U.S. patent application number 15/636952 was filed with the patent office on 2017-10-19 for anti-tumor therapy.
The applicant listed for this patent is THE UNIVERSITY OF CHICAGO. Invention is credited to Nikolai Khodarev, Bernard Roizman, Ravi Sood, Ralph Weichselbaum.
Application Number | 20170298362 15/636952 |
Document ID | / |
Family ID | 51934064 |
Filed Date | 2017-10-19 |
United States Patent
Application |
20170298362 |
Kind Code |
A1 |
Khodarev; Nikolai ; et
al. |
October 19, 2017 |
ANTI-TUMOR THERAPY
Abstract
Panels, compositions, and methods for treating cancer in a
subject in need thereof are disclosed involving one or more genes
the suppression of which renders the cancer chemosensitive and/or
radiosensitive.
Inventors: |
Khodarev; Nikolai; (Villa
Park, IL) ; Sood; Ravi; (Seattle, WA) ;
Roizman; Bernard; (Chicago, IL) ; Weichselbaum;
Ralph; (Chicago, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNIVERSITY OF CHICAGO |
Chicago |
IL |
US |
|
|
Family ID: |
51934064 |
Appl. No.: |
15/636952 |
Filed: |
June 29, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14893690 |
Nov 24, 2015 |
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PCT/US2014/038885 |
May 21, 2014 |
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15636952 |
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61827402 |
May 24, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/113 20130101;
C12N 2320/30 20130101; C12N 15/1137 20130101; C12Q 2600/158
20130101; C12N 2310/14 20130101; C12Q 1/6886 20130101; A61K 31/713
20130101; A61K 31/519 20130101; A61K 45/06 20130101; C12Q 2600/136
20130101; A61K 31/506 20130101; C12N 2320/12 20130101 |
International
Class: |
C12N 15/113 20100101
C12N015/113; A61K 31/713 20060101 A61K031/713; A61K 31/506 20060101
A61K031/506; C12N 15/113 20100101 C12N015/113; C12Q 1/68 20060101
C12Q001/68; A61K 31/519 20060101 A61K031/519; A61K 45/06 20060101
A61K045/06 |
Claims
1. A panel for treating a cancer in a subject in need thereof, the
panel comprising: one or more genes contributing to tumor
development or chemoresistance and/or radioresistance of a cancer
cell, wherein suppression of the one or more genes results in at
least one of: suppression of growth or proliferation of the cancer
cell, cell death of the cancer cell, or sensitization of the cancer
cell to chemotherapy and/or radiotherapy.
2. The panel of claim 1, wherein the cancer is at least one of
Jak/Stat dependent or associated with activation of a
Jak/Stat-related pathway; wherein the Jak comprises a Jak-1, a
Jak-2, a Jak-3 or a Tyk2 kinase, and the Stat comprises a Stat1, a
Stat2, a Stat 3, a Stat4, a Stat5, or a Stat6 transcriptional
factor.
3. The panel of claim 2, wherein the one or more genes is an
interferon stimulated gene.
4. The panel of claim 3, wherein the one or more genes is selected
from the group consisting of DHX58 (LGP2), PLSCR1, USP18, PSMB10,
IFITM1, OASL, EPSTL1, LGALS3BP, IFIH1, ABCC3, DTX3L, PSMB9, IRF9,
TAGLN, IFIT2, TPD52L1, CXCL9, GBP1, BST2, SP110, HERCS, CCL2, WARS,
MCL1, and TRIM14.
5. The panel of claim 1, wherein the one or more genes contributes
to chemoresistance and/or radioresistance of the cancer cell.
6. A panel for treating a cancer in a subject in need thereof, the
panel comprising: one or more inhibitors of expression and/or
functional activity specific for one or more genes selected from
the group consisting of DHX58, PLSCR1, USP18, PSMB10, IFITM1, OASL,
EPSTL1, LGALS3BP, IFIH1, ABCC3, DTX3L, PSMB9, IRF9, TAGLN, IFIT2,
TPD52L1, CXCL9, GBP1, BST2, SP110, HERCS, CCL2, WARS, MCL1, and
TRIM14, wherein administration of the one or more inhibitors of
expression to a cancer cell results in at least one of: suppression
of growth or proliferation of the cancer cell, cell death of the
cancer cell, or sensitization of the cancer cell to chemotherapy
and/or radiotherapy.
7. The panel of claim 6, wherein the one or more inhibitors of
expression and/or functional activity comprises an siRNA molecule,
an shRNA molecule, a micro-RNA molecule, a small molecule, a
peptide inhibitor, or a combination or a pharmaceutically
acceptable salt or prodrug thereof, and wherein the one or more
inhibitors of expression and/or functional activity is in a
therapeutically effective amount and formulated for administration
to the subject.
8. The panel of claim 7, wherein the panel comprises inhibitors of
expression and/or functional activity specific for at least two
genes selected from the group consisting of DHX58, PLSCR1, USP18,
PSMB10, IFITM1, OASL, EPSTL1, LGALS3BP, IFIH1, ABCC3, DTX3L, PSMB9,
IRF9, TAGLN, IFIT2, TPD52L1, CXCL9, GBP1, BST2, SP110, HERCS, CCL2,
WARS, MCL1, and TRIM14.
9. The panel of claim 6, wherein administration to the subject of
one or more antineoplastic agents and radio therapy results in at
least one of: suppression of growth or proliferation of the cancer
cell, or cell death of the cancer cell; wherein the administration
of the one or more antineoplastic agents or radio therapy is
subsequent to the administration of the inhibitor of expression
and/or functional activity.
10. A kit for treating cancer in a subject in need thereof,
comprising: a panel comprising one or more inhibitors of expression
and/or functional activity specific for one or more genes selected
from the group consisting of DHX58, PLSCR1, USP18, PSMB10, IFITM1,
OASL, EPSTL1, LGALS3BP, IFIH1, ABCC3, DTX3L, PSMB9, IRF9, TAGLN,
IFIT2, TPD52L1, CXCL9, GBP1, BST2, SP110, HERCS, CCL2, WARS, MCL1,
and TRIM14; and an optional antineoplastic agent.
11. The kit of claim 10 further comprising at least one of a Jak2
or a Jak1/Jak2 inhibitor in a therapeutically effective amount.
12. The kit of claim 11, wherein the Jak2 inhibitor is
SAR302503.
13. The kit of claim 11, wherein the Jak1/Jak2 inhibitor is
Ruxolitinib.
14. A pharmaceutical composition, comprising: a therapeutically
effective amount of an agent that suppresses at least one gene in a
subject, the gene selected from the group consisting of DHX58,
PLSCR1, USP18, PSMB10, IFITM1, OASL, EPSTL1, LGALS3BP, IFIH1,
ABCC3, DTX3L, PSMB9, IRF9, TAGLN, IFIT2, TPD52L1, CXCL9, GBP1,
BST2, SP110, HERCS, CCL2, WARS, MCL1, and TRIM14; and one or more
pharmaceutically acceptable carriers, diluents and excipients.
15. The pharmaceutical composition of claim 14, wherein the
composition further comprises a therapeutically effective amount of
at least one of an antineoplastic agent or a radiotherapy
agent.
16. The pharmaceutical composition of claim 15, wherein the
pharmaceutical composition is formulated to be administered to the
subject in at least one of an oral, inhalation, parental injection,
topical, or suppository dosage form.
17. A method of treating a cancer in a subject by increasing the
sensitivity of tumor cells of the subject to radiotherapy,
comprising: a) administering to the tumor cells a siRNA or shRNA to
directly target PSMB9 mRNA in an amount that is sufficient to
suppress expression of PSMB9 gene, thereby increasing the
sensitivity of the cells to radiotherapy; and b) administration to
the subject a therapeutically effective amount of at least one of
an antineoplastic agent or radiotherapy.
18. The method of claim 17 further comprising administration to the
subject a therapeutically effective amount of at least one of a
Jak2 or a Jak1/Jak2 inhibitor.
19. The method of claim 18, wherein the PSMB9 mRNA is decreased by
at least 25% in tumor cells compared to untreated cells of the same
tumor cells.
20. The method of claim 18, wherein the radio therapy comprises at
least one of brachytherapy, external beam radiation therapy, or
radiation from cesium, iridium, iodine, or cobalt.
21. The method of claim 17 further comprises administrating to the
tumor cells a siRNA or shRNA to directly target mRNA of a gene
selected from the group consisting of DHX58, PLSCR1, USP18, PSMB10,
IFITM1, OASL, EPSTL1, LGALS3BP, IFIH1, ABCC3, DTX3L, IRF9, TAGLN,
IFIT2, TPD52L1, CXCL9, GBP1, BST2, SP110, HERCS, CCL2, WARS, MCL1,
and TRIM14.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/893,690 filed on Nov. 24, 2015, which is
the national stage application of PCT/US2014/038885, which claims
priority to U.S. Application Ser. No. 61/827,402 filed May 24,
2013. The contents of each of these applications are incorporated
by reference.
STATEMENT CONCERNING GOVERNMENT INTEREST
[0002] Not applicable
SEQUENCE LISTING
[0003] Not applicable
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0004] The present invention relates to the identification and
control of gene targets for treatment of cancers, including
chemoresistant and/or radioresistant cancers.
2. Description of the Background of the Invention
[0005] Cancer is not fully understood on a molecular level and
remains a leading cause of death worldwide. One of the deadliest
forms of cancer is solid tumors. One such solid tumor is lung
cancer, the most common cancer worldwide and the leading cause of
cancer-related death in the United States. Approximately 219,000
new diagnoses and over 159,000 deaths from lung cancer occur
annually in the United States. Approximately 85% of lung cancers
are non-small cell histology (NSCLC), including lung
adenocarcinomas, which are the most common lung cancer type in the
U.S. Treatment of early and intermediate stage NSCLC usually
involves surgery, stereotactic radiotherapy, or conventional
radiotherapy with or without adjuvant chemotherapy. Chemotherapy
regimens for lung cancer, either concurrent with radiotherapy (RT)
or adjuvant to surgery, usually incorporate platinum-based drugs
such as cisplatin or carboplatin, as this has been shown to confer
a survival advantage when either combined with radiotherapy or in
the adjuvant setting.
[0006] Standard fractionated radiotherapy as the primary treatment
for NSCLC is reserved for patients with tumors too advanced to
resect, who are medically unstable, whose disease has spread beyond
the chest, or in the case of small or metastatic tumor
hypofractionated stereotacktic body radiotherapy. The utility of
postoperative radiotherapy is controversial and subsets of patients
who are likely to benefit have been proposed. These include
patients with advanced lymph node metastases (N2-N3 or
extra-capsular extension) and close or positive surgical margins.
However, clear clinical and/or molecular selection criteria for
patients who may benefit from postoperative radiotherapy remains
elusive. No prognostic or predictive signature to select patients
with NSCLC who may benefit from radiotherapy or chemotherapy is
consistently used in clinical practice at this time.
[0007] The activity of Jak/Stat dependent genes has been shown to
predict the outcome of patients with lung cancer and their response
to the adjuvant radiotherapy or chemotherapy. Stat1 (Signal
Transducer and Activator of Transcription 1) is a member of the
Stat family of proteins, which are mediators of Jak signaling.
Stat1 is phosphorylated at the tyrosine 701 position by Jak kinases
and translocates to the nucleus to activate the transcription of
hundreds of Interferon-Stimulated Genes (ISGs).
[0008] Further, clinical trials of Jak/Stat pathway inhibitors in
hematological malignancies are ongoing for the pharmacological
suppression of the Stat-related pathways. Jak inhibitors currently
available include either specific inhibitors of Jak2 or combined
inhibitors of Jak1 and Jak2. The radiosensitizing effects of the
Jak2 inhibitor TG101209 (TargeGen Inc., CAS 936091-14-4) were
recently described in two lung cancer cell lines and were
associated with suppression of the Stat3 pathway. TG101209 was
developed to potentially inhibit myeloproliferative
disorder-associated JAK2V617F and MPLW515L/K mutations. Activation
of Jak2/Stat3 signaling was demonstrated in several other lung
cancer cell lines and was associated with increased oncogenic
potential, tumor angiogenesis, and EGFR signaling associated with
progression of lung adenocarcinomas. Further, next-generation
sequencing recently revealed constitutively active Jak2 mutation
(V617F) in some lung cancer patients.
[0009] To date, few publications describe the application of these
drugs in lung cancer models, and mechanisms of their action in lung
cancer are still poorly understood. The majority of publications
regarding the application of Jak inhibitors in solid tumors,
including lung cancer, explain their action based on pathways
activated by Stat3, Stat5 or not directly related to Stat
signaling. Jak/Stat1 pathways in solid tumors are not described in
the context of therapeutic effects of Jak inhibitors, though they
are already described in some myelodysplastic diseases. It is
believed that Jak1 kinase is activated by Jak2 kinase and both are
necessary for activation of Stat1 and Stat3. It is also believed
that Stat1 and Stat3 can form heterodimers with transcriptional
activity. Additionally, genes induced by Jak2/Stat3 activation
overlap with IFN/Stat1-dependent genes. Finally, constitutively
active oncogenic Jak2 (Jak2V617F) induces genes overlapping with
the Stat1-dependent genes.
[0010] While the importance of Jak/Stat signaling, in general, for
cancers continues to be investigated, the role that downstream
effector genes may play in tumors remains undefined. Consequently,
there is an urgent and definite need to identify the downstream
effector genes that may potentially have a role in tumor
development associated with activation of the Jak/Stat pathway.
Such genes may provide new targets for Jak-related therapy of
cancers, including, for example, lung cancer, or for sensitization
of cancers for chemotherapies and/or radiotherapies. Therefore,
there is a need to determine the identities of downstream effector
genes in the Jak/Stat pathway of cancer, including solid tumors,
that may play a role in treating cancers, and to develop effective
cancer therapies around these downstream effector genes. More
effective and targeted cancer therapies with potentially fewer side
effects are also needed.
SUMMARY OF THE INVENTION
[0011] According to a first aspect, a panel for treating a cancer
in a subject in need thereof includes one or more genes
contributing to tumor development or chemoresistance and/or
radioresistance of a cancer cell. Suppression of the one or more
genes results in at least one of suppression of growth or
proliferation of the cancer cell, cell death of the cancer cell, or
sensitization of the cancer cell to chemotherapy and/or
radiotherapy.
[0012] In one embodiment, the cancer is Jak/Stat dependent.
[0013] In another embodiment, the cancer is associated with
activation of a Jak/Stat-related pathway.
[0014] Illustratively, Jak includes a Jak-1, a Jak-2, a Jak-3 or a
Tyk2 kinase, and the Stat includes a Stat1, a Stat2, a Stat 3, a
Stat4, a Stat5, or a Stat6 transcriptional factor
[0015] In another embodiment, the one or more genes of the panel is
an interferon stimulated gene.
[0016] In a further embodiment, the one or more genes of the panel
includes, for example, DHX58 (LGP2), PLSCR1, USP18, PSMB10, IFITM1,
OASL, EPSTL1, LGALS3BP, IFIH1, ABCC3, DTX3L, PSMB9, IRF9, TAGLN,
IFIT2, TPD52L1, CXCL9, GBP1, BST2, SP110, HERCS, CCL2, WARS, MCL1,
and TRIM14.
[0017] According to a second aspect, a panel for treating a cancer
in a subject in need thereof includes one or more inhibitors of
expression specific for one or more genes including, for example,
DHX58, PLSCR1, USP18, PSMB10, IFITM1, OASL, EPSTL1, LGALS3BP,
IFIH1, ABCC3, DTX3L, PSMB9, IRF9, TAGLN, IFIT2, TPD52L1, CXCL9,
GBP1, BST2, SP110, HERCS, CCL2, WARS, MCL1, or TRIM14.
Administration of the one or more inhibitors of expression to a
cancer cell results in, for example, at least one of: suppression
of growth or proliferation of the cancer cell, cell death of the
cancer cell, or sensitization of the cancer cell to chemotherapy
and/or radiotherapy.
[0018] In one embodiment, the one or more inhibitors of expression
and/or functional activity comprises an siRNA molecule, an shRNA
molecule, a micro-RNA molecule, a small molecule, a peptide
inhibitor, or a combination or a pharmaceutically acceptable salt
or prodrug thereof. The one or more inhibitors of expression and/or
functional activity is in a therapeutically effective amount and
formulated for administration to the subject.
[0019] In another embodiment, the panel comprises inhibitors of
expression and/or functional activity specific for at least two
genes including, for example, DHX58, PLSCR1, USP18, PSMB10, IFITM1,
OASL, EPSTL1, LGALS3BP, IFIH1, ABCC3, DTX3L, PSMB9, IRF9, TAGLN,
IFIT2, TPD52L1, CXCL9, GBP1, BST2, SP110, HERCS, CCL2, WARS, MCL1,
and TRIM14.
[0020] In one embodiment, administration to the subject of one or
more antineoplastic agents and radio therapy results in at least
one of suppression of growth or proliferation of the cancer cell,
or cell death of the cancer cell.
[0021] In a further embodiment, administration of the one or more
antineoplastic agents or radio therapy is subsequent to the
administration of the inhibitor of expression and/or functional
activity.
[0022] According to a third aspect, a kit for treating cancer in a
subject in need thereof includes a panel comprising one or more
inhibitors of expression and/or functional activity specific for
one or more genes including, for example, DHX58, PLSCR1, USP18,
PSMB10, IFITM1, OASL, EPSTL1, LGALS3BP, IFIH1, ABCC3, DTX3L, PSMB9,
IRF9, TAGLN, IFIT2, TPD52L1, CXCL9, GBP1, BST2, SP110, HERCS, CCL2,
WARS, MCL1, and TRIM14. The kit further includes an antineoplastic
agent, which may be optional in some embodiments.
[0023] In one embodiment, the kit further includes at least one of
a Jak2 or a Jak1/Jak2 inhibitor in a therapeutically effective
amount.
[0024] In another embodiment, the Jak 2 inhibitor is SAR302503.
[0025] In a further embodiment, the Jak1/Jak2 inhibitor is
Ruxolitinib.
[0026] In a fourth aspect, a method of treating a cancer in a
subject in need thereof includes: a) suppression of at least one
gene in the subject in a therapeutically effective amount, the gene
includes, for example, DHX58, PLSCR1, USP18, PSMB10, IFITM1, OASL,
EPSTL1, LGALS3BP, IFIH1, ABCC3, DTX3L, PSMB9, IRF9, TAGLN, IFIT2,
TPD52L1, CXCL9, GBP1, BST2, SP110, HERCS, CCL2, WARS, MCL1, and
TRIM14; and b) administration to the subject a therapeutically
effective amount of at least one of an antineoplastic agent or
radio therapy.
[0027] In one embodiment, the method further includes
administration to the subject a therapeutically effective amount of
at least one of a Jak2 or a Jak1/Jak2 inhibitor.
[0028] In a further embodiment, the therapeutically effective
amount of gene suppression is sufficient to render the cancer
chemosensitive or radiosensitive.
[0029] In another embodiment, the therapeutically effective amount
of gene suppression is less than or equal to about 75% of normal
gene activity.
[0030] In a fifth aspect, a pharmaceutical composition includes a
therapeutically effective amount of an agent that suppresses at
least one gene in a subject, the gene includes, for example, DHX58,
PLSCR1, USP18, PSMB10, IFITM1, OASL, EPSTL1, LGALS3BP, IFIH1,
ABCC3, DTX3L, PSMB9, IRF9, TAGLN, IFIT2, TPD52L1, CXCL9, GBP1,
BST2, SP110, HERCS, CCL2, WARS, MCL1, and TRIM14. The
pharmaceutical composition includes one or more pharmaceutically
acceptable carriers, diluents and excipients.
[0031] In one embodiment, the composition further includes a
therapeutically effective amount of at least one of an
antineoplastic agent or a radiotherapy agent.
[0032] In another embodiment, the pharmaceutical composition is
formulated to be administered to the subject in at least one of an
oral, inhalation, parental injection, topical, or suppository
dosage form.
BRIEF DESCRIPTION OF THE FIGURES
[0033] FIG. 1 shows a heat map of a phenotypic screen for selected
genes using a panel of siRNAs in 16 cell lines representing 7 types
of cancer (lung, breast, prostate, colon, bladder, head and neck
and glioblastoma multiforme (GBM)). Blue represents suppression of
cell viability in the result of suppression of the given gene and
yellow-increase of viability. Genes for which suppression leads to
lost viability potentially confer growth/survival of tumor cells.
Eight genes were selected based on their ability to suppress
survival in the majority of cell lines tested independently of
cancer type. One gene (MCL1) was selected as an example of
cancer-type specific gene (knock-down of MCL1 led to the maximal
suppression of survival in 2 cell lines representing
hormone-independent prostate cancer). Data indicate that
suppression of one or more of these genes alone or in combination
with ionizing radiation (IR) (and/or chemotherapy) may increase
killing of tumor cells as compared with ionizing radiation (or
chemotherapy);
[0034] FIG. 2 shows heat maps of Interferon-Related DNA Damage
Resistance Signature (IRDS);
[0035] FIG. 3 shows a Western blot of SCC61 cells treated with and
without the Jak2 inhibitor TG101348 (TG) and the Jak1/Jak2
inhibitor Ruxolitinib (Rux). Treatment with TG and Rux inhibited
.beta.- and .gamma.-interferon-mediated activation of Stat1 and
Stat3;
[0036] FIG. 4 illustrates the relative radioresistance of lung
cancer cell lines (y-axis). Cells lines were plated and irradiated
with 5 Gy. Two weeks post-irradiation, colonies were stained and
counted. Data are normalized to non-irradiated samples.
[0037] FIG. 5 shows a Western blot depicting the constitutive
activation of the Jak/Stat axis in lung cancer cells and ability of
Jak2 inhibitor to suppress this activation;
[0038] FIG. 6 shows grouping of lung cancer cell lines according to
their resistance/sensitivity to SAR and ionizing radiation;
[0039] FIG. 7 shows the grouping of lung cancer cell lines
according to their resistance/sensitivity to SAR and Etoposide;
[0040] FIG. 8A shows the cell line MCF10A plated at optimal cell
concentrations and reverse transfected on Day 0, incubated at
37.degree. C. in 5% CO2 and then viability assayed at 96 hours
post-transfection (no post-ionizing radiation);
[0041] FIG. 8B shows the cell line MCF10A plated at optimal cell
concentrations and reverse transfected on Day 0, incubated at
37.degree. C. in 5% CO2, irradiated with 3 Gy at 48 hours, and then
viability assayed at 96 hours post-transfection (48 hours
post-ionizing radiation);
[0042] FIG. 9 shows the comparison of (a) pooled versus (b)
deconvoluted siRNA suppression of viability for HCT116 treated with
3 Gy IR;
[0043] FIG. 10 shows cytometry validation of candidate target genes
identified in siRNA screen. Double-positive cells, presented in the
upper right quadrant of the each panel were quantified as the
measure of cell death induced by siRNA suppression of the given
gene without (e) or with (f) irradiation. Gene used in these
experiments is DHx58 (LGP2);
[0044] FIG. 11 shows radiosensitization of candidate genes,
suppressed by individual siRNAs identified in deconvoluted screen
(see FIGS. 8 and 9). Cell line HCT116 was reverse-transfected by
siRNAs against indicated genes (see X-axis). 48 hours
post-transfection cells were irradiated at 3Gy and 48 hours post-IR
cells were stained with propidium iodide (PI) and Abs against
Annexin V. Samples were analyzed on a FACSCanto flow cytometer (BD
Biosciences), and data were analyzed with FlowJo software
(TreeStar, Inc.). Shown are amounts of the double-positive dead
cells (see FIG. 10). All experiments were done in triplicates;
error bars are SDs;
[0045] FIG. 12 shows that individual siRNA against PSMB9 and PSMB10
inhibit expression of corresponding proteins in breast cancer tumor
cell line MDA-MB-231 and glioblastoma cell line D54. Cell lines
were transfected by corresponding siRNAs, lyzed 72 hours
post-transfection and proteins were separated and detected by
Western analysis as described in Methods. Panels A and C represent
gel images and panels B and D--quantification of PSMB9 and PSMB10
protein expression (normalized to non-targeting control);
[0046] FIG. 13 shows that inhibition of PSMB9 leads to the
suppression of cell growth of breast cancer cell line MDA-MB-231
and glioblastoma cell line D54; cells were transfected by siRNA
against PSMB9 (#1) or non-targeting control (NT2) and 24 or 48
hours post-transfection plated in 24-well plates. Cells were
counted after 24, 48, 72 and 96 hours after plating in 24 well
plates. Y axis-number of cells/well, normalized to day 1;
X-axis-time of cultivation, hours. Error bars are SDs between
triplicated measurements;
[0047] FIG. 14 shows inhibition of PSMB9 and PSMB10 that leads to
the increased radiation killing of breast cancer cell line
MDA-MB-231. Cells were transfected by siRNAs against PSMB9 or
PSMB10, or non-targeting control; 24 hours post-transfection cells
were irradiated at 5Gy and 48 hours post-IR analyzed by flow
cytometry as described in FIG. 10 and FIG. 11. Panel A represents
raw data and panels B and C-quantification of dead cells normalized
to un-irradiated controls; Y-axis in panels B and C-fold changes
related to un-irradiated cells transfected by non-targeting
control). Error bars are SDs; asterisks indicate differences with
p.ltoreq.0.05;
[0048] FIG. 15 shows inhibition of PSMB9 and PSMB10 that leads to
the increased radiation killing of glioblastoma cell line D54; all
indications are identical to FIG. 14;
[0049] FIG. 16 shows that overexpression of USP18 leads to
increased radioresistance of glioblastoma cell lines U87 and D54
and head & neck cancer cell line SCC61; differences between
wild type and USP18 overexpressors were significant at 3 and 7Gy in
U87; 7Gy in SCC61 and 3Gy in D54; and
[0050] FIG. 17 shows overexpression of USP18 and increased
radioresistance of xenografted D54 tumors in nude mice. Control
(empty vector) and USP18-transfected cells were injected in the
flanks of the nude mice; when tumors reached 200-300 mm3, they were
irradiated with 6 fractions of 5Gy each (30Gy total; day 0) or left
un-treated. Tumor volumes were measured once in 4 days and
represented as relative tumor volume (Y-axis).
DESCRIPTION
[0051] Treatment of a cancer in a subject in need thereof is
provided herein, as are compositions, kits, and methods for
treating cancer, and methods for identifying effector genes in the
Jak/Stat pathway having a role in the treatment of cancer and
therapies to treat cancer based on these effector genes. A Jak/Stat
dependent cancer may include any solid tumor, including lung,
prostate, head and neck, breast and colorectal cancer, melanomas
and gliomas, and the like. While the present disclosure may be
embodied in different forms, several specific embodiments are
discussed herein with the understanding that the present disclosure
is to be considered only an exemplification and is not intended to
limit the invention to the illustrated embodiments.
[0052] While not wishing to be bound by theory, it is believed that
downstream effector genes in the Jak/Stat pathway have a causal
role in treatment-resistant cancers, including solid tumors, such
as lung cancer. Therefore, if downstream effector genes could be
identified to have a direct relationship to treatment resistance,
new therapies could be developed for treatment resistant cancers.
One approach for determining downstream effector genes that have a
direct role in treatment resistance is to suppress the gene and
determine whether a treatment resistant cancer cell with the
suppressed gene becomes treatment sensitive. In experimental models
using knock-downs of Stat1, suppression of Stat1 and subsequent
suppression of down-stream genes activated by Stat1 lead to
radiosensitization, chemosensitization to doxorubicin, and/or
growth suppression of cancer. These genes in the Stat1 pathway may
provide targets for personalized therapy of cancer, including lung
cancer. Further, implementing siRNA screening technologies, several
Stat1-dependent genes were detected as candidates for conferring
tumor resistance to genotoxic stress and protection from
apoptosis.
[0053] Another potential step in this process is to identify gene
candidates. One approach is to use available microarray and
proteomics data to identify potential candidates. Criteria for
selection may include control by the Jak/Stat pathway, association
with oncogenesis and/or radio/chemoresistance, and/or dysregulation
in cancerous (or precancerous) tissues. Additional criteria may
also be chosen and combined for selection.
[0054] Another potential step is to suppress expression or
otherwise inhibit the gene or resultant protein of the candidate
gene. One method to achieve expression inhibition is siRNA. Other
methodologies known in the art may be used, such as, for example,
small hairpin RNA (shRNA), micro-RNAs, small molecules, peptide
inhibitors, combinations thereof, and the like.
[0055] A further step can include administering an antineoplastic
agent (e.g., chemotherapy) and/or radiotherapy to a treatment
resistant cancer cell in which the candidate downstream effector
gene is suppressed. A loss of viability of the treatment resistant
cancer cell reveals that that candidate downstream effector gene
may be an effective target for therapy.
[0056] An illustrative antineoplastic agent or chemotherapeutic
agent includes, for example, a standard taxane. Taxanes are
produced by the plants of the genus Taxus and are classified as
diterpenes and widely uses as chemotherapy agents including, for
example, paclitaxel, (Taxol.RTM., Bristol-Meyers Squibb, CAS
33069-62-4) and docetaxel (Taxotere.RTM., Sanofi-Aventis, CAS
114977-28-5). Other chemotherapeutic agents include semi-synthetic
derivatives of a natural taxoid such as cabazitaxel (Jevtana.RTM.,
Sanofi-Aventis, CAS 183133-96-2). Other chemotherapeutic agents
also include an androgen receptor inhibitor or mediator.
Illustrative androgen receptor inhibitors include, a steroidal
antiandrogen (for example, cyperterone, CAS 2098-66-0); a
non-steroidal antiandrogen (for example, flutamide, Eulexin.RTM.,
Schering-Plough, CAS 13311-84-7); nilutamide (Nilandron.RTM., CAS
63612-50-0); enzalutamide (Xtandi.RTM., Medivation.RTM., CAS
915087-33-1); bicalutamide (Casodex, AstraZeneca, CAS 90357-06-5);
a peptide antiandrogen; a small molecule antiandrogen (for example,
RU58642 (Roussel-Uclaf SA, CAS 143782-63-2); LG120907 and LG105
(Ligand Pharmaceuticals); RD162 (Medivation, CAS 915087-27-3);
BMS-641988 (Bristol-Meyers Squibb, CAS 573738-99-5); and CH5137291
(Chugai Pharmaceutical Co. Ltd., CAS 104344603904)); a natural
antiandrogen (for example, ataric acid (CAS 4707-47-5) and
N-butylbensensulfonamide (CAS 3622-84-2); a selective androgen
receptor modulator (for example, enobosarm (Ostarine.RTM., Merck
& Company, CAS 841205-47-8); BMS-564,929 (Bristol-Meyer Squibb,
CAS 627530-84-1); LGD-4033 (CAS 115910-22-4); AC-262,356 (Acadia
Pharmaceuticals); LGD-3303 (Ganolix Lifescience Co., Ltd.,
9-chloro-2-ethyl-1-methyl-3-(2,2,2-trifluoroethyl)-3H-pyrrolo[3,2-f]quino-
lin-7(6H)-one; 5-40503, Kaken Pharmaceuticals,
2-[4-(dimethylamino)-6-nitro-1,2,3,4-tetrahydroquinolin-2-yl]-2-methylpro-
pan-1-ol); andarine (GTx-007, S-4, GTX, Inc., CAS 401900-40-1); and
S-23 (GTX, Inc., (2
S)--N-(4-cyano-3-trifluoromethylphenyl)-3-(3-fluoro-4-chlorophenoxy)-2-hy-
droxy-2-methyl-propanamide)); or those described in U.S. Patent
Appln. No. 2009/0304663. Other neoplastic agents or
chemotherapeutic agents that may be used include, for example:
alkylating agents such as nitrogen mustards such as mechlorethamine
(HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcoly sin) and
chlorambucil; ethylenimines and methylmelamines such as
hexamethylmelamine, thiotepa; alkyl sulphonates such as busulfan;
nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine
(methyl-CCNU) and streptozocin (streptozotocin); and triazenes such
as decarbazine (DTIC; dimethyltriazenoimidazole-carboxamide);
antimetabolites including folic acid analogues such as methotrexate
(amethopterin); pyrimidine analogues such as fluorouracil
(5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and
cytarabine (cytosine arabinoside); and purine analogues and related
inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP),
thioguanine (6-thioguanine; TG) and pentostatin
(2'-deoxycoformycin); natural products including vinca alkaloids
such as vinblastine (VLB) and vincristine; epipodophyllotoxins such
as etoposide and teniposide; antibiotics such as dactinomycin
(actinomycin D), daunorubicin (daunomycin; rubidomycin),
doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin
(mitomycin C); enzymes such as L-asparaginase; biological response
modifiers such as interferon alphenomes; other agents such as
platinum coordination complexes such as cisplatin (cis-DDP) and
carboplatin; anthracenedione such as mitoxantrone and
anthracycline; substituted urea such as hydroxyurea; methyl
hydrazine derivative such as procarbazine (N-methylhydrazine, MTH);
adrenocortical suppressant such as mitotane (o,p'-DDD) and
aminoglutethimide; taxol analogues/derivatives; hormone
agonists/antagonists such as flutamide and tamoxifen; and GnRH and
analogues thereof. Examples of other chemotherapeutic can be found
in Cancer Principles and Practice of Oncology by V. T. Devita and
S. Hellman (editors), 6.sup.th edition (Feb. 15, 2001), Lippincott
Williams & Wilkins Publishers.
[0057] Radiotherapy is based on ionizing radiation delivered to a
target area that results in death of reproductive tumor cells. Some
examples of radiotherapy include the radiation of cesium,
palladium, iridium, iodine, or cobalt and is usually delivered as
ionizing radiation delivered from a linear accelerator or an
isotopic source such as a cobalt source. Also variations on linear
accelerators are Cyberkine and Tomotherapy. Particle radiotherapy
from cyclotrons such as Protons or Carbon nuclei may be employed.
Also radioisotopes delivered systemically such as p32 or radium 223
may be used. The external radiotherapy may be systemic radiation in
the form of sterotacktic radiotherapy total nodal radiotherapy or
whole body radiotherapy but is more likely focused to a particular
site, such as the location of the tumor or the solid cancer tissues
(for example, abdomen, lung, liver, lymph nodes, head, etc.). The
radiation dosage regimen is generally defined in terms of Gray or
Sieverts time and fractionation, and must be carefully defined by
the radiation oncologist. The amount of radiation a subject
receives will depend on various consideration but the two important
considerations are the location of the tumor in relation to other
critical structures or organs of the body, and the extent to which
the tumor has spread. One illustrative course of treatment for a
subject undergoing radiation therapy is a treatment schedule over a
5 to 8 week period, with a total dose of 50 to 80 Gray (Gy)
administered to the subject in a single daily fraction of 1.8 to
2.0 Gy, 5 days a week. A Gy is an abbreviation for Gray and refers
to 100 rad of dose.
[0058] Radiotherapy can also include implanting radioactive seeds
inside or next to an site designated for radiotherapy and is termed
brachytherapy (or internal radiotherapy, endocurietherapy or sealed
source therapy). For prostate cancer, there are currently two types
of brachytherapy: permanent and temporary. In permanent
brachytherapy, radioactive (iodine-125 or palladium-103) seeds are
implanted into the prostate gland using an ultrasound for guidance.
Illustratively, about 40 to 100 seeds are implanted and the number
and placement are generally determined by a computer-generated
treatment plan known in the art specific for each subject.
Temporary brachytherapy uses a hollow source placed into the
prostate gland that is filled with radioactive material
(iridium-192) for about 5 to about 15 minutes, for example.
Following treatment, the needle and radioactive material are
removed. This procedure is repeated two to three times over a
course of several days.
[0059] Radiotherapy can also include radiation delivered by
external beam radiation therapy (EBRT), including, for example, a
linear accelerator (a type of high-powered X-ray machine that
produces very powerful photons that penetrate deep into the body);
proton beam therapy where photons are derived from a radioactive
source such as iridium-192, caesium-137, radium-226 (no longer used
clinically), or colbalt-60; Hadron therapy; multi-leaf collimator
(MLC); and intensity modulated radiation therapy (IMRT). During
this type of therapy, a brief exposure to the radiation is given
for a duration of several minutes, and treatment is typically given
once per day, 5 days per week, for about 5 to 8 weeks. No radiation
remains in the subject after treatment. There are several ways to
deliver EBRT, including, for example, three-dimensional conformal
radiation therapy where the beam intensity of each beam is
determined by the shape of the tumor. Illustrative dosages used for
photon based radiation is measured in Gy, and in an otherwise
healthy subject (that is, little or no other disease states present
such as high blood pressure, infection, diabetes, etc.) for a solid
epithelial tumor ranges from about 60 to about 80 Gy, and for a
lymphoma ranges from about 20 to about 40 Gy. Illustrative
preventative (adjuvant) doses are typically given at about 45 to
about 60 Gy in about 1.8 to about 2 Gy fractions for breast, head,
and neck cancers.
[0060] When radiation therapy is a local modality, radiation
therapy as a single line of therapy is unlikely to provide a cure
for those tumors that have metastasized distantly outside the zone
of treatment. Thus, the use of radiation therapy with other
modality regimens, including chemotherapy, have important
beneficial effects for the treatment of metastasized cancers.
[0061] Radiation therapy has also been combined temporally with
chemotherapy to improve the outcome of treatment. There are various
terms to describe the temporal relationship of administering
radiation therapy and chemotherapy, and the following examples are
illustrative treatment regimens and are generally known by those
skilled in the art and are provided for illustration only and are
not intended to limit the use of other combinations. "Sequential"
radiation therapy and chemotherapy refers to the administration of
chemotherapy and radiation therapy separately in time in order to
allow the separate administration of either chemotherapy or
radiation therapy. "Concomitant" radiation therapy and chemotherapy
refers to the administration of chemotherapy and radiation therapy
on the same day. Finally, "alternating" radiation therapy and
chemotherapy refers to the administration of radiation therapy on
the days in which chemotherapy would not have been administered if
it were given alone.
[0062] It should be noted that other therapeutically effective
doses of radiotherapy can be determined by a radiation oncologist
skilled in the art and can be based on, for example, whether the
subject is receiving chemotherapy, if the radiation is given before
or after surgery, the type and/or stage of cancer, the location of
the tumor, and the age, weight and general health of the
subject.
[0063] It is further contemplated that subsets of gene targets,
including those identified or described herein, could be used as a
therapeutic tool for diagnosing and/or treating a tumor or cancer.
For example, siRNA pools (or other sets of molecules individually
specific for one or more predetermined targets including, for
example, shRNA pools, small molecules, and/or peptide inhibitors,
collectively "expression inhibitors" or "active ingredients" or
"active pharmaceutical ingredients") may be generated based on one
or more (e.g., 2 or 4 or 8 or 12, or any number) targets and used
to treat a subject in need thereof (e.g., a mammal having a
chemoresistant or radioresistant cancer). Upon rendering of the
subject's cancer chemosensitive and/or radiosensitive, therapeutic
intervention in the form of antineoplastic agents and/or ionizing
radiation as known in the art (see for example, U.S. Pat. No.
6,689,787, incorporated by reference) may be administered to reduce
and/or eliminate the cancer. It is contemplated that therapeutic
intervention may occur before, concurrent, and/or subsequent to the
treatment to render the subject chemosensitive or radiosensitive.
It is further envisioned that particular subsets of targets may be
advantageous over others based on the particular type of cancer
and/or tissue of origin for providing a therapeutic effect.
Administration of such therapies may be accomplished by any means
known in the art.
[0064] In one embodiment, a kit may include a panel of siRNA pools
directed at one or more targets as identified by or in the present
disclosure, including those targets identified in Table Nos. 4a and
4b, below. It is envisioned that a particular kit may be designed
for a particular type of cancer and/or a specific tissue. The kit
may further include means for administering the panel to a subject
in need thereof. In addition, the kit may also include one or more
antineoplastic agents directed at the specific type of cancer
against which the kit is directed and one or more compounds that
inhibit that Jak/Stat pathway.
[0065] Kits may further be a packaged collection of related
materials, including, for example, a single and/or a plurality of
dosage forms each approximating an therapeutically effective amount
of an active ingredient, such as, for example, an expression
inhibitor and/or a pharmaceutical compound as described herein that
slows, stops, or reverses the growth or proliferation of a tumor or
cancer or kills tumor or cancer cells, and/or an additional drug.
The included dosage forms may be taken at one time, or at a
prescribed interval. Contemplated kits may include any combination
of dosage forms.
[0066] In another embodiment, a method of treating a subject in
need thereof includes administering to the subject one or more
molecules that target one or more genes of Table Nos. 4a and 4b,
such as siRNA and/or shRNA pools. The method may further include,
for example, treatment of the subject with one or more
antineoplastic agents, ionizing radiation, and/or one or more
compounds that inhibit that Jak/Stat pathway.
[0067] Suppression of a gene refers to the absence of expression of
a gene or a decrease in expression of a gene as compared to the
activity of an untreated gene. Suppression of a gene may be
determined by detecting the presence or absence of expression of a
gene or by measuring a decrease of expression of a gene by any
means known in the art including, for example, detecting a decrease
in the level of the final gene product, such as a protein, or
detecting a decreased level of a precursor, such as mRNA, from
which gene expression levels may be inferred when compared to
normal gene activity, such as a negative (untreated) control. Any
molecular biological assay to detect mRNA or an immunoassay to
detect a protein known in the art can be used. A molecular
biological assay includes, for example, polymerase chain reaction
(PCR), Northern blot, Dot blot, or an analysis method with
microarray or macroarray. An immunological assay includes, for
example, ELISA (enzyme-linked immunosorbent assay) with a
microtiter plate, radioimmunoassay (RIA), a fluorescence antibody
technique, Western blotting, or an immune structure dyeing method.
Suppression of a gene may also be inferred biologically in vivo, in
situ, and/or in vitro, by the suppression of growth or
proliferation of a tumor or cancer cell, cell death of a tumor or
cancer cell, and/or the sensitization of a tumor or cancer cell to
chemotherapy and/or radiotherapy. Illustratively, a therapeutically
effective amount of gene suppression in a subject results in the
suppression of growth or proliferation of a tumor or cancer cell,
cell death of the tumor or cancer cell, and/or the sensitization of
the tumor or cancer cell to chemotherapy and/or radiotherapy. As
each subject is different and each cancer is different, the amount
of gene suppression to achieve a therapeutically effective amount
of gene suppression may be determined by a trained professional
skilled in the area on a case-by-case basis. Illustratively, a
therapeutically effective amount of gene suppression may include,
for example, less than or equal to about 95% of normal gene
activity, or less than or equal to about 90% of normal gene
activity, or less than or equal to about 85% of normal gene
activity, or less than or equal to about 80% of normal gene
activity, or less than or equal to about 75% of normal gene
activity, or less than or equal to about 65% of normal gene
activity, or less than or equal to about 50% of normal gene
activity, or less than or equal to about 35% of normal gene
activity, or less than or equal to about 25% of normal gene
activity, or less than or equal to about 15% of normal gene
activity, or less than or equal to about 10% of normal gene
activity, or less than or equal to about 7.5% of normal gene
activity, or less than or equal to about 5% of normal gene
activity, or less than or equal to about 2.5% of normal gene
activity, or less than or equal to about 1% of normal gene
activity, or less than or equal to about 0% of normal gene
activity.
[0068] Suppression of identified genes individually or in
combination combined with ionizing radiation and/or any
chemotherapeutic agents may improve the outcome of patients treated
with the ionizing radiation or any chemotherapy agent or any
treatment designed to improve outcome of the cancer patients (like
Jak1/Jak2 inhibitors) if such treatment is combined with the
suppression of any of these genes or their combination.
[0069] Based on the functional groups, we also contemplate that
suppression of the chemokine signaling, or suppression of negative
regulators of interferon response, or suppression of protein
degradation or mitochondria-related anti-apoptotic molecules or
anti-viral proteins or extracellular matrix proteins (ECM) alone or
in combination with ionizing radiation or any chemotherapy drug or
any treatment designed to improve outcome of the cancer patients
will improve cancer treatment. This is based on the functional
associations between detected targets. DHX58 (also known as LGP2)
is known as an apical suppressor of RNA dependent activation of the
Type I interferons alpha and beta. IFITM1 and OASL are known
anti-viral proteins. USP18 and HERCS are enzymes involved in
protein ISGylation/de-ISGylation, known to protect proteins from
ubiquitin-dependent degradation in proteosome complex, while PSMB9
and PSMB10 are proteasome subunits. EPSTL1, LGALS3P and TAGLN are
involved in the structure and functional regulation of ECM. CXCL9
and CCL2 are chemokines with multiple functions including
growth-promoting functions for tumor cells.
[0070] Jak (Janus kinase) refers to a family of intracellular,
nonreceptor tyrosine kinases and includes four family members,
Janus 1 (Jak-1), Janus 2 (Jak-2), Janus 3 (Jak-3), and Tyrosine
kinase 2 (Tyk2).
[0071] Stat (Signal Transducer and Activator of Transcription)
plays a role in regulating cell growth, survival and
differentiation and the family includes Stat1, Stat2, Stat3, Stat4,
Stat5 (Stat5a and Stat5b), and Stat6.
[0072] The term "subject" refers to any organism classified as a
mammal, including mice, rats, guinea pigs, rabbits, dogs, cats,
cows, horses, monkeys, and humans.
[0073] As used herein, the term "cancer" refers to a class of
diseases of mammals characterized by uncontrolled cellular growth.
The term "cancer" is used interchangeably with the terms "tumor,"
"solid tumor," "malignancy," "hyperproliferation" and "neoplasm."
Cancer includes all types of hyperproliferative growth, hyperplasic
growth, neoplastic growth, cancerous growth or oncogenic processes,
metastatic tissues or malignantly transformed cells, tissues, or
organs, irrespective of histopathologic type or stage of
invasiveness. Illustrative examples include, lung, prostate, head
and neck, breast and colorectal cancer, melanomas and gliomas (such
as a high grade glioma, including glioblastoma multiforme (GBM),
the most common and deadliest of malignant primary brain tumors in
adult humans).
[0074] As used herein, the phrase "solid tumor" includes, for
example, lung cancer, head and neck cancer, brain cancer, oral
cancer, colorectal cancer, breast cancer, prostate cancer,
pancreatic cancer, and liver cancer. Other types of solid tumors
are named for the particular cells that form them, for example,
sarcomas formed from connective tissue cells (for example, bone
cartilage, fat), carcinomas formed from epithelial tissue cells
(for example, breast, colon, pancreas) and lymphomas formed from
lymphatic tissue cells (for example, lymph nodes, spleen, thymus).
Treatment of all types of solid tumors regardless of naming
convention is within the scope of this invention.
[0075] As used herein, the term "chemoresistant" refers to a tumor
or cancer cell that shows little or no significant detectable
therapeutic response to an agent used in chemotherapy.
[0076] As used herein, the term "radioresistant" refers to a tumor
or cancer cell that shows little or no significant detectable
therapeutic response to an agent used in radiotherapy such as
ionizing radiation.
[0077] As used herein, the term "chemosensitive" refers to a tumor
or cancer cell that shows a detectable therapeutic response to an
agent used in chemotherapy.
[0078] As used herein, the term "radiosensitive" refers to a tumor
or cancer cell that shows a detectable therapeutic response to an
agent used in radiotherapy.
[0079] As used herein, the phrases "chemotherapeutic agent,"
"cytotoxic agent," "anticancer agent," "antineoplastic agent" and
"antitumor agent" are used interchangeably and refer to an agent
that has the effect of inhibiting the growth or proliferation, or
inducing the killing, of a tumor or cancer cell. The
chemotherapeutic agent may inhibit or reverse the development or
progression of a tumor or cancer, such as for example, a solid
tumor.
[0080] As used herein, the term "chemotherapy" refers to
administration of at least one chemotherapeutic agent to a subject
having a tumor or cancer.
[0081] As used herein, the term "radiotherapy" refers to
administration of at least one "radiotherapeutic agent" to a
subject having a tumor or cancer and refers to any manner of
treatment of a tumor or cancer with a radiotherapeutic agent. A
radiotherapeutic agent includes, for example, ionizing radiation
including, for example, external beam radiotherapy, stereotatic
radiotherapy, virtual simulation, 3-dimensional conformal
radiotherapy, intensity-modulated radiotherapy, ionizing particle
therapy and radioisotope therapy.
[0082] Compositions herein may be formulated for oral, rectal,
nasal, topical (including buccal and sublingual), transdermal,
vaginal, injection/injectable, and/or parental (including
subcutaneous, intramuscular, intravenous, and intradermal)
administration. Other suitable administration routes are
incorporated herein. The compositions may be presented conveniently
in unit dosage forms and may be prepared by any methods known in
the pharmaceutical arts. Examples of suitable drug formulations
and/or forms are discussed in, for example, Hoover, John E.
Remington's Pharmaceutical Sciences, Mack Publishing Co., Eston,
Pa.; 18.sup.th edition (1995); and Liberman, H. A. and Lachman, L.
Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.,
1980. Illustrative methods include the step of bringing one or more
active ingredients into association with a carrier that constitutes
one or more accessory ingredients. In general, the compositions may
be prepared by bringing into association uniformly and intimately
one or more active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0083] Pharmaceutical formulations may include those suitable for
oral, intramuscular, rectal, nasal, topical (including buccal and
sub-lingual), vaginal or parenteral (including intramuscular,
subcutaneous and intravenous) administration or in a form suitable
for administration by inhalation or insufflation. One or more of
the compounds of the invention, together with a conventional
adjuvant, carrier, or diluent, may thus be placed into the form of
pharmaceutical compositions and unit dosages thereof, and in such
form may be employed as solids, such as tablets or filled capsules,
or liquids such as solutions, suspensions, emulsions, elixirs, or
capsules filled with the same, all for oral use, in the form of
suppositories for rectal administration; or in the form of sterile
injectable solutions for parenteral (including subcutaneous) use.
Such pharmaceutical compositions and unit dosage forms thereof may
comprise conventional ingredients in conventional proportions, with
or without additional active compounds or principles, and such unit
dosage forms may contain any suitable effective amount of the
active ingredient commensurate with the intended daily dosage range
to be employed.
[0084] A salt may be a pharmaceutically suitable (i.e.,
pharmaceutically acceptable) salt including, but not limited to,
acid addition salts formed by mixing a solution of the instant
compound with a solution of a pharmaceutically acceptable acid. A
pharmaceutically acceptable acid may be, for example, hydrochloric
acid, methanesulphonic acid, fumaric acid, maleic acid, succinic
acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric
acid, carbonic acid or phosphoric acid.
[0085] Suitable pharmaceutically-acceptable salts may further
include, but are not limited to salts of
pharmaceutically-acceptable inorganic acids, including, for
example, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic,
and hydrobromic acids, or salts of pharmaceutically-acceptable
organic acids such propionic, butyric, maleic, hydroxymaleic,
lactic, mucic, gluconic, benzoic, succinic, phenylacetic,
toluenesulfonic, benezenesulfonic, salicyclic sulfanilic, aspartic,
glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic,
tannic, ascorbic, and valeric acids.
[0086] Various pharmaceutically acceptable salts include, for
example, the list of FDA-approved commercially marketed salts
including acetate, benzenesulfonate, benzoate, bicarbonate,
bitartrate, bromide, calcium edetate, camsylate, carbonate,
chloride, citrate, dihydrochloride, edetate, edisylate, estolate,
esylate, fumarate, gluceptate, gluconate, glutamate,
glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,
hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate,
lactobionate, malate, maleate, mandelate, mesylate, methylbromide,
methylnitrate, methylsulfate, mucate, napsylate, mitrate, pamoate,
pantothenate, phosphate, diphosphate, polygalacturonate,
salicylate, stearate, subacetate, succinate, sulfate, tannate,
tartrate, teoclate, and triethiodide.
[0087] A hydrate may be a pharmaceutically suitable (i.e.,
pharmaceutically acceptable) hydrate that is a compound formed by
the addition of water or its elements to a host molecule (for
example, the free form version of the compound) including, but not
limited to, monohydrates, dihydrates, etc. A solvate may be a
pharmaceutically suitable (i.e., pharmaceutically acceptable)
solvate, whereby solvation is an interaction of a solute with a
solvent that leads to stabilization of the solute species in a
solution, and whereby the solvated state is an ion in a solution
complexed by solvent molecules. Solvates and hydrates may also be
referred to as "analogues."
[0088] A prodrug may be a compound that is pharmacologically inert
but is converted by enzyme or chemical action to an active form of
the drug (i.e., an active pharmaceutical ingredient) at or near the
predetermined target site. In other words, prodrugs are inactive
compounds or partially active compounds that yield an active
compound upon metabolism in the body, which may or may not be
enzymatically controlled. Prodrugs may also be broadly classified
into two groups: bioprecursor and carrier prodrugs. Prodrugs may
also be subclassified according to the nature of their action.
Bioprecursor prodrugs are compounds that already contain the embryo
of the active species within their structure, whereby the active
species are produced upon metabolism.
[0089] Carrier prodrugs are formed by combining the active drug
(e.g., active ingredient) with a carrier species forming a compound
having desirable chemical and biological characteristics, whereby
the link is an ester or amide so that the carrier prodrug is easily
metabolized upon absorption or delivery to the target site. For
example, lipophilic moieties may be incorporated to improve
transport through membranes. Carrier prodrugs linked by a
functional group to carrier are referred to as bipartite prodrugs.
Prodrugs where the carrier is linked to the drug by a separate
structure are referred to as tripartite prodrugs, whereby the
carrier is removed by an enzyme-controlled metabolic process, and
whereby the linking structure is removed by an enzyme system or by
a chemical reaction. A hydroxy-protecting group includes, for
example, a tert-butyloxy-carbonyl (t-BOC) and
t-butyl-dimethyl-silyl (TBS). Other hydroxy protecting groups
contemplated are known in the art.
[0090] In another embodiment, a dosage form and/or composition may
include one or more active metabolites of the active ingredients in
place of or in addition to the active ingredients disclosed
herein.
[0091] Dosage form compositions containing the active ingredients
may also contain one or more inactive pharmaceutical ingredients
such as diluents, solubilizers, alcohols, binders, controlled
release polymers, enteric polymers, disintegrants, excipients,
colorants, flavorants, sweeteners, antioxidants, preservatives,
pigments, additives, fillers, suspension agents, surfactants (for
example, anionic, cationic, amphoteric and nonionic), and the like.
Various FDA-approved topical inactive ingredients are found at the
FDA's "The Inactive Ingredients Database" that contains inactive
ingredients specifically intended as such by the manufacturer,
whereby inactive ingredients can also be considered active
ingredients under certain circumstances, according to the
definition of an active ingredient given in 21 CFR 210.3(b)(7).
Alcohol is a good example of an ingredient that may be considered
either active or inactive depending on the product formulation.
[0092] As used herein, an oral dosage form may include capsules (a
solid oral dosage form consisting of a shell and a filling, whereby
the shell is composed of a single sealed enclosure, or two halves
that fit together and which are sometimes sealed with a band and
whereby capsule shells may be made from gelatin, starch, or
cellulose, or other suitable materials, may be soft or hard, and
are filled with solid or liquid ingredients that can be poured or
squeezed), capsule or coated pellets (solid dosage form in which
the drug is enclosed within either a hard or soft soluble container
or "shell" made from a suitable form of gelatin; the drug itself is
in the form of granules to which varying amounts of coating have
been applied), capsule coated extended release (a solid dosage form
in which the drug is enclosed within either a hard or soft soluble
container or "shell" made from a suitable form of gelatin;
additionally, the capsule is covered in a designated coating, and
which releases a drug or drugs in such a manner to allow at least a
reduction in dosing frequency as compared to that drug or drugs
presented as a conventional dosage form), capsule delayed release
(a solid dosage form in which the drug is enclosed within either a
hard or soft soluble container made from a suitable form of
gelatin, and which releases a drug (or drugs) at a time other than
promptly after administration, whereby enteric-coated articles are
delayed release dosage forms), capsule delayed release pellets
(solid dosage form in which the drug is enclosed within either a
hard or soft soluble container or "shell" made from a suitable form
of gelatin); the drug itself is in the form of granules to which
enteric coating has been applied, thus delaying release of the drug
until its passage into the intestines), capsule extended release (a
solid dosage form in which the drug is enclosed within either a
hard or soft soluble container made from a suitable form of
gelatin, and which releases a drug or drugs in such a manner to
allow a reduction in dosing frequency as compared to that drug or
drugs presented as a conventional dosage form), capsule film-coated
extended release (a solid dosage form in which the drug is enclosed
within either a hard or soft soluble container or "shell" made from
a suitable form of gelatin; additionally, the capsule is covered in
a designated film coating, and which releases a drug or drugs in
such a manner to allow at least a reduction in dosing frequency as
compared to that drug or drugs presented as a conventional dosage
form), capsule gelatin coated (a solid dosage form in which the
drug is enclosed within either a hard or soft soluble container
made from a suitable form of gelatin; through a banding process,
the capsule is coated with additional layers of gelatin so as to
form a complete seal), and capsule liquid filled (a solid dosage
form in which the drug is enclosed within a soluble, gelatin shell
which is plasticized by the addition of a polyol, such as sorbitol
or glycerin, and is therefore of a somewhat thicker consistency
than that of a hard shell capsule; typically, the active
ingredients are dissolved or suspended in a liquid vehicle).
[0093] Oral dosage forms contemplated herein also include granules
(a small particle or grain), pellet (a small sterile solid mass
consisting of a highly purified drug, with or without excipients,
made by the formation of granules, or by compression and molding),
pellets coated extended release (a solid dosage form in which the
drug itself is in the form of granules to which varying amounts of
coating have been applied, and which releases a drug or drugs in
such a manner to allow a reduction in dosing frequency as compared
to that drug or drugs presented as a conventional dosage form),
pill (a small, round solid dosage form containing a medicinal agent
intended for oral administration), powder (an intimate mixture of
dry, finely divided drugs and/or chemicals that may be intended for
internal or external use), elixir (a clear, pleasantly flavored,
sweetened hydroalcoholic liquid containing dissolved medicinal
agents; it is intended for oral use), chewing gum (a sweetened and
flavored insoluble plastic material of various shapes which when
chewed, releases a drug substance into the oral cavity), or syrup
(an oral solution containing high concentrations of sucrose or
other sugars; the term has also been used to include any other
liquid dosage form prepared in a sweet and viscid vehicle,
including oral suspensions).
[0094] Oral dosage forms contemplated herein may further include a
tablet (a solid dosage form containing medicinal substances with or
without suitable diluents), tablet chewable (a solid dosage form
containing medicinal substances with or without suitable diluents
that is intended to be chewed, producing a pleasant tasting residue
in the oral cavity that is easily swallowed and does not leave a
bitter or unpleasant after-taste), tablet coated (a solid dosage
form that contains medicinal substances with or without suitable
diluents and is covered with a designated coating), tablet coated
particles (a solid dosage form containing a conglomerate of
medicinal particles that have each been covered with a coating),
tablet delayed release (a solid dosage form which releases a drug
or drugs at a time other than promptly after administration,
whereby enteric-coated articles are delayed release dosage forms),
tablet delayed release particles (a solid dosage form containing a
conglomerate of medicinal particles that have been covered with a
coating which releases a drug or drugs at a time other than
promptly after administration, whereby enteric-coated articles are
delayed release dosage forms), tablet dispersible (a tablet that,
prior to administration, is intended to be placed in liquid, where
its contents will be distributed evenly throughout that liquid,
whereby term `tablet, dispersible` is no longer used for approved
drug products, and it has been replaced by the term `tablet, for
suspension`), tablet effervescent (a solid dosage form containing
mixtures of acids, for example, citric acid, tartaric acid, and
sodium bicarbonate, which release carbon dioxide when dissolved in
water, whereby it is intended to be dissolved or dispersed in water
before administration), tablet extended release (a solid dosage
form containing a drug which allows at least a reduction in dosing
frequency as compared to that drug presented in conventional dosage
form), tablet film coated (a solid dosage form that contains
medicinal substances with or without suitable diluents and is
coated with a thin layer of a water-insoluble or water-soluble
polymer), tablet film coated extended release (a solid dosage form
that contains medicinal substances with or without suitable
diluents and is coated with a thin layer of a water-insoluble or
water-soluble polymer; the tablet is formulated in such manner as
to make the contained medicament available over an extended period
of time following ingestion), tablet for solution (a tablet that
forms a solution when placed in a liquid), tablet for suspension (a
tablet that forms a suspension when placed in a liquid, which is
formerly referred to as a `dispersible tablet`), tablet multilayer
(a solid dosage form containing medicinal substances that have been
compressed to form a multiple-layered tablet or a
tablet-within-a-tablet, the inner tablet being the core and the
outer portion being the shell), tablet multilayer extended release
(a solid dosage form containing medicinal substances that have been
compressed to form a multiple-layered tablet or a
tablet-within-a-tablet, the inner tablet being the core and the
outer portion being the shell, which, additionally, is covered in a
designated coating; the tablet is formulated in such manner as to
allow at least a reduction in dosing frequency as compared to that
drug presented as a conventional dosage form), tablet orally
disintegrating (a solid dosage form containing medicinal substances
which disintegrates rapidly, usually within a matter of seconds,
when placed upon the tongue), tablet orally disintegrating delayed
release (a solid dosage form containing medicinal substances which
disintegrates rapidly, usually within a matter of seconds, when
placed upon the tongue, but which releases a drug or drugs at a
time other than promptly after administration), tablet soluble (a
solid dosage form that contains medicinal substances with or
without suitable diluents and possesses the ability to dissolve in
fluids), tablet sugar coated (a solid dosage form that contains
medicinal substances with or without suitable diluents and is
coated with a colored or an uncolored water-soluble sugar), and the
like.
[0095] Injection and infusion dosage forms (i.e., parenteral dosage
forms) include, but are not limited to, the following. Liposomal
injection includes or forms liposomes or a lipid bilayer vesicle
having phospholipids that encapsulate an active drug substance.
Injection includes a sterile preparation intended for parenteral
use. Five distinct classes of injections exist as defined by the
USP. Emulsion injection includes an emulsion comprising a sterile,
pyrogen-free preparation intended to be administered parenterally.
Lipid complex and powder for solution injection are sterile
preparations intended for reconstitution to form a solution for
parenteral use.
[0096] Powder for suspension injection is a sterile preparation
intended for reconstitution to form a suspension for parenteral
use. Powder lyophilized for liposomal suspension injection is a
sterile freeze dried preparation intended for reconstitution for
parenteral use that is formulated in a manner allowing
incorporation of liposomes, such as a lipid bilayer vesicle having
phospholipids used to encapsulate an active drug substance within a
lipid bilayer or in an aqueous space, whereby the formulation may
be formed upon reconstitution. Powder lyophilized for solution
injection is a dosage form intended for the solution prepared by
lyophilization ("freeze drying"), whereby the process involves
removing water from products in a frozen state at extremely low
pressures, and whereby subsequent addition of liquid creates a
solution that conforms in all respects to the requirements for
injections. Powder lyophilized for suspension injection is a liquid
preparation intended for parenteral use that contains solids
suspended in a suitable fluid medium, and it conforms in all
respects to the requirements for Sterile Suspensions, whereby the
medicinal agents intended for the suspension are prepared by
lyophilization.
[0097] Solution injection involves a liquid preparation containing
one or more drug substances dissolved in a suitable solvent or
mixture of mutually miscible solvents that is suitable for
injection. Solution concentrate injection involves a sterile
preparation for parenteral use that, upon addition of suitable
solvents, yields a solution suitable for injections. Suspension
injection involves a liquid preparation (suitable for injection)
containing solid particles dispersed throughout a liquid phase,
whereby the particles are insoluble, and whereby an oil phase is
dispersed throughout an aqueous phase or vice-versa. Suspension
liposomal injection is a liquid preparation (suitable for
injection) having an oil phase dispersed throughout an aqueous
phase in such a manner that liposomes (a lipid bilayer vesicle
usually containing phospholipids used to encapsulate an active drug
substance either within a lipid bilayer or in an aqueous space) are
formed. Suspension sonicated injection is a liquid preparation
(suitable for injection) containing solid particles dispersed
throughout a liquid phase, whereby the particles are insoluble. In
addition, the product may be sonicated as a gas is bubbled through
the suspension resulting in the formation of microspheres by the
solid particles.
[0098] A parenteral carrier system may include one or more
pharmaceutically suitable excipients, such as solvents and
co-solvents, solubilizing agents, wetting agents, suspending
agents, thickening agents, emulsifying agents, chelating agents,
buffers, pH adjusters, antioxidants, reducing agents, antimicrobial
preservatives, bulking agents, protectants, tonicity adjusters, and
special additives.
[0099] Inhalation dosage forms include, but are not limited to,
aerosol being a product that is packaged under pressure and
contains therapeutically active ingredients that are released upon
activation of an appropriate valve system intended for topical
application to the skin as well as local application into the nose
(nasal aerosols), mouth (lingual and sublingual aerosols), or lungs
(inhalation aerosols). Inhalation dosage forms further include foam
aerosol being a dosage form containing one or more active
ingredients, surfactants, aqueous or nonaqueous liquids, and the
propellants, whereby if the propellant is in the internal
(discontinuous) phase (i.e., of the oil-in-water type), a stable
foam is discharged, and if the propellant is in the external
(continuous) phase (i.e., of the water-in-oil type), a spray or a
quick-breaking foam is discharged. Inhalation dosage forms also
include metered aerosol being a pressurized dosage form consisting
of metered dose valves which allow for the delivery of a uniform
quantity of spray upon each activation; powder aerosol being a
product that is packaged under pressure and contains
therapeutically active ingredients, in the form of a powder, that
are released upon activation of an appropriate valve system; and
aerosol spray being an aerosol product which utilizes a compressed
gas as the propellant to provide the force necessary to expel the
product as a wet spray and being applicable to solutions of
medicinal agents in aqueous solvents.
[0100] Pharmaceutically suitable inhalation carrier systems may
include pharmaceutically suitable inactive ingredients known in the
art for use in various inhalation dosage forms, such as (but not
limited to) aerosol propellants (for example, hydrofluoroalkane
propellants), surfactants, additives, suspension agents, solvents,
stabilizers and the like.
[0101] A transdermal dosage form may include, but is not limited
to, a patch being a drug delivery system that often contains an
adhesive backing that is usually applied to an external site on the
body, whereby the ingredients either passively diffuse from, or are
actively transported from some portion of the patch, and whereby
depending upon the patch, the ingredients are either delivered to
the outer surface of the body or into the body; and other various
types of transdermal patches such as matrix, reservoir and others
known in the art. The "pharmaceutically suitable transdermal
carrier system" includes pharmaceutically suitable inactive
ingredients known in the art for use in various transdermal dosage
forms, such as (but not limited to) solvents, adhesives, diluents,
additives, permeation enhancing agents, surfactants, emulsifiers,
liposomes, and the like.
[0102] Suitable dosage amounts and dosing regimens may be selected
in accordance with a variety of factors, including one or more
particular conditions being treated, the severity of the one or
more conditions, the genetic profile, age, health, sex, diet, and
weight of the subject, the route of administration alone or in
combination with pharmacological considerations including the
activity, efficacy, bioavailability, pharmacokinetic, and
toxicological profiles of the particular compound employed, whether
a drug delivery system is utilized and whether the drug is
administered as part of a drug combination. Therefore, the dosage
regimen to be employed may vary widely and may necessarily deviate
from the dosage regimens set forth herein.
[0103] Contemplated dosage forms may include an amount of one or
more expression inhibitors (or inhibitors of expression) ranging
from about 1 to about 1200 mg, or about 5 to about 100 mg, or about
25 to about 800 mg, or about 100 to about 500 mg, or 0.1 to 50
milligrams (.+-.10%), or 10 to 100 milligrams (.+-.10%), or 5 to
500 milligrams (.+-.10%), or 0.1 to 200 milligrams (.+-.10%), or 1
to 100 milligrams (.+-.10%), or 5 to 50 milligrams (.+-.10%), or 30
milligrams (.+-.10%), or 20 milligrams (.+-.10%), or 10 milligrams
(.+-.10%), or 5 milligrams (.+-.10%), per dosage form, such as, for
example, a tablet, a pill, a bolus, and the like.
[0104] In another embodiment, a dosage form may be administered to
a subject in need thereof once per day, or twice per day, or once
every 6 hours, or once every 4 hours, or once every 2 hours, or
hourly, or twice an hour, or twice a day, or twice a week, or
monthly.
[0105] The phrase "therapeutically effective" is intended to
qualify the amount that will achieve the goal of improvement in
disease severity and/or the frequency of incidence over
non-treatment, while limiting, reducing, or avoiding adverse side
effects typically associated with disease therapies. A "therapeutic
effect" relieves to some extent one or more of the symptoms of a
cancer disease or disorder. In reference to the treatment of a
cancer, a therapeutic effect refers to one or more of the
following: 1) reduction in the number of cancer cells by, for
example, killing the cancer cells; 2) reduction in tumor size; 3)
inhibition (i.e., slowing to some extent, preferably stopping) of
cancer cell infiltration into peripheral organs; 4) inhibition
(i.e., slowing to some extent, preferably stopping) of tumor
metastasis; 5) inhibition, to some extent, of tumor growth; 6)
relieving or reducing to some extent one or more of the symptoms
associated with the disorder; and/or 7) relieving or reducing the
side effects associated with the administration of anticancer
agents. "Therapeutic effective amount" is intended to qualify the
amount required to achieve a therapeutic effect.
[0106] A therapeutically effective amount of an expression
inhibitor (or inhibitors of expression) may be any amount that
begins to improve cancer treatment in a subject. In one embodiment,
an effective amount of an expression inhibitor used in the
therapeutic regime described herein may be, for example, about 1
mg, or about 5 mg, or about 10 mg, or about 25 mg, or about 50 mg,
or about 100 mg, or about 200 mg, or about 400 mg, or about 500 mg,
or about 600 mg, or about 1000 mg, or about 1200 mg, or about 1400
mg, or from about 10 to about 60 mg, or about 50 mg to about 200
mg, or about 150 mg to about 600 mg per day. Further, another
effective amount of an expression inhibitor used herein may be that
which results in a detectable blood level of above about 1 ng/dL,
5, ng/dL, 10 ng/dL, 20, ng/dL, 35 ng/dL, or about 70 ng/dL, or
about 140 ng/dL, or about 280 ng/dL, or about 350 ng/dL, or lower
or higher.
[0107] The term "pharmaceutically acceptable" is used herein to
mean that the modified noun is appropriate for use in a
pharmaceutical product. Pharmaceutically acceptable cations include
metallic ions and organic ions. Other metallic ions include, but
are not limited to appropriate alkali metal salts, alkaline earth
metal salts and other physiological acceptable metal ions.
Exemplary ions include aluminium, calcium, lithium, magnesium,
potassium, sodium and zinc in their usual valences. Organic ions
include protonated tertiary amines and quaternary ammonium cations,
including in part, trimethylamine, diethylamine,
N,N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and
procaine. Pharmaceutically acceptable acids include without
limitation hydrochloric acid, hydrobromic acid, phosphoric acid,
sulfuric acid, methanesulfonic acid, acetic acid, formic acid,
tartaric acid, maleic acid, malic acid, citric acid, isocitric
acid, succinic acid, lactic acid, gluconic acid, glucuronic acid,
pyruvic acid oxalacetic acid, fumaric acid, propionic acid,
aspartic acid, glutamic acid, benzoic acid, and the like.
[0108] It is further contemplated that one active ingredient may be
in an extended release form, while an optional second, third, or
fourth other active ingredient, for example, may or may not be, so
the recipient experiences, for example, a spike in the second,
third, or fourth active ingredient that dissipates rapidly, while
the first active ingredient is maintained in a higher concentration
in the blood stream over a longer period of time. Similarly, one of
the active ingredients may be an active metabolite, while another
may be in an unmetabolized state, such that the active metabolite
has an immediate effect upon administration to a subject whereas
the unmetabolized active ingredient administered in a single dosage
form may need to be metabolized before taking effect in the
subject.
[0109] Also contemplated are solid form preparations that include
at least one active ingredient which are intended to be converted,
shortly before use, to liquid form preparations for oral
administration. Such liquid forms include solutions, suspensions,
and emulsions. These preparations may contain, in addition to the
active component, colorants, flavors, stabilizers, buffers,
artificial and natural sweeteners, dispersants, thickeners,
solubilizing agents, and the like. Solutions or suspensions may be
applied topically and/or directly to the nasal cavity, respiratory
tract, eye, or ear by conventional means, for example with a
dropper, pipette or spray.
[0110] Alternatively, one or more of the active ingredients may be
provided in the form of a dry powder, for example a powder mix of
the compound in a suitable powder base such as lactose, starch,
starch derivatives such as hydroxypropylmethyl cellulose and
polyvinylpyrrolidone (PVP). Conveniently the powder carrier may
form a gel in the nasal cavity. The powder composition may be
presented in unit dose form, for example, in capsules or cartridges
of, for example, gelatin, or blister packs from which the powder
may be administered by means of an inhaler.
[0111] The pharmaceutical preparations may be in unit dosage forms.
In such form, the preparation may be subdivided into unit doses
containing appropriate quantities of the active component. The unit
dosage form can be a packaged preparation, such as a kit or other
form, the package containing discrete quantities of preparation,
such as packeted tablets, capsules, liquids or powders in vials or
ampoules. Also, the unit dosage form can be a capsule, tablet,
cachet, or lozenge, or it can be the appropriate number of any of
these in packaged form.
[0112] The present disclosure is further illustrated by the
following examples, which should not be construed as limiting in
any way. The contents of all cited references throughout this
application are hereby expressly incorporated by reference. The
practice of the present invention will employ, unless otherwise
indicated, conventional techniques of pharmacology and
pharmaceutics, which are within the skill of the art.
Examples
Materials and Methods
[0113] Cell Lines
[0114] Cell lines were from the American Type Culture Collection
(Rockville, Md.), except for CW22Rv1 and CWRR1, which were kindly
provided by Dr. Donald VanderGriend, the University of Chicago.
[0115] Western Blotting and Flow Cytometry
[0116] Total cellular protein was extracted in
radioimmunoprecipitation assay buffer with protease inhibitors
added (1.times.PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1
mmol/L Na.sub.3VO.sub.4, 2 .mu.g/mL aprotinin, 1 mmol/L
phenylmethylsulfonyl fluoride). All samples were normalized by
protein concentration using Bradford reagent and standard solution
of bovine serum albumin (1 mg/mL). Concentration of all samples was
adjusted to 1 mg/mL and equal amount of protein was loaded in each
well. For total proteins, 10 .mu.g of protein was loaded per well.
Proteins were separated on 7.5%-12% SDS-PAGE (depending on
molecular mass of protein) and transferred to polyvinylidene
difluoride membranes (PVDF). Total proteins were detected using the
rabbit or goat primary Abs. For loading control, the antibodies for
actin (I-19) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH;
FL-335) were used. All antibodies were purchased from Santa Cruz
Biotechnology. Images were quantified with ImageJ software by
integration pixel values across the area of specific bands.
[0117] Real-Time PCR Analysis
[0118] cDNA was synthesized as described previously (see, Khodarev
N N, Yu J, Nodzenski E, et al. Method of RNA purification from
endothelial cells for DNA array experiments. Biotechniques 2002;
32: 316-20). For the internal control, GAPDH was used. PCR was done
for 40 cycles at 95.degree. C. for 15 s and 60.degree. C. for 1 min
after initial incubations at 50.degree. C. for 2 min and 95.degree.
C. for 10 min using SYBR Green PCR reagent in ABI 7700 System
(Applied Biosystems). .DELTA.Ct values were calculated according to
the manufacturer's instructions. Fold induction/suppression
relative to GAPDH was calculated as 2.sup.-.DELTA.ct. Fold
induction of gene Xs, in gene-specific siRNA transfected cell line
relative to gene X.sub.nt in the same cell line transfected by
non-targeting siRNA was calculated as 2.sup.-(.DELTA..DELTA.CtX),
where .DELTA.Ct values of all control replicates were averaged.
Number of replicates per each group varied from three to six in
different experiments.
Example 1. Identification of Downstream Effector Genes of the
Jak/Stat Pathway
[0119] To identify downstream effector genes in the Jak/Stat
pathway that may have a causal role in treatment-resistant cancers,
microarray and proteomics data were collected from available
literature demonstrating association of subset of these genes named
as interferon-stimulated-genes (ISG) with oncogenesis and tumor
radio/chemoresistance. Association in this case refers to
differential expression of interferon-stimulated genes in tumors
versus normal tissues or in chemoresistant and/or radioresistant
cell lines versus sensitive cell lines, or induction of
interferon-stimulated genes by chemotherapy and radiotherapy
[0120] To elucidate the downstream interferon-stimulated genes that
contributes to a more aggressive and therapy-resistant (ionizing
radiation and chemotherapy) phenotype, the Interferome and GEO
databases (see, respectively, Pitroda S P, Khodarev N N, Beckett M
A, Kufe D W, Weichselbaum R R. MUC1-induced alterations in a lipid
metabolic gene network predict response of human breast cancers to
tamoxifen treatment. Proc Natl Acad Sci USA 2009; 106:5837-41; and
Keller S M, Adak S, Wagner H, Herskovic A, Komaki R, Brooks B J, et
al. A randomized trial of postoperative adjuvant therapy in
patients with completely resected stage II or IIIA non-small-cell
lung cancer. Eastern Cooperative Oncology Group. N Engl J Med 2000;
343:1217-22) were initially screened to identify 787 genes that
were differentially expressed in chemoresistant and radioresistant
tumors or responded to radio-chemotherapy. A literature review and
review of the Interferome database was then used to identify
interferon-stimulated genes (ISGs) that have been shown to be
associated with an aggressive phenotype or resistance to therapy
(ionizing radiation or chemotherapy). Eleven studies were
identified (see Table No. 1) that described interferon-stimulated
genes associated with resistance to DNA-damage or a poor clinical
prognosis.
TABLE-US-00001 TABLE NO. 1 Studies Describing Interferon-stimulated
Genes Study No. ISG Study 1 Sawyer T E, Bonner J A, Gould P M,
Foote R L, Deschamps C, Trastek V F, et al. Effectiveness of
postoperative irradiation in stage IIIA non-small cell lung cancer
according to regression tree analyses of recurrence risks. Ann
Thorac Surg 1997; 64: 1402-7; discussion 7-8. 2 Stephens R J,
Girling D J, Bleehen N M, Moghissi K, Yosef H M, Machin D. The role
of post-operative radiotherapy in non-small-cell lung cancer: a
multicentre randomised trial in patients with pathologically staged
T1-2, N1-2, M0 disease. Medical Research Council Lung Cancer
Working Party. British journal of cancer 1996; 74: 632-9. 3
MacDermed D M, Khodarev N N, Pitroda S P, Edwards D C, Pelizzari C
A, Huang L, et al. MUC1-associated proliferation signature predicts
outcomes in lung adenocarcinoma patients. BMC Med Genomics 2010; 3:
16. 4 Khodarev N N, Pitroda S P, Beckett M A, MacDermed D M, Huang
L, Kufe D W, et al. MUC1-induced transcriptional programs
associated with tumorigenesis predict outcome in breast and lung
cancer. Cancer Res 2009; 69: 2833-7. 5 Khodarev N N, Minn A J,
Efimova E V, Darga T E, Labay E, Beckett M, et al. Signal
transducer and activator of transcription 1 regulates both
cytotoxic and prosurvival functions in tumor cells. Cancer Res
2007; 67: 9214-20. 6 Khodarev N N, Roach P, Pitroda S P, Golden D
W, Bhayani M, Shao M Y, et al. STAT1 pathway mediates amplification
of metastatic potential and resistance to therapy. PLoS One 2009;
4: e5821. 7 Pardanani A, Vannucchi A M, Passamonti F, Cervantes F,
Barbui T, Tefferi A. JAK inhibitor therapy for myelofibrosis:
critical assessment of value and limitations. Leukemia; 25: 218-25.
8 Wernig G, Kharas M G, Okabe R, Moore S A, Leeman D S, Cullen D E,
et al. Efficacy of TG101348, a selective JAK2 inhibitor, in
treatment of a murine model of JAK2V617F- induced polycythemia
vera. Cancer Cell 2008; 13: 311-20. 9 Fridman J S, Scherle P A,
Collins R, Burn T C, Li Y, Li J, et al. Selective inhibition of
JAK1 and JAK2 is efficacious in rodent models of arthritis:
preclinical characterization of INCB028050. J Immunol; 184:
5298-307. 10 Garber K. Pfizer's JAK inhibitor sails through phase 3
in rheumatoid arthritis. Nat Biotechnol 2011 29: 467-8. 11 Sun Y,
Moretti L, Giacalone N J, Schleicher S, Speirs C K, Carbone D P, et
al. Inhibition of JAK2 signaling by TG101209 enhances radiotherapy
in lung cancer models. J Thorac Oncol 20116: 699-706.
[0121] In addition, two published papers (Hsu H S, Lin J H, Hsu T
W, Su K, Wang C W, Yang K Y, et al. Mesenchymal stem cells enhance
lung cancer initiation through activation of IL-6/JAK2/STAT3
pathway. Lung cancer (Amsterdam, Netherlands) 2012; 75:167-77; Zhao
M, Gao F H, Wang J Y, Liu F, Yuan H H, Zhang W Y, et al. JAK2/STAT3
signaling pathway activation mediates tumor angiogenesis by
upregulation of VEGF and bFGF in non-small-cell lung cancer. Lung
cancer (Amsterdam, Netherlands) 2011; 73:366-74) and unpublished
data from our laboratory were used to identify candidate downstream
interferon-stimulated genes. Genes were included in the final
screening set if they were identified in the IRDS or if they were
reported in greater than or equal to two other studies. In total,
89 candidate interferon-stimulated genes were identified as shown
below in Table No. 2.
TABLE-US-00002 TABLE NO. 2 Candidate Interferon-stimulated Genes
Gene Entrez Gene Symbol Entrez Gene Name ID for Human ABCC3
ATP-binding cassette, sub-family C (CFTR/MRP), 8714 member 3 B2M
beta-2-microglobulin 567 BST2 bone marrow stromal cell antigen 2
684 CCL2 chemokine (C-C motif) ligand 2 6347 CCL5 chemokine (C-C
motif) ligand 5 6352 CCNA1 cyclin A1 8900 CD74 CD74 molecule, major
histocompatibility complex, class II 972 invariant chain CMPK2
cytidine monophosphate (UMP-CMP) kinase 2, 129607 mitochondrial
CTSS cathepsin S 1520 CXCL1 chemokine (C-X-C motif) ligand 1
(melanoma growth 2919 stimulating activity, alpha) CXCL10 chemokine
(C-X-C motif) ligand 10 3627 CXCL3 chemokine (C-X-C motif) ligand 3
2921 CXCL9 chemokine (C-X-C motif) ligand 9 4283 DAZ1 deleted in
azoospermia 1 1617 DDX58 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58
23586 DDX60 DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 55601 DDX60L
DEAD (Asp-Glu-Ala-Asp) box polypeptide 60-like 91351 DHX58 DEXH
(Asp-Glu-X-His) box polypeptide 58 79132 DTX3L deltex 3-like
(Drosophila) 151636 EIF2AK2 eukaryotic translation initiation
factor 2-alpha kinase 2 5610 EPSTI1 epithelial stromal interaction
1 (breast) 94240 GBP1 guanylate binding protein 1,
interferon-inducible, 67 kDa 2633 GBP2 guanylate binding protein 2,
interferon-inducible 2634 HERC5 hect domain and RLD 5 51191 HERC6
hect domain and RLD 6 55008 HNMT histamine N-methyltransferase 3176
IFI16 interferon, gamma-inducible protein 16 3428 IFI27 interferon,
alpha-inducible protein 27 3429 IFI35 interferon-induced protein 35
3430 IFI44 interferon-induced protein 44 10561 IFI44L
interferon-induced protein 44-like 10964 IFI6 interferon,
alpha-inducible protein 6 2537 IFIH1 interferon induced with
helicase C domain 1 64135 IFIT1 interferon-induced protein with
tetratricopeptide repeats 1 3434 IFIT2 interferon-induced protein
with tetratricopeptide repeats 2 3433 IFIT3 interferon-induced
protein with tetratricopeptide repeats 3 3437 IFITM1 interferon
induced transmembrane protein 1 (9-27) 8519 IFITM2 interferon
induced transmembrane protein 2 (1-8D) 10581 IFITM3 interferon
induced transmembrane protein 3 (1-8U) 10410 IGFBP3 insulin-like
growth factor binding protein 3 3486 IRF1 interferon regulatory
factor 1 3659 IRF7 interferon regulatory factor 7 3665 IRF9
interferon regulatory factor 9 10379 ISG15 ISG15 ubiquitin-like
modifier 9636 LAMP3 lysosomal-associated membrane protein 3 27074
LGALS3BP lectin, galactoside-binding, soluble, 3 binding protein
3959 LTK leukocyte receptor tyrosine kinase 4058 LY6E lymphocyte
antigen 6 complex, locus E 4061 LY96 lymphocyte antigen 96 23643
MARCKS myristoylated alanine-rich protein kinase C substrate 4082
MCL1 myeloid cell leukemia sequence 1 (BCL2-related) 4170 MGP
matrix Gla protein 4256 MX1 myxovirus (influenza virus) resistance
1, interferon- 4599 inducible protein p78 (mouse) MX2 myxovirus
(influenza virus) resistance 2 (mouse) 4600 NLRC5 NLR family, CARD
domain containing 5 84166 NMI N-myc (and STAT) interactor 9111 OAS1
2',5'-oligoadenylate synthetase 1, 40/46 kDa 4938 OAS2
2'-5'-oligoadenylate synthetase 2, 69/71 kDa 4939 OAS3
2'-5'-oligoadenylate synthetase 3, 100 kDa 4940 OASL
2'-5'-oligoadenylate synthetase-like 8638 PARP12 poly (ADP-ribose)
polymerase family, member 12 64761 PLSCR1 phospholipid scramblase 1
5359 PRIC285 peroxisomal proliferator-activated receptor A
interacting 85441 complex 285 PSMB10 proteasome (prosome,
macropain) subunit, beta type, 10 5699 PSMB8 proteasome (prosome,
macropain) subunit, beta type, 8 5696 (large multifunctional
peptidase 7) PSMB9 proteasome (prosome, macropain) subunit, beta
type, 9 5698 (large multifunctional peptidase 2) RNF213 ring finger
protein 213 57674 RSAD2 radical S-adenosyl methionine domain
containing 2 91543 RTP4 receptor (chemosensory) transporter protein
4 64108 SAMD9 sterile alpha motif domain containing 9 54809 SAMD9L
sterile alpha motif domain containing 9-like 219285 SAMHD1 SAM
domain and HD domain 1 25939 SP110 SP110 nuclear body protein 3431
SRGN serglycin 5552 STAT1 signal transducer and activator of
transcription 1, 91 kDa 6772 TAGLN transgelin 6876 TAP1 transporter
1, ATP-binding cassette, sub-family B 6890 (MDR/TAP) THBS1
thrombospondin 1 7057 TIMP3 TIMP metallopeptidase inhibitor 3 7078
TNFSF10 tumor necrosis factor (ligand) superfamily, member 10 8743
TPD52L1 tumor protein D52-like 1 7164 TRIM14 tripartite
motif-containing 14 9830 TRIM21 tripartite motif-containing 21 6737
UBA7 ubiquitin-like modifier activating enzyme 7 7318 UBE2L6
ubiquitin-conjugating enzyme E2L 6 9246 USP18 ubiquitin specific
peptidase 18 11274 VAMP5 vesicle-associated membrane protein 5
(myobrevin) 10791 WARS tryptophanyl-tRNA synthetase 7453 XAF1 XIAP
associated factor 1 54739
[0122] To screen the candidate interferon-stimulated genes, a panel
of fifteen individual cancer cell lines were selected from those
available in our lab, the ATCC, or affiliated laboratories (see
Table No. 3, below). These cell lines were characterized for
relative radioresistance using a clonogenic assay. Briefly, the
cells were cultured in their appropriate media at 5% CO2 at
37.degree. C. Optimal cell plating concentrations for a p60 dish
were determined for each cell line. The majority of cell lines were
plated in triplicate at concentrations of 100 cells/plate for
control, 1000 cells/plate for 2 Gy, 2000 cells/plate for 5 Gy, and
3000 cells/plate for 8 Gy. Plates were irradiated 24 hours after
plating with either 2, 5, or 8 Gy using a Nordion Gammacell
.sup.60Co irradiator operating at a dose rate of -18 Gy/minute.
Cells were allowed to grow for 10-21 days (depending on the cell
line) until colonies were >50 cells. Plates were then fixed with
formalin and stained with crystal violet. Colonies with more than
50 cells were counted. Clonogenic survival for Clonogenic data was
obtained for all cell lines tested in screen.
[0123] An siRNA screen of 86 interferon stimulated genes (ISGs) was
performed in a series of cancer cell lines to determine which of
the candidate genes may be associated with treatment resistance in
the cell lines. The final screen was conducted as follows: On day
1, Lipofectamine RNAiMAX 0.075 .mu.L/well diluted in Opti-MEM (Life
Technologies) was added using a Tecan Freedom EVO 200 robotic
liquid handling station to previously prepared 384-well microplates
(Corning/3712) containing immobilized pooled siRNAs (Dharmacon
siGENOME) plated in triplicate for each target gene. Cells were
then added using a Thermo Electron MultiDrop Combi dispenser at 500
cells/well in 50 .mu.L of RPMI 1640 media supplemented with 10%
FCS. The final siRNA concentration in each well was 50 nM. Plates
were be incubated overnight at 37.degree. C., and on day 2 were
treated with ionizing radiation at a dose of either 3Gy or left
untreated. Plates were incubated at 37.degree. C. and then assayed
for viability on days 3 and 4 using the highly sensitive
luciferase-based CellTiterGlo assay (Promega, Madison, Wis.).
Luminescent reagent was added using a Thermo Electron MultiDrop
Combi, and luminescent measurements was taken 90 minutes later
using Molecular Devices Analyst GT.
[0124] Cell lines tested were screened under two conditions: (A) no
treatment (basal); and (B) treatment by ionizing radiation (3 Gy).
A total of fifteen (15) cell lines available from the American Type
Culture Collection (ATCC), representing 7 cancer types, were
screened (see Table No. 3 below). Experimental endpoint was loss of
cell viability as assessed by CelTiterGlo.RTM. assay (Promega,
Madison, Wis.) following manufacturer's recommendations. A heat map
of the screen is shown in FIG. 1.
TABLE-US-00003 TABLE NO. 3 Cell lines. Cell Line Primary Tumor A549
Lung NCI-H226 Lung D54 Glioblastoma multiforme T98G Glioblastoma
multiforme U251 Glioblastoma multiforme DU-145 Prostate CWRR1
Prostate CW22Rv1 Prostate MCF7 Breast MCF-10A Breast WiDR Colon
HCT116 Colon SCC-61 Head & Neck Nu61 Head & Neck T24
Bladder
[0125] Interferon-stimulated genes, for which suppression led to
the maximal loss of viability in the maximal amount of cell lines,
were selected for further validation with individual siRNA
(deconvolution).
[0126] The HCT116 and MCF10A cell lines were selected for the
confirmation experiment as they consistently had the highest level
of viability suppression for the candidate genes. The siRNA's were
deconvoluted (4 individual siRNA's per gene) and plated in
triplicate. The transfection conditions used were the same as the
primary screen for each cell line. The cell lines were plated at
optimal cell concentrations and reverse transfected on Day 0,
incubated at 37.degree. C. in 5% CO2, irradiated with 3 Gy at 48
hours, and then viability was assayed at 120 hours
post-transfection (72 hours post-ionizing radiation) using the
CellTiter-Glo.RTM. Luminescent Cell Viability Assay (Promega,
Madison, Wis.). This experiment was repeated to confirm
reproducibility of the data (FIGS. 8A and 8B). Comparison of the
pooled to the deconvoluted siRNA's demonstrates improved
suppression of viability (FIG. 9). The top two siRNA's for each
gene were selected for subsequent qRT-PCR confirmation experiments
to exclude off-target effects. All selected candidate genes were
confirmed with individual siRNAs phenotipically and by the ability
to suppress gene-specific mRNA.
[0127] Based on these data, new targets were identified for the
suppression of tumor growth and radiosensitization that may serve
as companion targets for improved tumor response to the
Jak1/Jak2-based therapy, see Table Nos. 4a and 4b.
TABLE-US-00004 TABLE NO. 4a Interferon-Stimulated Gene Targets for
Suppression of Tumor Growth and Radiosensitization in Order of
Ranking. List of Genes in Order of Their Ranking) Function DHX58
Cytoplasmic DNA sensors PLSCR1 Bacterial toxin defense USP18
protein modification/degradation PSMB10 protein
modification/degradation IFITM1 Anti-viral defense OASL Anti-viral
defense EPSTL1 Extracellular matrix protein LGALS3BP Extracellular
matrix protein IFIH1 Cytoplasmic DNA sensors ABCC3 Drug transporter
DTX3L Other PSMB9 protein modification/degradation IRF9 Anti-viral
defense TAGLN Other IFIT2 Anti-viral defense TPD52L1 Other CXCL9
Chemokine GBP1 Other BST2 Anti-viral defense SP110 Other HERC5
protein modification/degradation CCL2 Chemokine WARS Other MCL1
Anti-apoptotic mitochondria-related proteins TRIM14 Other
TABLE-US-00005 TABLE NO. 4b Interferon-Stimulated Gene Targets for
Suppression of Tumor Growth and Radiosensitization in Order of
Functional Groups. List of Genes According to Functional Groups
Functions DHX58 anti-viral defense (recognition of viral RNA)
IFITM1 anti-viral defense OASL anti-viral defense IRF9 anti-viral
defense, transcription BST2 anti-viral defense IFIT2 anti-viral
defense IFIH1 anti-viral defense (recognition of viral RNA) PLSCR1
bacterial toxin defense PSMB9 protein modification/degradation
PSMB10 protein modification/degradation USP18 protein
modification/degradation HERC5 protein modification/degradation
EPSTI1 cell-ECM interaction/cytoskeleton LGALS3BP cell-ECM
interaction/cytoskeleton TAGLN cell-ECM interaction/cytoskeleton
CXCL9 chemokine CCL2 chemokine ABCC3 drug transporter MCL1
anti-apoptotic mitochondrial protein DTX3L other TPD52L1 other GBP1
other SP110 other WARS other TRIM14 other
[0128] Genes in the Table No. 4a are distributed according to their
rank of suppression, with the highest rank for DHX58 and a lowest
rank for TRIM14. Genes in the Table No. 4b are distributed
according to their functions, with bold font indicating genes that
were validated in independent experiments using flow cytometry or
clonogenics assays or/and in vivo xenograft models.
[0129] Results
[0130] Using expressional profiling of experimental tumors in nude
mice, analysis of published databases and bioinformatics
approaches, constitutive expression of genes activated by Jak/Sat
signaling was observed in various types of tumors and associated
with aggressive tumor phenotype and radio/chemoresistance (FIG. 2).
It was found that ionizing radiation activated the Jak/Stat axis in
tumor cells (FIG. 3). This activation involved Stat1, Stat2, Stat3
and Stat6. Further, many down-stream genes activated by these
transcription factors overlapped indicating that different Stat
proteins can activate the same sets of genes or operate on the same
promoter sequences. Indeed, the data herein show that Stat1 can
bind to the GAS sequence in the promoter region of the Muc1 gene
and activate its transcription after IFN.gamma. stimulation
(Khodarev N, Ahmad R, Rajabi H, Pitroda S, Kufe T, McClary C, et
al. Cooperativity of the MUC1 oncoprotein and STAT1 pathway in poor
prognosis human breast cancer. Oncogene 2010; 29:920-9). The same
GAS sequence in the promoter region of Muc1 can also be occupied by
Stat3 after IL6 stimulation and lead to the activation of the same
oncogene (Ahmad R, Rajabi H, Kosugi M, Joshi M D, Alam M, Vasir B,
et al. MUC1-C oncoprotein promotes STAT3 activation in an
autoinductive regulatory loop. Science signaling 2011; 4:ra9).
Therefore, Stat1 and Stat3 can operate on the same promoters
thereby activating the same oncogenes, although they are triggered
to respond by different signaling systems.
[0131] The effects of Jak1/2 and Jak2 inhibitors were investigated
on the activation of Stat1 and Stat3 in the context of Type I and
Type II IFN signaling. FIG. 4 shows that the Jak2 inhibitor TG
(SAR302503) and Jak1/Jak2 inhibitor Ruxolitinib (Rux) (Incyte
Pharmaceuticals and Novarts) (CAS 941678-49-5) suppressed
phosphorylation of both Stat1 and Stat3. TG101348 (SAR302503)
(Sanofi-Aventis) (CAS 936091-26-8) was developed for the treatment
of patients with myeloproliferative diseases including
myelofibrosis, and acts as a competitive inhibitor of protein
kinase JAK-2. Myelofibrosis is a myeloid malignancy associated with
anemia, splenomegaly, and constitutional symptoms. TG101348 was
originally discovered by TargeGen and is now under development by
Sanofi-Aventis under company code SAR302503. These data suggest
that the therapeutic effects of Jak2 inhibitors are associated with
the suppression of the Stat1/Stat3 signaling pathways. This
observation is consistent with previous observations, shown in
FIGS. 3 and 4. TG more effectively suppressed tumor growth compared
to Ruxolitinib in different cell lines (data not shown) and was
used in all subsequent experiments.
[0132] To further characterize the effects of Jak2 inhibition on
radioresistance, the radiosensitivity of 24 lung cancer cell lines
was tested using a clonogenic assay. Based on clonogenic survival
at 5 Gy (SF5=surviving fraction at 5 Gy), lung cancer cell lines
were arbitrarily separated into radioresistant (RR) and
radiosensitive (RS) categories (FIG. 5). The relatively
radioresistant (RR) cell line A549 and the relatively
radiosensitive (RS) cell line H460 were further tested using
Western blot analysis of the Jak/Stat pathway. Both cell lines were
treated with IFN.gamma. (10 ng/ml) and Jak2 inhibitor TG in
concentrations of 0.5, 1, and 5 (see FIG. 5). This experiment
demonstrated that both radioresistant and radiosensitive lung
cancer cell lines have intact upstream Jak2 signaling leading to
phosphorylation of both Stat1 and Stat3 in response to the
administration of IFN.gamma..
[0133] Further, Stat3 was shown to respond to Type I IFN in both
the radioresistant and radiosensitive cell lines. Activation of
Stat3 in response to Type I IFNs is not described as the
"traditional" Stat3 activating pathway and may be considered in the
explanation of phenotypes associated with overexpression of the
Jak/Stat axis. Moreover, these data reveal the absence of
constitutively phosphorylated Stat3 in the radioresistant lung
cancer cell line A549. Contrary to these observations,
radioresistant A549 cells demonstrate constitutively active Stat1.
However, H460, the radiosensitive lung cancer cell line, did not
express pStat1 without IFN stimulation but demonstrated
constitutively phosphorylated Stat3. These data suggest that in
lung cancer cell lines constitutive activation of Stat1 is
associated with the radioresistant phenotype, as is described
elsewhere.
[0134] It was also demonstrated that in both the A549 and H460 lung
cancer cell lines, Jak2 is constitutively activated via
phosphorylation (FIG. 6). These data represent important
observations suggesting that Jak/Stat signaling can be activated in
some lung cancer cell lines providing the rationale for further
investigations using Jak2 inhibitors as a treatment modality. In
subsequent experiments, 18 lung cancer cell lines were tested for
sensitivity to the Jak2 inhibitor SAR302503 (TG) using a clonogenic
assay. Similar to ionizing radiation resistance, it was found that
lung cancer cell lines can be separated based on their relative
resistance to TG (FIG. 7). Further, radioresistance was positively
correlated with resistance to Jak2 suppression in 11 cell lines.
However, 7 cell lines were relatively radioresistant but
demonstrated high sensitivity to TG. Further, H2030 cells were the
most radioresistant cells (see FIG. 5) but following treatment with
5 Gy+1 .mu.M TG, no clonogenic colonies formed. These data suggest
that some lung cancer cells with intrinsic radioresistance may be
suppressed and/or radiosensitized by TG.
[0135] The relative sensitivity of lung cancer cell lines to TG and
etoposide--one of the most common drugs in adjuvant chemotherapy of
lung cancer was also tested. Subgroups of cell lines were found
with relatively high resistance to etoposide (SF>0.5) but
relatively sensitive to TG (0.1<SF<0.2; see FIG. 8). As proof
of principle, two isogenic cell lines CWRR1 (pStat3-negative,
without constitutive activation of Jak/Stat-signaling) and CW22Rv1
(with constitutively active Jak/Stat signaling) were established as
xenografts in nude mice and treated by ionizing radiation, TG, or
their combination. As is shown in FIG. 9, pStat3-negative CWRR1 was
more radioresistant and was not sensitized by SAR302503. Contrary
to this, pStat3-positive CW22Rv1 was successfully sensitized to
ionizing radiation by TG.
[0136] Further, flow cytometry approaches may be used for
independent validation of the siRNA screen (see FIG. 10). The gene
used in these experiments shown in FIG. 10 was DHx58 (LGP2). Cells
were transfected by individual siRNA, detected in deconvoluted
screen or by non-targeting control (nt) and 24 hours
post-transfection irradiated at 5Gy. Forty-eight (48) hours
post-IR, cells were stained with propidium iodide (PI, vertical
axis) as marker of membrane destabilization and Annexin V
(horizontal axis) as marker of apoptotic cell death. Proportion of
double-positive cells, presented in the upper-right quadrant of the
each panel was used as a measure of cell death induced either by
suppression of the targeted gene alone (e) or IR alone (c) or their
combination (f).
[0137] We have also shown that the inhibition of identified target
genes leads to significant Radiosensitization of the tumor cell
line HCT116 (see FIG. 11). In this experiment the amount of cells
killed by IR (5Gy) was increased in colorectal cancer cells HCT116
transfected by siRNAs against indicated genes as compared to the
same cells transfected by non-targeting siRNA. Shown are
double-positive cells detected as is described in FIG. 10.
[0138] Individual siRNA against PSMB9 and PSMB10 also was shown to
inhibit expression of corresponding proteins in the breast cancer
tumor cell line MDA-MB-231 and glioblastoma cell line D54. As seen
in FIG. 12, transfection of tumor cells by individual siRNAs
against PSMB9 and PSMB10 leads to the suppression of proteins
encoded by these genes in breast cancer cell line MDA-MB-231 and
glioblastoma cell line D54. Further, as seen in FIGS. 13-15
suppression of PSMB9 in MDA-MB-231 and D54 cell lines leads to the
inhibition of their proliferation (FIG. 13); suppression of PSMB9
and PSMB10 in the breast cancer cell line MDA-MB-231 leads to the
increased killing of tumor cells by IR (FIG. 14); and suppression
of PSMB9 and PSMB10 in the glioblastoma cell line D54 leads to the
increased killing of tumor cells by IR (FIG. 15).
[0139] It was also demonstrated that ectopic expression of the
USP18-gene, selected as a potential candidate in siRNA screen and
involved in protein modifications, led to increased radioresistance
of the tumor cell lines U87, D54 and SCC61 in vitro (FIG. 16).
Further, it was demonstrated that D54 tumors established in nude
mice and stably overexpressing USP18 were more resistant to the
fractionated IR (5Gyx6 days) as compared with mock-transfected
cells (FIG. 12).
[0140] It is believed that these observations provide a unique
opportunity to detect the molecular properties of resistant and
sensitive lung tumors to therapy with Jak2 inhibitors alone or in
combination with ionizing radiation and chemotherapy.
Correspondingly, these investigations may lead to the detection of
biomarkers predicting individual response to
Jak2/radio/chemotherapy of lung cancer.
[0141] The invention has been described in an illustrative manner
and it is to be understood the terminology used is intended to be
in the nature of description rather than of limitation. All patents
and other references cited herein are incorporated herein by
reference in their entirety. It is also understood that many
modifications, equivalents, and variations of the present invention
are possible in light of the above teachings. Therefore, it is to
be understood that within the scope of the appended claims, the
invention may be practiced other than as specifically
described.
* * * * *