U.S. patent application number 09/265350 was filed with the patent office on 2003-03-27 for inhibition of stress activated protein kinase (sapk) pathway and sensitization of cells to cancer therapies.
Invention is credited to DEAN, NICHOLAS M., MCKAY, ROBERT, MERCOLA, DANIEL A..
Application Number | 20030060433 09/265350 |
Document ID | / |
Family ID | 25343644 |
Filed Date | 2003-03-27 |
United States Patent
Application |
20030060433 |
Kind Code |
A1 |
MERCOLA, DANIEL A. ; et
al. |
March 27, 2003 |
INHIBITION OF STRESS ACTIVATED PROTEIN KINASE (SAPK) PATHWAY AND
SENSITIZATION OF CELLS TO CANCER THERAPIES
Abstract
The present invention provides compositions that can be
administered to an individual and inhibit a stress activated
protein kinase pathway. The invention also provides methods of
increasing the sensitivity of cancer cells to a cancer therapy by
contacting the cancer cells with a stress activated protein kinase
pathway inhibitor. The invention further provides methods of
reducing the severity of cancer in a patient by administering to
the patients a stress activated protein kinase pathway inhibitor
and treating the patient with a conventional cancer therapy.
Inventors: |
MERCOLA, DANIEL A.; (RANCHO
SANTO FE, CA) ; DEAN, NICHOLAS M.; (OLIVENHAIN,
CA) ; MCKAY, ROBERT; (SAN DIEGO, CA) |
Correspondence
Address: |
JANE MASSEY LICATA
LAW OFFICES OF JANE MASSEY LICATA
66 E MAIN STREET
MARLTON
NJ
08053
|
Family ID: |
25343644 |
Appl. No.: |
09/265350 |
Filed: |
March 9, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09265350 |
Mar 9, 1999 |
|
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08864603 |
May 28, 1997 |
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Current U.S.
Class: |
514/44A ;
536/23.2 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/82 20130101; C12N 9/1205 20130101; A61P 35/00 20180101;
A61P 43/00 20180101 |
Class at
Publication: |
514/44 ;
536/23.2 |
International
Class: |
A61K 048/00; C07H
021/04 |
Goverment Interests
[0001] This invention was made with government support under CA
63783 awarded by the National Cancer Institute. The government has
certain rights in the invention.
Claims
What is claimed is:
1. A method of increasing the sensitivity of cancer cells to a
cancer therapy, comprising contacting the cancer cells with a
stress activated protein kinase (SAPK) pathway inhibitor.
2. The method of claim 1, wherein said SAPK pathway inhibitor is an
antisense nucleic acid molecule.
3. The method of claim 2, wherein said antisense nucleic acid
molecule is an antisense SAPK nucleic acid molecule.
4. The method of claim 2, wherein said SAPK nucleic acid molecule
is selected from the group consisting of SAPK1.alpha.1,
SAPK1.alpha.2, SAPK1.beta.1, SAPK1.beta.2, SAPK2.alpha.1,
SAPK2.beta.1, SAPK2.beta.2, SAPK3.alpha.1 and SAPK3.alpha.2.
5. The method of claim 2, wherein said antisense nucleic acid
molecule is an antisense SAPK kinase kinase (MEKK1) nucleic acid
molecule.
6. The method of claim 1, wherein said SAPK pathway inhibitor is a
ribozyme
7. The method of claim 1, wherein said SAPK pathway inhibitor is a
dominant negative mutant.
8. The method of claim 8, wherein said dominant negative mutant is
a dominant negative c-jun mutant.
9. The method of claim 8, wherein said SAPK pathway inhibitor is a
dominant negative SAPK mutant.
10. The method of claim 8, wherein said SAPK pathway inhibitor is a
dominant negative SAPK kinase kinase (MEKK1) mutant.
11. A method of reducing the severity of a cancer in a patient,
comprising administering to the patient a stress activated protein
kinase pathway (SAPK) inhibitor and treating the patient with a
cancer therapy.
12. The method of claim 11, wherein said SAPK pathway inhibitor is
an antisense nucleic acid molecule.
13. The method of claim 12, wherein said antisense nucleic acid
molecule is an antisense SAPK nucleic acid molecule.
14. The method of claim 12, wherein said SAPK nucleic acid molecule
is selected from the group consisting of SAPK1.alpha.1,
SAPK1.alpha.2, SAPK1.beta.1, SAPK1.beta.2, SAPK2.alpha.1,
SAPK2.beta.1, SAPK2.beta.2, SAPK3.alpha.1 and SAPK3.alpha.2.
15. The method of claim 12, wherein said antisense nucleic acid
molecule is an antisense SAPK kinase kinase (MEKK1) nucleic acid
molecule.
16. The method of claim 11, wherein said SAPK pathway inhibitor is
a ribozyme.
17. The method of claim 11, wherein said SAPK pathway inhibitor is
a dominant negative mutant.
18. The method of claim 17, wherein said dominant negative mutant
is a dominant negative c-jun mutant.
19. The method of claim 17, wherein said SAPK pathway inhibitor is
a dominant negative SAPK mutant.
20. The method of claim 17, wherein said SAPK pathway inhibitor is
a dominant negative SAPK kinase kinase (MEKK1) mutant.
21. A composition, comprising a stress activated protein kinase
(SAPK) pathway inhibitor and a carrier acceptable for
administration to an individual.
22. The composition of claim 21, wherein said SAPK pathway
inhibitor is an antisense nucleic acid molecule.
23. The composition of claim 22, wherein said antisense nucleic
acid molecule is an antisense SAPK nucleic acid molecule.
24. The composition of claim 22, wherein said SAPK nucleic acid
molecule is selected from the group SAPK2.alpha.1, SAPK2.beta.1,
SAPK2.beta.2, SAPK3.alpha.1 and SAPK3.alpha.2.
25. The composition of claim 22, wherein said antisense nucleic
acid molecule is an antisense SAPK kinase kinase (MEKK1) nucleic
acid molecule.
26. The composition of claim 21, wherein said SAPK pathway
inhibitor a ribozyme.
27. The composition of claim 21, wherein said SAPK pathway
inhibitor is a dominant negative mutant.
28. The composition of claim 27, wherein said dominant negative
mutant is a dominant negative c-jun mutant.
29. The method of claim 27, wherein said SAPK pathway inhibitor is
a dominant negative SAPK mutant.
30. The method of claim 27, wherein said SAPK pathway inhibitor is
a dominant negative SAPK kinase kinase (MEKK1) mutant.
Description
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to molecular
medicine and cancer therapy and more specifically to compositions
that inhibit the stress activated protein kinase pathway and
methods of using such compositions to sensitize cancer cells to a
cancer therapy.
[0004] 2. Background Information
[0005] In renewing tissues such as bone marrow, the intestine and
the skin, a steady state .s maintained between the rate of cell
growth and the rate of cell death. In particular, the rate of cell
death in renewing tissue occurs through programmed cell death
pathways and are characterized by the process of apoptosis.
Disruption of this steady state often is associated with the
development of cancer. For example, where the rate of programmed
cell death is lower than normal, an increased number of cells
occurs in a tissue, resulting in the formation or a tumor.
[0006] Cancer cells are characterized by an ability to proliferate
indefinitely and to invade into normal tissue surrounding the
tumor. In addition, many types of cancer cells can metastasize
throughout the body such that the tumor is disseminated in the
cancer patient.
[0007] Conventional methods for treating cancer have increased the
survival and quality of life of cancer patients. Such conventional
methods include surgery, radiotherapy and chemotherapy. In
addition, bone marrow transplantation is becoming useful in
treating patients with certain types of cancers.
[0008] Surgery generally is the first choice for treating patients
having a tumor that is localized to a specific area of the human
body. Tumor excision is quick and quite effective, accounting for
the majority of cures. However, surgery has several disadvantages.
One major obstacle to this form of treatment occurs when the tumor
is in an inoperable location such that resection of the tumor is
not possible. In addition, the cancer already may have spread to
other parts of the body, but is not yet detectable at the time of
surgery. While surgical removal of the localized tumor can improve
the quality of the patient's life, the cancer is destined to recur
in the other locations. Similarly, even when a tumor is localized
and has not yet spread, failure to remove all of the cancer cells
can result in recurrence of the tumor. Finally, surgery is, by
nature, an invasive procedure and can cause loss of function of a
normal tissue or organ or affect the patient's appearance.
[0009] Radiotherapy often is used in combination with, or as an
alternative, to surgery. Radiation primarily causes damage to the
tumor cell DNA, thus inducing apoptosis and death of the cells. Of
course, normal cells in the radiation field also are damaged, but
normal cells generally have a greater ability than cancer cells to
recover from radiation damage and, therefore, a therapeutic benefit
can be obtained. However, radiation therapy, like surgery, is a
localized treatment and suffers from the same inadequacies, for
example, failure to kill tumor cells that are outside of the
treatment field, particularly metastatic lesions. In addition,
radiation damage occurs to particularly susceptible tissues such as
bone marrow, skin and intestine, thus causing patient
morbidity.
[0010] In contrast to surgery and radiotherapy, chemotherapy
provides a systemic method of treating cancer. Chemotherapy
utilizes various classes of chemotherapeutic agents that have
different modes of action. For example, anti-metabolite
chemotherapeutic agents generally share structural similarities
with normal cellular components and exert their toxic effect by
inhibiting a normal cellular process. For example, methotrexate is
a chemical analogue of folic acid, which is a vitamin required for
DNA synthesis. Methotrexate functions by competing with folic acid
for binding to an enzyme normally involved in the conversion of
folic acid into adenine and guanine, which are two building blocks
of DNA. As a result of the competition, methotrexate prevents cells
from dividing by inhibiting their ability to synthesize DNA.
[0011] Other chemotherapeutic agents, such as topoisomerase analogs
or inhibitors and alkylating agents, also function by disrupting
normal DNA synthesis in cells, resulting in death of the cells.
Since tumor cells generally divide more rapidly than normal
tissues, tumor cells are somewhat preferentially killed by such
chemotherapeutic agents. However, as discussed above, cells such as
bone marrow cells, intestinal epithelial cells and skin cells also
are rapidly dividing and, therefore, susceptible to the toxic
effects of such chemotherapeutic agents. In fact, it is the
toxicity to normal cells that generally limits the dose of
chemotherapeutic agent that can be administered to a patient. In
addition, tumor cells have a propensity to acquire resistance to
certain chemotherapeutic agents, further limiting the usefulness of
such agents for treating cancer.
[0012] More recently, biochemical agents that are expressed
normally in individuals and act as natural defense agents or as
agents that induce natural immunity against diseased cells have
been used as cancer therapeutic agents. In particular, the
cytokines are a class of naturally occurring biochemicals that are
involved in stimulation and activation of the immune response
system. Such cytokines, including, for example, the interferons and
interleukins can kill cells directly and provide the additional
advantage that they can stimulate the patient's immune response.
However, the normal expression of such biochemicals in the body is
tightly regulated and the usefulness of agents is limited by the
toxic effects that occur when higher than physiological amounts of
these agents are administered to an individual.
[0013] In order to improve the therapeutic advantage of the various
conventional cancer therapeutic modalities, the therapies often are
used in combination. Thus, as toxicity to normal cells or tissues
begins to occur due to the use of one modality, that modality is
terminated and a second treatment using a different type of
modality is initiated. Such first and second modalities can be, for
example, surgery or radiotherapy, followed by chemotherapy, or a
first type of chemotherapy followed by a second type of
chemotherapy or a biochemical agent therapy.
[0014] In addition, a therapeutic advantage can be obtained by
combining a therapeutic modality with treatment using an agent that
modifies the effectiveness of the modality to a greater extent
against cancer cells than normal cells. Such chemical modifiers
generally are not toxic at the doses used, but act to modify or
enhance the responsiveness of cancer cells to a conventional
therapy. The effectiveness of a such chemical modifiers to
sensitize tumor cells to a cytotoxic therapy generally is expressed
as the sensitizer enhancement ratio, which is a ratio of the dose
of a therapy required to produce a defined level of killing in the
absence of the sensitizer to the dose required to produce the same
level of cell killing in the presence of the sensitizer.
[0015] The use of chemical sensitizers is exemplified by the use of
oxygen mimetics to increase the sensitivity of tumor cells to
radiotherapy. Generally, the cancer cells forming a tumor grow
faster than the cells that produce blood vessels in the tumor. As a
result, as the tumor increases in size, it develops regions that
are relatively deficient in oxygen. Such hypoxic tumor cells are
relatively resistant to radiation damage and, therefore, limit the
effectiveness of radiotherapy. However, chemical sensitizers have
been developed that act as oxygen mimetics. The administration of
such sensitizers to a cancer patient increases the "oxygenation" of
the otherwise hypoxic tumor cells, thus rendering them more
sensitive to a given dose of radiation. Since normal tissue
generally is normally oxygenated, the use of such chemical
sensitizers essentially has no effect on the normal cells. Of
course, as discussed above, the use of radiotherapy nevertheless
remains limited to treatment of patients having relatively
localized tumors. Thus, while methods for treating cancer continue
to improve, a need exists for compositions and methods that can
generally increase the effectiveness of the variety of conventional
cancer therapies currently available. The present invention
satisfies this need and provides additional advantages.
SUMMARY OF THE INVENTION
[0016] The present invention provides compositions that inhibit a
stress activated protein kinase (SAPK) pathway and are suitable for
administration to an individual. For example, the invention
provides compositions containing an antisense SAPK1, SAPK2 or SAPK3
nucleic acid molecule and a carrier, such that the composition is
acceptable for administration to a human individual.
[0017] The invention also provides methods of increasing the
sensitivity of cancer cells to a cancer therapeutic modality by
inhibiting an SAPK in the cells. For example, the invention
provides a method of increasing the sensitivity of cancer cells to
a cancer therapeutic modality by expressing an antisense SAPK1,
SAPK2 or SAPK3 nucleic acid molecule in the cells, wherein the
antisense molecule inhibits the SAPK. The invention further
provides methods of reducing the severity of a cancer in a patient
by administering to the patient an SAPK pathway inhibitor. In
addition, the patient can be treated with a conventional cancer
therapy.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present invention provides compositions that inhibit a
stress activated protein kinase (SAPK) pathway and are suitable for
administration to an individual. The SAPK's (also called "jun
N-terminal kinases" or "JNK's") are a family of protein kinases
that represent the penultimate step in signal transduction pathways
that result in activation of the c-jun transcription factor and
expression of genes regulated by c-jun. In particular, c-jun is
involved in the transcription of genes that encode proteins
involved in the repair of DNA that is damaged due to genotoxic
insults. As disclosed herein, agents that inhibit SAPK activity in
a cell prevent DNA repair and sensitize the cell to those cancer
therapeutic modalities that act by inducing DNA damage.
[0019] Various cancer therapeutic modalities act by causing damage
to DNA in the cancer cells or by adversely affecting DNA synthesis
or replication in the cells and inducing apoptosis and cell death.
Such genotoxic cancer therapeutic modalities include, for example,
ionizing radiation; chemical agents that crosslink or otherwise
directly damage DNA including cis-platinum and alkylating agents
such as N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) and
methylmethanesulphonate (MMS); and agents that interfere with DNA
synthesis including DNA chain terminating agents such as
1-.beta.-arabinofuranosylcytosine (AraC), topoisomerase inhibitors
such as camptothecin, and nucleoside analogs or precursors of such
analogs such as methotrexate (MTX) and 5-flurouracil (5-FU).
[0020] The various cancer therapeutic modalities discussed above
act by directly damaging DNA or by inhibiting DNA synthesis,
thereby inducing apoptosis and death of the cancer cells. In
addition, the SAPK pathway is involved in the mitogenic response of
certain cells, including cancer cells. For example, human A549
tumor cells, which express an EGF receptor on their cell surface,
respond mitogenically to EGF. However, the mitogenic response, but
not basal growth, is inhibited when the SAPK pathway is inhibited
by expressing a dominant negative c-jun mutant in the cells. Thus,
in addition to sensitizing tumor cells to a cancer therapeutic
modality, inhibition of the SAPK pathway also can block mitogenesis
of tumor cells, for example, in response to an autocrine growth
factor, thereby providing a therapeutic advantage to an individual
treated with a cancer therapeutic modality. It should be
recognized, however, that a SAPK inhibitory agent as disclosed
herein can be useful, alone, for inhibiting proliferation of cancer
cells and, therefore, for reducing the severity of a cancer in an
individual.
[0021] For purposes of the present invention, the term "cancer
therapeutic modality," unless specifically indicated otherwise, is
used herein to mean those agents that induce DNA damage or inhibit
DNA synthesis and induce apoptosis of a cell or that inhibit cell
proliferation. It should be recognized that DNA damage or the
inhibition of DNA synthesis can be caused directly by a cancer
therapeutic modality, for example, as occurs due to alkylating
agents, or can be caused indirectly, for example, by inhibiting or
otherwise interfering with DNA synthesis or, as with radiation, by
inducing the formation of free radicals, which damage DNA.
[0022] The SAPK pathway is activated in response to genotoxic
agents such as ultraviolet radiation and various cancer therapeutic
modalities (see, for example, Derijard et al., Cell 76:1025-1037
(1994); Adler et al., J. Biol. Chem. 270:26071-26077 (1995); van
Dam et al, EMBO J. 14:1798-1811 (1995); Kharabanda et al., Proc.
Natl. Acad. Sci., USA 93:6898-6901 (1996)). SAPK (JNK)
phosphorylates c-jun at serine residues 63 and 73 (Smeal et al.,
Nature 354:494-496 (1991)). In turn, working backwards from c-jun
activation in the SAPK pathway, SAPK is activated by
phosphorylation of a SAPK kinase (SAPKK; JNKK), which, itself, is
activated by phosphorylation of a SAPKK kinase (SAPKKK; JNKKK; also
called MEKK1 and referred to herein as "MEKK1;" GenBank Accession
No. U29671, which is incorporated herein by reference; see, also,
U.S. Pat. No. 5,405,941, which is incorporated herein by
reference). Additional steps of the pathway precede the activation
of MEKK1 (Liu et al., Cell 87:565-576 (1996)) and, as discussed
below, MEKK1 also acts as a branch point for a second pathway.
[0023] Various SAPK's, including SAPK1 (JNK; SAPK1.alpha.1; GenBank
Accession No. 226318; see, also, U.S. Pat. No. 5,534,426, which is
incorporated herein by reference), SAPK2 (SAPK2.alpha.1; U34821)
and SAPK3 (SAPK3.alpha.1; U34820), and related isozymes,
SAPK1.alpha.2 (U34822), SAPK1.beta.1 (U35004), SAPK1.beta.2
(U35005) , SAPK2.beta.1 (U35002) , SAPK2.beta.2 (U35003) and
SAPK3.alpha.2 (U34819), each of which is incorporated herein by
reference, have been described (see Gupta et al., EMBO J.
15:2760-2770 (1996); see, also, Cuenda et al., EMBO J. 16:295-305
(1997)). Activation of one or more SAPK's in a cell is associated
with the induction of expression of various genes involved in DNA
repair and cell survival following a stress, including the genes
encoding c-jun (Chu et al., Mol. Endocrinol. 8:59 (1994)),
p21(Waf1/Cip1) (El-Deiry et al., Cancer Res. 55:2910 (1995)), ATF2,
ATF3 (Gately et al., Brit. J. Cancer 70:1102 (1994)), PCNA (Huang
et al., Mol. Cell. Biol. 14:4233 (1994)), cyclin-A, cyclin-D1
(Herbert et al., Oncogene 9:1295 (1994)), cyclin-G and GADD153
(Luethy and Holbrook, Cancer Res. 54:1902S (1994); Gately et al.,
supra, 1994).
[0024] The SAPK pathway likely is active in cancer cells. For
example, tumors provide a stress-inducing environment for the
cancer cells due to hypoxia in regions distant from blood vessels
and to exposure to cell breakdown products and inflammatory signals
that occur in regions of necrosis. In this regard, DNA damage
commonly is observed in cancer cells. In addition, the SAPK pathway
leads to activation of various transcription factors, some of which
are involved in cell growth and proliferation. Thus, increased SAPK
activity can be present in cancer cells that are proliferating
relatively rapidly. Accordingly, a composition that inhibits a SAPK
pathway as disclosed herein can be useful to inhibit proliferation,
growth or DNA repair in cancer cells, thereby increasing the
likelihood that cancer cells containing such damage will die.
Furthermore, particular cancer cells can express one or more
specific SAPK isozymes or unique SAPK kinases or MEKK1 kinases.
Thus, the skilled artisan will recognize that the specific SAPK,
SAPK kinase or MEKK1 isozyme or isozymes expressed in specific
cancer cells can be selectively inhibited using a composition of
the invention that is specific for the isozyme. Antisense molecules
or ribozymes, for example, can exhibit such selectivity.
[0025] In addition to the prevalence of DNA damage that occurs in
cancer cells in a tumor, administration of a cancer therapeutic
modality to an individual results in DNA damage to the cancer
cells, as well as to normal cells, and induces the SAPK pathway.
Depending on the level of DNA damage, either the cells repair the
damage and survive or the cells die. In general, the amount of a
cancer therapeutic modality that an individual receives is limited
by toxicity to normal tissues. The present invention provides a
therapeutic benefit to an individual receiving a cancer therapeutic
modality because the amount of the modality that is administered to
the individual can be reduced due to inhibition of SAPK activity
and, therefore, failure of the damaged cells to repair DNA damage
due to the modality.
[0026] The present invention can provide a therapeutic benefit to
an individual receiving a cancer therapeutic modality in various
ways. For example, the amount of the modality that must be
administered to the individual can be reduced due to inhibition of
DNA repair due to inhibition of SAPK activity. Thus, where a side
effect due to the modality is due to an intrinsic sensitivity of
the particular patient, the sensitivity can be ameliorated due to
administration of a lesser amount of cancer therapeutic modality.
In addition, it is recognized that SAPK proteins constitute a
family of at least three SAPK proteins, as well as isozymes of each
SAPK protein, indicating that various cells, including cancer
cells, likely differentially express one or more particular SAPK
proteins. Thus, a method of the invention can be practiced so as to
specifically inhibit the activity of the SAPK or SAPK proteins
expressed by the cancer cells, thus inhibiting DNA repair in those
cells but not in other cells such as normal cells expressing
different SAPK protein. The identification of SAPK proteins
expressed in cancer cells of an individual to be treated can be
determined, for example, by western blot analysis using antibodies
specific for SAPK isozymes. Furthermore, certain cancer cells can
express SAPK constitutively, resulting in a greater than normal
steady-state SAPK activity For example, whereas normal lung cells
do not express SAPK, non-small cell lung carcinoma cells either
exhibit constitutive SAPK activity or SAPK activity can be induced
by exposure to ultraviolet irradiation, which induces the SAPK
pathway. These observations indicate that such cancer cells have an
enhanced capacity for DNA repair and, therefore, an increased
resistance to a cancer therapeutic modality. Thus, inhibition of
SAPK activity in such cells can preferentially sensitize the cancer
cells to a cancer therapeutic modality.
[0027] In one embodiment of the invention, SAPK activity is
directly inhibited in a cancer cell, thereby decreasing the ability
of the cell to repair DNA damage caused by a cancer therapeutic
modality and increasing the sensitivity of the cancer cell to the
cancer therapeutic modality As used herein, the term "SAPK
activity" means the ability of a SAPK to phosphorylate its
substrate such as the ability of SAPK1 to phosphorylate c-jun on
serine-63 and serine-73, or to mediate phosphorylation-dependent
activation of transcription, specifically that mediated by
phosphorylation of serine-63 and serine-73. In this regard, it is
recognized that c-jun associates with a Fos family member to form
heterodimers that bind to specific promoters and modestly increase
the level of gene transcription. If, however, serine-63 and
serine-73 of c-jun are phosphorylated by SAPK, a high level of
transcriptional activity occurs. Thus, a SAPK mediates
phosphorylation-dependent activation of transcription. Methods for
identifying SAPK activity are well known in the art (see, for
example, Hibi et al., Genes Devel. 7:2135-2148 (1993), which is
incorporated herein by reference).
[0028] The present invention provides compositions containing a
SAPK inhibitory agent that directly inhibits the activity of a SAPK
in a cell. Such an agent can be an antisense SAPK nucleic acid
molecule or a SAPK ribozyme, including a "padlock ribozyme," such
agents which directly inhibit SAPK activity by preventing the
expression of the SAPK in the cell; can be a dominant negative
mutant, such as a nonphosphorylatable form of c-jun, which directly
inhibits SAPK activity by preventing SAPK from phosphorylating
normal c-jun in a cell; or can be an small molecule such as a drug,
which directly alters the interaction of proteins in the SAPK
pathway. Although no mechanism of action is proposed as to how a
dominant negative c-jun mutant can inhibit a SAPK pathway and,
therefore, SAPK activity, the mutant c-jun may bind to and
competitively inhibit the activity of SAPK in all its forms, or the
mutant c-jun may form heterodimers with Fos family members or with
ATF2, but lack the ability to participate in
phosphorylation-dependent activation of transcription due to the
inability of the mutant c-jun to be phosphorylated. The inhibition
of SAPK activity can be determined, for example, by measuring the
phosphorylation of a substrate such as c-jun (Hibi et al., supra,
1993) or, where SAPK activity is inhibited using an antisense SAPK
nucleic acid, by performing an immunoassay using anti-SAPK
antibodies, which are commercially available (Santa Cruz
Biotechnology, Inc.; Santa Cruz Calif.).
[0029] As discussed above, activation of MEKK1 leads to c-jun
activation. In addition, MEKK1 represents a branch point for a
second pathway that leads to inactivation of the inhibitor of
NF-.kappa.B (I.kappa.B) and activation of the NF-.kappa.B
transcription factor (Lee et al., Cell 88:213-222 (1997)).
Activation of NF-.kappa.B suppresses apoptosis (Beg et al., Science
274:782-789 (1996); van Antwerp et al., Science 274:787-789 (1996);
Liu et al., supra, 1996) and it has been proposed that inhibiting
NF-.kappa.B can potentiate killing of tumor cells by
chemotherapeutic agents (Wang et al., Science 274:784-787 (1996)).
Accordingly, in a second embodiment of the invention, SAPK activity
in a cell is inhibited indirectly by inhibiting the activity of
MEKK1 in the cell.
[0030] By inhibiting MEKK1 activity in a cancer cell, SAPKK is not
activated and, therefore, does not activate SAPK, thus increasing
the sensitivity of the cell to a cancer therapeutic modality, as
discussed above. As an additional advantage, the inhibition of
MEKK1 activity results in the inhibition of NF-.kappa.B activation,
thus increasing the sensitivity of the cells to apoptosis. Thus,
the invention provides a composition comprising a SAPK inhibitory
agent that inhibits MEKK1 activity, for example, an antisense MEKK1
nucleic acid molecule.
[0031] For example, the invention provides compositions containing
an antisense SAPK1, SAPK2 or SAPK3 nucleic acid molecule and a
carrier that is acceptable for administration to a human
individual. An antisense nucleic acid molecule useful in the
invention is a polymer of about twelve to fifty nucleotides,
generally about fifteen to thirty-five nucleotides and usually
about twenty to twenty-five nucleotides, which are linked by a
covalent bond, such as a phosphodiester bond, a thioester bond, or
any of various other bonds known in the art as useful and effective
for linking nucleotides.
[0032] A nucleic acid molecule encoding an antisense SAPK isozyme
or antisense MEKK1 useful in the invention, or a ribozyme or a
dominant negative mutant, as discussed below, can be expressed from
a vector, which is introduced into a cell in which it is desired to
express the antisense molecule or ribozyme or dominant negative
mutant. Antisense SAPK or MEKK1 nucleic acid molecules or SAPK or
MEKK1 ribozymes also conveniently can be chemically synthesized
(Seimiya et al., J. Biol. Chem. 272:4631-4636 (1997)).
[0033] An advantage of expressing the antisense molecule or
ribozyme or dominant negative mutant in the desired cells is that
it can be expressed at a relatively high level, increasing the
ability of the molecule to inhibit SAPK activity. An expression
vector expressing, for example, an antisense SAPK or MEKK1 nucleic
acid molecule can be introduced into cells using well known
transfection methods (see, for example, Sambrook et al., Molecular
Cloning: A laboratory manual (Cold Spring Harbor Laboratory Press
1989); Ausubel et al., Current Protocols in Molecular Biology
(Green Publ., N.Y. 1989), each of which is incorporated herein by
reference).
[0034] In general, an expression vector contains the expression
elements necessary to achieve, for example, sustained transcription
of the antisense SAPK or MEKK1 molecule or ribozyme or dominant
negative mutant. In particular, an expression vector contains or
encodes a promoter sequence, which can provide constitutive or, if
desired, inducible expression of the encoding nucleic acid
molecule, and a poly-A recognition sequence, and can contain other
regulatory elements such as an enhancer, which can be tissue
specific. For example, when the nucleic acid molecule contained in
the vector encodes a dominant negative mutant c-jun, the vector can
contain translation regulatory sequences, including a ribosome
binding site. of course, such regulatory elements can be a part of
the nucleic acid molecule encoding the dominant negative
mutant.
[0035] A vector also can contain elements required for replication
in a procaryotic or eukaryotic host system or both, as desired.
Such vectors, which include plasmid vectors and viral vectors such
as bacteriophage, baculovirus, retrovirus, lentivirus, poliovirus,
rhinovirus, vaccinia virus, influenza virus, adenovirus,
adeno-associated virus, herpes simplex virus, measles coronavirus,
Sindbis virus, and semliki forest virus vectors, are well known and
can be purchased from a commercial source (Promega, Madison, Wis.;
Stratagene, La. Jolla, Calif.; GIBCO/BRL, Gaithersburg, Md.) or can
be constructed by one skilled in the art (see, for example, Meth.
Enzymol., Vol. 185, D. V. Goeddel, ed. (Academic Press, Inc.,
1990); Jolly, Canc. Gene Ther. 1:51-64 (1994); Flotte, J. Bioenerg.
Biomemb. 25:37-42 (1993); Kirshenbaum et al., J. Clin. Invest
92:381-387 (1993), which is incorporated herein by reference).
[0036] Introduction of a nucleic acid molecule encoding, for
example, an antisense SAPK or a SAPK ribozyme or a dominant
negative c-jun by infection with a viral vector is particularly
advantageous in that it can efficiently introduce the encoding
nucleic acid molecule to a cell ex vivo or in vivo. Moreover,
viruses are very specialized and typically infect and propagate in
specific cell types. Thus, their natural specificity can be used to
target the encoding nucleic acid molecule Viral or non-viral
vectors also can be modified with specific receptors or ligands to
alter target specificity through receptor mediated events.
[0037] In comparison to expressing an encoding nucleic acid
molecule, an advantage of chemically synthesizing antisense nucleic
acid molecules or ribozymes is that they can be stabilized against
degradation by nucleases by the incorporation of a non-naturally
occurring nucleoside analog or by using, for example,
phosphorothioate bonds to link the nucleotides. An antisense
nucleic acid molecule or ribozyme comprising a ribonucleotide
containing a 2-methyl group, instead of the normal hydroxyl group,
bonded to the 2'-carbon atom of ribose residues, is an example of
an RNA molecule that is resistant to enzymatic and chemical
degradation and, therefore, is relatively stable in vivo. Other
examples of stable, chemically synthesized nucleic acid molecules
include RNA containing 2'-aminopyrimidines, such RNA being
1000.times. more stable in human serum and urine as compared to
naturally occurring RNA (see Lin et al., Nucl. Acids Res.,
22:5229-5234 (1994); and Jellinek et al., Biochemistry,
34:11363-11372 (1995), each of which is incorporated herein by
reference), and RNA containing 2'-amino-2'-deoxypyrimidines or
2'-fluro-2'-deoxypyrimidines, which are less susceptible to
nuclease activity (Pagratis et al., Nature Biotechnol., 15:68-73
(1997), which is incorporated herein by reference).
[0038] Antisense RNA molecules or ribozymes containing
2'-O-methylpurine substitutions on the ribose residues and short
phosphorothioate caps at the 3'- and 5'-ends also exhibit enhanced
resistance to nucleases (Green et al., Chem. Biol., 2:683-695
(1995), which is incorporated herein by reference), as do L-RNA
molecules, which are a stereoisomer of naturally occurring D-RNA
(Nolte et al., Nature Biotechnol., 14:1116-1119 (1996), and
Klobmann et al., Nature Biotechnol., 14:1112-1115 (1996); each of
which is incorporated herein by reference). Such RNA molecules and
methods of producing them are well known and routine (see Eaton and
Piekern, Ann. Rev. Biochem., 64:837-863 (1995), which is
incorporated herein by reference). Similarly, phosphorothioate
linked oligodeoxynucleotides are nuclease resistant DNA molecules
that are useful as antisense nucleic acid molecules in the present
invention (Reed et al., Cancer Res 50:6565-6570 (1990), which is
incorporated herein by reference).
Phosphorothioate-3'-hydroxypropylamine modification of the
phosphodiester bond also reduces the susceptibility of a nucleic
acid molecule to nuclease degradation (see Tam et al., Nucl. Acids
Res., 22:977-986 (1994), which is incorporated herein by
reference). Of course, antisense nucleic acid molecules or
ribozymes having naturally occurring nucleotides and phosphodiester
bonds also can be chemically synthesized.
[0039] Chirally pure antisense molecules or ribozymes containing at
least one chirally pure internucleosidyl linkage also are useful in
the invention because such molecules form the appropriate angles to
bind to a complementary nucleic acid molecule, thus improving the
efficiency of hybridization. Such nucleic acid molecules are
prepared using known methods (see, for example, Lesnikowski et al.,
Nucl, Acids Res. 18:2109-2115 (1990); Stec et al., Nucl. Acids Res.
19:5883-5888 (1991), each of which is incorporated herein by
reference).
[0040] Methylphosphonate antisense molecules or ribozymes also can
be useful (see Lee et al., Biochemistry 27:3197-3203 (1988); and
Miller et al., Biochemistry 25:5092-5097 (198G); PCT applications
WO 92/07864 and WO/07882, each of which is incorporated herein by
reference), as are antisense molecules or ribozymes that are
nucleoside/non-nucleoside polymers or chimeric oligonucleotides
that are composite RNA, DNA analogs (Inoue et al., FEBS Lett.
2115:327 (1987)), which can have chimeric backbones. Antisense
nucleic acid molecules or ribozymes having chimeric backbone
include those having mixed phosphate backbones, including
nucleoside sequences that are capable of activating RNase H, which
allows site directed cleavage of an RNA molecule (see U.S. Pat. No.
5,149,797, which is incorporated herein by reference). Antisense
nucleic acid molecules or ribozymes having chimeric backbones also
include those having a mixture of internucleosidyl linkages and
those having a neutral backbone, for example, methylphosphonate
nucleic acid molecules. Such nucleic acid molecules can have a
longer half-life in vivo, since the neutral structure reduces the
rate of nuclease digestion. The addition of a cleaving or
cross-linking moiety also can be useful and can promote
inactivation of target polynucleotide sequences. Conjugation
partners also can be introduced into the antisense molecules or
ribozymes by incorporating modified nucleosides or nucleoside
analogs using enzymatic or chemical modification of the nucleic
acid molecule, for example, by the use of non-nucleotide linker
groups.
[0041] A chemically synthesized antisense nucleic acid molecule or
ribozyme can be introduced into a cell by any of a variety of
methods known in the art (Sambrook et al., supra, 1989, and in
Ausubel et al., Current Protocols in Molecular Biology, John Wiley
and Sons, Baltimore, Md. (1994), which is incorporated herein by
reference), including, for example, transfection, lipofection,
microinjection, electroporation or the use of liposomes. In
addition, it is recognized that naked nucleic acid molecules are
taken up by cells in vivo and, therefore, that the antisense
nucleic acid molecule or ribozyme of the invention simply can be
administered directly to the region containing the cancer cells,
where appropriate. In particular, antisense nucleic acid molecules
or ribozymes can be introduced into a cell using methods that do
not require the initial introduction of an encoding nucleic acid
molecule into a vector. For example, a nucleic acid molecule
encoding an antisense SAPK isozyme or MEKK1 can be introduced into
a cell using a cationic liposomes, which also can be modified with
specific receptors or ligands as described above (Morishita et al.,
J. Clin. Invest., 91:2580-2585 (1993), which is incorporated herein
by reference; see, also, Nabel et al., supra, 1993)). In addition,
a nucleic acid molecule can be introduced into a cell using
adenovirus-polylysine DNA complexes (see, for example, Michael et
al., J. Biol. Chem., 268:6866-6869 (1993), which is incorporated
herein by reference) Specific portions of a SAPK nucleic acid
molecule or a MEKK1 nucleic acid molecule to be targeted by the
antisense nucleic acid or the ribozyme ("target nucleic acid") can
be selected based on the sequence of the target nucleic acid (see
GenBank Accession numbers, as disclosed above). In addition, based
on comparisons of the disclosed MEKK1 and SAPK nucleic acid
sequences, an antisense nucleic acid molecule or ribozyme can be
designed such that it is specific only for MEKK1 or only for a
single SAPK isozyme or can be more promiscuous, inhibiting the
expression of MEKK1 and a SAPK or various SAPK isozymes or all SAPK
isozymes.
[0042] Antisense nucleic acid molecules useful in the invention can
be selected using well known and routine methods. For example, a
panel of antisense nucleic acid molecules that are complementary to
various 5'-untranslated, coding and 3'-untranslated regions of the
target nucleic acid molecule, for example, SAPK1.alpha.1 or MEKK1,
can be prepared and can be examined using in vitro assays to select
those antisense molecules that have the desired specificity (see,
for example, Monia et al., Nature Med. 2:668-674 (1996); Dean et
al., Cancer Res. 56:3499-3507 (1996), each of which is incorporated
herein by reference). It is recognized that the ability of an
antisense nucleic acid molecule (or a ribozyme) to hybridize to the
target nucleic acid depends, for example, on the degree of
complementarity shared between the sequences, the GC content of the
hybridizing molecules, and the length of the antisense nucleic acid
molecule or complementary portion of the ribozyme. In particular,
specificity of hybridization can be such that an antisense SAPK or
a ribozyme binds to one or a selected few SAPK isozymes, but not to
others (see, for example, Dean et al., supra, 1996).
[0043] In view of the above disclosure relating to the selection of
target sequences in a SAPK or MEKK1, the skilled artisan would
recognize that ribozymes also can be used to inhibit the activity
of a SAPK or MEKK1. Ribozymes comprise two ribonucleic acid
sequences, which are complementary to a target nucleic acid
sequence, flanking an RNA sequence that can cleave a specific RNA
sequence (Cech, J. Amer. Med. Assoc., 260:3030 (1988)). Two basic
types of ribozymes, "tetrahymena-type" (Hasselhoff, Nature 334:585
(1988)) and "hammerhead-type," are known. Tetrahymena-type
ribozymes specifically recognize sequences that are four bases in
length, while hammerhead ribozymes recognize sequences that are 11
to 18 bases in length. The location of specific ribozyme target
sequences can be identified by examination of the SAPK and MEKK1
sequences disclosed above and ribozymes can be constructed
containing the ribozyme sequence flanked by additional target
sequences such that the ribozyme specifically hybridizes to the
target sequence. Ribozymes can be constructed and introduced into
cells as disclosed above.
[0044] Since cells, including normal cells and cancer cells can
express one or more specific SAPK isozymes, the identification of
the specific isozymes expressed provides specific targets for which
the antisense nucleic acid molecules or ribozymes are designed. The
use of antisense nucleic acid molecules or ribozymes that target
specific SAPK isozymes also can provide a means to further spare
normal tissue from the effects of the cancer therapeutic modality,
since, where normal cells that are particularly sensitive to the
cancer therapeutic modality express a SAPK that is different from
at least one SAPK expressed in the cancer cells, the antisense SAPK
nucleic acid molecule can be designed to inhibit the expression of
the cancer cell SAPK but not the normal cell SAPK. For example,
where the cancer therapeutic modality is radiotherapy, the SAPK
isozyme expressed in normal cells that are within the radiation
field can be identified and, where the SAPK isozyme in the cancer
cells is different, the antisense SAPK nucleic acid molecule can be
designed to inhibit expression of the cancer cell SAPK isozyme, but
not the normal cell SAPK isozyme. In this regard, it is noted that
cancer therapeutic modalities generally are toxic to rapidly
renewing tissues, including blood cells and epithelial cells. The
identification of SAPK isozymes that generally are expressed, or
not expressed in such tissues, therefore, can be informative to
practicing the methods with most cancer therapeutic modalities.
[0045] Methods for identifying which specific SAPK isozymes are
expressed in cancer cells and normal cells are routine and can
utilize, for example, nucleic acid hybridization using SAPK isozyme
specific probes. For example, northern blot or dot blot analysis
provides rapid and simple assays for determining the specific SAPK
isozymes expressed in a cell. Probes that are selective for one or
more SAPK isozymes can be obtained by performing computerized
searches of the nucleic acid sequences encoding the various
isozymes (see GenBank Accession numbers, above) and identifying
unique sequences that will specifically hybridize to the desired
nucleic acid molecule under stringent hybridization conditions.
[0046] As disclosed herein, a dominant negative mutant of c-jun
such as dn-jun, which lacks serine-63 and serine-73 and cannot be
phosphorylated by SAPK, provides an additional example of a SAPK
inhibitory agent that can inhibit SAPK activity and can increase
the sensitivity of various cancer cells, including glioblastoma
cells, prostate cancer cells and breast cancer cells, to a cancer
therapeutic modality, cis-platinum (see Examples I and II). For
example, the expression of a dn-jun, which contained alanine-63 and
alanine-73 substitutions, in glioblastoma cells inhibited
phosphorylation-dependent activation of transcription, but did not
have a significant effect on SAPK activity, suggesting that dn-jun
does not strongly bind to or competitively inhibit the SAPK enzyme,
but competes with normal c-jun for binding to a Fos family member
or to ATF2. These results indicate that a dominant negative c-jun
inhibits the ability of SAPK to promote phosphorylation-dependent
activation of transcription. In particular, expression of dn-jun in
the glioblastoma cells sensitized the cells to cis-platinum and
decreased the concentration of cis-platinum required to kill 50% of
the cells (IC-50) seven-fold (Example I). In addition, expression
of a different dominant negative c-jun mutant, Tam-67, sensitized
prostate cancer cells to cis-platinum. Thus, dominant negative
c-jun mutants are useful generally for sensitizing various cancer
cells to a cancer therapeutic modality.
[0047] A dominant negative c-jun can be any of various c-jun
mutants, provided the mutation results in an inability of
phosphorylation of serine-63 and serine-73. Thus, a dn-jun can
contain amino acid substitutions such as the alanine-63 and
alanine-73 substitutions in the dn-jun disclosed herein (see Smeal
et al., Nature 354:494-496 (1991), which is incorporated herein by
reference). In addition, a dn-jun can be a deletion mutant of
c-jun, such as the Tam-67 c-jun mutant, which contains a deletion
of amino acids 3 to 122 (Lenczowski et al, Mol. Cell. Biol.
17:170-181 (1997); Brown et al., Oncogene 8:877-886 (1993); Alani
et al., Mol. Cell. Biol. 11:6286-6295 (1991), each of which is
incorporated herein by reference). Furthermore, the SAPK pathway
and, therefore, SAPK activity can be inhibited by expressing,
instead of a dominant negative c-jun, a dominant negative ATF2 (van
Dam et al., EMBO J. 14:1798-1811 (1995), which is incorporated
herein by reference) or a dominant negative SAPK (see, for example,
Gupta et al., Science 267:389-391 (1995); Chen et al., J. Biol.
Chem. 271:31929-31936 (1996); Clark et al., J. Biol. Chem.
272:1677-1681 (1997), each of which is incorporated herein by
reference). Methods for producing such dominant negative mutants
are routine in view of the ready availability of the nucleic acid
sequences encoding the relevant proteins and in view of the cited
references, which generally describe the regions of the proteins,
such as SAPK and c-jun, that are required for SAPK activity.
[0048] A small molecule also can act as a SAPK inhibitory agent,
for example, by inhibiting the activity of a kinase in the pathway
or by interfering with a step of the pathway. For example, a small
molecule can alter the association of two proteins such as SAPK
kinase with a SAPK in the SAPK pathway, thus preventing
phosphorylation of the SAPK and terminating the SAPK pathway. Small
molecules that can inhibit SAPK activity generally are organic
molecules, including peptides and drugs, which contain a reactive
group that can be varied. Thus, libraries of peptides can be
prepared, wherein each amino acid is a reactive group that can be
varied (see, for example, U.S. Pat. No. 5,264,563, issued Nov. 23,
1993; U.S. Pat. No. 5,223,409, issued Jun. 29, 1993, each of which
is incorporated herein by reference). For example, the sequence
comprising amino acids 33 to 79 of c-Jun binds a cleft associated
with the active site of SAPK (Derijard et al., supra, 1994; Gupta,
supra, 1996). Accordingly, a library of diverse peptides based on
this sequence of c-jun can be prepared and the peptides can be
screened to identify those that alter SAPK activity, particularly
the ability of a SAPK to phosphorylate c-jun on serine-63 and
serine-73. In addition, libraries of small organic molecules such
as drugs can be prepared by combinatorial organic synthesis such
that the molecules that share a common structure but vary in a
reactive group are produced (see, for example, Gordon et al., J.
Med. Chem. 37:1385-1401 (1994)). Such libraries of molecules can be
synthesized using known methods and are commercially available.
[0049] It should be recognized that a library of small molecules
can be screened to identify those agents useful for inhibiting a
SAPK pathway. For example, the library of molecules can be screened
against particular cancer cells and the ability of the molecules to
increase or decrease the activity of a SAPK in the cells can be
determined using methods as disclosed herein. Those molecules that
inhibit SAPK activity are identified as agents that can increase
the sensitivity of a cancer cell to a cancer therapeutic modality
and, therefore, are s useful in the invention. It should further be
recognized that molecules that increase SAPK activity also will be
identified by such screening methods and that such agents are
particularly useful, for example, for protecting normal cells from
a cancer therapeutic modality.
[0050] In addition, methods of rational drug design have been
developed and can be used to prepare small molecules useful for
inhibiting a SAPK pathway and SAPK activity (Jackson, Sem. Oncol.
24:264-172 (1997); Webber et al., J. Med. Chem. 39:5072-5082
(1996); Hopkins et al., J. Med. Chem. 39:1589-1600 (1996), each of
which is incorporated herein by reference). For example, the
crystal structure of a SAPK or MEKK1 can be determined to a
resolution of about 3 angstroms and the active site of the kinase
can be defined with respect to positive and negative charges. A
small molecule presenting the appropriate opposite charges in the
appropriate orientation and configuration then can be designed. If
desired, appropriate hydrophobic groups can be incorporated into
the molecule. Computer programs are available for determining, for
example, correct chemical bond lengths and angles and steric
hindrance and attraction forces. For example, the choice of a small
molecule can be based on a derivative of ATP, which is bound by
SAPK and MEKK1.
[0051] Since a SAPK inhibitory agent such as a small organic
molecule, an antisense SAPK or MEKK1 nucleic acid molecule, a SAPK
or MEKK1 specific ribozyme, or a dominant negative mutant inhibitor
of activity of a SAPK generally is used to sensitize cancer cells,
the agent can be formulated into a composition that is convenient
for contacting the agent with the cancer cells. Such contacting can
be to cells in culture or can be to an individual.
[0052] As used herein, the term "SAPK inhibitory agent" is used
broadly to mean a small organic molecule or a DNA, RNA or
polypeptide that inhibits a SAPK pathway. Thus, as disclosed
herein, a SAPK inhibitory agent can be a small molecule, i.e., a
drug; an antisense SAPK or MEKK1 molecule; a SAPK or MEKK1 specific
ribozyme; a nucleic acid molecule encoding such an antisense
molecule or ribozyme; or a nucleic acid encoding a dominant
negative mutant such as a dominant negative c-jun mutant or the
encoded polypeptide. Furthermore, a nucleic acid molecule agent can
be in various forms, including naked DNA or RNA, either alone or in
a vector, and, when part of a vector, can be, for example, a naked
vector, or a vector encapsulated in a liposome or microemulsion or
the like, or a vector contained within or associated with a virus
particle. The term "SAPK inhibitory agent" encompasses these forms
and others known in the art.
[0053] A SAPK inhibitory agent can be formulated as a
pharmaceutical composition, which contains the agent and a
pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers are well known in the art and include aqueous solutions
such as water, physiologically buffered saline or other solvents or
vehicles such as glycols, glycerol, oils such as olive oil or
injectable organic esters.
[0054] A pharmaceutically acceptable carrier can contain
physiologically acceptable compounds that act, for example, to
stabilize the or increase the absorption of the SAPK inhibitory
agent. Such physiologically acceptable compounds include, for
example, carbohydrates, such as glucose, sucrose or dextrans,
antioxidants, such as ascorbic acid or glutathione, chelating
agents, low molecular weight proteins or other stabilizers or
excipients. One skilled in the art would know that the choice of a
pharmaceutically acceptable carrier, including a physiologically
acceptable compound, depends, for example, on the route of
administration of the SAPK inhibitory agent and on the particular
physico-chemical characteristics of the specific agent, i.e.,
whether it is DNA or RNA or a polypeptide and, where DNA or RNA,
whether the molecule contains naturally occurring nucleotides and
phosphodiester bonds or analogs of such nucleotides and bonds.
[0055] A SAPK inhibitory agent can be administered to an individual
by various routes including, for example, orally or parenterally,
such as intravenously, intramuscularly, subcutaneously,
intraorbitally, intracapsularly, intraperitoneally, intrarectally
intracisternally or by passive or facilitated absorption through
the skin using, for example, a skin patch or transdermal
iontophoresis, respectively. Furthermore, a composition comprising
a SAPK inhibitory agent can be administered by injection,
incubation, orally or topically, the latter of which can be
passive, for example, by direct application of an ointment or
powder, or active, for example, using a nasal spray or inhalant. A
SAPK inhibitory agent also can be administered as a topical spray
or an inhalant, in which case one component of the composition is
an appropriate propellant.
[0056] The pharmaceutical composition also can be incorporated, if
desired, into oil-in-water emulsions, microemulsions, micelles,
mixed micelles, liposomes, microspheres or other polymer matrices
(Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, Boca Raton,
Fla. 1984); Fraley, et al., Trends Biochem. Sci , 6:77 (1981), each
of which is incorporated herein by reference). Liposomes, for
example, which consist of phospholipids or other lipids, are
nontoxic, physiologically acceptable and metabolizable carriers
that are relatively simple to make and administer. In addition,
liposomes are particularly useful because they can 1) encapsulate a
SAPK inhibitory agent with high efficiency while not compromising
the biological activity of the agent; 2) preferentially and
substantially bind to a target cell; and 3) deliver the aqueous
contents of the vesicle into the target cell with high efficiency
(see Mannino et al., Biotechniques 6:6082 (1988))
[0057] Targeting of liposomes to a cancer in an individual can be
passive or active. Passive targeting, for example, utilizes the
tendency of liposomes to accumulate in cells of the
reticuloendothelial system (RES) and in organs such as the liver,
which contain sinusoidal capillaries. Active targeting, in
comparison, involves alteration of the liposome by coupling a
specific ligand such as a monoclonal antibody, a sugar, a
glycolipid or a protein such as a ligand for a receptor expressed
by the target cells. Either method of targeting can be selected,
based on the type and location as of the cancer. For example,
passive targeting can be an effective means for delivering a SAPK
inhibitory agent to a liver cancer due to the concentration of RES
cells in the liver and the sinusoidal nature of the circulatory
system in the liver.
[0058] In addition, it is recognized that tumors, as they increase
in size, develop necrotic centers and that blood vessels in the
region of the necrotic centers become "incompetent" or "leaky" (see
Maragoudakis et al., "Angiogenesis: Molecular biology, clinical
aspects" (Plenum Press 1994); Walmsley et al., Scan. Microsc.
1:823-830 (1987); Zama et al., J. Cancer Res. Clin. Oncol.
117:396-402 (1991)). Thus, intravenous or intra-arterial
administration of a SAPK inhibitory agent, particularly into a
blood vessel that carries blood to the tumor, preferentially should
permit accumulation of the agent in the tumor.
[0059] In order to sensitize cancer cells to a cancer therapeutic
modality, the SAPK inhibitory agent is administered in an amount
that can inhibit SAPK activity or expression. In general, the
amount of an antisense molecule or a nucleic acid encoding a
dominant negative inhibitor of the SAPK pathway administered to an
individual and the frequency of administrations is determined,
initially, in Phase I and Phase II clinical trials. For example,
antisense SAPK nucleic acid molecules can be administered in graded
steps of increasing dose in the range of 1 mg/kg to 10 mg/kg in
Phase I trials in order to determine a dose useful for inhibiting
SAPK activity or expression without producing unacceptable toxicity
to the individual (see Dean et al., supra, 1996). In additional
cohorts, the graded series can be combined with a conventional
cancer therapeutic modality in order to establish combination doses
useful for inhibiting SAPK activity or expression without toxicity
to the individual. In addition, where the antisense nucleic acid
molecule is expressed, for example, from a viral vector, or where a
dominant negative mutant is expressed from a viral vector, Phase I
trials can be performed using 10.sup.6 to 10.sup.10 colony forming
units. The viral vector is purified to a concentration ranging from
0.25% to 25%, preferably about 5% to 20% before formulation. After
formulation, a dose of about 1 pg to 100 ng viral vector is
contained in approximately 0.1 ml to 1.0 ml of the pharmaceutical
composition.
[0060] Where a SAPK inhibitory agent is administered to an
individual, the total dose can be administered as a single dose,
either as a bolus or by infusion over a relatively short period of
time, or can be administered using a fractionated treatment
protocol, in which the multiple doses are administered over a more
prolonged period of time. The skilled artisan would know that the
concentration of a SAPK inhibitory agent required to inhibit SAPK
activity in a subject depends on many factors including the age and
general health of the subject as well as the route of
administration and the number of treatments to be administered. In
view of these factors, the skilled artisan would adjust the
particular dose as necessary. For example, where the SAPK
inhibitory agent is to be administered in order to sensitize cancer
cells to a cancer therapeutic modality, the SAPK inhibitory agent
can be administered prior to or in concert with the cancer
therapeutic modality Methods for administering conventional cancer
therapeutic modalities are well known in the art.
[0061] Although the invention is particularly useful for increasing
the sensitivity of cancer cells to a cancer therapeutic modality,
the invention also can be practiced on essentially any type of cell
where the goal is to increase the genotoxic effect of a cancer
therapeutic modality as defined herein. For example, a method of
the invention can be practiced against immunoeffector cells so as
to sensitize the cells to an immunosuppressive agent, which is used
for treating a patient prior to transplantation or for treating a
patient with an auto-immune disease. Furthermore, a SAPK inhibitory
agent as disclosed herein can be used to sensitize essentially any
type of cancer cell, including carcinoma cells and sarcoma cells,
as well as cancer cells in a central nervous tumor, a melanoma, a
leukemia, a lymphoma, ovarian cancer, bone cancer, lung cancer,
colorectal cancer, hepatocellular carcinoma, glioblastoma, prostate
cancer, breast cancer, bladder cancer, kidney cancer, pancreatic
cancer, gastric cancer, biliary cancer, urogenital cancer, and head
and neck cancer. The usefulness of the methods of the invention
against such a variety of cancers or other population of cells
where it is desired that the cells be sensitized to a genotoxic
drug is apparent in view of the conservation of the SAPK pathway in
all mammalian cells. Accordingly, the present invention provides
methods of increasing the sensitivity of a cell to a genotoxic drug
by inhibiting the activity of a SAPK in the cell, for example,
increasing the sensitivity of cancer cells to a cancer therapeutic
modality.
[0062] The invention further provides methods to reduce the
severity of a pathology in an individual, wherein the pathology is
due to a population of cells, by administering a SAPK inhibitory
agent to the individual and treating the individual with a
genotoxic drug. For example, the invention provides a method of
reducing the severity of a cancer in a patient, comprising
administering to the patient a SAPK inhibitory agent and treating
the patient with a cancer therapeutic modality. As used herein, the
term "reduce the severity," when used in reference to a pathology
such as cancer, means that the clinical symptoms or signs of the
disease are lessened.
[0063] A method of the invention provides a means, for example, to
reduce the severity of a cancer by increasing the sensitivity of
the cancer cells to a cancer therapeutic agent. For example,
expression of a dominant negative c-jun mutant in various tumor
cells, including glioblastoma cells, breast cancer cells (Example
I) and prostate cancer cells (Example II), reduced the dose of
cis-platinum required to kill 50% of the cells (IC-50), as compared
to the dose required to kill cells not expressing the dominant
negative c-jun. Thus, a method of the invention allows the
administration of a lower dose of a conventional cancer therapeutic
modality such as a chemotherapeutic agent or radiation than would
be required if SAPK activity was not inhibited.
[0064] This method also improves the efficacy of a therapy that
leads to an increase in the expression of cytokines that are known
to activate c-jun, particularly in tumor cells. Such cytokines
including, but not limited to, interleukins (IL) such as IL-2,
IL-6, IL-7 and IL-12, the tumor necrosis factors, interferons, and
various growth factors, including EGF, PDGF and TGF-.alpha., GM-CSF
and G-CSF (see, for example, Kyriakis et al., Nature 369:156-160
(1994)). In addition, a method of the invention improves a therapy
that has, as an effect, the release of inflammatory intermediates
that activate SAPK in tumor cells. Furthermore, introduction of
antisense oligonucleotides or expression vectors that encode them
makes tumor cells better targets for the immune system by restoring
the apoptotic pathways required for killing by cytotoxic immune T
cells, lymphokine activated killer cells, natural killer cells,
macrophages, monocytes, and granulocytes.
[0065] The following examples are intended to illustrate but not
limit the present invention.
EXAMPLE I
Dominant Negative C-Jun Sensitizes Glioblastoma Cells To Killing By
Cis-Platinum
[0066] This example demonstrates that inhibition of SAPK activity
by expression of a dominant negative c-jun (dn-jun) in glioblastoma
cells sensitizes the cells to killing by cis-platinum.
[0067] T98G glioblastoma cells were maintained in Dulbecco's
modified minimal medium containing 5% fetal calf serum. SAPK (JNK)
assays were performed as described by Hibi et al. (supra, 1993).
c-jun and dn-jun expression were quantitated using the methods and
antibodies described by Grover-Bardwick et al. (Carcinogenesis
15:1667-1674 (1994), which is incorporated herein by
reference).
[0068] Cisplatin, but not transplatin, forms covalent crosslinks
between adjacent guanine or guanine-adenine residues. Incubation of
T98G cells with 250, 500 or 1000 .mu.M cisplatin for 1 hr produced
a dose-dependent increase (up to 10-fold) in SAPK (JNK) activity,
whereas no effect was observed with transplatin. As a control, UV-C
irradiation was performed and similarly produced a dose-dependent
increase in SAPK activity. Similar results were observed using two
human non-small cell lung carcinoma cell lines (A549 and M103).
These results indicate that cisplatin, but not transplatin,
increase SAPK activity in various cancer cell lines.
[0069] T98G cells were stably transfected with a cDNA encoding the
dn-jun (Smeal et al., supra, 1991; Smeal et al., supra, 1992) and
clonal lines were obtained. Expression of dn-jun had no effect on
basal AP-1 activity or on the enzyme activity of SAPK. In contrast,
dn-jun expression inhibited phosphorylation-dependent activation of
transcription.
[0070] Cell viability was determined using the MTS method (Promega
Corp.; Madison WI), measuring the A590 of the formazan product 1 hr
after adding the MTS (Gjerset et al., Mol. Carcin. 14:275-285
(1995), which is incorporated herein by reference). Cells were
seeded into 96 well plates. After 24 hr, various concentrations of
cisplatin or transplatin was added and incubation was continued for
1 hr. Medium was then removed and replaced with fresh medium and
cell viability was determined 5 days later. Experiments were
performed in quadruplicate; cell viability is expressed as the
ratio of the viable cells following treatment to viable untreated
cells.
[0071] The IC-50 for cisplatin was determined using the T98G cells
that express dn-jun, control T98G cells and T98G cells stably
transfected with the corresponding empty vector. No significant
difference was observed between the control (untransfected) T98G
cells or the vector transfected T98G cells (IC-50=147 .mu.M and 154
.mu.M, respectively) . In comparison, expression of dn-jun in T98G
cells decreased the IC-50 to 21 .mu.M, which represents a 7-fold
increase in sensitivity. Furthermore, when various clonal dn-jun
transfected T98G cells were examined, the IC-50 correlated to the
steady-state level of dn-jun expressed in the cells
(r.sub.pearson=0.98) . In comparison, increased expression of c-jun
in T98G cells somewhat increased the viability of cells to
cisplatin exposure, suggesting that an increased amount of the SAPK
substrate in cells augments their viability.
[0072] In other experiments, U87 human glioblastoma cells and MCF-7
breast cancer cells were stably transfected with the vector
expressing dn-jun and clonal lines were isolated. U87 cells
expressing dn-jun showed a 2.5-fold increased sensitivity to
cisplatin as compared to parental U87 cells, and MCF-7 cells
expressing dn-jun showed a 3-fold increased sensitivity as compared
to the parental cells. These results indicate that expression of
dn-jun in various cancer cell lines sensitizes the cells to
cisplatin.
[0073] The PCR-stop assay was used to quantitate cisplatin-DNA
adduct formation and subsequent repair (Jennerwein and Eastman,
Nucl. Acids. Res. 19:6209-6214 (1991), which is incorporated herein
by reference). The assay is based on the observation that the
efficiency of amplification of cisplatin-treated DNA is inversely
proportional to the degree of platination. Cells were treated with
various concentrations of cis-platinum (cisplatin) or transplatin
for 1.25 hr, then genomic DNA was isolated immediately or 6 hr
later. DNA was amplified quantitatively to produce a 2.7 kb product
of the hypoxanthine phosphoribosyl transferase (HPRT) gene and a
0.15 kb nested fragment of the HPRT gene using .sup.32P-end labeled
primers: 5'-TGGGATTACACGTGTGAACCAACC-3'(5' primer; SEQ ID NO: 1)
and 5'-GATCCACAGTCTGCCTGAGTCACT-3' (3'primer; SEQ ID NO: 2), for
the 2.7 kb product; 5'-CCTAGAAAGCACATGGAGAGCTAG-3'(5' nested
primer; SEQ ID NO: 3) and the above 3' primer (SEQ ID NO: 2) for
the 0.15 kb product. The 0.15 kb product contains undetectable
levels of DNA damage under the present conditions and serves as an
internal PCR control and as the basis for normalization of the
amount of amplification of the 2.7 kb product. The number of
lesions per 2.7 kb product was calculated as 1 minus (cpm damaged
DNA/cpm undamaged DNA).
[0074] DNA isolated for T98G cells immediately after a 1 hr
treatment with 0, 100 or 200 AM cisplatin showed increasing levels
of DNA damage. However, if a 6 hr recovery period was allowed prior
to isolating the DNA, damage was markedly and significantly
(p=0.003) reduced. As a control, 2-aminobenzidine (ABZ), which
inhibits DNA repair by inhibiting ADP-ribosylation, was added with
cisplatin. Following the 6 hr recovery period, no repair was
observed in the ABZ treated cells and the level of damage was
substantially increased. In experiments performed using T98G cells
expressing dn-jun, DNA damage remained completely unrepaired,
following the 6 hr "recovery" period, in cells treated with either
100 .mu.M or 200 .mu.M cisplatin (p>0.53). These results
indicate that dn-jun inhibits DNA repair in cisplatin treated
glioblastoma cells.
EXAMPLE II
Dominant Negative C-Jun Sensitizes Prostate Carcinoma Cells to
Killing by Cis-Platinum
[0075] This example demonstrates that expression of a dn-jun in
prostate carcinoma cells sensitizes the cells to killing by
cis-platinum, thus confirming the general applicability of the
claimed invention.
[0076] PC3 prostate carcinoma cells were genetically modified to
express dn-jun or a second dominant negative c-jun, Tam-67, both of
which were under control of the metallothionein promoter (Brown et
al., Oncogene 9:791-799 (1994), which is incorporated herein by
reference) Viability studies showed an IC-50 of 109 .mu.M for the
control PC3 cells and 154 .mu.M for PC3 cells transfected with the
corresponding empty vector. In comparison, PC3 cells expressing
dn-jun had an IC-50 of 18 .mu.M, representing an increased
sensitivity of greater than 7-fold and greater than 9-fold above
the control and vector control cells, respectively. Similarly,
expression of Tam-67 strongly enhanced the sensitivity of the PC3
cells to cisplatin. These results demonstrate that various dominant
negative c-jun mutants can sensitize cancer cells to cisplatin and
confirm the general applicability of the claimed invention.
[0077] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
claims.
Sequence CWU 1
1
3 1 24 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 1 tgggattaca cgtgtgaacc aacc 24 2 24 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 2
gatccacagt ctgcctgagt cact 24 3 24 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 3 cctagaaagc acatggagag
ctag 24
* * * * *