FIG. 18E) were generated in the
right flank of nude Balb/c mice. When tumors had reached
approximately 0.5 cm in diameter, tumors were injected every other
day a total of three times (arrows) with 1E7 PFU HSV1.gamma.34.5 in
50 .mu.l PBS (N=7) or 50 .mu.l PBS only (N=3) and tumor growth
measured every other day. Statistical analysis was carried out
comparing the two treatment groups at the last time point using the
unpaired Student's t-test. P values are as indicated.
[0046] FIG. 19 shows dsDNA90 transfection efficiency into colon
cancer cell lines monitored with FITC-dsDNA90 3 hours post
Lipofectamine 2000 transfection under fluorescent microscopy.
Images shows at 400.times..
[0047] FIG. 20A-20D. Normal and colon cancer cell cells were
treated with non-specific siRNA (si-NT) or STING siRNA (si-STING)
for 3 days followed by dsDNA90 transfection at 3 .mu.g/ml for 3
hours. Cells were then analyzed for STING siRNA efficiency by
immunoblot (FIG. 20A) and by qPCR for IFNB expression (FIG. 20B)
and CXCL10 expression (FIG. 20C). FIG. 20D, cells were similarly
treated with siRNA as above followed by HSV.gamma.34.5 infection at
MOI 5 for 3 hours. Cells were then analyzed by qPCR for IFNB
expression.
[0048] FIG. 21A-21C. FIG. 21A, schematic representation of CpG
islands located in the proximal promoter regions of cGAS. FIG. 21B,
Bisulfite sequencing analysis of cGAS promoter region. Each box
represents one CpG dinucleotide located within the promoter region
indicated by the position marker at the bottom. Grayscale compares
methylated, unmethylated and not sequenced. FIG. 21C, colon cancer
cells were treated with 5AZADC (DNA methyltransferase inhibitor),
SAHA (histone deacetylase inhibitor) and BIX01294 (histone-lysine
methyltransferase inhibitor) at 1 .mu.M for 5 days. cGAS expression
was then examined by qPCR. Error bars indicate s.d.
[0049] FIG. 22. Normal and colon cancer cells were treated with AOM
or DMH at 15 mM for 20 hours. IFNB induction was analyzed by qPCR.
STING, IRF3 and NF-kB translocation was examined: +, translocation;
-, no translocation.
[0050] FIG. 23A-23C. FIG. 23A, immunoblot of STING in various
transformed or cancer derived human cell lines. HUVEC was used as
positive control. FIG. 23B, Northern blot analysis of STING mRNA
expression in cell lines. FIG. 23C, ELISA analysis of IFNB
production in the media of cells transfected with 3 .mu.g/ml
polyI:C or dsDNA90 or mock transfected for 16 hours. PASMC, NHDF-ad
and hTERT used as positive control.
[0051] FIG. 24 shows sequencing of STING in colon cancer cell
lines.
[0052] FIG. 25 shows sequencing of cGAS in colon cancer cell
lines.
[0053] FIG. 26A-26D. STING expression is suppressed and dsDNA
induced innate immune activation is impaired in majority of human
melanoma cell lines. FIG. 26A, hTERT fibroblasts, normal human
epidermal melanocytes (HEMa) and a series of human melanoma cell
lines were analyzed for STING expression by immunoblot (top) and
cGAS expression by qPCR (bottom). FIG. 26B, ELISA analysis of human
Interferon .beta. production in the media of cells (same as A)
transfected with 3 .mu.g/ml polyIC or dsDNA90 or mock transfected
for 16 hours. qPCR analysis of human CXCL10 (FIG. 26C) and IFNB
(FIG. 26D) induction in cells (same as FIG. 26A) transfected with 3
.mu.g/ml dsDNA90 or mock transfected for 3 hours.
[0054] FIG. 27A-27D. dsDNA induced STING signaling pathway is
defective in majority of human melanoma cell lines.
Immunofluorescence Microscopy analysis of STING translocation (FIG.
27A), IRF3 translocation (FIG. 27B) and p65 translocation (FIG.
27C) in normal and human melanoma cell lines transfected with 3
.mu.g/ml dsDNA90 or mock transfected for 3 hours. Original
magnification, 1260.times.. Bar size, 1 .mu.m. FIG. 27D, Immunoblot
analysis of STING signal activation in cells (same as above)
transfected with 3 .mu.g/ml dsDNA90 for indicated time periods.
[0055] FIG. 28A-28C. RNA in situ hybridization and
immunohistochemistry analysis of STING and cGAS in human melanoma
cell lines. FIG. 28A, RNA fluorescence in situ hybridization (RNA
FISH) analysis of STING and cGAS expression in normal and human
melanoma cell lines. Representative images are shown at
1260.times.. Bar size, 500 nm. Quantitation of STING and cGAS RNA
copy number are shown in bar graph. FIG. 28B, Chromogenic RNA in
situ hybridization (RNA CISH) analysis of STING and cGAS expression
in formalin-fixed paraffin-embedded (FFPE) normal and human
melanoma cell lines. Representative images are shown at 600.times..
Bar size, 1 .mu.m. Quantitation of STING and cGAS RNA copy number
are shown in bar graph. FIG. 28C, Immunohistochemistry analysis of
STING and cGAS expression in melanoma cells. Images were shown at
400.times.. Bar size, 20 .mu.m.
[0056] FIG. 29. STING and cGAS expression were suppressed in high
percentage of human melanomas. Immunohistochemistry analysis of
STING and cGAS in human melanoma tissue microarray containing
normal human epidermal and human melanoma tissues. Representative
images of normal human epidermal and human melanoma tissues stained
for STING and cGAS. Images are shown at 400.times.. Bar size, 50
.mu.m. STING and cGAS expression status is summarized and shown in
bottom panel.
[0057] FIG. 30A-30G. DNA demethylation partially recapitulated
STING and cGAS expression in human melanoma cell lines. FIG. 30A,
qPCR analysis of cGAS expression in indicated human melanoma cells
mock treated or treated with 1 .mu.M 5-Azacytidine (5AZADC) for 5
days. FIG. 30B, Immunoblot analysis of STING in indicated human
melanoma cells treated same as above. FIG. 30C, RNA FISH analysis
of STING and cGAS in cells (same as above) treated with 5AZADC same
as above. Representative images are shown at 1260.times.. Bar size,
400 nm. qPCR analysis of IFNB (FIG. 30D) and CXCL10 (FIG. 30E) in
cells (same as above) treated with 5AZADC followed by dsDNA
transfection at 3 .mu.g/ml dsDNA90 for 3 hours. Immunofluorescence
Microscopy analysis of IRF3 translocation (FIG. 30F) and STING
translocation (FIG. 30G) in indicated cells treated same as in FIG.
30D. Representative images are shown at 1260.times.. Bar size, 500
nm.
[0058] FIG. 31A-31D. STING signal defect leads melanoma cells more
susceptible to HSV1 infection. Cells (same as in FIG. 1) were
infected with HSV1.gamma.34.5 at M.O.I. 5 for 1 hour and human IFNB
(FIG. 31A) and CXCL10 (FIG. 31B) induction was analyzed by qPCR 3
hours post infection. FIG. 31C, normal human hTERT cells and
selected human melanoma cell lines were infected with
HSV1.gamma.34.5 at indicated M.O.I. or M.O.I. 10 for 1 hour, and
titration of HSV1.gamma.34.5 was analyzed by standard plaque assay
in vero cells 24 hours later. FIG. 31D, Cells (same as in FIG. 31C)
were infected with HSV1.gamma.34.5 at M.O.I. 10 for 1 hour, and
cell viability was analyzed by trypan blue staining 24 hours and 48
hours later.
[0059] FIG. 32A-32D. Increased HSV1.gamma.34.5 oncolytic effect was
observed in melanoma xenografts with impaired STING signal in vivo.
FIG. 32A, A375; FIG. 32B, SK-MEL-5; FIG. 32C, RPMI7951; and FIG.
32D, SK-MEL-3 melanoma xenografts were generated in the right flank
of nude Balb/c mice. When tumors had reached approximately 0.5 cm
in diameter, tumors were injected every other day a total of three
times (arrows) with 1E7 PFU HSV1.gamma.34.5 in 50 .mu.l PBS or 50
.mu.l PBS only and tumor growth measured every other day.
Statistical analysis was carried out comparing the two treatment
groups at the last time point using the unpaired Student's t-test.
P values are as indicated.
DETAILED DESCRIPTION
[0060] The present disclosure provides methods for selecting a
cancer treatment therapy which involves assessing a cell of the
cancer for STING activity and treating cancer with an indicated
therapy. The below described embodiments illustrate representative
examples of these methods. Nonetheless, from the description of
these embodiments, other aspects of the invention can be made
and/or practiced based on the description provided below.
General Methods
[0061] Methods involving conventional immunological and molecular
biological techniques are described herein. Immunological methods
are generally known in the art and described in methodology
treatises such as Current Protocols in Immunology, Coligan et al.,
ed., John Wiley & Sons, New York. Techniques of molecular
biology are described in detail in treatises such as Molecular
Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Sambrook et al.,
ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
2001; and Current Protocols in Molecular Biology, Ausubel et al.,
ed., Greene Publishing and Wiley-Interscience, New York. General
methods of medical treatment are described in McPhee and Papadakis,
Current Medical Diagnosis and Treatment 2010, 49.sup.th Edition,
McGraw-Hill Medical, 2010; and Fauci et al., Harrison's Principles
of Internal Medicine, 17.sup.th Edition, McGraw-Hill Professional,
2008.
[0062] An analysis of the function of STING in colon cancer cells
was conducted and found that STING was frequently expressed but
STING function was ablated in approximately 86% of cells analyzed
(n=12). However, the cGAS was not detectable in 30-50% of cells
analyzed. In colon cancer cells lacking cGAS, STING function was
completely ablated. In cancer colon cells with detectable cGAS,
STING function was dramatically reduced. It was also noted that
STING and cGAS were gone in a variety of other cancers including
melanoma.
[0063] The innate immune system provides the first line of defense
against pathogen infection though can also influence pathways that
can control tumorigenesis. For example, it is known that the
cellular adaptor MyD88 (Myeloid differentiation primary response
gene 88) that facilitates Toll-like receptor (TLR) and IL-1
receptor (IL-1R) signaling pathway in the innate immune response
can regulate tumorigenesis through control of NF-.kappa.B
activation, cytokine secretion and inflammatory responses. Mice
lacking MyD88 are susceptible to colitis-associated carcinogenesis
(CAC) induced by the drugs azoxymethane (AOM) and dextran sulfate
sodium (DSS). In this situation, MyD88 exerts a protective effect
in part by facilitating the production of IL-18, in epithelial
cells, which downregulates dendritic cell production of the IL-22
binding protein (IL-22-BP). IL-22-BP suppresses the function of
IL-22 which is produced from innate lymphoid cells in response to
cellular/tissue damage and which potently stimulates the
proliferation of intestinal epithelial cells.
[0064] Azoxymethane (AOM) is the metabolite of
1,2-dimethylhydrazine (DMH) and is converted to methylazoxymethanol
(MAM) which mediates O-methyl-guanine formation to trigger DNA
damage responses. A single injection of AOM into mice, followed by
administration of the inflammatory agent dextran sulfate sodium
(DSS) via drinking water induces almost 100% colon cancer. It was
previously demonstrated that the cellular protein STING (stimulator
of cellular genes) facilitates cytosolic DNA-triggered innate
immune signaling pathways, independent of Toll-Receptor 9 or the
DNA sensor AIM II. In humans, STING is a 348 amino acid endoplasmic
reticulum (ER) associated molecule predominantly expressed in
epithelial cells as well as cells of the hematopoietic lineage,
that has been shown to play a key role in triggering innate immune
signaling pathways in response to infection by viruses such as
herpes simplex virus 1 (HSV1), and even bacteria. STING has also
been shown to be responsible for triggering vascular and pulmonary
syndrome, self-DNA-induced inflammatory diseases such as Aicardi
Goutieres syndrome (AGS) perhaps forms of severe systemic lupus
erythematosus (SLE). STING may be associated with dsDNA-species
directly and is highly activated by cyclic dinucleotides (CDN)
generated by certain bacteria or by cytosolic dsDNA triggering the
activation of a synthase, referred to as cGAS (Cyclic GMP-AMP
Synthase, C6orf150, Mab-21 Domain-Containing Protein).
[0065] Given that STING appears to play a pivotal role in
controlling a variety of inflammation driven events, the methods
described herein address the role of STING in inflammation
aggravated cancer. Using the AOM/DSS model, observations similar to
MyD88, STING-deficient mice (SKO) are sensitive to CAC suggesting a
protective role for STING in tumorigenesis. Subsequent analysis
indicated that STING signaling and cytokine production was ablated
in numerous colon cancer cells analyzed. Data indicates that STING
may be a key sensor that promotes the elimination of damaged
intestinal epithelial cells. Loss of STING signaling may be a
common event in colon-associated cancer, an event that may enable
such cells to escape surveillance from the immune system.
Definitions
[0066] The term "about" or "approximately" means within an
acceptable error range for the particular value as determined by
one of ordinary skill in the art, which will depend in part on how
the value is measured or determined, i.e., the limitations of the
measurement system. For example, "about" can mean within 1 or more
than 1 standard deviation, per the practice in the art.
Alternatively, "about" can mean a range of up to 20%, preferably up
to 10%, more preferably up to 5%, and more preferably still up to
1% of a given value. Alternatively, particularly with respect to
biological systems or processes, the term can mean within an order
of magnitude, preferably within 5-fold, and more preferably within
2-fold, of a value. Where particular values are described in the
application and claims, unless otherwise stated the term "about"
meaning within an acceptable error range for the particular value
should be assumed.
[0067] The term "induces or enhances an immune response" is meant
causing a statistically measurable induction or increase in an
immune response over a control sample to which a therapeutic has
not been administered. Preferably the induction or enhancement of
the immune response results in a prophylactic or therapeutic
response in a subject. Examples of immune responses are increased
production of type I IFN, increased resistance to viral and other
types of infection by alternate pathogens. The enhancement of
immune responses to tumors (anti-tumor responses), or the
development of vaccines to prevent tumors or eliminate existing
tumors.
[0068] The term "STING" is meant to include, without limitation,
nucleic acids, polynucleotides, oligonucleotides, sense and
antisense polynucleotide strands, complementary sequences,
peptides, polypeptides, proteins, homologous and/or orthologous
STING molecules, isoforms, precursors, mutants, variants,
derivatives, splice variants, alleles, different species, and
active fragments thereof. STING polynucleotides and polypeptides
are described in U.S. Patent Publications 20130039933 and
20110262485.
[0069] The term "lacks a functional STING gene" is meant that a
transgenic animal lacks a gene that encodes STING, or lacks other
genetic components (e.g. promoters) required for expression of
STING.
[0070] Unless otherwise indicated, the terms "peptide",
"polypeptide" or "protein" are used interchangeably herein,
although typically they refer to peptide sequences of varying
sizes.
[0071] The term "variant," when used in the context of a
polynucleotide sequence, may encompass a polynucleotide sequence
related to a wild type gene. This definition may also include, for
example, "allelic," "splice," "species," or "polymorphic" variants.
A splice variant may have significant identity to a reference
molecule, but will generally have a greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA
processing. The corresponding polypeptide may possess additional
functional domains or an absence of domains. Species variants are
polynucleotide sequences that vary from one species to another. Of
particular utility in the invention are variants of wild type gene
products. Variants may result from at least one mutation in the
nucleic acid sequence and may result in altered mRNAs or in
polypeptides whose structure or function may or may not be altered.
Any given natural or recombinant gene may have none, one, or many
allelic forms. Common mutational changes that give rise to variants
are generally ascribed to natural deletions, additions, or
substitutions of nucleotides. Each of these types of changes may
occur alone, or in combination with the others, one or more times
in a given sequence.
[0072] The resulting polypeptides generally will have significant
amino acid identity relative to each other. A polymorphic variant
is a variation in the polynucleotide sequence of a particular gene
between individuals of a given species. Polymorphic variants also
may encompass "single nucleotide polymorphisms" (SNPs) or single
base mutations in which the polynucleotide sequence varies by one
base. The presence of SNPs may be indicative of, for example, a
certain population with a propensity for a disease state, that is
susceptibility versus resistance.
[0073] Derivative polynucleotides include nucleic acids subjected
to chemical modification, for example, replacement of hydrogen by
an alkyl, acyl, or amino group. Derivatives, e.g., derivative
oligonucleotides, may comprise non-naturally-occurring portions,
such as altered sugar moieties or inter-sugar linkages. Exemplary
among these are phosphorothioate and other sulfur containing
species which are known in the art. Derivative nucleic acids may
also contain labels, including radionucleotides, enzymes,
fluorescent agents, chemiluminescent agents, chromogenic agents,
substrates, cofactors, inhibitors, magnetic particles, and the
like.
[0074] A "derivative" polypeptide or peptide is one that is
modified, for example, by glycosylation, pegylation,
phosphorylation, sulfation, reduction/alkylation, acylation,
chemical coupling, or mild formalin treatment. A derivative may
also be modified to contain a detectable label, either directly or
indirectly, including, but not limited to, a radioisotope,
fluorescent, and enzyme label.
[0075] The term "immunoregulatory" is meant a compound, composition
or substance that is immunogenic (i.e. stimulates or increases an
immune response) or immunosuppressive (i.e. reduces or suppresses
an immune response).
[0076] "An antigen presenting cell" (APC) is a cell that is capable
of activating T cells, and includes, but is not limited to,
monocytes/macrophages, B cells and dendritic cells (DCs). The term
"dendritic cell" or "DC" refers to any member of a diverse
population of morphologically similar cell types found in lymphoid
or non-lymphoid tissues. These cells are characterized by their
distinctive morphology, high levels of surface MHC-class II
expression. DCs can be isolated from a number of tissue sources.
DCs have a high capacity for sensitizing MHC-restricted T cells and
are very effective at presenting antigens to T cells in situ. The
antigens may be self-antigens that are expressed during T cell
development and tolerance, and foreign antigens that are present
during normal immune processes.
[0077] The term "expression vector" as used herein refers to a
vector containing a nucleic acid sequence coding for at least part
of a gene product capable of being transcribed. In some cases, RNA
molecules are then translated into a protein, polypeptide, or
peptide. In other cases, these sequences are not translated, for
example, in the production of antisense molecules, siRNA,
ribozymes, and the like. Expression vectors can contain a variety
of control sequences, which refer to nucleic acid sequences
necessary for the transcription and possibly translation of an
operatively linked coding sequence in a particular host organism.
In addition to control sequences that govern transcription and
translation, vectors and expression vectors may contain nucleic
acid sequences that serve other functions as well.
[0078] By "encoding" or "encoded", "encodes", with respect to a
specified nucleic acid, is meant comprising the information for
translation into the specified protein. A nucleic acid encoding a
protein may comprise non-translated sequences (e.g., introns)
within translated regions of the nucleic acid, or may lack such
intervening non-translated sequences (e.g., as in cDNA). The
information by which a protein is encoded is specified by the use
of codons. Typically, the amino acid sequence is encoded by the
nucleic acid using the "universal" genetic code.
[0079] As used herein, "heterologous" in reference to a nucleic
acid is a nucleic acid that originates from a foreign species, or,
if from the same species, is substantially modified from its native
form in composition and/or genomic locus by deliberate human
intervention. For example, a promoter operably linked to a
heterologous structural gene is from a species different from that
from which the structural gene was derived, or, if from the same
species, one or both are substantially modified from their original
form. A heterologous protein may originate from a foreign species
or, if from the same species, is substantially modified from its
original form by deliberate human intervention.
[0080] "Sample" is used herein in its broadest sense. A sample
comprising polynucleotides, polypeptides, peptides, antibodies and
the like may comprise a bodily fluid; a soluble fraction of a cell
preparation, or media in which cells were grown; a chromosome, an
organelle, or membrane isolated or extracted from a cell; genomic
DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound
to a substrate; a cell; a tissue; a tissue print; a fingerprint,
skin or hair; and the like.
[0081] The terms "patient", "subject" or "individual" are used
interchangeably herein, and refers to a mammalian subject to be
treated, with human patients being preferred. In some cases, the
methods of the disclosure find use in experimental animals, in
veterinary application, and in the development of animal models for
disease, including, but not limited to, rodents including mice,
rats, and hamsters; primates, cats and dogs.
[0082] "Diagnostic" or "diagnosed" means identifying the presence
or nature of a pathologic condition. Diagnostic methods differ in
their sensitivity and specificity. The "sensitivity" of a
diagnostic assay is the percentage of diseased individuals who test
positive (percent of "true positives"). Diseased individuals not
detected by the assay are "false negatives." Subjects who are not
diseased and who test negative in the assay, are termed "true
negatives." The "specificity" of a diagnostic assay is 1 minus the
false positive rate, where the "false positive" rate is defined as
the proportion of those without the disease who test positive.
While a particular diagnostic method may not provide a definitive
diagnosis of a condition, it suffices if the method provides a
positive indication that aids in diagnosis.
[0083] The terms "treat", "treated", "treating" and "treatment", as
used with respect to methods herein refer to eliminating, reducing,
suppressing or ameliorating, either temporarily or permanently,
either partially or completely, a clinical symptom, manifestation
or progression of an event, disease or condition. Such treating
need not be absolute to be useful. Those in need of treatment
include those already with the disorder as well as those in which
the disorder is to be prevented. In tumor (e.g., cancer) treatment,
a therapeutic agent may directly decrease the pathology of tumor
cells, or render the tumor cells more susceptible to treatment by
other therapeutic agents, e.g., radiation and/or chemotherapy. As
used herein, "ameliorated" or "treatment" refers to a symptom which
is approaches a normalized value (for example a value obtained in a
healthy patient or individual), e.g., is less than 50% different
from a normalized value, preferably is less than about 25%
different from a normalized value, more preferably, is less than
10% different from a normalized value, and still more preferably,
is not significantly different from a normalized value as
determined using routine statistical tests. For example the term
"treat" or "treating" with respect to tumor cells refers to
stopping the progression of said cells, slowing down growth,
inducing regression, or amelioration of symptoms associated with
the presence of said cells. Treatment of an individual suffering
from an infectious disease organism refers to a decrease and
elimination of the disease organism from an individual. For
example, a decrease of viral particles as measured by plaque
forming units or other automated diagnostic methods such as ELISA
etc.
[0084] The "treatment of cancer", refers to one or more of the
following effects: (1) inhibition, to some extent, of tumor growth,
including, (i) slowing down and (ii) complete growth arrest; (2)
reduction in the number of tumor cells; (3) maintaining tumor size;
(4) reduction in tumor size; (5) inhibition, including (i)
reduction, (ii) slowing down or (iii) complete prevention, of tumor
cell infiltration into peripheral organs; (6) inhibition, including
(i) reduction, (ii) slowing down or (iii) complete prevention, of
metastasis; (7) enhancement of anti-tumor immune response, which
may result in (i) maintaining tumor size, (ii) reducing tumor size,
(iii) slowing the growth of a tumor, (iv) reducing, slowing or
preventing invasion and/or (8) relief, to some extent, of the
severity or number of one or more symptoms associated with the
disorder.
[0085] As used herein, the term "safe and effective amount" refers
to the quantity of a component which is sufficient to yield a
desired therapeutic response without undue adverse side effects
(such as toxicity, irritation, or allergic response) commensurate
with a reasonable benefit/risk ratio when used in the manner of
this invention. By "therapeutically effective amount" is meant an
amount of a compound of the present invention effective to yield
the desired therapeutic response. For example, an amount effective
to delay the growth of or to cause a cancer, or to shrink the
cancer or prevent metastasis. The specific safe and effective
amount or therapeutically effective amount will vary with such
factors as the particular condition being treated, the physical
condition of the patient, the type of mammal or animal being
treated, the duration of the treatment, the nature of concurrent
therapy (if any), and the specific formulations employed and the
structure of the compounds or its derivatives.
[0086] "Cells of the immune system" or "immune cells", is meant to
include any cells of the immune system that may be assayed,
including, but not limited to, B lymphocytes, also called B cells,
T lymphocytes, also called T cells, natural killer (NK) cells,
natural killer T (NK) cells, lymphokine-activated killer (LAK)
cells, monocytes, macrophages, neutrophils, granulocytes, mast
cells, platelets, Langerhans cells, stem cells, dendritic cells,
peripheral blood mononuclear cells, tumor-infiltrating (TIL) cells,
gene modified immune cells including hybridomas, drug modified
immune cells, and derivatives, precursors or progenitors of the
above cell types.
[0087] "Immune effector cells" refers to cells capable of binding
an antigen and which mediate an immune response selective for the
antigen. These cells include, but are not limited to, T cells (T
lymphocytes), B cells (B lymphocytes), monocytes, macrophages,
natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), for
example CTL lines, CTL clones, and CTLs from tumor, inflammatory,
or other infiltrates.
[0088] "Immune related molecules" refers to any molecule identified
in any immune cell, whether in a resting ("non-stimulated") or
activated state, and includes any receptor, ligand, cell surface
molecules, nucleic acid molecules, polypeptides, variants and
fragments thereof.
[0089] "T cells" or "T lymphocytes" are a subset of lymphocytes
originating in the thymus and having heterodimeric receptors
associated with proteins of the CD3 complex (e.g., a rearranged T
cell receptor, the heterodimeric protein on the T cell surfaces
responsible for antigen/MHC specificity of the cells). T cell
responses may be detected by assays for their effects on other
cells (e.g., target cell killing, activation of other immune cells,
such as B-cells) or for the cytokines they produce.
[0090] The phrase "T cell response" means an immunological response
involving T cells. The T cells that are "activated" divide to
produce antigen specific memory T cells or antigen specific
cytotoxic T cells. The cytotoxic T cells bind to and destroy cells
recognized as containing the antigen. The memory T cells are
activated by the antigen and thus provide a response to an antigen
already encountered. This overall response to the antigen is the
antigen specific T cell response, e.g. tumor specific.
[0091] A "secondary immune response" or "adaptive immune response"
may be active or passive, and may be humoral (antibody based) or
cellular that is established during the life of an animal, is
specific for an inducing antigen, and is marked by an enhanced
immune response on repeated encounters with said antigen. A key
feature of the T lymphocytes of the adaptive immune system is their
ability to detect minute concentrations of pathogen-derived
peptides presented by MHC molecules on the cell surface.
[0092] As used herein, "pharmaceutical composition" refers to a
composition suitable for administration to a subject animal,
including humans and mammals. A pharmaceutical composition
comprises a pharmacologically effective amount of a virus or
antigenic composition of the invention and also comprises a
pharmaceutically acceptable carrier. A pharmaceutical composition
encompasses a composition comprising the active ingredient(s), and
the inert ingredient(s) that make up the pharmaceutically
acceptable carrier, as well as any product which results, directly
or indirectly, from combination, complexation or aggregation of any
two or more of the ingredients. Accordingly, the pharmaceutical
compositions of the present invention encompass any composition
made by admixing a compound or conjugate of the present invention
and a pharmaceutically acceptable carrier.
[0093] As used herein, "pharmaceutically acceptable carrier"
include any and all clinically useful solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, buffers, and excipients, such as a
phosphate buffered saline solution, 5% aqueous solution of dextrose
or mannitol, and emulsions, such as an oil/water or water/oil
emulsion, and various types of wetting agents and/or adjuvants.
Suitable pharmaceutical carriers and formulations are described in
Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co.,
Easton, 1995). Pharmaceutical carriers useful for the composition
depend upon the intended mode of administration of the active
agent. Typical modes of administration include, but are not limited
to, enteral (e.g., oral) or parenteral (e.g., subcutaneous,
intramuscular, intravenous or intraperitoneal injection; or
topical, transdermal, or transmucosal administration). A
"pharmaceutically acceptable salt" is a salt that can be formulated
into a compound or conjugate for pharmaceutical use including,
e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) and
salts of ammonia or organic amines.
[0094] As used herein, "pharmaceutically acceptable" or
"pharmacologically acceptable" refers to a material which is not
biologically or otherwise undesirable, i.e., the material may be
administered to an individual without causing any undesirable
biological effects or interacting in a deleterious manner with any
of the components of the composition in which it is contained, or
when administered using routes well-known in the art, as described
below.
[0095] "Detectable moiety" or a "label" refers to a composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, or chemical means. For example, useful labels
include 32P, 35S, fluorescent dyes, electron-dense reagents,
enzymes (e.g., as commonly used in an ELISA), biotin-streptavadin,
dioxigenin, haptens and proteins for which antisera or monoclonal
antibodies are available, or nucleic acid molecules with a sequence
complementary to a target. The detectable moiety often generates a
measurable signal, such as a radioactive, chromogenic, or
fluorescent signal, that can be used to quantitate the amount of
bound detectable moiety in a sample.
Labels
[0096] In some embodiments, the STING, cGAS or other molecule is
labeled to facilitate its detection. A "label" or a "detectable
moiety" is a composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, chemical, or other
physical means. For example, labels suitable for use in the present
invention include, but are not limited to, radioactive labels
(e.g., .sup.32P), fluorophores (e.g., fluorescein), electron-dense
reagents, enzymes (e.g., as commonly used in an ELISA), biotin,
digoxigenin, or haptens as well as proteins which can be made
detectable, e.g., by incorporating a radiolabel into the hapten or
peptide, or used to detect antibodies specifically reactive with
the hapten or peptide.
[0097] Examples of labels suitable for use in the present invention
include, but are not limited to, fluorescent dyes (e.g.,
fluorescein isothiocyanate, Texas red, rhodamine, and the like),
radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C,
.sup.32P), or enzymes (e.g., horse radish peroxidase, alkaline
phosphatase and others commonly used in an ELISA), and colorimetric
labels such as colloidal gold, colored glass or plastic beads
(e.g., polystyrene, polypropylene, latex, etc.).
[0098] The label may be coupled directly or indirectly to the
desired component according to methods well known in the art.
Preferably, the label in one embodiment is covalently bound to the
molecule using an isocyanate reagent for conjugation of an active
agent according to the invention. In one aspect of the invention,
the bifunctional isocyanate reagents of the invention can be used
to conjugate a label to a target molecule to form a label target
molecule conjugate without an active agent attached thereto. The
label target molecule conjugate may be used as an intermediate for
the synthesis of a labeled conjugate according to the invention or
may be used to detect the target molecule conjugate. As indicated
above, a wide variety of labels can be used, with the choice of
label depending on sensitivity required, ease of conjugation with
the desired component, stability requirements, available
instrumentation, and disposal provisions. Non-radioactive labels
are often attached by indirect means. Generally, a ligand molecule
(e.g., biotin) is covalently bound to the target molecule. The
ligand then binds to another molecule (e.g., streptavidin), which
is either inherently detectable or covalently bound to a signal
system, such as a detectable enzyme, a fluorescent compound, or a
chemiluminescent compound.
[0099] The STING, cGAS or other molecule contemplated herein for
use in the methods can also be conjugated directly to
signal-generating compounds, e.g., by conjugation with an enzyme or
fluorophore. Enzymes suitable for use as labels include, but are
not limited to, hydrolases, particularly phosphatases, esterases
and glycosidases, or oxidotases, particularly peroxidases.
Fluorescent compounds, i.e., fluorophores, suitable for use as
labels include, but are not limited to, fluorescein and its
derivatives, rhodamine and its derivatives, dansyl, umbelliferone,
etc. Further examples of suitable fluorophores include, but are not
limited to, eosin, TRITC-amine, quinine, fluorescein W, acridine
yellow, lissamine rhodamine, B sulfonyl chloride erythroscein,
ruthenium (tris, bipyridinium), Texas Red, nicotinamide adenine
dinucleotide, flavin adenine dinucleotide, etc. Chemiluminescent
compounds suitable for use as labels include, but are not limited
to, luciferin and 2,3-dihydrophthalazinediones, e.g., luminol. For
a review of various labeling or signal producing systems that can
be used in the methods of the present invention, see U.S. Pat. No.
4,391,904.
[0100] Means for detecting labels are well known to those of skill
in the art. Thus, for example, where the label is radioactive,
means for detection include a scintillation counter or photographic
film, as in autoradiography. Where the label is a fluorescent
label, it may be detected by exciting the fluorochrome with the
appropriate wavelength of light and detecting the resulting
fluorescence. The fluorescence may be detected visually, by the use
of electronic detectors such as charge coupled devices (CCDs) or
photomultipliers and the like. Similarly, enzymatic labels may be
detected by providing the appropriate substrates for the enzyme and
detecting the resulting reaction product. Colorimetric or
chemiluminescent labels may be detected simply by observing the
color associated with the label. Other labeling and detection
systems suitable for use in the methods of the present invention
will be readily apparent to those of skill in the art. Such labeled
modulators and ligands can be used in the diagnosis of a disease or
health condition.
Formulation of Pharmaceutical Compositions
[0101] To administer compositions of the present disclosure to
human or test animals, it is preferable to formulate the active
agent in a composition comprising one or more pharmaceutically
acceptable carriers. The phrase "pharmaceutically or
pharmacologically acceptable" refer to molecular entities and
compositions that do not produce allergic, or other adverse
reactions when administered using routes well-known in the art, as
described below. "Pharmaceutically acceptable carriers" include any
and all clinically useful solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like.
[0102] In addition, compounds may form solvates with water or
common organic solvents. Such solvates are contemplated as
well.
[0103] The compositions are administered by any suitable means,
including parenteral, subcutaneous, intraperitoneal,
intrapulmonary, and intranasal, and, if desired for local
treatment, intralesional administration. Parenteral infusions
include intravenous, intraarterial, intraperitoneal, intramuscular,
intradermal or subcutaneous administration. Preferably the dosing
is given by injections, most preferably intravenous or subcutaneous
injections, depending in part on whether the administration is
brief or chronic. Other administration methods are contemplated,
including topical, particularly transdermal, transmucosal, rectal,
oral or local administration, e.g. through a catheter placed close
to the desired site.
[0104] Pharmaceutical compositions of the present disclosure
containing the active agent described herein may contain
pharmaceutically acceptable carriers or additives depending on the
route of administration. Examples of such carriers or additives
include water, a pharmaceutical acceptable organic solvent,
collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl
polymer, carboxymethylcellulose sodium, polyacrylic sodium, sodium
alginate, water-soluble dextran, carboxymethyl starch sodium,
pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic,
casein, gelatin, agar, diglycerin, glycerin, propylene glycol,
polyethylene glycol, Vaseline, paraffin, stearyl alcohol, stearic
acid, human serum albumin (HSA), mannitol, sorbitol, lactose, a
pharmaceutically acceptable surfactant and the like. Additives used
are chosen from, but not limited to, the above or combinations
thereof, as appropriate, depending on the dosage form of the
present disclosure.
[0105] Formulation of the pharmaceutical composition will vary
according to the route of administration selected (e.g., solution,
emulsion). An appropriate composition comprising the composition to
be administered can be prepared in a physiologically acceptable
vehicle or carrier. For solutions or emulsions, suitable carriers
include, for example, aqueous or alcoholic/aqueous solutions,
emulsions or suspensions, including saline and buffered media.
Parenteral vehicles can include sodium chloride solution, Ringer's
dextrose, dextrose and sodium chloride, lactated Ringer's or fixed
oils. Intravenous vehicles can include various additives,
preservatives, or fluid, nutrient or electrolyte replenishers.
[0106] A variety of aqueous carriers, e.g., sterile phosphate
buffered saline solutions, bacteriostatic water, water, buffered
water, 0.4% saline, 0.3% glycine, and the like, and may include
other proteins for enhanced stability, such as albumin,
lipoprotein, globulin, etc., subjected to mild chemical
modifications or the like.
[0107] Therapeutic formulations are prepared for storage by mixing
the active agent having the desired degree of purity with optional
physiologically acceptable carriers, excipients or stabilizers
(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980)), in the form of lyophilized formulations or aqueous
solutions. Acceptable carriers, excipients, or stabilizers are
nontoxic to recipients at the dosages and concentrations employed,
and include buffers such as phosphate, citrate, and other organic
acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0108] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0109] The active ingredients may also be entrapped in microcapsule
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0110] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0111] Aqueous suspensions may contain the active compound in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents may be a naturally-occurring phosphatide, for
example lecithin, or condensation products of an alkylene oxide
with fatty acids, for example polyoxyethylene stearate, or
condensation products of ethylene oxide with long chain aliphatic
alcohols, for example heptadecaethyl-eneoxycetanol, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and a hexitol such as polyoxyethylene sorbitol monooleate, or
condensation products of ethylene oxide with partial esters derived
from fatty acids and hexitol anhydrides, for example polyethylene
sorbitan monooleate. The aqueous suspensions may also contain one
or more preservatives, for example ethyl, or n-propyl,
p-hydroxybenzoate.
[0112] The active agents described herein can be lyophilized for
storage and reconstituted in a suitable carrier prior to use. This
technique has been shown to be effective with conventional
immunoglobulins. Any suitable lyophilization and reconstitution
techniques can be employed. It will be appreciated by those skilled
in the art that lyophilization and reconstitution can lead to
varying degrees of antibody activity loss and that use levels may
have to be adjusted to compensate.
[0113] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
compound in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above.
[0114] The concentration of active agent in these formulations can
vary widely, for example from less than about 0.5%, usually at or
at least about 1% to as much as 15 or 20% by weight and will be
selected primarily based on fluid volumes, viscosities, etc., in
accordance with the particular mode of administration selected.
Thus, a typical pharmaceutical composition for parenteral injection
could be made up to contain 1 ml sterile buffered water, and 50 mg
of active agent. A typical composition for intravenous infusion
could be made up to contain 250 ml of sterile Ringer's solution,
and 150 mg of antibody. Actual methods for preparing parenterally
administrable compositions will be known or apparent to those
skilled in the art and are described in more detail in, for
example, Remington's Pharmaceutical Science, 15th ed., Mack
Publishing Company, Easton, Pa. (1980). An effective dosage of
active agent is within the range of 0.01 mg to 1000 mg per kg of
body weight per administration.
[0115] The pharmaceutical compositions may be in the form of a
sterile injectable aqueous, oleaginous suspension, dispersions or
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions. The suspension may be
formulated according to the known art using those suitable
dispersing or wetting agents and suspending agents which have been
mentioned above. The sterile injectable preparation may also be a
sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example as a
solution in 1,3-butane diol. The carrier can be a solvent or
dispersion medium containing, for example, water, ethanol, polyol
(for example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, vegetable oils,
Ringer's solution and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose any bland fixed oil
may be employed including synthetic mono- or diglycerides. In
addition, fatty acids such as oleic acid find use in the
preparation of injectables.
[0116] In all cases the form must be sterile and must be fluid to
the extent that easy syringability exists. The proper fluidity can
be maintained, for example, by the use of a coating, such as
lecithin, by the maintenance of the required particle size in the
case of dispersion and by the use of surfactants. It must be stable
under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The prevention of the action of
microorganisms can be brought about by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
desirable to include isotonic agents, for example, sugars or sodium
chloride. Prolonged absorption of the injectable compositions can
be brought about by the use in the compositions of agents delaying
absorption, for example, aluminum monostearate and gelatin.
[0117] Compositions useful for administration may be formulated
with uptake or absorption enhancers to increase their efficacy.
Such enhancers include for example, salicylate,
glycocholate/linoleate, glycholate, aprotinin, bacitracin, SDS,
caprate and the like. See, e.g., Fix (J. Pharm. Sci., 85:1282-1285
(1996)) and Oliyai and Stella (Ann. Rev. Pharmacol. Toxicol.,
32:521-544 (1993)).
[0118] In addition, the properties of hydrophilicity and
hydrophobicity of the compositions contemplated for use in the
present disclosure are well balanced, thereby enhancing their
utility for both in vitro and especially in vivo uses, while other
compositions lacking such balance are of substantially less
utility. Specifically, compositions contemplated for use in the
disclosure have an appropriate degree of solubility in aqueous
media which permits absorption and bioavailability in the body,
while also having a degree of solubility in lipids which permits
the compounds to traverse the cell membrane to a putative site of
action. Thus, antibody compositions contemplated are maximally
effective when they can be delivered to the site of target antigen
activity.
Administration and Dosing
[0119] In one aspect, methods of the present disclosure include a
step of administering a pharmaceutical composition. In certain
embodiments, the pharmaceutical composition is a sterile
composition.
[0120] Methods of the present disclosure are performed using any
medically-accepted means for introducing therapeutics directly or
indirectly into a mammalian subject, including but not limited to
injections, oral ingestion, intranasal, topical, transdermal,
parenteral, inhalation spray, vaginal, or rectal administration.
The term parenteral as used herein includes subcutaneous,
intravenous, intramuscular, and intracisternal injections, as well
as catheter or infusion techniques. Administration by, intradermal,
intramammary, intraperitoneal, intrathecal, retrobulbar,
intrapulmonary injection and or surgical implantation at a
particular site is contemplated as well.
[0121] In one embodiment, administration is performed at the site
of a cancer or affected tissue needing treatment by direct
injection into the site or via a sustained delivery or sustained
release mechanism, which can deliver the formulation internally.
For example, biodegradable microspheres or capsules or other
biodegradable polymer configurations capable of sustained delivery
of a composition (e.g., a soluble polypeptide, antibody, or small
molecule) can be included in the formulations of the disclosure
implanted near or at site of the cancer.
[0122] Therapeutic compositions may also be delivered to the
patient at multiple sites. The multiple administrations may be
rendered simultaneously or may be administered over a period of
time. In certain cases it is beneficial to provide a continuous
flow of the therapeutic composition. Additional therapy may be
administered on a period basis, for example, hourly, daily, every
other day, twice weekly, three times weekly, weekly, every 2 weeks,
every 3 weeks, monthly, or at a longer interval.
[0123] Also contemplated in the present disclosure is the
administration of multiple agents, such as the active agent
compositions in conjunction with a second agent as described
herein, including but not limited to a chemotherapeutic agent.
Suitable chemotherapeutic agents include: daunomycin, doxorubicin,
methotrexate, and vindesine (Rowland et al., (1986) supra) and
those listed in the Table below.
TABLE-US-00001 Alkylating agents Nitrogen mustards mechlorethamine
cyclophosphamide ifosfamide melphalan chlorambucil Nitrosoureas
carmustine (BCNU) lomustine (CCNU) semustine (methyl-CCNU)
Ethylenimine/Methyl-melamine thriethylenemelamine (TEM) triethylene
thiophosphoramide (thiotepa) hexamethylmelamine (HMM, altretamine)
Alkyl sulfonates busulfan Triazines dacarbazine (DTIC)
Antimetabolites Folic Acid analogs methotrexate Trimetrexate
Pemetrexed (Multi-targeted antifolate) Pyrimidine analogs
5-fluorouracil fluorodeoxyuridine gemcitabine cytosine arabinoside
(AraC, cytarabine) 5-azacytidine 2,2'-difluorodeoxy-cytidine Purine
analogs 6-mercaptopurine 6-thioguanine azathioprine
2'-deoxycoformycin (pentostatin) erythrohydroxynonyl-adenine (EHNA)
fludarabine phosphate 2-chlorodeoxyadenosine (cladribine, 2-CdA)
Type I Topoisomerase Inhibitors camptothecin topotecan irinotecan
Biological response modifiers G-CSF GM-CSF Differentiation Agents
retinoic acid derivatives Hormones and antagonists
Adrenocorticosteroids/antagonists prednisone and equivalents
dexamethasone ainoglutethimide Progestins hydroxyprogesterone
caproate medroxyprogesterone acetate megestrol acetate Estrogens
diethylstilbestrol ethynyl estradiol/equivalents Antiestrogen
tamoxifen Androgens testosterone propionate
fluoxymesterone/equivalents Antiandrogens flutamide
gonadotropin-releasing hormone analogs leuprolide Nonsteroidal
antiandrogens flutamide Natural products Antimitotic drugs Taxanes
paclitaxel Vinca alkaloids vinblastine (VLB) vincristine
vinorelbine Taxotere .RTM. (docetaxel) estramustine estramustine
phosphate Epipodophylotoxins etoposide teniposide Antibiotics
actimomycin D daunomycin (rubido-mycin) doxorubicin (adria-mycin)
mitoxantroneidarubicin bleomycin splicamycin (mithramycin)
mitomycinC dactinomycin aphidicolin Enzymes L-asparaginase
L-arginase Radiosensitizers metronidazole misonidazole
desmethylmisonidazole pimonidazole etanidazole nimorazole RSU 1069
EO9 RB 6145 SR4233 nicotinamide 5-bromodeozyuridine
5-iododeoxyuridine bromodeoxycytidine Miscellaneous agents
Platinium coordination complexes cisplatin Carboplatin oxaliplatin
Anthracenedione mitoxantrone Substituted urea hydroxyurea
Methylhydrazine derivatives N-methylhydrazine (MIH) procarbazine
Adrenocortical suppressant mitotane (o,p'-DDD) ainoglutethimide
Cytokines interferon (.alpha., .beta., .gamma.) interleukin-2
Photosensitizers hematoporphyrin derivatives Photofrin .RTM.
benzoporphyrin derivatives Npe6 tin etioporphyrin (SnET2)
pheoboride-a bacteriochlorophyll-a naphthalocyanines
phthalocyanines zinc phthalocyanines Radiation X-ray ultraviolet
light gamma radiation visible light infrared radiation microwave
radiation
[0124] The amounts of active agent composition in a given dosage
may vary according to the size of the individual to whom the
therapy is being administered as well as the characteristics of the
disorder being treated. In exemplary treatments, it may be
necessary to administer about 1 mg/day, 5 mg/day, 10 mg/day, 20
mg/day, 50 mg/day, 75 mg/day, 100 mg/day, 150 mg/day, 200 mg/day,
250 mg/day, 500 mg/day or 1000 mg/day. These concentrations may be
administered as a single dosage form or as multiple doses. Standard
dose-response studies, first in animal models and then in clinical
testing, reveals optimal dosages for particular disease states and
patient populations.
[0125] Also contemplated in the present disclosure, the amounts of
active agent in a given dosage may vary according to the size of
the individual to whom the therapy is being administered as well as
the characteristics of the disorder being treated. It will also be
apparent that dosing may be modified if traditional therapeutics
are administered in combination with therapeutics of the
disclosure.
[0126] Exemplary conditions or disorders that can be treated using
the present methods include cancers, such as esophageal cancer,
pancreatic cancer, metastatic pancreatic cancer, metastatic
adenocarcinoma of the pancreas, bladder cancer, stomach cancer,
fibrotic cancer, glioma, malignant glioma, diffuse intrinsic
pontine glioma, recurrent childhood brain neoplasm renal cell
carcinoma, clear-cell metastatic renal cell carcinoma, kidney
cancer, prostate cancer, metastatic castration resistant prostate
cancer, stage IV prostate cancer, metastatic melanoma, melanoma,
malignant melanoma, recurrent melanoma of the skin, melanoma brain
metastases, stage IIIA skin melanoma; stage IIIB skin melanoma,
stage IIIC skin melanoma; stage IV skin melanoma, malignant
melanoma of head and neck, lung cancer, non small cell lung cancer
(NSCLC), squamous cell non-small cell lung cancer, breast cancer,
recurrent metastatic breast cancer, hepatocellular carcinoma,
hodgkin's lymphoma, follicular lymphoma, non-hodgkin's lymphoma,
advanced B-cell NHL, HL including diffuse large B-cell lymphoma
(DLBCL), multiple myeloma, chronic myeloid leukemia, adult acute
myeloid leukemia in remission; adult acute myeloid leukemia with
Inv(16)(p13.1q22); CBFB-MYH11; adult acute myeloid leukemia with
t(16;16)(p13.1;q22); CBFB-MYH11; adult acute myeloid leukemia with
t(8;21)(q22;q22); RUNX1-RUNX1T1; adult acute myeloid leukemia with
t(9;11)(p22;q23); MLLT3-MLL; adult acute promyelocytic leukemia
with t(15;17)(q22;q12); PML-RARA; alkylating agent-related acute
myeloid leukemia, chronic lymphocytic leukemia, richter's syndrome;
waldenstrom macroglobulinemia, adult glioblastoma; adult
gliosarcoma, recurrent glioblastoma, recurrent childhood
rhabdomyosarcoma, recurrent ewing sarcoma/peripheral primitive
neuroectodermal tumor, recurrent neuroblastoma; recurrent
osteosarcoma, colorectal cancer, MSI positive colorectal cancer;
MSI negative colorectal cancer, nasopharyngeal nonkeratinizing
carcinoma; recurrent nasopharyngeal undifferentiated carcinoma,
cervical adenocarcinoma; cervical adenosquamous carcinoma; cervical
squamous cell carcinoma; recurrent cervical carcinoma; stage IVA
cervical cancer; stage IVB cervical cancer, anal canal squamous
cell carcinoma; metastatic anal canal carcinoma; recurrent anal
canal carcinoma, recurrent head and neck cancer; carcinoma,
squamous cell of head and neck, head and neck squamous cell
carcinoma (HNSCC), ovarian carcinoma, colon cancer, gastric cancer,
advanced GI cancer, gastric adenocarcinoma; gastroesophageal
junction adenocarcinoma, bone neoplasms, soft tissue sarcoma; bone
sarcoma, thymic carcinoma, urothelial carcinoma, recurrent merkel
cell carcinoma; stage III merkel cell carcinoma; stage IV merkel
cell carcinoma, and myelodysplastic syndrome.
Example 1
[0127] Activation of STING-Dependent Genes by AOM.
[0128] Given that chronic inflammation is known to aggravate colon
cancer and that STING has been shown to influence inflammatory
responses, especially that invoked by cytosolic self or pathogen
related DNA, the role of STING in the control of inflammatory
colitis-associated carcinogenesis (CAC) was examined. Towards these
objectives, the AOM/DSS model, as described above, was utilized and
analyzed the effects of AOM and precursor DMH upon STING signaling.
Principally, wild type (WT) or STING-deficient (SKO) murine
embryonic fibroblasts (MEF) were treated in vitro with DMH or
metabolite AOM for 8 hours and microarray analysis performed to
analyze the consequences to gene expression. This study indicated
that AOM activated mRNA production of a wide array of innate immune
related genes in WT cells, including IFIT3 and Cxcl2 (FIG. 1A; FIG.
7). However, there was a marked decrease in the production of the
same genes in cells lacking STING (SKO) indicating that AOM was
indeed capable of activating the STING pathway (FIG. 1A, left
panel). A similar effect was observed following the treatment of
cells with DMH (FIG. 1A, right panel). The observed STING-dependent
gene expression was confirmed following RT-PCR analysis of select
mRNA such as Cxcl10 and IFIT3 (FIG. 1B). Similarly normal human
colon epithelial cells (FHC) were treated with AOM and a similar
induction of innate immune genes was observed, controlled by STING,
including Cxcl10 (FIG. 1C). The production of Cxcl10 by AOM was
similarly reduced in FHC's treated with RNAi to STING (FIG. 1D).
Thus, the DNA damaging agent DMH/AOM can invoke STING-dependent
signaling.
[0129] To start determining the mechanisms underlining the cause of
DMH/AOM-induced STING activity, MEF or FHC cells were treated with
these drugs and observed by two different approaches, DAPI staining
and immunofluorescence (IF) using anti-dsDNA antibody, the leakage
of DNA into the cytosol (FIGS. 8A and 8B). Cytosolic DNA activates
STING and stimulates STING/TBK1 trafficking via autophagy to
endosomal regions harboring the transcription factors IRF3 and
NF-.kappa.B, which triggers cytokine production. Thus, to determine
the consequences of DMH/AOM treatment upon STING's ability to
activate these key transcription factors, IF analysis on FHC'S
treated with these drugs was carried out. This study indicated that
DMH/AOM could instigate the translocation of IRF3/NF-.kappa.B in
treated cells (FIG. 8C). Thus, DMH/AOM induces STING-dependent
signaling conceivably through the leaking of DNA into the cytyosol
(FIG. 1A; FIGS. 8A and 8B).
[0130] Loss of STING Renders Mice Susceptible to CAC:
[0131] The data indicates that DMH/AOM can activate STING in vitro.
To examine the consequences of this in vivo, mice were treated once
with AOM and subsequently orally with 4 treatments of DSS. Prior to
this, STING expression in the intestine was analyzed by IHC. This
study showed that STING was expressed in lamina propria cells as
well as in endothelial and epithelial cells of the gastrointestinal
tract (FIG. 1E). After 13 weeks the mice were analyzed for tumor
development in the colon. Surprisingly, the mice lacking STING
(SKO) developed colonic tumors at a much higher frequency compared
to wild type mice (WT) (FIG. 2A-C; FIG. 9A). Indeed, 4/7 WT mice
exhibited tumor formation compared to 7/7 SKO within the same time
period (FIGS. 2A and 2B; FIG. 9A), Hematoxylin and eosin (HE)
analysis confirmed that AOM/DSS treated SKO mice exhibited
significant inflammatory cell infiltration and development of
adenocarcinoma in colon, compared to WT mice (FIG. 2B; FIG. 9B).
However, microarray analysis indicated that tumors from WT mice
exhibited higher levels of select gene expression, such as Cxcl13
and Ccr6, compared to tumors retrieved from SKO mice, perhaps since
loss of STING suppressed immunomodulatory transcriptional events
(FIG. 2E). It is postulated that STING may recognize damaged DNA
and activate the production of cytokines that conceivably could
promote tissue repair or stimulate the immune system to eradicate
such cells. Thus, loss of STING may enable damaged cells to escape
immune surveillance processes and progress more readily into
tumors.
[0132] Chronic STING Activation is Responsible for Inflammatory
Bowel Disease.
[0133] Transient STING activation in response to cell damaging
agents such as AOM and dextran sulphate (DSS) facilitates wound
healing. Thus, loss of STING may lead to a lack of colonic repair
and the infiltration of genotoxic microbiota that aggravate
STING-independent inflammatory responses. However, chronic
irritation of STING in wild type mice by agents such as DSS can
lead to inflammatory bowel disease (IBD). Thus, suppression of
STING activity by inhibitors/drugs/compounds may lead to a
reduction in IBD such as Crohn's disease and ulcerative
colitis.
[0134] Suppression of IL22BP Expression in STING-Deficient
Mice.
[0135] In demonstrating that loss of STING facilitated colon cancer
development, the tumor suppressive mechanisms associated with STING
activity remain to be clarified. It remains plausible that STING
could exert direct tumor suppressive, growth inhibitory or
pro-apoptotic properties similar to tumor suppressor p53. Further,
AOM treatment has been known to induce frequent Ras mutations,
which in the context of loss of STING, could facilitate cellular
transformation. Expression of oncogenic Ras in an environment where
p53 function is lost renders normal cells the ability to form foci
in soft agar and to become tumorigenic. To evaluate this
possibility, WT, SKO, or p53-deficient MEFs were transfected as
positive controls, with Myc or activated Ras and cellular
transformation monitored. MEF's lacking p53 were found to be
readily transformed by the introduction of Myc or activated Ras.
However, MEF's lacking STING did not appear appreciably sensitive
to transformation by overexpression of Myc, or activated Ras. Thus,
the absence of STING does not appear to exert an oncogenic
stimulus, at least in vitro or to cooperate with Myc or Ras in the
cellular transformation process.
[0136] However, it has been demonstrated that mice lacking certain
cytokines such as IL-18, IL-22 or the innate immune adaptor MyD88
are similarly susceptible to AOM/DSS induced CAC. In this
situation, MyD88 exerts a protective effect by facilitating the
production of IL-18 through the IL-18R, which is required to
inhibit IL-22BP. IL-22BP is necessary to suppress IL-22 function,
which can promote the proliferation of intestinal epithelial cells
following damage by carcinogens or inflammatory agents. Mice
lacking IL-18 or IL-22BP are highly susceptible to CAC, similar to
STING-deficient mice. It was noted from the microarray analyses
that IL-18 levels were reduced in SKO MEFs treated with
STING-activating dsDNA (dsDNA90 base pairs) (FIG. 3A). Therefore
investigation of the involvement of STING on the possible
regulation of the IL-18/IL-22BP axis was made. First, a
confirmation of the influence of STING upon IL-18 expression was
made since it was additionally noted that the promoter of this
cytokine is known to harbor numerous sites recognized by innate
immune gene activating transcription factors such as STAT1,
NF-.kappa.B, IRF1 and IRF7. The analysis indicated that IL-18,
which is expressed in a wide variety of cell types, is a STING
inducible gene, as determined following treatment of MEF cells with
dsDNA or STING-activating CDN's (cGAMP) (FIG. 3A). A similar study
indicated that DMH/AOM could also trigger the production of IL-18
in dendritic cells, in a STING-dependent manner, confirming that
IL-18 can indeed be induced through the STING pathway.
[0137] Following the confirmation, an examination of whether DSS
treatment could affect IL-18 and IL-22BP expression in vivo was
made. Following 7 days of DSS treatment, colons were retrieved from
WT or SKO mice and IL-18, IL-22BP or IL-22 expression analyzed by
RT-PCR. This study indicated that IL-18 expression was reduced in
mice lacking STING (SKO) after 2 days treatment (FIG. 3B). However,
a much more pronounced decrease in IL-22BP expression, a protein
predominantly expressed from CD11c.sup.+ dendritic cells, was
observed in SKO mice compared to WT mice (FIG. 3B). The expression
of IL-22, in contrast, remained relatively unaffected. It was
surprising to note that IL-22BP levels were suppressed in the
absence of STING, where IL-18 levels were noted to be also
relatively low. However, it has been reported that downregulation
of IL22-BP can occur even in the absence of IL-18, indicating that
other STING-dependent factors may also contribute to the regulation
of IL-22BP. To complement this study, control or SKO mice with
AOM/DSS regimes were treated and after 13 weeks again analyzed
IL-22BP expression levels in normal or tumor tissue taken from the
treated mice. Analogous to DSS treatment alone, observations noted
the greatly reduced expression of IL-22BP in the tumors of SKO mice
compared to WT mice (FIG. 3C). However, IL-18 and IL-22 levels
appeared less dramatically affected. Taken together, it is
conceivable that DNA damage or the sensing of microbial ligands
that may invade colon tissue after intestinal damage (for example
by DSS) can trigger STING activity leading to the production of
IL-18. This event would suppress IL-22BP production and enable
IL-22 to facilitate tissue repair. However, it appears that loss of
STING function in the long term also influences IL-22BP production
which is critical for controlling the growth stimulator properties
of IL-22. This event may be mediated by microbes triggering
STING-dependent innate immune pathways that control IL-22BP
production. Data thus indicates that similar to mice lacking MyD88,
IL-18 or IL-22BP, STING-deficient mice are also prone to CAC
induced by AOM/DSS.
[0138] STING Activity is Suppressed in Colonic Tumor Cells.
[0139] Data indicates that STING is required to protect against
carcinogens and perhaps microbes that facilitate inflammation
driven CAC. STING conceivably senses DNA damage and signals the
event to anti-tumor immunosurveillance cells. Dendritic cells (DC)
such as CD8 alpha DCs engulf tumor cells or necrotic tumor cell
debris, and the DNA from the engulfed cell or debris activates
STING extrinsically in the DC. This leads to the production of
cytokines that are essential for anti-tumor T cell responses. Given
this, STING signaling in human colonic cells was analyzed, since it
was feasible that defects in STING function may enable damaged
cells to evade the immune system. To thus evaluate STING signaling
in human colonic cancer cells, STING expression in hTERT and (FHC)
intestinal epithelial cells as controls were examined. STING was
found to be expressed in these cells and to produce type I IFN in
response to transfected cytosolic DNA (dsDNA 90 base pairs), which
is known to be a STING-dependent event (FIG. 4A-C). A similar
inducible effect by dsDNA was noted following measurement by
Cxcl10, also a highly dsDNA-inducible, STING regulated gene (FIG.
4D). Next, examination of the expression of STING in a variety of
tumor cells isolated from various stages of colon cancer was done
and it was found that 10/11 cell lines expressed STING (FIG. 4A;
FIG. 12). However, the STING pathway was defective in the majority
of cells analyzed (>80%) and such cells could not efficiently
produce type I IFN in response to cytosolic dsDNA (FIG. 4A-C). Only
two out the 11 cell lines analyzed (SW116 and LS123) appeared only
somewhat able to produce type I IFN in response to cytosolic DNA
stimulation. In contrast, transfected synthetic dsRNA (polyI:C) was
able to stimulate the production of type I IFN relatively well in
all but 3 of the cancer cells, likely indicating that the RIGI/MDA5
pathway was functional. To confirm this analyses DNA microarray
analyses on the colon cancer cells following stimulation of the
STING pathway using transfected cytosolic DNA was carried out. This
data indicated significant suppression of STING-dependent primary
innate immune gene activation in the colon cancer cells analyzed,
including type I IFN, as well as a host of other key innate immune
modulatory genes such as CXCL10, CXCL11 and CC15 (FIGS. 4E and 4F).
LoVo cells were observed to retain some ability to induce cytosolic
DNA-induced cells, while HT116 cells appeared significantly
defective in STING-dependent signaling. The data indicates that
STING-dependent innate immune pathways appear to be preferentially
deregulated in colonic tumor lines.
[0140] To analyze the mechanisms of STING inactivation further,
immunohistochemical (MC) analyses were carried out on the normal or
the colon tumor cells. It had been previously shown that in the
presence of cytosolic DNA, STING becomes activated and translocates
via a non-canonical autophagy associated process required for the
activation of transcription factors NF-.kappa.B and IRF3
(interferon regulatory factor 3). Thus, blockage of STING
translocation through use of Brefeldin A or through suppression of
the key autophagy modulator VPS34 inhibits STING signaling
function. Thus, normal or human colon cancer cells were transfected
with dsDNA to activate STING and it was observed that only 4/11
cell-lines (LS123, SW480, LoVo and HT29) exhibited evidence of
STING trafficking (FIG. 5A). This study was then completed by
analyzing the translocation of the transcription factors
NF-.kappa.B and IRF3 in similarly treated cells using IHC
techniques. It was observed that IRF3 was able to translocate in
cells that facilitated STING trafficking, except for SW480 (FIG.
5B). This may explain, in part, the partial stimulation of innate
immune genes in some of the cells analyzed by microarray, such as
LoVo. However, only two of the cells (LS1116, LS123) exhibited
translocation of the NF-.kappa.B subunit p65 (FIG. 5C). Since both
IRF3 and NF-.kappa.B are required for optimal transcription of type
I IFN, it may explain why LS1116 and LS123 remained partially able
to stimulate type I IFN production, following treatment with
cytosolic DNA, while the remainder did not (FIGS. 4B and 4C).
However, cytosolic DNA-dependent, STING-controlled signaling
remained severely defective in all colon cancer types examined as
shown (FIG. 4).
[0141] To extend these findings further, immunoblot analyses were
carried out on the normal or colon tumor cells. In the presence of
cytosolic DNA, STING undergoes phosphorylation and is then
degraded, an event that facilitates its activity, perhaps through
releasing TBK1 to phosphorylate IRF3. First, STING
phosphorylation/degradation after activation was impeded in many
cell lines analyzed, which may affect STING function (for example,
LS123, SW480, SW1417, HT116) (FIG. 5D). Confirmation was made in
tumor cells that exhibited some STING trafficking (LS1116, LS123,
LoVo and HT29) that the IRF3 kinase activator Tank binding kinase 1
(TBK1) underwent phosphorylation in the presence of cytosolic DNA
(FIG. 5D). Accordingly, observations of phospho-IRF3 activity in
cells with active TBK1 (FIG. 5D) were made. The remainder of the
tumor cells, such as SW480, SW1417, SW48 and HT116 did not exhibit
phospho-TBK1 activity or IRF3 translocation, likely due to an
inability of STING to undergo autophagy, or since STING expression
was completely absent as in the case of SW48 (FIG. 5D).
Surprisingly, observations showed that the vast majority of cells
lacked p65 translocation, phosphorylation of p65 was evident (for
example LoVo, HT29, SW480 and SW1417). This may suggest that
NF-.kappa.B signaling could be defective at the level of p65
translocation. Taken together the study clearly indicates that
STING-signaling is defective in a wide variety of colon cancer
cells examined.
[0142] Cyclic Dinucleotides (CDN's) have been Shown to Activate
STING.
[0143] CDN's have been shown to be generated through cytosolic
dsDNA species triggering the activation of a synthase, referred to
as cGAS (Cyclic GMP-AMP Synthase, C6orf150, Mab-21
Domain-Containing Protein). Loss of cGAS has thus been shown to
affect STING signaling. To complement the above study, the
expression of cGAS in the colon cancer cell-lines was examined.
This analysis indicated that 5/11 colon cancer cells had
undetectable levels of cGAS expression, an event that correlated
with loss of STING translocation and TBK1/IRF3 activity (FIGS. 5D
and 5E highlighted by dashed line or box). Interestingly, loss of
cGAS expression could be rescued using de-methylating agents lines
(SW480, HT116) indicating that some cells exhibited suppressed cGAS
promoter activity (FIG. 5F). However, rescue of cGAS expression did
not robustly rescue STING activity as determined (FIGS. 13A and
13B) indicating that further defects in STING signaling may exist
in these cells. Given these findings, examination of the expression
of cGAS in 47 human colon cancers at various stages of
tumorigenesis was analyzed (FIG. 13C). Expression of cGAS was low
to undetectable in approximately 30% of cells analyzed. Thus,
defects in cGAS or STING expression, or signaling appear defective
in a large number of tumor colon cancer cells lines and could
constitute a major cause of tumorigenesis. It should be noted,
however, that similarly defective STING-signaling was found in a
wide variety of other tumor cells examined, indicating that defects
in STING function could be common in cells other than those of the
colon (FIG. 14).
[0144] Cancer Cells with Defective STING Signaling are Susceptible
to Viral Oncolysis.
[0145] Numerous cancer cells have been shown to be defective in
antiviral responses, although the mechanisms remain to be fully
determined. Indeed, a variety of viruses are now being used in the
clinic to determine their efficacy as anti-tumor therapeutics,
including herpes simplex 1 (HSV1) which harbors a dsDNA genome.
HSV1 has been shown to potently trigger innate immune responses
through activating the STING. Mice deficient in STING signaling are
extremely sensitive to lethal HSV1 infection since they lack the
ability to mount an appropriate innate immune response, including
the generation of type I IFN. Given the findings that STING
signaling is defective in a large number of cancer cells, their
susceptibility to HSV1 infection was examined. First, analysis of
the response to a recombinant HSV1 that expresses luciferase
(HSV1-Luc) was made. This analysis indicated that colon cancer
cells exhibiting defective STING signaling enabled high levels of
HSV1-Luc expression (FIG. 6A). However, the two cancer lines (SW116
and LS123), which exhibited partial STING-dependent innate immune
responses (FIG. 4B-D), as well as the control hTERT and FHC's did
not facilitate robust HSV-Luc gene expression (FIG. 6A). This
coincided with the normal cells and SW116 and LS123 responding to
infection by producing CXCL10, similar to their response to dsDNA
(FIGS. 6B and 6C; FIG. 4C). None of the tumor cells harboring
severely defective STING function could robustly produce type I IFN
after infection. To extend this study, an HSV construct that lacked
the .gamma.34.5 gene (HSV1 .gamma.34.5) was used, encoding viral
protein that has been reported to inhibit host defense in part
through preventing host cell translational shut-off. A similar
virus that lacks .gamma.34.5 is being examined in the clinic as an
anti-cancer agent. It was observed that colon cancer cells
defective in STING-signaling were unable to mount an efficient type
I IFN response following infection with HSV1 .gamma.34.5 (FIG. 6D).
Thus, the examination of STING-signaling may be a useful prognostic
marker for whether HSV1 or other viral based anti-cancer therapies
will be efficacious for the treatment of malignant disease.
Experimental Procedure
[0146] Mice:
[0147] STING knockout mice (SKO, Sting.sup.-/-) were generated in
the University of Miami laboratory (Ishikawa 2008). Wild type mice
(WT) were used as control groups. Mice care and study were
conducted under approval from the Institutional Animal Care and Use
Committee (IACUC) of the University of Miami.
[0148] Acute DSS Colitis.
[0149] WT and SKO mice 6-8 weeks of ages were divided into
experimental and control groups. Mice in experimental group
received 3% Dextran sodium sulfate (DSS, MP 160110; MW 36000-5000)
for 5 days, followed by 2 days of regular drinking water. Distilled
water was administered into control group mice.
[0150] AOM/DSS Induced Colitis-Associated Tumor Induction:
[0151] WT and SKO mice were injected intraperitoneally with
Azoxymethane (AOM; MP 180139; MW 74.08) at a dose of 10 mg/kg. DSS
at 5% which was administered in the drinking water for 7 days every
3 weeks. DSS cycle was repeated 4 times. On 91 days, micro
endoscopic procedure was performed in a blinded fashion for
counting number of polyps. Mice were sacrificed at day 121 and
colon was resected, flushed with PBS, fixed in formalin for
histology and frozen for RNA expression analysis.
[0152] Primary Cell Culture:
[0153] Mouse embryonic fibroblasts (MEFs) were obtained from e15
embryos by a standard procedure as described. Bone marrow derived
dendritic cells were isolated from hind-limb femurs of 8-10 weeks
old mice. Briefly, the marrow cells were flushed from the bones
with Dulbecco's modified eagle medium (DMEM, Invitrogen), 10%
heat-inactivated fetal calf serum (FCS, Invitrogen) with a 23 gauge
needle and incubated at 37.degree. C. for 4 hours. After harvesting
floating cells, approximately 2.times.10.sup.7 cells were seeded in
10 cm dish with complete DMEM including 10 ng/ml of Recombinant
mouse GM-CSF (GM-CSF, BioLegend) for CD11c positive dendritic
cells. After 1 week, bone marrow derived dendritic cells were
obtained. Normal human colon epithelial cells and colon cancer cell
lines were purchased from ATCC and cultured in their appropriate
growth media according to the ATCC instructions. Media and
supplements are from Invitrogen. hTERT-BJ1 Telomerase Fibroblasts
(hTERT) were originally purchased from Clontech and were cultured
in 4:1 ratio of DMEM:Medium 199 supplement with 10% FBS, 4 mM
L-Glutamine and 1 mM sodium pyruvate at 37.degree. C. in a 5%
CO.sub.2-humidified atmosphere.
[0154] Gene Array Analysis:
[0155] Total RNA was isolated from cells or tissues with RNeasy
Mini kit (74104, Qiagen, Valencia, Calif.). RNA quality was
analyzed by Bionalyzer RNA 6000 Nano (Agilent Technologies, Santa
Clara Calif.). Gene array analysis was examined by Illumina Sentrix
BeadChip Array (Mouse WG6 version 2) (Affymetrix, Santa Clara
Calif.) at the Oncogenomics Core Facility, University of Miami. Raw
intensity values from Illumina array are uploaded on GeneSpring.TM.
software from Agilent. Values are Quantile normalized and log 2
transformed to the median of all samples. Significantly
differential expressed genes are computed using the Student's
t-test and selected using threshold of P-value .ltoreq.0.05.
Hierarchical Clustering and visualization of selected
differentially expressed genes is performed on GeneSpring using
Pearson Correlation distance method and linkage was computed using
the Ward method. Gene expression profiles were processed and
statistical analysis was performed at the Sylvester Comprehensive
Cancer Center Bioinformatics Core Facility University of Miami.
[0156] Histopathology.
[0157] Mice were sacrificed and the colon tissues were fixed in 10%
formalin for 48 hours. All processes for paraffin block and
Hematoxylin and Eosin staining (H&E) were performed at the
pathology research resources histology laboratory in University of
Miami.
[0158] Statistical Analysis:
[0159] All statistical analysis was performed by Student's t test
unless specified. The data was considered to be significantly
different when P<0.05.
[0160] Supplemental Information
[0161] Quantitative Real Time PCR (qPCR):
[0162] Total RNA were reverse-transcribed using M-MLV Reverse
Transcriptase (Promega). Real-time PCR was performed using Taqman
Gene Expression Assay (Applied Biosystems) for innate immune genes
and inflammatory cytokines (Cxcl10: Mm00445235, Ifit3:
Mm0170846).
[0163] Immunoblot Analysis:
[0164] Equal amounts of proteins were resolved on sodium dodecyl
sulfate (SDS)--Polyacrylamide gels and then transferred to
polyvinylidene fluoride (PVDF) membranes (Millipore). After
blocking with 5% Blocking Reagent, membranes were incubated with
various primary antibodies (and appropriate secondary antibodies).
The image was resolved using an enhanced chemiluminescence system
ECL (Thermo Scientific) and detected by autoradiography.
Antibodies: rabbit poyclonal antibody against STING was developed
in the laboratory; other antibodies were obtained from following
sources: .beta.-actin (Sigma Aldrich), p-IRF3 (Cell Signaling),
IRF3 (Santa Cruz Biotechnology), p-TBK1, TBK1, p-p65, p65.
[0165] Interferon .beta. ELISA Analysis:
[0166] Interferon .beta. (IFNB, IFN.beta.) Elisa was performed
using either the IFN.beta. human ELISA Kit from Invitrogen or the
Human IFN.beta. ELISA Kit from PBL Interferon Source following the
manufacturer's protocol.
[0167] Immunofluorescence Microscopy:
[0168] Cells were cultured and treated in their appropriate media
on Lab-Tek II chamber slides. Cell were fixed with 4%
paraformaldehyde for 15 minutes in at 37.degree. C. and
permeabilized with 0.05% Triton X-100 for 5 minutes at room
temperature. Immunostaining was performed with rabbit-anti-STING
polyclonal followed by fluorescence conjugated secondary antibodies
(FITC-goat-anti-rabbit) (Invitrogen). Images were taken with Leica
SP5 confocal microscope at the Image Core Facility, University of
Miami.
[0169] Northern Blot Analysis:
[0170] Northern blot was performed with 5 .mu.g of polyA RNA using
NorthernMax.RTM.-Gly Kit (Ambion). Briefly, RNA was resolved in 1%
Glyoxal gels, transferred to the BrightStar.RTM.-Plus Nylon
[0171] Discussion
[0172] Demonstrated here is a protective role for STING in the
prevention of CAC induced by AOM/DSS carcinogenic treatment. Data
indicates that this event may occur in large part through STINGS
ability to control the production of IL-22BP. Following tissue
damage, for example by DSS, IL-22 is induced and manifests
protective, wound healing effects, including the promotion of
tissue regeneration. However, if left uncontrolled, IL-22 can also
endorse tumor development. Thus, IL-22 is tightly regulated by
secreted IL-22BP, which is expressed by CD11c.sup.+ dendritic
cells. The importance of IL-22BP in controlling IL-22 has been
emphasized through observing that IL-22BP-deficient mice are also
susceptible to AOM/DSS induced CAC, similar to STING-deficient
mice. Nevertheless, IL-22 may have dual functions since mice
lacking IL-22 have also been reported to exhibit enhanced
inflammatory responses when treated repeatedly with DSS, plausibly
because complete loss of IL-22 may cause a delay in intestinal
repair which in turn may actually aggravate inflammatory processes.
The production of IL-22 BP can be suppressed by IL-18, which is
known to be induced early after DSS-induced intestinal damage.
Accordingly, IL-18-deficient mice are also susceptible to colon
cancer, presumably through chronic suppression of IL-22 activity,
by unregulated IL-22BP, which may mimic the situation observed with
IL-22-deficient mice. Nevertheless, the control of IL-22BP remains
to be fully clarified since down regulation of IL-22BP has also
been reported to occur in the absence of IL-18. In addition, it is
known that loss of the TLR and IL-1R/IL-18R adaptor MyD88 also
renders mice sensitive to CAC, in part due to loss of IL-18R
signaling. Finally, susceptibility to AOM/DDS-induced CAC has been
shown to be enhanced in mice lacking Caspase-1, the adaptor PYCARD
(Apoptosis-associated speck-like protein containing a CARD; ASC) or
nucleotide-binding domain, leucine rich repeat and pyrin domain
containing proteins 3 and 6 (NLRP3/6), presumably since Pro-IL-18
produced by epithelial cells or dendritic cells requires cleavage
prior to secretion into an active form.
[0173] Data here indicates that IL-18 is inducible by dsDNA, or
CDN's, or by AOM/DMH in a STING-dependent manner. Similar to the
situation with IL-22, it is proposed that intestinal damage
triggers STING activity (as a consequence of DNA damage or even
from microbial ligands such as CDN's or DNA). This results in the
up-regulation of IL-18 and down-regulation of IL-22BP, which would
enable IL-22 to promote tissue repair. However, similar to the
situation with IL-22, long term loss of STING may delay wound
repair, facilitate microbial invasion trigger inflammation which
would actually aggravate tumorigenesis. It was noted that IL-18
expression was not totally ablated in tumors from SKO mice,
presumably since the expression of this cytokine could be induced
by other pathways. Despite this, IL-22BP levels remained low in SKO
mice demonstrating the importance of STING in IL-22BP regulation.
Collectively, the data indicates that STING plays a key role in
controlling intestinal tissue damage and CAC through regulating
IL-22BP's suppression of IL-22.
[0174] Given that loss of STING invokes a pro-tumorigenic state, at
least in part through an inability to transiently promote tissue
repair or to signal DNA damaging events to the immune system via
secretion of cytokines, the expression and function of STING in
normal and cancer-related colon cells was explored. The study
indicated that STING was expressed in the majority of colon cancer
cells analyzed. However, it was observed that STING function was
almost completely defective in greater than 80% of the examined
cells. Defects in STING signaling were also observed in a wide
variety of other tumor cells studied (FIG. 14). STING may associate
with nucleic acids while CDN's are potent stimulators of STING
activity. Cytoplasmic DNA can bind to the synthase cGAS and
generate CDN's which then bind to and activate STING. This event
invokes STING trafficking with TBK1 via non-canonical autophagy
processes, to endosomal regions harboring the transcription factors
IRF3 and NF-.kappa.B, resulting in cytokine activation. The data
indicates that STING did not respond to transfected DNA and in many
instances failed to translocate. In these situations a lack of IRF3
activity and translocation was observed. Interestingly, loss of
STING trafficking coincided with a loss of cGAS expression (in
greater than 30% of cases), presumably since CDN's were unavailable
to facilitate STING function. In other situations, defects in
NF-.kappa.B activity were observed. Since both NF-.kappa.B and IRF3
activity are required for the optimal production of type I IFN and
other cytokines, loss of either or both of these pathways would
have detrimental effects on STING's ability to stimulate the
transcription of host defense genes, such as IL-18 or type I IFN,
required for efficient anti-tumor T cells responses. It is proposed
that loss of STING signaling may enable DNA damaged cells to escape
immune surveillance and even promote inflammatory events due to an
inability to repair damaged intestinal walls which may be
vulnerable to invading microbes.
[0175] Finally, it has been previously shown that STING plays a key
role in protecting against DNA virus infection. Since it was
observed that STING function was ablated in nearly all tumor
cell-lines examined thus far, these cells' susceptibility to HSV1
and vaccinia virus (VV) infection was examined. The study indicated
that colon cancer cells harboring defects in STING function were
highly sensitive to HSV1 and vaccinia virus infection. A number of
oncolytic viruses, including HSV1, are being considered in the
clinic as anti-tumor therapeutics, although understanding the
mechanisms of action remain to be fully determined. The data here
provides information on the causes of intestinal tumorigenesis and
may provide prognostic information to dictate the success of
oncolytic viral therapy, and even disease outcome including
response to chemotherapeutic treatments.
Example 2
STING Function in Colorectal Adenocarcinoma
[0176] Defective STING Signaling in Colorectal Adenocarcinoma
Cells:
[0177] STING-deficient mice have been reported to be prone to
AOM/DSS-associated CAC. However, whether STING function is
deregulated to any extent in human colorectal adenocarcinoma (CA)
is unknown. To start to evaluate this, STING expression was
examined by immunoblot in a variety of CA cells, generated from
cancers diagnosed at various stages as described by Duke's system.
Results indicated that STING was expressed in 10 out of 11 cell
lines examined, albeit at varying levels (FIG. 4A). To correlate
expression levels with STING function, cells were transfected with
dsDNA to activate STING signaling, or with dsRNA (polyI:C) to
activate the RIG-I like pathway. Type I IFN expression was measured
by ELISA, which is known to be STING-inducible. It was noted that
all 11 CA cells responded poorly to dsDNA-triggered type I IFN
production (FIG. 4B). It was confirmed that all cells were
transfected adequately using FITC-labeled dsDNA activator and
immunofluorescence analysis (FIG. 18). This was in contrast to
control hTERT cells or normal colon epithelial cells (FHC), which
when transfected with dsDNA did express IFN.beta.. In contrast, 8
of the 11 CA cells were able to produce type I IFN, in various
amounts, in response to dsRNA, indicating that the RIG-I-Like
pathway retained function in the majority of cases examined (FIG.
4B). A similar finding was noted upon examination of CXCL10 mRNA
production by RT-PCR, although some CXCL10 was detected, albeit in
low levels, in LoVo and HT29 in response to STING-dependent dsDNA
transfection (FIG. 4D). To extend these findings further,
IL-1.beta. production was measured in the CA cells since it was
previously noted that carcinogen triggered DNA damage can induce
IL-1.beta. through STING-signaling. Loss of IL-1.beta. has been
shown to render mice susceptible to CAC due to wound healing
responses being impaired. This study indicated that IL-1.beta. was
produced in the normal hTERT and FHC cells by dsDNA, indicating the
importance of STING-activity in this process. However, only 3 out
of the 11 CA cells appeared able to produce IL-1.beta. in response
to dsDNA treatment, again suggesting that STING function is
defective in the majority of CA cells examined (FIG. 4G). SW48,
which lacked STING expression, did not appear responsive to dsDNA
transfection in any capacity. RNAi treatment confirmed that the
upregulation of these cytokines was STING-dependent (FIG. 19A-C).
Given this data, a more detailed analysis of dsDNA-dependent STING
signaling in CA cells was performed, by microarray analysis. CA
cells were selected based on their ability to exhibit some STING
function or not. For example, data from FIG. 4D, indicated that
HT29 and LoVo cells were partially able to produce CXCL10 in
response to dsDNA. In contrast, SW480 and HT116 were noted to be
unable to produce CXCL10 to any significant level. Microarray
analysis revealed that all the CA cells examined did not respond to
dsDNA signaling as efficiently as control FHC cells, and confirmed
the RT-PCR analysis (FIG. 4E, 4F). For instance, the level of
CXCL10 was significantly higher in the control FHC cells compared
to the CA cells analyzed. However, HT29 cells did appear able to
retain some response to cytosolic dsDNA, more than any of the other
CA cells examined, especially when compared to SW480 or HT116 (FIG.
4E, F). While HT29 was able to produce IFN.beta. moderately as
determined by microarray analysis, IFN.beta. protein production was
not readily evident by ELISA, perhaps due to low level expression,
which was similarly observed even in the FHC controls (FIG. 4B).
Nevertheless, taken together, the data indicates that a majority of
CA cells exhibit defective STING-dependent signaling with only
SW1116, LS123, LoVo and HT29 exhibiting some low level
activity.
[0178] Loss of IRF3 Function in CA Cells:
[0179] To examine the extent of defective STING signaling in CA
cells, immunofluorescence and immunoblot analysis was performed to
evaluate NF-.kappa.B and IRF3 function. In the presence of dsDNA,
STING rapidly undergoes trafficking from the ER, along with TBK1,
to perinuclear-associated endosomal regions, containing NF-kB and
IRF3, in a process resembling autophagy (Ishikawa and Barber, 2008;
Konno et al., 2013). This event accompanies STING phosphorylation
and degradation, likely to avoid sustained STING-activated cytokine
production which can manifest inflammation. This approach confirmed
that STING could traffic and undergo phosphorylation and
degradation in the control hTERT and FHC cells, following treatment
with dsDNA (FIGS. 5A and 5D, left panel). In these cells, TBK1
became phosphorylated as well as its cognate target IRF3 and the
p65 subunit of NF-.kappa.B (FIG. 5D, left panel). IRF3 and p65 were
also noted to translocate into the nucleus, as expected (FIG. 5B,
5C). A comparable effect was observed using SW1116 and LS123 CA
cells which exhibited modest dsDNA-dependent IL-1.beta. induction,
confirming that the STING pathway retained some function in these
two cells (FIG. 5A-D and FIG. 4C, D). Similarly, HT29 and LoVo
displayed similar IRF3 translocation, but lacked p65 translocation.
This likely explained that the defect in dsDNA-mediated innate
immune gene induction rested in the inability of STING to trigger
p65 translocation (FIG. 5A-D and FIG. 4E,4F). However, it was noted
that the other CA cells, such as SW480, SW1417, SW48 and HT116,
exhibited very little STING activity or trafficking. Similarly,
little evidence of TBK1 or IRF3 phosphorylation/translocation was
noted. Some indication of p65 phosphorylation was revealed, for
example in SW480, but translocation of this transcription factor
was not evident in any of these cells. STING expression was not
observed in SW48 cells as previously described (FIG. 4A, 5A, 5D).
This data indicates that dsDNA-signaling is affected at various
points of the STING pathway. For example, STING retains some
activity and ability to traffic and escort TBK1 to IRF3, as in HT29
or LoVo cells, but NF-kB signaling is affected. In contrast, STING
does not appear to undergo any phosphorylation or trafficking in
SW480, SW1417, SW48 or HT116 cells, indicating that STING function
is impeded upstream of IRF3/NF-kB interaction.
[0180] CA Cells Exhibit Defective cGAS Expression:
[0181] Loss of STING trafficking in SW480, SW1417, SW48 or HT116
cells could indicate a problem with STING function in the ER,
perhaps involving a mutation that would render STING unable to
interact with CDNs. Conversely, the breakdown in STING-signaling
could occur upstream and involve the synthase cGAS, which generates
CDNs following association with dsDNA, to augment STING function.
To evaluate this, the entire STING genome within all 11 CA cells
was sequenced (introns and exons comprise approximately 7.2 kb on
chromosome 5q31.2). Sequence analysis indicated that 2 of the 11 CA
cells (LoVo and SW480) exhibited a previously reported HAQ STING
variant (Jin et al., 2011; Yi et al., 2013), which occurs in
approximately 20% of the population, and which has been reported to
be partially defective when overexpressed in 293T cells, yet is
able to function normally in the presence of CDNs (FIG. 23). The
remainder of the STING genes analyzed represented the R272 encoded
product, which has not been reported to exert any defects in
function and which represent approximately 85% of the population.
Collectively, these findings do not suggest the existence of a
major mutation in the STING gene contained within the CA cells and
suggest that a defect upstream of STING, for example at the level
of cGAS could plausibly be prevalent. We thus started to examine
the expression and activity of cGAS in CA cells. An RT-PCR assay
was developed and principally measured cGAS mRNA levels. The
results indicated that, of the 11 CA cells examined, cGAS
expression was absent in 5 (55%) of them (LS174T, SW480, SW1417,
SW48 and HT116) (FIG. 13A). This data was confirmed via immunoblot
and immunohistochemistry analysis using an antibody to cGAS (FIG.
13A, FIG. 13C). A qPCR examination of 48 human colon adenocarcinoma
samples similarly indicated low to undetectable level of cGAS in 15
of 48 samples (31%) (Supplemental FIG. 13B). Our findings could be
explained through loss of the cGAS gene. However, sequencing
analysis similarly indicated that no major mutations or deletions
existed within the genome encoding the cGAS gene (FIG. 24). In view
of this, it was examined whether cGAS expression was suppressed by
epigenetic phenomena, such as by hypermethylation of the cGAS
promoter region (Lao and Grady, 2011; Mitchell et al., 2014).
Indeed, databank analysis indicated the presence of considerable
CpG islands within the cGAS promoter region (FIG. 20A). Control
hTERT, or cGAS-defective LS174T, SW480, SW1417, SW48 or HT116 cells
were thus treated with the de-methylating agent
5-Aza-2'-deoxycytidine (5AZADC) for 5 days, and cGAS mRNA levels
again evaluated. The study indicated that cGAS expression was
rescued in 2 of the 5 cells examined (SW480 and HT116) (FIG. 13B).
The sequencing of bisulfite converted genomic DNA retrieved from
normal and CA cells confirmed significant hypermethylation within
the cGAS promoter region of CA cells where cGAS expression is
suppressed (FIG. 20B). It is not yet clear why expression levels of
cGAS are muted in the remainder of the CA cells (LS174T, SW1417,
SW48) but suppression could speculatively involve other epigenetic
modifications such as histone modifications (Jin and Robertson,
2013). Accordingly, treatment of these cells with histone
deacetylase or histone-lysine methyltransferases inhibitors
partially rescued cGAS mRNA expression in CA cells examined (FIG.
20C). It may also be apparent that alternate mechanisms of cGAS
suppression exist, such as those involving miRNAs. To determine if
reconstitution of cGAS expression rescued STING-dependent dsDNA
signaling, control hTERT or SW480, HT116 (cGAS rescued by 5AZADC)
or LS174T (cGAS not rescued by 5AZADC) CA cells were examined. It
was observed that the 5AZADC-treated cGAS-rescued SW480, HT116 CA
cells, but not LS174T cells regained phosphorylation of TBK1 and
IRF3, with concomitant phospho-IRF3 translocation (FIG. 15C, D).
These effects were reflected in modest expression of type I IFN and
IL-1.beta. in the 5AZADC treated SW480 and HT116 CA cells (FIG.
15E, F). Thus, demethylating agents may be able to partially rescue
STING-dependent innate immune gene induction in select CAs.
[0182] The question arises as to why the STING-signaling pathway
may be inhibited in colon adenocarcinoma. Recently, it was shown
that STING-deficient cells and mice are sensitive to AOM-induced
DNA damage. In this situation, the STING pathway may play a role in
the DNA-damage response pathway, to induce the production of
cytokines which facilitate tissue repair or damaged cell removal
(Chatzinikolaou et al., 2014; Kidane et al., 2014; Lord and
Ashworth, 2012). As such, innate immune induction of CA cells in
response to DNA damaging agents was examined. As shown in FIG. 21,
the carcinogens AOM and DMH were able to induce the production of
type I IFN in normal colon epithelial (CCD841) and in LS123 (which
exhibited partial STING activity; FIGS. 4C and 4D). However, CA
cells which exhibited defective STING activated IRF3 or NF-kB
activity were unable to generate type I IFN in response to AOM or
DMH. Thus, the inhibition of the STING pathway may enable DNA
damaged cells, harboring mutations, to escape part of the DNA
damage response and the immune surveillance machinery to progress
into a tumorigenic state.
[0183] Tumors with Defective STING-Signaling are Sensitive to Viral
Oncolysis:
[0184] The inventors have previously shown that loss of STING
signaling in vitro or in vivo renders cells or mice, respectively,
extremely sensitive to Herpes simplex virus (HSV) infection. HSV,
containing a dsDNA genome of 375 kb is presently being evaluated in
clinical trials as a therapeutic agent for the treatment of cancer
(Kolodkin-Gal et al., 2009). However, the mechanisms of oncolysis
remain to be fully determined and there is no evaluation,
presently, for determining the efficacy of HSV antitumor treatment.
Given that it has been previously shown that STING signaling plays
a critical role in host defense responses to HSV infection, and
that STING activity is defective in numerous CA cells, it was
postulated that the ability of STING to signal may affect outcome
to HSV therapy. To start to address this CA cells or control hTERT
and FHC were infected with HSV1 lacking the .lamda.34.5 encoding
product that is presently being evaluated as an oncolytic agent,
including against colon cancer as well as melanoma. The .lamda.34.5
viral protein has been proposed to suppress host defense responses,
although the mechanisms remain to be fully clarified. Thus,
HSV1.lamda.34.5 does not robustly repress innate immune signaling
events and potently triggers STING-dependent innate immune gene
induction, including type I IFNs. This analysis indicated that
similar to our dsDNA transfection results, HSV1.lamda.34.5 induced
the production of IFN.beta. mRNA significantly in control hTERT and
FHC cells, as well as SW1116 and LS123 CA cells (FIG. 16A).
However, little type I IFN was induced in the remainder of the CA
cells, including SW480 and HT116, deficient in cGAS expression. The
ability to induce type I IFN inversely correlated with
HSV1.lamda.34.5 replication, due to the induced anti-viral effects
(FIG. 16B). Furthermore, cells such as SW480 and HT116 underwent
rapid cell death, likely due to robust viral replication, while
control cells and cells with partial STING function (SW1116 and
HT29) were significantly more refractory (FIG. 15C). The
experiments were followed up by infecting CA cells with HSV
expressing the luciferase gene that contained .lamda.34.5
(HSV-Luc). This data confirmed that CA cells exhibiting defective
STING-signaling such as SW480 and HT116 enabled more viral-induced
luciferase expression (FIG. 16D). siRNA treatment further confirmed
that the IFN.beta. responses induced by HSV1.lamda.34.5 in normal
and STING functional CA cells are STING dependent (FIG. 19D). Of
note is that HSV1 is not the only DNA virus to be considered as an
oncolytic therapeutic agent to treat cancer. Other candidate
viruses under consideration, including as a therapeutic against
colon cancer, comprise Vaccinia Virus (VV), a dsDNA virus with 190
kb genome that replicates in the cytoplasm of infected cells. To
evaluate whether VV can trigger host innate immune response in the
absence of functional STING signaling, we infected CA cells with
partial STING signaling capacity (SW116 and HT29) or completely
defective STING signaling (SW480, HT116) with VV. Similar to the
situation using HSV1.lamda.34.5, VV triggered type I IFN and CXCL10
production only in the control cells or CA cells with partial STING
signaling ability and not in cells with loss of STING function
(SW480 and HT116) (FIG. 16E, F). The data herein indicates that CA
cells with defective STING-signaling are highly susceptible to HSV1
and VV oncolytic activity. Thus, it is plausible that being able to
measure the presence or absence of STING/cGAS expression may help
predict the response of patients with certain cancers to viral
oncolytic therapy.
[0185] Predicting Outcome to Viral Oncolytic Therapy.
[0186] The present data indicates that the outcome of oncolytic
virotherapy involving DNA-based viruses such as HSV1 may be
predicted by the presence or absence of STING/cGAS expression.
Since the STING pathway naturally requires the presence of STING
and cGAS to function, and since it has been observed that STING
and/or cGAS may be absent in 30-55% of colon cancer, being able to
measure the presence of these two gene products may therefore
indicate the effectiveness of DNA-viral oncotherapy. This could be
achieved using RT-PCR methodology but biopsied tissue may contain
infiltrating hematopoietic cells that contain normal STING/cGAS
expression. Thus, analysis of STING and/or cGAS protein or RNA
expression within the cancer cell itself would provide more
accurate information into the status of STING function. Since an
effective antibody to detect cGAS protein was not identified, a RNA
in situ hybridization assay was designed using RNAscope technology
that can detect the single levels copies of an mRNA within
individual cells. By labeling the STING probe green (FITC), and the
cGAS probe red (Cy5), both probes were detectable in the same assay
and the mRNA levels of STING and cGAS within the identical cell
could be effectively quantitated. To test the assay, control cells
or cGAS positive (SW1116 or HT29) or negative (SW480 and HT116) CA
cells were incubated with RNA probes recognizing cGAS (red) or
STING (green) mRNA. This study indicated that STING and cGAS
expression could be detected and quantitated in the control (hTERT
and FHC) and STING/cGAS positive (SW1116 or HT29) CA cells using
the RNAscope (FIG. 17A, C). However, only STING was observed in the
cGAS negative (SW480 and HT116) CA cells (FIG. 17A, C). STING was
not detectable in SW48 cells, as expected, using this assay (FIGS.
5A and 17A, C). This data also correlated with our previous
expression analysis of cGAS in these cells by RT-PCR (FIG. 13A).
Moreover, cGAS expression was observed by RNAscope in those CA
cells where cGAS mRNA production was rescued following treatment
with 5AZADC (SW480 and HT116); FIG. 17B, D). Thus, fluorescence in
situ hybridization analysis may be able to predict the outcome to
oncolytic viral therapy depending on the presence or absence of
cGAS or STING.
[0187] To further follow up on this assay, normal hTERT, or cGAS
positive (SW1116 or HT29) or negative (SW480 and HT116) CA cells
were paraffin embedded, as well as SW48 which had both cGAS and
STING expression missing. This situation may mimic situations where
biopsied and paraffin embedded patient derived material required
analysis. The experiment was again readily able to detect using the
RNA probes both STING and cGAS expression in control, SW1116 and
HT29 cells, as before, and only STING in the cGAS negative SW480
and HT116 CA cells (FIG. 17E, F). Neither cGAS nor STING was
observed in the double negative SW48 line (FIGS. 4A and 17E, F).
This assay was further tested on 12 normal and 80 CA samples in
paraffin embedded tissue microarray (TMA) and it was observed that
STING was lost in 14% of CA samples and cGAS 15% of CA samples.
Both STING and cGAS were lost in 9% of CA samples (FIG. 17G, H).
However, it was noted that STING expression and/or function was
absent in a variety of other tumors, indicating that suppression of
this pathway may be widespread in human cancer (FIG. 23). Thus,
RNAscope analysis of STING/cGAS duel expression from paraffin
embedded tissue may help predict the outcome of select viral
oncolytic therapy in vivo, as determined next.
[0188] In Vivo Analysis of CA Cells with Defective STING Signaling
to HSV11.34.5 Therapy.
[0189] To correlate the in vitro oncolytic effect of HSV1
.lamda.34.5 in vivo, nude mice were subcutaneously inoculated with
CA cells harboring partial (SW1116 or HT29) or defective (SW480 and
HT116) STING signaling. HSV1.lamda.34.5 was then administered
intratumorally (FIG. 18A). It was observed that tumors exhibiting
partial STING-signaling (SW1116 and HT29) were refractory to viral
oncolytic treatment (FIG. 18B, C). However, tumors derived from CA
cells with defective STING-signaling were noted to be extremely
susceptible to virus treatment (FIG. 18D, E). This data indicates
that the activity of the STING pathway may predict the outcome of
HSV-related oncolytic therapy against colon as well as other
cancers.
[0190] Discussion
[0191] The STING controlled signaling pathway is essential for
facilitating innate immune gene transcription in response to the
recognition of cytosolic DNA species. STING activity can be
triggered by CDNs such as cyclic-di-AMP or cyclic-di-GMP produced
from intracellular bacteria such as Listeria monocytogenes or by
cyclic-di-GMP-AMP (cGAMP) manufactured by the synthase cGAS
following association with cytosolic dsDNA species (Sun et al.,
2013; Woodward et al., 2010). Such DNA can represent the genome of
DNA pathogens, such as HSV-1 or bacteria such as mycobacterium
tuberculosis, as well as self DNA leaked from the nucleus of DNA
damaged cells. STING-deficient mice, while viable, are extremely
sensitive to lethal infection by a variety of pathogens. However,
chronic STING activity has been shown to cause a diversity of
autoinflammatory disease, through the overproduction of
pro-inflammatory cytokines. Indeed, inappropriate overstimulation
of STING has even been shown to aggravate inflammation driven skin
cancer (Ahn et al., 2014). However, transient STING activity has
been shown to be essential for mediating the generation of
anti-tumor T-cell responses. Data suggests that STING, in
professional antigen presenting cells (CD8+ dendritic cells)
becomes extrinsically activated by the DNA of engulfed dying tumor
cells which results in the triggering of cytokines such as type I
IFN, which facilitates cross-presentation and CTL priming.
Correspondingly, the therapeutic administration of CDNs,
intratumorally, has been shown to repress tumor growth, presumably
by facilitating DC-dependent CTL production (Corrales et al., 2015;
Woo et al., 2014). STING may also play a role in influencing the
anti-tumor effects of checkpoint inhibitors such as PD1, although
the mechanisms remain to be determined.
[0192] The inventors have also recently demonstrated that
STING-deficient mice are susceptible to carcinogen-aggravated CAC
(Ahn et al., 2015). In this situation, evidence indicates that
damaged DNA can trigger STING intrinsic activity, perhaps by
leaking out of the nucleus or through other mechanisms that remain
to be clarified. Presumably, this event would augment cytokine
production that would attract the immune system to the damaged
cell(s). Eradication of such cells may ensue, as well as the
stimulation of cytokine and growth factor dependent tissue repair.
Data suggests that STING can trigger the production of cytokines
that facilitate wound repair in the gut, such as IL-1.beta.. Such
cytokines are processed by nucleotide-binding
oligomerization-domain protein like receptors (NLRs) such as NLRP3
and NLRP6, which interact with inflammasome-associated ASC and
caspase-1 to process IL-1.beta. and IL-18. These pro-inflammatory
cytokines are secreted and bind to receptors mainly requiring MyD88
for signaling. IL-18 production can suppress IL-22BP, which is
responsible for inhibiting the wound repair activity of IL-22. Loss
of ASC, caspase-I, MyD88 or IL22BP can increase tumorigenesis in
colitis-associated colon cancer models, similar to loss of STING
(Elinav et al., 2011; Huber et al., 2012; Salcedo et al., 2010).
STING may thus work in concert with inflammasome processing.
[0193] Thus, loss of STING suppresses tissue healing and damaged
mucosal lining may enable the infiltration and expansion of
bacteria with enhanced genotoxic ability which would aggravate
STING-independent inflammatory responses. The generation of ROS by
overactive, infiltrating immune cells may enhance DNA damaging
processes and facilitate mutational inactivation of TSGs or the
mutational activation of growth stimulatory proteins such as k-ras.
Thus, intrinsic STING-signaling may play a key role in preventing
the development of cancer through responding to DNA damage and
alerting the immune surveillance machinery. In addition, extrinsic
STING activity in DCs is also required for the generation of
anti-tumor CTLs. This places STING in a pivotal role in the host
anti-cancer defense arsenal.
[0194] Given this, the expression and regulation of STING signaling
in colon cancer was analyzed and found frequent suppression of
STING activity. These events inhibited the production of DNA-damage
dependent cytokine production, which may enable the damaged cell to
escape the attention of the immune surveillance system. Such cells
may evade eradication and further genetic mutation events may
accrue to enhance the tumorigenic process. The inhibition of STING
signaling was observed to mainly involve the suppression of STING
expression, or of the synthase cGAS. Significant mutation or
deletion events involving the STING or cGAS genes were not
observed, but rather observed frequent transcriptional suppression
involving hypermethylation of the promoter regions. Cytosolic DNA
signaling was partially rescued using demethylating agents which
regained cGAS expression in some but not other CA cells. However,
it remains unclear whether the rescue of STING signaling in cancer
cells may afford better responses to anti-cancer agents. Further,
that cGAS and in some cases STING expression was not rescued by
demethylating agents may indicate other forms of epigenetic
silencing that requires additional characterization. In other CA
types, it was observed that the ability of STING to activate the
transcription factors NF-.kappa.B or IRF3 was impaired, by
molecular mechanisms that also remain to be determined. It is
noteworthy that a number of other genes involved in DNA repair,
such as the mismatch repair proteins MHS2 and MLH1 are also
reported to be frequently silenced in colon cancer (Chatzinikolaou
et al., 2014; Lord and Ashworth, 2012). Thus, targeting the DNA
repair machinery maybe a common requirement in cancer development.
Collectively, it was observed that STING-dependent signaling was
defective in approximately 80% or more of colon related tumors
examined. This may indicate that suppression of STING function is
also a key obligation for the tumorigenic process.
[0195] Since loss of STING may be common in tumors and may even
predict outcome to anti-cancer therapy, the inventors developed
assays to evaluate the expression levels of both STING and cGAS.
Loss of either of these two proteins appears to repress cytosolic
DNA mediated innate immune signaling. The present ability to
measure STING and cGAS mRNA expression in situ, and STING protein
expression using antibody enabled us to develop a screen that
indicated loss of one or other of these proteins in over 40% of
CAC. Such assays may be useful in predicting the effective response
rates of cancers to select therapeutic interventions. Further,
recapitulating STING signaling in tumors, via novel antitumor gene
therapy approaches, might enable such cells to reactivate host
antitumor immunity.
[0196] Accordingly, it was noticed that loss of STING signaling in
CA cells enabled the robust replication of DNA-based viruses such
as HSV1. Viruses, such as HSV1 and vaccinia virus, are presently
being used as oncolytic agents for the treatment of cancer. Such
viruses may directly destroy the tumor cell by lysis as well as
create a tumor antigen source for engulfment by APCs for the
generation of CTLs. Data indicates that STING plays a key role in
both these processes. However, the efficacy of successful oncoviral
therapy remains low, for reasons that remain unclear. Mainly,
assays based on molecular insight, that may help predict treatment
outcome have not been developed. This is because the molecular
mechanisms that explain oncolysis in cancer cells rather than
normal cells remains to be fully appraised. Evidence suggests that
innate immune signaling pathways that exert anti-viral activity may
be defective in cancer cells. Our data presented here is amongst
the first clear indication that loss of an innate signaling pathway
can predict the outcome to oncoviral therapy. Thus, utilization of
molecular biomarker assays similar to the ones portrayed here may
enable a more predictive response to the use of microbes for the
treatment of cancer. Such assays may also shed insight into whether
other STING-dependent anti-tumor therapies based on CDNs, or even
DNA-adduct based chemotherapeutic regimes, may work or not
(Mansour, 2014; Rowe and Cen, 2014). In this light, we have
recently described that the immunological benefits of using
chemotherapeutic agents such as cisplatin and etoposide
significantly involved the STING-signaling pathway. Thus, further
studies on the regulation and function of STING in cancer may
provide acumen into the molecular mechanisms of tumorigenesis as
well as provide a therapeutic target that may help in the treatment
of cancer.
Experimental Procedures
[0197] Materials.
[0198] All reagents were from Invitrogen, ThermoScientific or Sigma
unless specified.
[0199] Cell Culture.
[0200] Normal human cell and human cancer cell lines were purchased
from Lozna and ATCC respectively and cultured in their appropriate
growth media according to the instructions. Media and supplements
are from Invitrogen. hTERT-BJ1 Telomerase Fibroblasts (hTERT) were
originally purchased from Clontech and were cultured in 4:1 ratio
of DMEM:Medium 199 supplement with 10% FBS, 4 mM L-Glutamine and 1
mM sodium pyruvate at 37.degree. C. in a 5% CO2-humidified
atmosphere.
[0201] Immunoblot Analysis.
[0202] Equal amounts of proteins were resolved on sodium dodecyl
sulfate (SDS)-Polyacrylamide gels and then transferred to
polyvinylidene fluoride (PVDF) membranes (Millipore). After
blocking with 5% Blocking Reagent, membranes were incubated with
various primary antibodies (and appropriate secondary antibodies).
The image was resolved using an enhanced chemiluminescence system
ECL (Thermo Scientific) and detected by autoradiography (Kodak).
Antibodies: rabbit poyclonal antibody against STING was developed
in our laboratory as described previously in Ishikawa et al, 2008;
other antibodies were obtained from following sources: .beta.-actin
(Sigma Aldrich), p-IRF3 (Cell Signaling), IRF3 (Santa Cruz
Biotechnology), p-p65 (Cell Signaling), p65 (Cell Signaling),
p-TBK1 (Cell Signaling), TBK1 (Abcam), cGAS (Cell Signaling).
[0203] Interferon .beta. Elisa analysis.
[0204] Interferon .beta. Elisa was performed using either the
IFN.beta. human ELISA Kit from Invitrogen or the Human IFN.beta.
ELISA Kit from PBL InterferonSource following the manufacturer's
protocol.
[0205] Immunofluorescence Microscopy.
[0206] Cells were cultured and treated in their appropriate media
on Lab-Tek II chamber slides. Cell were fixed with 4%
parsformaldehyde for 15 minutes in at 37.degree. C. and
permeabilized with 0.05% Triton X-100 for 5 minutes at room
temperature. Immunostaining was performed with rabbit-anti-STING
polyclonal, rabbit-anti-IRF3 (Santa Cruz Biotechnology) or
rabbit-anti-p65 (Cell Signaling) followed by fluorescence
conjugated secondary antibodies (FITC-goat-anti-rabbit)
(Invitrogen). Images were taken with Leika LSM confocal microscope
at the Image Core Facility, University of Miami.
[0207] Microarray Analysis.
[0208] Total RNA was isolated from cells or tissues with RNeasy
Mini kit (Qiagen). RNA quality was analyzed by Bionalyzer RNA 6000
Nano (Agilent Technologies). Gene array analysis was examined by
Illumina Sentrix BeadChip Array (Human HT-12_V4_Bead Chip) at the
Oncogenomics Core Facility, University of Miami. Gene expression
profiles were processed and statistical analysis was performed at
the Bioinformatics Core Facility, University of Miami. Briefly, raw
intensity values from Illumina array are uploaded on GeneSpring.TM.
software from Agilent. Values are Quantile normalized and log 2
transformed to the median of all samples. Significantly
differential expressed genes from a two-class comparison are
computed using the Student's t-test and selected using threshold of
P-value .ltoreq.0.05. Hierarchical Clustering and visualization of
selected differentially expressed genes is performed on GeneSpring
using Pearson Correlation distance method and linkage was computed
using the Ward method. Fold Change analysis was performed between
two groups and differentially expressed genes were selected based
on threshold of Fold Changes.
[0209] Quantitative Real-Time PCR (qPCR).
[0210] Total RNA was reverse transcribed using QuantiTect Reverse
Transcription Kit (Qiagen). Real-time PCR was performed with the
TaqMan gene Expression Assay (Applied Biosystems).
[0211] Immunohistochemistry and Histological Analysis.
[0212] Tissue Microarray was purchased from Origene.
Immunohistochemistry staining was performed with rabbit-anti-cGAS
antibody following standard protocol.
[0213] HSV1.gamma.34.5 Amplification, Purification, Titration and
Infection.
[0214] HSV1.gamma.34.5 was amplified in Vero cells and purified by
sucrose gradient ultracentrifugation following standard protocol.
Plague assay using serial diluted virus was performed in Vero cells
following standard protocol. Cells were infected with
HSV1.gamma.34.5 at specific M.O.I. for 1 hours, washed and then
incubated for designated period for specific assay examination.
[0215] RNA In Situ Hybridization.
[0216] STING and cGAS RNA probed was custom designed by ACD and RNA
in situ Hybridization was performed using RNAscope.RTM. Multiplex
Fluorescent Reagent Kit for cultured cells and 2-plex RNAscope.RTM.
Reagent Kit for FFPE cells and tissue following the manufacturer's
instruction.
[0217] Mouse Treatment.
[0218] Balb/C nu/nu mice were purchased from Charles River. Tumor
cells were introduced in the flanks of Balb/c nude mice by
subcutaneous injection of 2E106 of the appropriate tumor cells and
tumors allowed to develop to an average diameter of approximately
0.5 cm. HSV1.gamma.34.5 was then be injected into the tumors every
other day for a total of three times at appropriate dosage (i.e. 50
.mu.l at 1E7). PBS was used as vehicle control. Effects on tumor
growth was monitored. Mice were euthanized when tumor diameter
exceeds 10 mm.
[0219] Bisulfite Sequencing Analysis.
[0220] Bisulfite conversion of genomic DNA was performed using
EZDNA Methylation Kit from Zymo Research followed by PCR
amplification. PCR products were then gel purified and
sequenced.
[0221] Statistical Analysis.
[0222] All statistical analysis was performed by Student's t test
unless specified. The data were considered to be significantly
different when P<0.05.
Example 3
STING Function in Melanoma
[0223] Given the findings above, the studies were extended into
evaluating STING function in melanoma, in part because such cancers
appear to be susceptible to viral oncolytic treatment, which
suggests defects in innate immune pathways. Here it is reported
that STING mediated innate immune signaling is largely impaired
both in human melanoma derived cells and in primary patient
melanoma-derived tissues. Loss of STING and/or cGAS expression in
melanoma was recurrently found, predominantly through epigenetic
hypermethylation silencing. These findings suggest that suppression
of STING signaling may be an important part of tumor development.
Moreover, loss of STING function rendered melanoma cells more
susceptible to HSV1 and vaccinia virus-mediated oncolysis.
Therefore, the development of a prognostic assay that enables the
measurement of STING or cGAS expression may lead to a better
indication of the efficacy of viral oncolytic treatment.
[0224] Recurrent Loss of STING Signaling in Human Melanoma Derived
Cell Lines.
[0225] The STING-controlled innate immune pathway has been reported
to be largely impaired in human colon cancers, an event which may
facilitate tumorigenesis (Xia T, Konno H, Ahn J, Barber G N.
Deregulation of STING Signaling in Colorectal Carcinoma Constrains
DNA Damage Responses and Correlates With Tumorigenesis. Cell
reports. 2016; 14:282-97). To evaluate whether this key pathway is
similarly defective in other types of cancer STING expression was
further examined by immunoblot in a panel of human malignant
melanomas. These results showed that STING expression was not
detectable in 3 out of 11 cell lines examined (G361, MeWo and
SK-MEL-5) and STING expression level was dramatically suppressed in
a further 3 cell lines (SK-MEL-2, SK-MEL-28 and WM115) (FIG. 26A).
The synthase cGAS resides upstream of STING and generates CDN's
capable of triggering STING function. Next, the expression of cGAS
by immunoblot was examined and it was found that this synthase was
absent in 4 out of 11 cell lines examined (A375, G361, SK-MEL-5 and
SK-MEL-24) (FIG. 26A). Real-time PCR analysis using cGAS probe
confirmed that cGAS was not detectable in A375 and SK-MEL-5, but
low level of cGAS was detected in G361 and SK-MEL-24 (FIG. 26A). To
correlate STING/cGAS expression analysis with functional STING
signaling, cells were transfected with dsDNA to activate
STING-dependent cytokine production, or with dsRNA (polyI:C) to
activate the STING-independent RIG-I like signaling pathway and
measured type I IFN expression by ELISA (Ishikawa et al., 2008).
This study indicated that all 11 melanoma cells responded poorly to
STING-dependent, dsDNA-triggered type I IFN production. Using
fluorescence microscopy analysis, it was confirmed that all cells
were indeed transfected with FITC-labeled dsDNA activator. However,
control hTERT cells and normal human melanocytes (HEMa) were able
to express high levels of IFN.beta. when transfected with dsDNA,
suggesting the STING mediated type I interferon responses were
suppressed specifically in the melanoma cells (FIG. 26B). This
finding was further supported by real-time PCR analysis, in which
dsDNA stimulated IFNB and CXCL10 induction was suppressed in
majority of the melanoma cells examined, although weak activity
were detected in SK-MEL-24 and SK-MEL-31 cells (FIG. 26C-D). In
contrast, 6 of the 11 Melanoma cells were able to produce type I
IFN and CXCL10, albeit at various levels, in response to dsRNA,
indicating that the RIG-I-Like RNA signal pathway were mostly
intact in majority of melanoma cells examined (FIG. 26C-D). Using
siRNA treatment to knock down STING expression in normal cells and
2 melanomas cell-lines (SK-MEL-24, SK-MEL31) that appeared to
retain partial STING activity, it was confirmed that the
upregulation of these dsDNA-induced cytokines was STING-dependent.
Taken together, our data indicates that STING-dependent signaling
is largely impaired in a majority of melanoma cells with only
SK-MEL-24 and SK-MEL-31 exhibiting weak STING activity.
[0226] Loss of STING Dependent TBK1-IRF3 Activation in Melanoma
Cells.
[0227] To examine the extent of STING signaling defect in melanoma
cells, IRF3 and NF-.kappa.B activation were evaluated by
immunofluorescence microscopy and immunoblot analysis. When
stimulated with dsDNA, STING rapidly undergoes translocation from
the ER, along with TBK1, to perinuclear-associated endosomal
regions, containing NF-kB and IRF3, in a procedure similar to
autophagy (Ishikawa et al., 2008, Konno et al., 2013). This
incident accompanies STING phosphorylation and degradation, almost
certainly to avoid prolonged STING-induced cytokine production
which is now known to provoke chronic inflammation (Ahn et al.,
2014). Our results confirmed that, following dsDNA treatment in
normal hTERT cells, STING translocated to perinuclear region and
underwent phosphorylation and degradation events, (FIGS. 27A and
27D, left panel). During this process, TBK1 was phosphorylated in
hTERT cells as well as its cognate target IRF3 and the p65 subunit
of NF-.kappa.B (FIG. 27D). IRF3 and p65 translocation into the
nucleus was also observed, indicating normal activation (FIGS. 27B,
C and D). A similar effect was observed in SK-MEL-24 and SK-MEL-31
cells which exhibited partial dsDNA-dependent cytokine production,
confirming that these two cell lines retained some STING function
(FIG. 27A-D and FIG. 26B-D). However, while RPMI7951 and SK-MEL-3
retained STING/cGAS expression and displayed similar IRF3
activation upon dsDNA treatment, these cells lacked p65
translocation. This observation would explain why dsDNA failed to
trigger type I IFN production, which requires both IRF3 and NF-kB
for its transcriptional activation (FIG. 27A-D and FIG. 26B-D). In
addition, in cells where STING and/or cGAS expression were not
detected (such as A375, G361, MeWo and SK-MEL-5), no evidence of
TBK1 or IRF3 phosphorylation/translocation was detected in these
cells following dsDNA treatment (highlighted by boxes) (FIG. 27B,
D). Although phosphorylated p65 was observed, no translocation of
this transcription factor into the nucleus was evident in any of
the RPMI7951, SK-MEL-3, A375, G361, MeWo or SK-MEL-5 cells (FIG.
27C-D). These results indicate that dsDNA induced STING signaling
is deregulated at various points along the pathway in many of the
melanoma cell lines examined. For example, while STING retained
some signaling activity and ability to induce the translocation of
IRF3, as in RPMI7951 and SK-MEL-3 cells, NF-kB signaling was
observed to be affected. In contrast, STING did not appear to
undergo any phosphorylation or translocation in A375, G361, MeWo or
SK-MEL-5 cells, suggesting that STING function is affected upstream
of IRF3/NF-kB activation, likely due to loss of STING and/or cGAS
expression.
[0228] RNAscope and IHC Analysis of STING/cGAS Expression.
[0229] Since the STING pathway requires the presence of STING and
cGAS, and since STING and/or cGAS expression was observed to be
absent in .about.40% melanoma cells examined, being able to measure
the presence of STING and cGAS could be useful in predicting
functional STING signaling in melanoma. Although immunoblot and
RT-PCR methodology is effective in examining STING/cGAS expression
in cultured cell lines, biopsied tissue often contains not only
tumor cells but also other cell types including infiltrating immune
cells that could retain normal STING/cGAS expression (Ishikawa et
al., 2009). Thus, analysis of STING and/or cGAS protein or RNA
expression within the cancer cell itself is necessary for accurate
evaluation into the presence of these products. As described above,
an RNA in situ hybridization assay using RNAscope technology that
can efficiently detect STING/cGAS mRNA copies within individual
cells. By using FITC-labelled STING probe (green), and Cy5-labelled
cGAS probe (red), melanoma cells were examined using RNA
fluorescent in situ hybridization (RNA FISH). Results showed that
both probes combined within the same assay effectively detected
STING and cGAS mRNA in control HEMa cells. STING mRNA was also
detected in A375, SK-MEL-24 and SK-MEL-31 cells but not in G361,
MeWo or SK-MEL-5 cells whereas cGAS mRNA was not detected in A375
or SK-MEL-5 cells (FIG. 28A). mRNA copy numbers were quantitated
with results being consistent with our previous results obtained
using our expression analysis (FIG. 26A, 28A). Thus, RNA
fluorescence in situ hybridization analysis can effectively
quantitate STING/cGAS expression simultaneously in single
cells.
[0230] mRNA expression by chromogenic in situ hybridization (RNA
CISH) of paraffin embedded melanoma cells was also evaluated. This
situation may mimic situations where biopsied and paraffin embedded
patient derived material are generally used for biomarker analysis.
This study was able to detect and quantitate both STING and cGAS
mRNA expression in SK-MEL-24 and SK-MEL-31 cells as before. In A375
cells, only STING was detected whereas cGAS was absent. STING was
not detected in G361 or MeWo cells. Both STING and cGAS were absent
in SK-MEL-5 cells (FIG. 28B). Overall RNA CISH analysis generated
similar results to RNA FISH evaluation.
[0231] Using antibody to cGAS and STING, immunohistochemistry (IHC)
analysis on paraffin embedded cells was also performed, which
confirmed cGAS and STING protein expression status in accord with
our immunoblot and RNAscope studies (FIG. 28C).
[0232] IHC Analysis of STING/cGAS Expression in Melanoma TMA.
[0233] To evaluate STING/cGAS expression in patient-derived
melanoma samples, we subsequently examined by IHC analysis a
paraffin embedded melanoma tissue microarray (TMA, MEL961,
Pantomics) that contains 8 normal skin tissues, 8 benign nevus
tissues, 56 malignant melanoma tissues and 24 metastatic melanoma
tissues. It was observed that all normal tissues expressed both
STING and cGAS. cGAS was not detected in 2 benign nevus tissues,
while STING was noted to be present in all 8 nevi. In malignant
melanoma tissues, 23.2% of melanoma samples lost STING expression,
while 16.1% of melanoma samples did not express cGAS, and both
STING and cGAS were absent in 14.3% of melanoma tissues. In more
advanced metastatic melanoma tissue, loss of both STING and cGAS
was more profound (41.7%) (FIG. 29). Given this data, suppression
of STING or cGAS expression may commonly occur in human melanoma
and plausibly other human cancers (Xia et al, 2016). In summary,
our IHC procedures may be useful for the analysis of cGAS and STING
expression in FFPE preserved clinical tumor samples.
[0234] STING/cGAS Expression May be Suppressed Through DNA
Hypermethylation in Melanoma Cells.
[0235] Loss of STING/cGAS expression could occur through either
genetic alteration or mutation. To evaluate the gene status of
STING and cGAS in melanoma cells, we sequenced the STING and cGAS
gene within all 11 melanoma cells. Sequence analysis of the entire
STING gene (introns and exons comprise approximately 7.2 kb on
chromosome 5q31.2) indicated that 7 of the 11 melanoma cells
exhibited HAQ STING variant (Jin et al., 2011, Yi et al., PloS One,
2013), which was previously reported to occur in approximately 20%
of the population. STING gene in all melanoma cells as well as
normal HEMa cell contains the R272 polymorphism, which was reported
to represent approximately 85% of the population but does not exert
any defects in STING function. Collectively, sequence analysis did
not reveal any major genetic defect in the STING gene within the
melanoma cells. Similar sequence analysis was also carried out on
cGAS exons. However, no major mutations or deletions were noted.
Taken together, genetic mutations or deletions do not seem to be
involved in STING/cGAS defective expression in melanoma cells.
[0236] In view of this, it was examined whether STING or cGAS
expression was suppressed by epigenetic processes, such as by
hypermethylation of the promoter regions (20, 21). Indeed, databank
analysis indicated the presence of considerable CpG islands within
the STING and cGAS promoter region. As such, melanoma cells lacking
either STING or cGAS expression were treated with the
de-methylating agent 5-Aza-2'-deoxycytidine (5AZADC) for 5 days and
evaluated recapitulation of STING or cGAS expression. Real-time PCR
analysis showed that cGAS expression was recovered in A375 cells as
well as SK-MEL-5 cells although at lower extent. Although SK-MEL-24
exhibited low cGAS expression by RT-PCR, 5AZADC treatment did not
seem to affect cGAS expression level of SK-MEL-24 cells
significantly (FIG. 30A). This result was again confirmed by both
immunoblot and RNA FISH analysis, in which cGAS expression was
apparently recapitulated in A375 and SK-MEL-5 cells following
5AZADC demethylation (FIG. 30B-C). In MeWo cells, STING expression
was restored by 5AZADC treatment as shown by both immunoblot and
RNA FISH analysis. However, STING remained absent in similarly
treated G361 cells as well as in SK-MEL-5 cells, although cGAS
expression was partially restored in the same treated SK-MEL-5
cells (FIG. 30A-C). Therefore DNA hypermethylation is involved in
silencing STING or cGAS expression in some melanoma cells (A375 and
MeWo). However it is not yet clear why expression levels of STING
are muted in the remainder melanoma cells (G361, SK-MEL-5). Other
epigenetic modifications such as histone modifications or other
transcription regulator factors such as miRNA could be involved in
suppressing STING and/or cGAS expression (Jin et al., 2013,
Yarbrough et al., 2014). To determine if reconstitution of
STING/cGAS expression rescued STING-dependent dsDNA signaling, IFNB
and CXCL10 induction was examined in 5AZADC treated melanoma cells
following dsDNA stimulation. Induction of both IFNB and CXCL10
production was observed in cGAS rescued A375 cells, as well as
modest expression of IFNB in STING rescued MeWo cells, concomitant
with IRF3 and STING translocation (FIG. 30D-G). Whereas no STING
function was observed in G361 or SK-MEL-5 cells following 5AZADC
treatment, confirmed that both STING and cGAS are necessary for
dsDNA stimulated cytokine production (FIG. 30D-E). Thus,
demethylating agents may be able to partially rescue
STING-dependent innate immune gene induction in select melanoma
cells.
[0237] Defect in STING Signal Renders Melanoma Cells Susceptible to
DNA Virus Infection.
[0238] STING innate immune signaling plays a critical role in host
defense responses to DNA viruses. For example, mice lacking STING
are extremely sensitive to Herpes simplex virus (HSV) infection
(Ishikawa et al., 2008, Ishikawa et al., 2009). A strain of HSV1
lacking the .gamma.34.5 gene, referred to as talimogene
laherparepvec (OncoVex, T-VEC) is presently being evaluated in
clinical trials as a therapeutic agent for the treatment of cancer
including melanoma (Andtbacka et al., 2015; Lawler et al., 2015;
Kolodkin-Gal et al., 2009). However, the mechanisms of oncolysis
remain to be fully determined and there is no evaluation,
presently, for determining the likely efficacy of HSV-based
antitumor treatment. Is was previously shown that STING activity is
defective in numerous colon cancer cells which renders cells
sensitive to DNA virus infection including HSV1. We postulated that
lack of STING function in melanomas cells may correlate with an
increased susceptibility to DNA virus infection and replication.
Plausibly, the ability of STING to effectively signal may affect
outcome to HSV-based oncoviral therapy. To start addressing this we
infected the melanoma cells or control hTERT and HEMa with HSV1
lacking the .gamma.34.5 gene similar to the strain presently being
investigated as an oncolytic agent against human melanoma. The
.gamma.34.5 viral protein has been proposed to suppress host
defense responses, although the mechanisms need to be fully
clarified. Thus, without the robust repression of the host innate
immune signaling, HSV1.gamma.34.5 is able to potently trigger
STING-dependent innate immune activation, including type I IFN
production (Ishikawa et al., 2009). Similar to dsDNA treatment,
HSV1.gamma.34.5 induced robust production of IFNB and CXCl10 mRNAs
in control hTERT and HEMa cells, as well as in SK-MEL-24 and
SK-MEL-31 cells that retained partial STING signaling (FIG. 31A-B).
However, little type I IFN production was observed in the remainder
of the melanoma cells. Loss of the ability to induce type I IFN
correlated with increased HSV1.gamma.34.5 replication, likely due
to the impaired anti-viral effects, especially in melanoma cells
lacking STING/cGAS expression (A375, G361, MeWo and SK-MEL-5) (FIG.
31C). Furthermore, cells with defective STING signal underwent
rapid cell death, likely due to robust viral replication whereas
control cells and cells with partial STING function (SK-MEL-24 and
SK-MEL-31) were significantly more resistant (FIG. 31D). This data
confirmed that melanoma cells exhibiting defective STING-signaling
enabled more HSV1 replication and lysis.
[0239] The ability of Vaccinia Virus (VV) to activate host innate
immune signaling in the absence of STING function in melanoma cells
was also examined. VV, a dsDNA virus with 190 kb genome that
replicates in the cytoplasm of infected cells, is another candidate
DNA virus that is currently under evaluation as an oncolytic
therapeutic agent to treat cancer (Rowe et al., 2014). Similar to
our observations using HSV1.gamma.34.5, VV triggered type I IFN and
CXCL10 production only in the control cells and melanoma cells with
partial STING function but not in cells with loss of STING/cGAS
expression (A375, G361, MeWo and SK-MEL-5). Our results indicate
that melanoma cells with defective STING-signaling are highly
susceptible to HSV1 and VV infection. Thus, it is plausible that
melanoma lacking STING/cGAS expression are more sensitive to DNA
virus oncolytic activity and being able to measure STING/cGAS
expression in melanoma tissue may help predict the response of
patients to selected viral oncolytic therapy.
[0240] In Vivo Analysis of Melanoma Cells to HSV1.gamma.34.5
Therapy.
[0241] Our in vitro analysis indicated that loss of STING signaling
may affect the outcome of select oncoviral therapy (FIG. 31A-D). To
further evaluate this possibility, in vivo, melanoma xenografts
were generated by subcutaneously inoculating nude mice with
melanoma cells harboring partial (RPMI7951 and SK-MEL-3) or
defective (A375, MeWo and SK-MEL-5) STING signaling.
HSV1.gamma.34.5 was then administered intratumorally and tumor
growth monitored (FIG. 32). Results showed that tumors derived from
melanoma cells with defective STING-signaling were extremely
susceptible to HSV1.gamma.34.5 treatment (FIG. 32A-B). Tumor size
decreased rapidly after HSV1.gamma.34.5 treatment. 4 out of 6 A375
tumors and 3 out of 5 SK-MEL-5 tumors diminished 2-3 weeks after
treatment (FIG. 32A-B). In contrast tumors derived from melanoma
cells exhibiting partial STING signaling (RPMI7951 and SK-MEL-3)
were refractory to viral oncolytic treatment (FIG. 32C-D). While
these tumors are slow growing in vivo, majority of mice did not
respond to HSV1.gamma.34.5 therapy at all and the animals were
sacked after the tumor burden became significant. Therefore, our
findings complement our previous studies and indicate that the
ability of measure STING function in melanoma may predict the
outcome of DNA virus-related oncolytic therapy against human
melanoma and perhaps other type of cancers.
[0242] Discussion
[0243] As reported above, STING signaling is frequently suppressed
in human colon cancer. As mentioned, loss of intrinsic STING signal
may play a key role in preventing cancer development through
inability to respond to DNA damage and alert the immune
surveillance machinery (Chatzinikolaou et al., 2014, Kondo et al.,
2013). To extend these studies, the expression and regulation of
STING signaling in melanoma was analyzed and it was similarly found
that STING-dependent cytokine production was frequently suppressed
in human melanoma. Although no significant mutation or deletion
events involving the STING or cGAS genes was observed, the
inhibition of STING signaling was found to mainly occur through
epigenetic suppression of STING and or cGAS expression. Cytosolic
DNA mediated STING signaling was partially rescued by demethylating
agent (5AZADC) treatment in some STING-defective melanoma cells,
suggesting DNA hypermethylation is one of the mechanisms for
STING/cGAS suppression. However, in other STING-defective melanoma
cells, demethylation was not effective in being able to restore
STING expression. STING and/or cGAS may selectively become targets
for suppression at various stages of cancer development, the
suppression of either being sufficient to impede STING function. It
was also noticed in some melanoma cells, that although both
STING/cGAS were expressed, the ability of STING to effectively
activate the transcription factors NF-.kappa.B or IRF3 was impaired
by molecular mechanisms that remain to be determined. Thus, STING
function can be impaired at different steps along the signaling
pathway, although epigenetic suppression of either STING/cGAS
expression seems to be common. Collectively, it was observed that
STING-dependent signaling was defective in numerous melanomas which
indicated that inhibiting STING function maybe a key obligation for
the development of melanoma, plausibly enabling such cells to evade
the immune system.
[0244] Loss of STING may be common in tumors and may even predict
outcomes to anti-cancer therapy. Accordingly, assays were developed
herein to evaluate the expression levels of both STING and cGAS,
loss of either of which will affect STING function. These assays
were validated in melanoma and showed that both RNAish based and
IHC based assays were able to measure STING and cGAS mRNA or
protein expression in melanoma cells accurately and sensitively.
Using IHC, a melanoma TMA was screened which showed loss of either
STING or cGAS in over 50% malignant and over 60% metastatic
melanomas. Loss of STING function may not be a key tumor onset
factor. However, STING does appear to be important in the
generation of cytokines in response to DNA damage (Ahn et al.,
2015, Xia et al., 2016, Ahn et al., 2014). Loss of STING function
is almost certainly important in later stages of cancer development
to escape immunosurveillance and host anti-tumor immunity,
especially beneficial in tumor metastasis. The assays described may
be useful in predicting the effective response rates of cancers to
select therapeutic interventions. Furthermore, recapitulating STING
signal in tumors, via novel antitumor gene therapy approaches, may
reactivate host antitumor immunity against escaped tumor cells.
[0245] Accordingly, it was noticed that loss of STING function in
melanoma cells rendered cells highly sensitive to DNA-virus
mediated oncolytic effect (such as HSV1). Oncolytic HSV1 is one
viral therapeutic agent in clinical application. For example,
talimogene laherparepvec (T-VEC) (Amgen) is a herpes simplex virus
type 1 (HSV-1) based OV that has been engineered to express
granulocyte-macrophage colony-stimulating factor (GM-CSF) to
increase immune recognition. Although T-VEC has shown improved
effect over traditional immune therapies for advanced melanoma, the
overall response rate is still limited. This phenomena could be
potentially due to diverse STING/cGAS expression status among
melanoma cases. Oncolytic viruses may directly destroy the tumor
cell by lysis as well as create a tumor antigen source for
activation of anti-tumor immune response (Woo et al., 2015). STING
may play key roles in both of these processes. Therefore,
utilization of STING/cGAS as molecular biomarker may enable a more
predictive response to the use of microbes for the treatment of
cancer. Such assays may also shed insight into the efficacy of
other STING-dependent anti-tumor therapies based on CDNs, or even
DNA-adduct based chemotherapeutic regimes (Zitvogel et al., 2013).
Further, gene therapies involving modification of the STING/cGAS
status may provide advantages of utilizing host innate and adaptive
defense mechanism to facilitate antitumor effects in combination
with traditional anti-tumor therapies. Thus, further studies on
STING signal in cancer development may provide insight into the
molecular mechanisms of human carcinogenesis as well as provide
novel anti-tumor therapeutic approaches.
Experimental Procedure
[0246] Materials.
[0247] All reagents were from ThermoFisher Scientific or Sigma
unless specified.
[0248] Cell Culture.
[0249] Normal human melanocytes (HEMa) and human melanoma cell
lines were purchased from ThermoFisher Scientific and ATCC
respectively and cultured in their appropriate growth media
according to the instructions. hTERT-BJ1 Telomerase Fibroblasts
(hTERT) were originally purchased from Clontech and were cultured
in 4:1 ratio of DMEM:Medium 199 supplement with 10% FBS, 4 mM
L-Glutamine and 1 mM sodium pyruvate at 37.degree. C. in a 5%
CO2-humidified atmosphere.
[0250] Immunoblot Analysis.
[0251] Equal amounts of proteins were resolved on sodium dodecyl
sulfate (SDS)-Polyacrylamide gels and then transferred to
polyvinylidene fluoride (PVDF) membranes (Millipore). After
blocking with 5% Blocking Reagent, membranes were incubated with
various primary antibodies (and appropriate secondary antibodies).
The image was resolved using an enhanced chemiluminescence system
ECL (Thermo Scientific) and detected by autoradiography (Kodak).
Antibodies: rabbit poyclonal antibody against STING was developed
in our laboratory as described previously in Ishikawa et al, 2008;
other antibodies were obtained from following sources: .beta.-actin
(Sigma Aldrich), p-IRF3 (Cell Signaling), IRF3 (Santa Cruz
Biotechnology), p-p65 (Cell Signaling), p65 (Cell Signaling),
p-TBK1 (Cell Signaling), TBK1 (Abcam), cGAS (Cell Signaling).
[0252] Interferon .beta. ELISA Analysis. Interferon .beta. ELISA
was Performed as Above.
[0253] Immunofluorescence Microscopy.
[0254] Cells were cultured and treated in their appropriate media
on Lab-Tek II chamber slides. Cell were fixed with 4%
paraformaldehyde for 15 minutes in at 37.degree. C. and
permeabilized with 0.05% Triton X-100 for 5 minutes at room
temperature. Immunostaining was performed with rabbit-anti-STING
polyclonal, rabbit-anti-IRF3 (Santa Cruz Biotechnology) or
rabbit-anti-p65 (Cell Signaling) followed by fluorescence
conjugated secondary antibodies (FITC-goat-anti-rabbit). Images
were taken with Leika LSM confocal microscope at the Image Core
Facility, University of Miami.
[0255] Quantitative Real-Time PCR (qPCR).
[0256] Total RNA was reverse-transcribed using QuantiTect Reverse
Transcription Kit (Qiagen). Real-time PCR was performed with the
TaqMan gene Expression Assay (Applied Biosystems).
[0257] Immunohistochemistry and Histological Analysis.
[0258] Tissue Microarray was purchased from Pantomics.
Immunohistochemistry staining was performed with rabbit-anti-cGAS
antibody or rabbit-anti-STING antibody following standard
protocol.
[0259] Virus Amplification, Purification, Titration and
Infection.
[0260] HSV-1 .gamma.34.5 was kindly provided by Bernard Roizman.
Vaccinia virus (vTF7-3) was kindly provided by John Rose. Virus was
amplified in Vero cells and purified by sucrose gradient
ultracentrifugation following standard protocol. Plague assay using
serial diluted virus was performed in Vero cells following standard
protocol. Cells were infected with virus at specific M.O.I. for 1
hour, washed and then incubated for designated period for specific
assay examination.
[0261] RNA In Situ Hybridization.
[0262] STING and cGAS RNA probed was custom designed by ACD and RNA
in situ Hybridization was performed using RNAscope.RTM. Multiplex
Fluorescent Reagent Kit for cultured cells and 2-plex RNAscope.RTM.
Reagent Kit for FFPE cells and tissue following the manufacturer's
instruction.
[0263] Mouse Treatment.
[0264] Balb/C nu/nu mice were purchased from Charles River and
maintained in the institutional Division of Veterinary Resources
(DVR). All experiments were performed with institutional animal
care and use committee (IACUC) approval and in compliance with
IACUC guidelines. Tumor cells were introduced in the flanks of
Balb/c nude mice by subcutaneous injection of 2E106 of the
appropriate tumor cells and tumors allowed to develop to an average
diameter of approximately 0.5 cm. HSV1.gamma.34.5 was then be
injected into the tumors every other day for a total of three times
at 1E7PFU. PBS was used as vehicle control. Effects on tumor growth
were monitored. Mice were euthanized when tumor diameter exceeds 10
mm.
[0265] Genomic DNA Sequencing.
[0266] Genomic DNA was extracted from melanoma cells as well as
normal cells using Qiagen DNeasy Kit and specific locus was
sequenced by Polymorphic DNA Technologies.
[0267] Statistical Analysis.
[0268] All statistical analysis was performed by Student's t test
unless specified. The data were considered to be significantly
different when P<0.05.
[0269] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
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Sequence CWU 1
1
8141DNAArtificial SequenceSynthetic polynucleotidemisc_featureIRF-1
Position 112025721 1taaagtaaag cgtattgtca ataaatttca ttgccacaaa g
41241DNAArtificial SequenceSynthetic
polynucleotidemisc_featureIRF-1 Position 112030068 2gaaactctgt
caacaccacc accaccacca agaacaaaag a 41341DNAArtificial
SequenceSynthetic polynucleotidemisc_featureIRF-1 Position
112034210 3gaggctacat tctgtgcaat attgcaatag tctgaatgca a
41441DNAArtificial SequenceSynthetic
polynucleotidemisc_featureNF-KB Position 112038354 4cgcgttgaga
agagggagaa gactagaagg agacagctgc a 41541DNAArtificial
SequenceSynthetic polynucleotidemisc_featureNF-KB Position
112039068 5ccgaatttat ggaaaagtaa aagtaaaatt tgaagctact t
41641DNAArtificial SequenceSynthetic
polynucleotidemisc_featureSTAT1 Position 112046243 6gagggacagt
ccaggcagtt ctgtgcgtgt tcactgttta g 41741DNAArtificial
SequenceSynthetic polynucleotidemisc_featureIRF-7 Position
112027291 7tctcccatct tagcctggga ctcccatctg ggaccacaga t
41841DNAArtificial SequenceSynthetic
polynucleotidemisc_featureIRF-7 Position 112036090 8gataagaata
catacgtgac tcaagttgaa atagtaagtt t 41
* * * * *