U.S. patent application number 14/830194 was filed with the patent office on 2016-02-18 for compositions and methods for inhibiting tumor cells by inhibiting the transcription factor atf5.
This patent application is currently assigned to THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK. The applicant listed for this patent is THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK. Invention is credited to James Angelastro, Lloyd A. Greene.
Application Number | 20160046686 14/830194 |
Document ID | / |
Family ID | 51391834 |
Filed Date | 2016-02-18 |
United States Patent
Application |
20160046686 |
Kind Code |
A1 |
Greene; Lloyd A. ; et
al. |
February 18, 2016 |
COMPOSITIONS AND METHODS FOR INHIBITING TUMOR CELLS BY INHIBITING
THE TRANSCRIPTION FACTOR ATF5
Abstract
The present invention relates to methods for treating and/or
preventing tumors and/or promoting apoptosis in a neoplastic cell
comprising contacting the neoplastic cell with an cell-penetrating
dominant-negative ATF5 ("CP-d/n-ATF5"), wherein the CP-d/n-ATF5 is
capable of inhibiting ATF5 function and/or activity.
Inventors: |
Greene; Lloyd A.;
(Larchmont, NY) ; Angelastro; James; (Davis,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW
YORK |
New York |
NY |
US |
|
|
Assignee: |
THE TRUSTEES OF COLUMBIA UNIVERSITY
IN THE CITY OF NEW YORK
New York
NY
|
Family ID: |
51391834 |
Appl. No.: |
14/830194 |
Filed: |
August 19, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US2014/017550 |
Feb 21, 2014 |
|
|
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14830194 |
|
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61768390 |
Feb 22, 2013 |
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Current U.S.
Class: |
514/19.3 ;
530/350 |
Current CPC
Class: |
A61K 31/495 20130101;
A61K 38/00 20130101; A61P 35/00 20180101; A61K 38/1709 20130101;
C07K 14/4705 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made with government support under Grant
No. RCA126924A awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method for treating and/or preventing tumors and/or promoting
apoptosis in a neoplastic cell comprising contacting the neoplastic
cell with an cell-penetrating dominant-negative ATF5, wherein the
cell-penetrating dominant-negative ATF5 is capable of inhibiting
ATF5 function and/or activity.
2. The method of claim 1, wherein the neoplastic cell is selected
from the group consisting of: breast, ovary, endometrium, gastric,
colon, liver, pancreas, kidney, bladder, prostate, testis, skin,
esophagus, tongue, mouth, parotid, larynx, pharynx, lymph node,
lung, peripheral nervous system and brain.
3. The method of claim 2, wherein the neoplastic brain cell is
selected from the group consisting of glioblastoma, astrocytoma,
glioma, medulloblastoma mesothelioma, and neuroblastoma, and the
neoplastic brain cell is associated with a primary or a recurrent
brain tumor.
4. The method of claim 1, wherein the cell-penetrating
dominant-negative ATF5 is administered orally, parenterally, and/or
transdermally.
5. A composition comprising a cell-penetrating dominant-negative
ATF5, wherein the cell-penetrating dominant-negative ATF5 consists
of a sequence selected from the group consisting of: TABLE-US-00025
LEQENAELEGECQGLEARNRELKERAES, LEKEAEELEQENAELEGECQGLEARNRE LKERAES,
LARENEELLEKEAEELEQENAELEGECQGLEARNRELKERAES,
LEQRAEELARENEELLEKEAEELEQENAELEGECQGLEARNRELKERAES,
LEQRAEELARENEELLEKEAEELEQENAELEGECQGLEARNRELKERAESV,
where the underlined sequence is the dominant-negative sequence and
the remainder of the sequence is the ATF5 leucine zipper, and the
sequence is operably lined to a cell-penetrating sequence.
6. A composition comprising a cell-penetrating dominant-negative
ATF5, wherein the cell-penetrating dominant-negative ATF5 consists
of a sequence selected from the group TABLE-US-00026
LEQENAELEGECQGLEARNRELRERAES, LEKEAEELEQENAELEGECQGLEARNRE LRERAES,
LARENEELLEKEAEELEQENAELEGECQGLEARNRELRERAES,
LEQRAEELARENEELLEKEAEELEQENAELEGECQGLEARNRELRERAES,
LEQRAEELARENEELLEKEAEELEQENAELEGECQGLEARNRELRERAESV,
consisting of: where the underlined sequence is the
dominant-negative sequence and the remainder of the sequence is the
ATF5 leucine zipper, and the sequence is operably lined to a
cell-penetrating sequence.
7. The composition of claim 6, wherein the cell-penetrating
dominant-negative ATF5 comprises a sequence selected from the group
consisting In certain embodiments, the cell-penetrating
dominant-negative ATF5 comprises a sequence selected from the group
consisting of: TABLE-US-00027 (1)
MGSSHHHHHHSSGLVPRGSHMRQIKIWFQNRRMKWKKDYKDDDDKMAS MTGGQQMGRDPD
LEGECQGLEARNRELRERAES V,
where the underlined residues (MG-HM) are a 6.times.His-tag leader
sequence, the bold residues (RQ-KK) are a Penetratin sequence, the
italicized residues (DY-DK) are a Flag tag, the residues with no
font modification (MA-PD) are spacer amino acids, the bold and
italicized residues (LE-AE) are a d/n sequence, and the bold and
underlined residues (LE-SV) are an ATF5 leucine zipper truncated
after its first Valine; TABLE-US-00028 (2)
MGSSHHHHHHSSGLVPRGSHMLEYGRKKRRQRRRYPYDVPDYAMASMTG GQQMGRDPD
LEGECQGLE ARNRELRERAESV,
where the underlined residues (MG-LE) are a 6.times.His-tag leader
sequence, the bold residues (YC-RR) are a TAT sequence, the
italicized residues (YP-YA) are an HA tag, the residues with no
font modification (MA-PD) are spacer amino acids, the bold and
italicized residues (LE-AE) are a d/n sequence, and the bold and
underlined residues (LE-SV) are an ATF5 leucine zipper truncated
after its first Valine; TABLE-US-00029 (3)
MGSSHHHHHHSSGLVPRGSHMRQIKIWFQNRRMKWKKLEQRAEELAREN E
ELLEKEAEELEQENAELEGECQGLEARNRELKERAESV
where the where the underlined residues (MG-HM) are a
6.times.His-tag leader sequence, the bold residues (RQ-KK) are a
Penetratin sequence, the italicized residues (LE-AE) are a din
sequence, and the bold and underlined residues (LE-SV) are an ATF5
leucine zipper truncated after its first Valine; and TABLE-US-00030
(4) RQIKIWFQNRRMKWKKLEQRAEELARENEELLEKEAEELEQENAELEGE CQ
GLEARNRELKERAESV
where the bold residues (RQ-KK) are a Penetratin sequence, the
italicized residues (LE-AE) are a d/n sequence, and the bold and
underlined residues (LE-SV) are an ATF5 leucine zipper truncated
after its first Valine.
8. A kit comprising a composition comprising a cell-penetrating
dominant-negative ATF5 of any one of claims 5-7.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of International Patent
Application No. PCT/US2014/017550, filed Feb. 21, 2014 and claims
priority to U.S. Provisional Application Ser. No. 61/768,390, filed
Feb. 22, 2013, to both of which priority is claimed and the
contents of both of which are incorporated herein in their
entirety.
1. BACKGROUND OF THE INVENTION
[0003] Approximately one million people are diagnosed with cancer
each year, and many millions of Americans of all ages are currently
living with some form of cancer. At some time during the course of
their lifetime, one out of every two American men and one out of
every three American women will be diagnosed with some form of
cancer. Of the one million Americans diagnosed with cancer
annually, 17,000 are diagnosed with brain tumors. Brain tumors
invade and destroy normal tissue, producing such effects as
impaired sensorimotor and cognitive function, increased
intracranial pressure, cerebral edema, and compression of brain
tissue, cranial nerves, and cerebral vessels. Drowsiness, lethargy,
obtuseness, personality changes, disordered conduct, and impaired
mental faculties are the initial symptoms in 25% of patients with
malignant brain tumors. Treatment of brain tumors is often
multimodal, and depends on pathology and location of the tumors.
For malignant gliomas, multimodal therapy, including chemotherapy,
radiation therapy, and surgery, is used to try to reduce tumor
mass. Regardless of approach, however, prognosis for patients
suffering from these tumors is guarded: the median term of survival
after chemotherapy, radiation therapy, and surgery is only about 1
year, and only 25% of these patients survive for 2 years.
[0004] The prevalence of cancer, and in particular brain tumors
refractory to existing therapies, has led to the identification of
transcription factors impacting cell cycle control of neuronal
cells, including ATF5 (Acharay et al., J Struct Biol 155:130-139
(2006)). ATF5 belongs to the activating transcription factor/CREB
family of basic leucine zipper transcription factors (Acharay et
al., J Struct Biol 155:130-139 (2006); Greene et al., J Neurochem
108:11-22 (2009)). ATF5 is highly expressed by neural stem and
progenitor cells for neuronal and glial lineages and its expression
plummets when these differentiate (Angelastro et al., J Neurosci
23:4590-4600 (2003); Angelastro et al., J Neurosci 25:3889-3899
(2005); Mason et al., Mol Cell Neurosci 29:372-380 (2005)). Because
constitutive ATF5 expression in neural progenitor cells causes them
to remain in cell cycle and blocks their differentiation,
(Angelastro et al., J Neurosci 23:4590-4600 (2003); Angelastro et
al., J Neurosci 25:3889-3899 (2005); Mason et al., Mol Cell
Neurosci 29:372-380 (2005)), ATF5 expression in GBM was assayed as
GBMs are thought to be derived from neural stem and progenitor
cells (Tanaka et al., Nat Rev Clin Oncol 10:14-26 (2012)).
Examination of 29 resected GBMs revealed high ATF5 expression by
all and by all 9 rodent and human GBM lines examined (Angelastro et
al., Oncogene 25:907-916 (2006)). These findings have been
corroborated and additional data has indicated a correlation
between ATF5 levels and GBM prognosis (Dong et al., J Neuropathol
Exp Neurol 64:948-955 (2005); Sheng et al., Nat Med 16:671-677
(2010)).
[0005] To examine the role of ATF5 in GBM, a dominant-negative
inhibitor of the protein was created to interfere with ATF5
function (Acharay et al., J Struct Biol 155:130-139 (2006),
Angelastro et al., Oncogene 25:907-916 (2006)), and si/shRNAs were
developed to silence its expression. Culture experiments with human
and rat GBM lines showed that both the d/n-ATF5 and the ATF5
si/shRNAs cause their massive apoptotic death (Angelastro et al.,
Oncogene 25:907-916 (2006)). In contrast, ATF5+ proliferating
neural progenitor cells and astrocytes did not show this apoptotic
response. In an initial in vivo study, it was found that if the
d/n-ATF5 was retrovirally-delivered it would selectively and with
very high efficiency kill tumor cells generated from implanted C6
rat GBM cells, but not normal proliferating brain cells (Angelastro
et al., Oncogene 25:907-916 (2006)). In subsequent studies, an
adult mouse model was used in which gliomas (of grades ranging form
low-grade gliomas to GBMs) are efficiently generated by infection
with a retrovirus expressing PDGF-B and a p53 shRNA (Arias et al.,
Oncogene 31:739-751 (2012)). Using mice engineered to conditionally
express the d/n-ATF5 from the human GFAP promoter (which is
expressed in neural stem/progenitor cells, astrocytes and GBMs),
induction of that d/n-ATF5 led to complete regression/eradication
of tumors and survival of all 24 treated mice. Likewise, expression
of the d/n-ATF5, prior to injection of the PDGF-B/shRNA-p53
retrovirus, prevented tumor development in 85.7% of the mice. In
contrast, for mice in which the d/n-ATF5 was not induced, 15/16 had
tumors and 40% died within the test period. There were no apparent
effects on normal cells (Arias et al., Oncogene 31:739-751
(2012)).
2. SUMMARY OF THE INVENTION
[0006] In certain embodiments, the present invention relates to
methods for treating and/or preventing tumors and/or promoting
apoptosis in a neoplastic cell comprising contacting the neoplastic
cell with an cell-penetrating dominant-negative ATF5
("CP-d/n-ATF5"), wherein the CP-d/n-ATF5 is capable of inhibiting
ATF5 function and/or activity.
[0007] In certain embodiments, the neoplastic cell is selected from
the group consisting of: breast, ovary, endometrium, gastric,
colon, liver, pancreas, kidney, bladder, prostate, testis, skin
(e.g., melanocyte/melanoma cell), esophagus, tongue, mouth,
parotid, larynx, pharynx, lymph node, lung, blood (e.g.,
hematological cancers), peripheral nervous system, and brain. In
certain embodiments, the neoplastic cell is selected from the group
consisting of glioblastoma, astrocytoma, glioma, medulloblastoma,
meningioma, mesothelioma, and neuroblastoma. In certain
embodiments, the neoplastic cell is associated with a primary or a
recurrent brain tumor.
[0008] In certain embodiments the CP-d/n-ATF5 is administered
orally, parenterally (e.g., subcutaneously), intranasally, and/or
transdermally.
[0009] In certain embodiments the CP-d/n-ATF5 comprises a portion
of the human, rat, or mouse ATF5 peptide sequence or a combination
thereof. In certain embodiments, the cell-penetrating
dominant-negative ATF5 comprises a sequence selected from the group
consisting of:
TABLE-US-00001 LEQENAE, LEKEAEELEQENAE, LARENEELLEKEAEELEQENAE,
LEQRAEELAREN EELLEKEAEELEQENAE, or
LEQRAEELARENEELLEKEAEELEQENAE,
linked to a peptide that forms a leucine zipper and to a
cell-penetrating sequence. In certain embodiments, the CP-d/n-ATF5
comprises a sequence selected from the group consisting of:
TABLE-US-00002 LEQENAELEGECQGLEARNRELKERAES,
LEKEAEELEQENAELEGECQGLEARNRELK ERAES,
LARENEELLEKEAEELEQENAELEGECQGLEARNRELKERAES,
LEQRAEELARNEELLEKEAEELEQENAELEGECQGLEARNRELKERAES,
LEQRAEELARENEELLEKEAEELEQENAELEGECQGLEARNRELKERAESV,
where the underlined sequence is the dominant-negative sequence and
the remainder of the sequence is the ATF5 leucine zipper, and the
sequence is operably linked to (in frame) a cell-penetrating
sequence. In certain embodiments, the cell-penetrating
dominant-negative ATF5 comprises a sequence selected from the group
consisting of:
TABLE-US-00003 LEQENAELEGECQGLEARNRELRERAES,
LEKEAEELEQENAELEGECQGLEARNRELRERAES,
LARENEELLEKEAEELEQENAELEGECQGLEARNREL RERAES,
LEQRAEELARENEELLEKEAEELEQENAELEGECQGLEARNRELRERAES,
LEQRAEELARENEELLEKEAEELEQENAELEGECQGLEARNRELRERAESV,
where the underlined sequence is the dominant-negative sequence and
the remainder of the sequence is the ATF5 leucine zipper, and the
sequence is operably lined to a cell-penetrating sequence.
[0010] In certain embodiments, the cell-penetrating
dominant-negative ATF5 comprises a sequence selected from the group
consisting of: (1)
TABLE-US-00004 MGSSHHHHHHSSGLVPRGSHMRQIKIWFQNRRMKWKKDYKDDDDKMAS
MTGGQQMGRDPD LEARNRELRERAES V,
where the underlined residues ( MG-HM) are a 6.times.His-tag leader
sequence, the bold residues (RQ-KK) are a Penetratin sequence, the
italicized residues (DY-DK) are a Flag tag, the residues with no
font modification (MA-PD) are spacer amino acids, the bold and
italicized residues (LE-AE) are a d/n sequence, and the bold and
underlined residues ( LE-SV) are an ATF5 leucine zipper truncated
after its first Valine; (2)
TABLE-US-00005 MGSSHHHHHHSSGLVPRGSHMLEYGRKKRRQRRRYPYDVPDYAMASMT
GGQQMGRDPD LEGECQGLE ARNRELRERAESV,
where the underlined residues (MG-LE) are a 6.times.His-tag leader
sequence, the bold residues (YG-RR) are a TAT sequence, the
italicized residues (YP-YA) are an HA tag, the residues with no
font modification (MA-PD) are spacer amino acids, the bold and
italicized residues (LE-AE) are a d/n sequence, and the bold and
underlined residues ( LE-SV) are an ATF5 leucine zipper truncated
after its first Valine; (3)
TABLE-US-00006 MGSSHHHHHHSSGLVPRGSHMRQIKIWFQNRRMKWKKLEQRAEELARE
NEELLEKEAEELEQENAELEGECQGLEARNRELKERAESV
where the where the underlined residues (MG-HM) are a
6.times.His-tag leader sequence, the bold residues (RQ-KK) are a
Penetratin sequence, the italicized residues (LE-AE) are a d/n
sequence, and the bold and underlined residues ( LE-SV) are an ATF5
leucine zipper truncated after its first Valine; and (4)
TABLE-US-00007 RQIKIWFQNRRMKWKKLEQRAEELARENEELLEKEAEELEQENAELEG
ECQGLEARNRELKERAESV
where the bold residues (RQ-KK) are a Penetratin sequence, the
italicized residues (LE-AE) are a d/n sequence, and the bold and
underlined residues ( LE-SV) are an ATF5 leucine zipper truncated
after its first Valine. In certain embodiments, the
cell-penetrating dominant-negative ATF5 is chemically
synthesized.
[0011] In certain embodiments, the invention also relates to kits
for use in treating and/or preventing tumors and/or promoting
apoptosis in a neoplastic cell. Additional aspects of the present
invention will be apparent in view of the description which
follows.
3. BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1 GFP-d/n-ATF5 C-terminally truncated fusion protein
(GFP-d/n-ATF5-Tr) promotes the same level of apoptosis as
full-length GFP-d/n-ATF5 protein in C6 glioma cells. C6 cells were
transfected with pQC-X-I-eGFP, pQC-d/n-GFPATF5, or pQC-GFPATF5-tr.
The percentages (mean.+-.SEM, n=4; total of approximately 200 cells
scored per condition) of condensed apoptotic nuclei in GFP+
transfected cells were determined 2 days later. Student's t-test;
GFP+ cells versus GFP-d/n-ATF5+ cells or GFP-d/n-ATF5-tr cells,
(*p<0.05); GFP-d/n-ATF5+ cells versus GFP-d/n-ATF5-tr cells,
(Not Significant).
[0013] FIG. 2A-2C Purity and molecular properties of
bacterially-expressed and purified 6.times.histidine-Flag-Tagged
Penetratin-Flag-D/N-ATF5-tr (Pen-d/n-ATF5-RP) and
6.times.histidine-Flag-Tagged Penetratin-Flag-Control
(Pen-control-RP) peptides. (FIG. 2A) Coomassie stained SDS-PAGE of
purified Pen-d/n-ATF5-RP and Pen-control-RP (5 .mu.g per lane).
Molecular weight markers are shown on the left, and a linear scheme
of each peptide is shown above each lane. Purification was as
described in Methods. (FIG. 2B) Deconvoluted mass spectra from
LC-high Resolution mass spectrometry of purified Pen-d/n-ATF5-RP.
The most abundant species is the 12,948.88 Da monomer form without
formyl-methionine followed by the formyl-methionine 13,127 Da
monomer form (isoform). The spectrum also reveals a small amount of
the 25,897.5 Da dimer. (FIG. 2C) Stability of Pen-d/n-ATF5-RP in
Human Serum. Pen-d/n-ATF5-RP (36 .mu.M) was incubated with human
serum (25% v/v in PBS) at 37.degree. C. for 0 to 48 h. Aliquots
were withdrawn at various times and the Pen-d/n-ATF5-RP peptide was
resolved by SDS-PAGE, transferred to PVDF membrane and probed with
anti-Flag antibody. The anti-Flag signal was detected by near IR
using LiCor software and densitometry of the band at the expected
size of Pen-d/n-ATF5-RP was performed and quantified using ImageJ.
Values are mean.+-.SEM, n=3.
[0014] FIG. 3A-3B Uptake and retention of Pen-d/n-ATF5-RP by
cultured glioblastoma cells. (FIG. 3A) Confocal images of C6 rat
glioblastoma cells incubated for 4 hours with either 200 nM
Pen-control-RP (left) or Pen-d/n-ATF5-RP (right). Cells were
washed, fixed and stained with anti-Flag (red) and DAPI (blue).
Scale bar=2 .mu.m. (FIG. 3B) Rat C6 and human U87 glioblastoma
cells were incubated for the indicated times with 3 .mu.M
Pen-d/n-ATF5-RP, washed, fixed and immunostained with anti-Flag
(green) and DAPI (blue). Scale bar=5 .mu.m.
[0015] FIG. 4 Pen-d/n-ATF5-RP promotes apoptosis of C6 glioblastoma
cells. C6 cells were treated with 3 .mu.M Pen-d/n-ATF5-RP or 3
.mu.M Pen-Control-RP, or were untreated. The percentage
(mean.+-.SEM; n=4 in 2 independent experiments; approximately 200
cells scored) of condensed apoptotic nuclei in cells was determined
5 days later. Student's t-test; Pen-d/n-ATF5-RP versus
Pen-Control-RP cells or nontreated, (*p<0.05); Pen-Control-RP
cells versus nontreated cells, (p=0.29).
[0016] FIG. 5A-5F Pen-d/n-ATF5-RP enters the mouse brain and causes
targeted apoptosis of glioma cells. (FIG. 5A-5F) Representative
brain sections stained with Flag antibody to indicate presence of
Pen-d/n-ATF5-RP or HA to identify presence of tumor-inducing
retrovirus (red); TUNEL to identify apoptosis (green) and DAPI to
localize nuclei (blue). (FIG. 5A) Murine brain tumor 24 h
post-treatment (16 h after last injection) with Pen-d/n-ATF5-RP (52
days post-retrovirus injection). (FIG. 5B) Normal contralateral
cerebral hemisphere of the same mouse in (FIG. 5A). (FIG. 5C)
Murine brain tumor 24 h post-injection with saline (59 days
post-retrovirus injection). Presence of Pen-d/n-ATF5 within cells
is confirmed in the treated mouse (FIG. 5A,5B) versus saline
control (FIG. 5C) by increased Flag antibody staining Glioma
cell-specific induction of apoptosis by Pen-d/n-ATF5-RP is
illustrated by increased TUNEL staining (green) in (FIG. 5A) as
compared to (FIG. 5B) and (FIG. 5C). (FIG. 5D) TUNEL and DAPI
staining of a tumor-containing brain section 160 days
post-retrovirus injection and 3 days after injection of
Pen-control-RP. Note HA+ cells identifying tumor cells and absence
of TUNEL staining (FIG. 5E) Staining as in (FIG. 5D) of a
tumor-containing section (143 days post-retrovirus injection) and 3
days after Pen-d/n-ATF5-RP treatment. Note the presence of TUNEL
staining in HA+ tumor cells and fragmented appearance of the
staining as compared to (FIG. 5A) and (FIG. 5D). (FIG. 5F) Staining
as in (FIG. 5D) of a tumor-containing section 150 days after
retrovirus injection and 2 days after 2 treatments of subcutaneous
Pen-d/n-ATF5-tr-RP injections. Note the qualitative similarity of
staining pattern to (FIG. 5E) with fragmented PDGF-B-HA and TUNEL
staining Scale bars equal 20 .mu.m.
[0017] FIG. 6A-6D Retention of Pen-d/n-ATF5-RP in mouse brain at
various times after administration. Mice received 4 intraperitoneal
injections of either saline (FIG. 6A) or Pen-d/n-ATF5-RP (FIG. 6B,
6C) as described in the text. Animals were sacrificed at either 40
(FIG. 6A, 6B) or 64 (FIG. 6C) h after the last injection and
sections of their fixed brains were stained with either anti-Flag
(Red; to visualize Pen-d/n-ATF5-RP) or DAPI (blue; to visualize
nuclei). (FIG. 6D) Densitometry of anti-Flag Immunostaining. The
optical densities (red channel) of fifteen random 0.176 inch2 areas
were determined in each of the images and averaged.+-.SD using
Image J. Student's t-test; Pen-d/n-ATF5-RP (64 hours) or (40 hours)
versus saline, (*p<0.05). Scale bar is 10 .mu.m.
[0018] FIG. 7A-7D' H&E staining of the SVZ and hippocampal
dentate gyms shows no detectable difference between these
structures in Pen-d/n-ATF5-RP-treated and non-treated mice. (FIG.
7A,7A') Lateral ventricle/SVZ (FIG. 7A) and hippocampal dentate
gyms (FIG. 7A') from a tumor-bearing mouse 183 days after the
second set of subcutaneous treatments with Pen-d/n-ATF5-RP (see
also Fig S8 for further data on the same mouse). (FIG. 7B,7B')
Lateral ventricle/SVZ (FIG. 7B) and hippocampal dentate gyms (FIG.
7B') from an age-matched control mouse not treated with
Pen-d/n-ATF5-RP and not injected with retrovirus. (FIG. 7C,7C')
Lateral ventricle/SVZ (FIG. 7C) and hippocampal dentate gyms (FIG.
7C') from a non-tumor-bearing mouse 1 day after the second set
(given 5 days after the first set) of subcutaneous treatments with
Pen-d/n-ATF5-RP. (FIG. 7D,7D') Lateral ventricle/SVZ (FIG. 7D) and
hippocampal dentate gyms (FIG. 7D') from an age-matched untreated
non-tumor-bearing control mouse. Scale for A-C is 20 .mu.m and 50
.mu.m for D-F.
[0019] FIG. 8A-8F Example of MRI and histopathology of a mouse
glioma treated with Pen-Control-RP peptide. (FIG. 8A) Post-contrast
3D FLASH MRI coronal image of the cerebrum of a control mouse that
was not injected with PDGF-B-HA/sh-p53 retrovirus. (FIG. 8B)
Post-contrast 3D FLASH MRI coronal image of mouse cerebrum showing
a bilateral tumor (white contrast) 246 days after PDGF-B-HA/shp53
retrovirus injection and prior to treatment with Pen-Control-RP
peptide. (FIG. 8C) Post-contrast 3D FLASH MRI image of the same
mouse brain 40 days after subcutaneous treatment with
Pen-Control-RP peptide (as described in the text) reveals
persistence of the tumor (arrows). (FIG. 8D) H&E stained
sections of the same mouse brain at tumor-containing areas 1 and 2
shown by arrows in panel (FIG. 8C). The mouse was sacrificed 116
days after the second treatment with Pen-Control-RP peptide due to
moribund behavior. Presence of tumor is indicated in both sections
by hyperchromatic nuclei and higher cellularity. (FIG. 8E)
Immunostaining for HA tag in sections from areas 1 and 2 shown in
Panel (FIG. 8C) reveals presence of virally-delivered PDGF-B-HA in
induced tumor cells. (FIG. 8F) Immunostaining of sections from
areas 1 and 2 shown in Panel (FIG. 8C) reveals a high index of
Ki67+/dividing cells indicative of tumor. Scale bars in d-f are 20
.mu.m.
[0020] FIG. 9A-9E Pen-d/n-ATF5-RP promotes rapid and long-term
regression/eradication of mouse glioma as indicated by MRI and
histology. (FIG. 9A) Post-contrast 3D FLASH MRI scans of a mouse
brain before and at various times after treatment (as described in
text) with Pen-d/n-ATF5-RP. Pretreatment shows image of cortex 243
days after PDGF-B-HA/shp53 retrovirus injection. Yellow arrows
indicate location of the bilateral tumor. Post-treatment images of
the same position of the mouse cortex are at the indicated times
after the second administration of Pen-d/n-ATF5-RP. Yellow arrows
in post-treatment images show location of original tumor. (FIG. 9B)
H&E image of the same mouse brain harvested 192 days after the
second Pen-d/n-ATF5-RP treatment. Region 1 represents the location
of the section as shown in the final time point in (FIG. 9A) and at
which the tumor was present before treatment. Note the absence of
hyperchromatic nuclei and higher cellularity that characterize
gliomas. (FIG. 9C) Ki67 staining in region 2 (from Panel A/176 days
post-treatment). Note the absence of Ki67+/proliferating cells seen
in gliomas. (FIG. 9D) HA/DAPI staining of section from region 1.
Note the absence of cells expressing exogenous PDGF-B-HA. (FIG.
9E). GFAP/DAPI staining of section region 1. Note clusters of GFAP+
cells consistent with the presence of a glial scar where the tumor
was formerly present. Lack of HA staining of a nearby section
confirmed the absence of tumor cells. Diagonal green stripes are
due to tissue folds. Scale bar is 20 .mu.m.
[0021] FIG. 10 MRI and histopathological images of an untreated
mouse with a bilateral tumor. Middle panel shows post-contrast 3D
FLASH MRI image of a tumor-bearing mouse brain at 112 days after
PDGF-B-HA/shp53 retrovirus injection. Panels (A) and (B) show
images for sections stained with HA to reveal tumor cells and with
DAPI to show nuclei. The yellow arrows on the MRI along with the
letters show the relative locations of the HA+ sections shown in
(A) and (B). Retroviral injection was on side B. Scale bar is 20
.mu.m. DAPI (40,6-diamidino-2-phenylindole).
[0022] FIG. 11A-11E Second example illustrating that
Pen-d/n-ATF5-RP promotes rapid and long-term regression/eradication
of a mouse glioma as indicated by MRI and histology. FIG. 11A
Post-contrast 3D FLASH MRI images of a tumor-bearing mouse brain
before and at various times after treatment with Pen-d/n-ATF5-RP.
Pretreatment coronal and transverse images (74 days after
PDGF-B-HA/shp53 retrovirus injection) show multifocal tumors within
the cortex (arrows). Images from the same mouse brain are shown at
8, 21 and 181 days after two sets of subcutaneous treatments with
Pen-d/n-ATF5-RP as described in the text. Note decreased signal at
8 days and absence of detectable signals at 21 and 181 days
following treatment. (FIG. 11A', FIG. 11A'') Estimates of tumor
volume corroborate loss of signal by 8 days after Pen-d/n-ATF5-RP
treatment. The same images as in (FIG. 11A) for pretreatment and 8
days post-treatment with arrows pointing to tumor foci (yellow
circles) for which volumetric measurements were obtained using the
region of interest elliptic cylinder tool (yellow circles). At
pretreatment, the calculated volumes in (FIG. 11A') are 0.597 mm3,
0.164 mm3, and 0.760 mm3 for foci 1, 2, and 3, respectively. For 8
days post-treatment (FIG. 11A''), volumes of the same tumors
decreased to 0.106 mm3, 0.0302 mm3, and 0.0895 mm3 for foci 1, 2,
and 3, respectively. After 21 days of treatment the tumors could
not be visualized for measurement. (FIG. 11B) H&E staining of
the same sacrificed mouse brain (183 days after treatment; 190 days
after initial tumor detection) corroborates the absence of
detectable tumor with the arrow pointing to the remnant scar
corresponding to tumor focus 1 shown in A' and corroborates absence
of detectable tumor. (FIG. 11C) HA immunostaining of the same brain
(for PDGF-B-HA) indicates the absence of detectable tumor cells in
the same focus 1 region as in FIG. 11A' and (FIG. 11B). (FIG. 11D)
GFAP immunostaining of the same brain at focus 1 shows a remnant
GFAP+ glial scar. (FIG. 11E) Ki67 immunostaining of the focus 1
region of the same brain reveals the absence of dividing cells.
Scale bar is 20 .mu.m for B-E.
[0023] FIG. 12A-12C Long-term survival and tumor presence outcomes
for glioma-bearing mice treated with Pen-d/n-ATF5-RP. (FIG. 12A)
Survival of glioma-bearing mice (verified by MRI) with or without
treatment with Pen-d/n-ATF5-RP (subcutaneous delivery as described
in the text). Of the nine control mice, four control mice were
treated with Pen-Control-RP peptide and five were untreated. The
experimental endpoint was 200 days after initial tumor detection by
MRI. Survival analysis achieved by log-rank test showed a
p-value=0.0007 (http://in-silico.net/tools/statistics/survivor).
(FIG. 12B) MRI outcomes for tumor-bearing mice before and after
subcutaneous treatment with Pen-d/n-ATF5-RP as described in the
text. The latter times range from 176-225 days after tumor
treatment (183-230 days after tumor detection). (FIG. 12C) Brain
histopathological outcomes for tumors in control and
Pen-d/n-ATF5-RP treated mice. In all cases, MRI verified the
presence of tumors prior to treatment. Control animals were as
described in (FIG. 12A) and brains were harvested either after
death (6 controls), after the 6 month experimental endpoint (4
treated animals) or after sacrifice for non-tumor related health
problems (2 treated animals). For treated animals, histological
analysis was carried out 260-438 days after tumor initiation
(183-259 days after Pen-d/n-ATF5-RP administration and 190-305 days
after initial tumor detection). Sections of brain were prepared as
described in Methods and were stained with H&E and
immunostained for Ki67 and HA (to identify PDGF-B-HA+ tumor cells).
The presence/absence of tumors was based on observations of
hyperchromatic nuclei, high cellularity, elevated Ki67 staining and
HA immunostaining.
[0024] FIG. 13 indicates that TAT-d/n-ATF5 (TAT-ZIP) promotes
apoptotic death of cultured melanoma MEL501 cells. TAT-linked
dominant-negative ATF5 peptide at the indicated concentrations (in
.mu.M) was added to medium of MEL501 melanoma cells. Four days
later the cells were stained with Hoescht dye and the cells were
stained for proportion with apoptotic nuclei.
[0025] FIG. 14 indicates that TAT-d/n-ATF5 (TAT-ZIP) reduces the
expression of endogenous ATF5 in cultured U373 glioblastoma cells.
TAT-linked dominant-negative ATF5 peptide at the indicated
concentrations (in .mu.M) was added to medium of U373 glioblastoma
cells for 17 hrs day and the cells were then harvested and analyzed
by Western immunoblotting for levels of endogenous ATF5. Note that
the TAT-d/n-ATF5 greatly reduces expression of endogenous ATF5. As
previous studies have shown that tumor cells require endogenous
ATF5 to survive, the mechanism of action by which the
cell-penetrating TAT-ZIP peptide kills may be by causing loss of
the endogenous ATF5 protein. Note also the smear above the
endogenous ATF5 when the TAT-ZIP peptide is present. This suggests
that TAT-ZIP reduces endogenous ATF5 by causing its ubiquitination
and proteasomal degradation.
[0026] FIG. 15 indicates that TAT-d/n-ATF5 (TAT-ZIP) peptide
induces expression of the pro-death gene DDIT3 (CHOP) in various
tumor cell lines. Cells were treated with TAT-d/n-ATF5 for the
indicated times at the indicated doses (in .mu.M) and then
harvested and analyzed by Western immunoblotting for expression of
CHOP and other non-responsive proteins. Note the elevation of CHOP
in all cases. Since CHOP may promote cell death, these data
indicate that induction of CHOP protein may be one mechanism by
which TAT-d/n-ATF5 kills tumor cells.
[0027] FIG. 16 indicates that silencing of CHOP protein with siRNA
(top Western immunoblot) partially protects U87 cells from death
caused by TAT-d/n-ATF5 peptide. Cells were treated with siCHOP to
silence CHOP expression (top Western immunoblot) or with control
siRNA. They were then exposed to TAT-d/n-ATF5 for 2 days and
assessed for proportion of cells with apoptotic nuclei. The data
support the idea that part of the mechanism by which TAT-d/n-ATF5
kills tumor cells is by increasing their expression of CHOP which
in turn mediates death.
[0028] FIG. 17 indicates that TAT-D/N-ATF5 down-regulates BCL2
survival protein. Cultured U87 human glioblastoma cells were
treated with the indicated concentrations of TATZIP (TAT-d/n-ATF5
peptide) (in .mu.M) for 30 hrs. The cells were then harvested and
assessed by Western immunoblotting for expression of the survival
protein BCL2. These findings indicate that in addition to elevating
pro-death CHOP, TAT-d/n-ATF5 may also kill tumor cells by reducing
their levels of the BCL2 survival protein.
[0029] FIG. 18 indicates that TAT-D/N-ATF5 synergizes with
temozolomide (TMZ) to kill cultured U87 glioblastoma cells. Cells
were cultured for one day with sub-lethal levels of TAT-d/n-ATF5
(TZIP 1 .mu.M) and TMZ (50 .mu.M) either separately or in
combination, and then assessed for proportion of cells with
apoptotic nuclei. TMZ is presently the first-line treatment for
human GBM. The data reveal that TAT-d/n-ATF5 not only functions in
presence of TMZ, but that the two drugs act in synergy to kill GBM
cells. This suggests that TAT-d/n-ATF5 can be administered to
patients who are taking TMZ.
[0030] FIG. 19 depicts recombinant TAT-d/n-ATF5 (3 .mu.M) treatment
for 3-5 days decreasing viability of two human and one mouse GMB
cell line as established by MTA assay.
[0031] FIG. 20 depicts synthetic PEN-d/n-ATF5 treatment decreasing
cell viability of cultured U87 human glioblastoma cells as
established by MTA assay. 5 days treatment at indicated
concentrations (.mu.M).
[0032] FIG. 21 depicts recombinant TAT-d/n-ATF5 treatment promoting
death of cultured U87 human glioblastoma cells as indicated by
Annexin V/PI staining and flow cytometry. Proportions of viable
cells are shown in lower left quadrant (88% control vs 58%
treated). Dying cell proportions are in the lower right and upper
right quadrants (9% in controls vs 36% in treated).
[0033] FIG. 22 depicts synthetic PEN-d/n-ATF5 promoting apoptotic
death of primary GS9-6 human glioblastoma stem cells growing in
culture as spheres. 6 days of treatment and data determined by
Annexin V/PI staining and flow cytometry
[0034] FIG. 23 depicts recombinant PEN-d/n-ATF5 promoting apoptotic
death of primary GS9-6 human glioblastoma stem cells growing in
culture as spheres. 5 days of treatment and data determined by
Annexin V/PI staining and flow cytometry.
4. DETAILED DESCRIPTION OF THE INVENTION
[0035] 4.1 ATF5 & D/N-ATF5 Compositions
[0036] ATF5 is widely expressed by various tumor types. In
particular, ATF5 is expressed not only in highly proliferative
neural tumors, e.g., glioblastomas, but is also expressed in
multiple neoplasias including, but not necessarily limited to:
breast, ovary, endometrium, gastric, colon, liver, pancreas,
kidney, bladder, prostate, testis, skin, esophagus, tongue, mouth,
parotid, larynx, pharynx, lymph node, lung, hematological cancers,
peripheral nervous system, and brain tumors.
[0037] As used herein, "ATF5" includes both an "ATF5 protein" and
an "ATF5 analogue". Unless otherwise indicated, "protein" shall
include a protein, protein domain, polypeptide, or peptide, and any
fragment thereof. The ATF5 protein has the amino acid sequence set
forth in NCBI Accession No. NP 001180575 (human ATF5) or NCBI
Accession No. NP.sub.--109618 (murine ATF5), including conservative
substitutions thereof. As used herein, "conservative substitutions"
are those amino acid substitutions which are functionally
equivalent to a substituted amino acid residue, either because they
have similar polarity or steric arrangement, or because they belong
to the same class as the substituted residue (e.g., hydrophobic,
acidic, or basic). As described below, Western immunoblotting has
permitted the identification of the major cellular form of ATF5
protein. The ATF5 cDNA sequence predicts two potential in-frame
methionine start sites that would lead to proteins of approximately
30 and 20 kDa. Observation that the major form of ATF5 in cells has
an apparent molecular mass of 20-22 kDa indicates favored
utilization of the second site. When a canonical Kozak initiation
consensus sequence was included upstream of the first methionine,
the larger protein was expressed, thereby indicating that the
22-kDa form is not formed by cleavage of a 30-kDa precursor.
Accordingly, the ATF5 protein of the present invention further
includes both the 22-kDa and 30-kDa isomers thereof.
[0038] An "ATF5 analogue", as used herein, is a functional variant
of the ATF5 protein, having ATF5 biological activity, that has 60%
or greater (in certain embodiments, 70% or greater or 80% or
greater or 90% or greater or 95% or greater) amino-acid-sequence
homology with the ATF5 protein. As further used herein, the term
"ATF5 biological activity" refers to the activity of an ATF5
protein or ATF5 analogue to associate physically with, or bind
with, CRE (i.e., binding of approximately two fold, or, more
preferably, approximately five fold, above the background binding
of a negative control), under the conditions of the assays
described herein, although affinity may be different from that of
native ATF5.
[0039] The skilled practitioner understands that the numbering of
amino acid residues in ATF5 may be different than that set forth
herein, or may contain certain conservative amino acid
substitutions that produce the same ATF5-CRE associating activity
as that described herein. Corresponding amino acids and
conservative substitutions in other isoforms or analogues are
easily identified by visually inspecting the relevant amino acid
sequences, or by using commercially available homology software
programs.
[0040] As outlined in the Examples section, interference with the
function and/or activity of ATF5 promote apoptosis of glioblastoma
multiforme tumors (GBM) in vitro and in vivo. Furthermore,
selective interference with ATF5 function and/or activity in other
carcinoma types is shown to triggers cell death. Culture and animal
studies also show that the transcription factor ATF5 is required
for survival of GBM cells and that limited subcutaneous treatment
with a CP-d/n-ATF5 causes apparent tumor eradication in a mouse
model of endogenous gliomas without apparent toxicity or side
effects. As highlighted in the attached examples, the effect of
such ATF5 interference by administration of a CP-d/n-ATF5 is indeed
specific, in that interfering with ATF5 function and/or activity
triggers increased cell death in neoplastic cells, but not normal
cells.
[0041] As used herein, "dominant-negative ATF5" or "d/n-ATF5" is a
peptide comprising a portion of the human ATF5 amino acid sequence.
In certain embodiments, the d/n-ATF5 peptide comprises the sequence
LEQENAELEGECQGLEARNRELKERAES, where the underlined sequence is the
dominant-negative sequence and the remainder of the sequence is the
ATF5 leucine zipper. In certain embodiments the d/n-ATF5 is encoded
by a nucleic acid comprising the sequence
CTGGAACAGGAAAACGCGGAACTGGAAGGCGAATGCCAGGGCCTGG
AAGCGCGCAACCGCGAACTGAAAGAACGCGCGGAAAGCTAA. In certain embodiments
the d/n-ATF5 peptide comprises
TABLE-US-00008 LEKEAEELEQENAELEGECQGLEARNRELKERAES.
[0042] In certain embodiments the d/n-ATF5 is encoded by a nucleic
acid comprising the sequence
CTGGAAAAAGAAGCGGAAGAACTGGAACAGGAAAACGCGGAACTGGAAGG
CGAATGCCAGGGCCTGGAAGCGCGCAACCGCGAACTGAAAGAACGCGCGGA AAGCTAA. In
certain embodiments, the d/n-ATF5 peptide comprises the
sequence
TABLE-US-00009 LARENEELLEKEA EELEQENAELEGECQGLEARNRELKERAES,
where the underlined sequence is the dominant-negative sequence and
the remainder of the sequence is the ATF5 leucine zipper. In
certain embodiments the d/n-ATF5 is encoded by a nucleic acid
comprising the sequence
CTGGCGCGCGAAAACGAAGAACTGCTGGAAAAAGAAGCGGAAGAACTGGAA
CAGGAAAACGCGGAACTGGAAGGCGAATGCCAGGGCCTGGAAGCGCGCAAC
CGCGAACTGAAAGAACGCGCGGAAAGCTAA. In certain embodiments, the
d/n-ATF5 peptide comprises the sequence
TABLE-US-00010 LEQRAEELARENEELLEKEAEELEQENAELEGECQGLEARN
RELKERAES,
the underlined sequence is the dominant-negative sequence and the
remainder of the sequence is the ATF5 leucine zipper. In certain
embodiments the d/n-ATF5 is encoded by a nucleic acid comprising
the sequence CTGGAACAGCGCGCGGA
AGAACTGGCGCGCGAAAACGAAGAACTGCTGGAAAAAGAAGCGGAAGAACT
GGAACAGGAAAACGCGGAACTGGAAGGCGAATGCCAGGGCCTGGAAGCGCG
CAACCGCGAACTGAAAGAACGCGCGGAAAGCTAA. In certain embodiments, the
d/n-ATF5 peptide comprises the sequence
TABLE-US-00011 LEQRAEELARENEELLEKEAEELEQENAELEGECQGLEARNREL
KERAESV,
where the underlined sequence is the dominant-negative sequence and
the remainder of the sequence is the ATF5 leucine zipper. In
certain embodiments the d/n-ATF5 is encoded by a nucleic acid
comprising the sequence CTGGAACAGCGCGCGGAAGAACTGGCGCG
CGAAAACGAAGAACTGCTGGAAAAAGAAGCGGAAGAACTGGAACAGGAAA
ACGCGGAACTGGAAGGCGAATGCCAGGGCCTGGAAGCGCGCAACCGCGAAC
TGAAAGAACGCGCGGAAAGCGTGTAA. In certain embodiments, a d/n-ATF5
comprising the ATF5 leucin zipper sequence LEGECQGLEARNRELKERAESV,
will further comprise, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 additional C-terminal
ATF5 leucine zipper residues. In certain embodiments, the d/n-ATF5
peptide consists of one of the foregoing peptide sequences.
[0043] As used herein, "dominant-negative ATF5" or "d/n-ATF5" is a
peptide comprising a portion of the rat or mouse ATF5 amino acid
sequence. In certain embodiments, the d/n-ATF5 peptide comprises
the sequence LEQENAELEGECQGLEARNRELRERAES, where the underlined
sequence is the dominant-negative sequence and the remainder of the
sequence is the ATF5 leucine zipper. In certain embodiments the
d/n-ATF5 is encoded by a nucleic acid comprising the sequence
CTGGAACAGGAAAACGCGGAACTGGAAGGCGAATGCCAGGG
CCTGGAAGCGCGCAACCGCGAACTGCGCGAACGCGCGGAAAGCTAA. In certain
embodiments, the d/n-ATF5 peptide comprises the sequence
TABLE-US-00012 LEKEAEELEQENAELEGECQ GLEARNRELRERAES,
where the underlined sequence is the dominant-negative sequence and
the remainder of the sequence is the ATF5 leucine zipper. In
certain embodiments the d/n-ATF5 is encoded by a nucleic acid
comprising the sequence CTGGAAAAAGAAGCG
GAAGAACTGGAACAGGAAAACGCGGAACTGGAAGGCGAATGCCAGGGCCTG
GAAGCGCGCAACCGCGAACTGCGCGAACGCGCGGAAAGCTAA. In certain embodiments,
the d/n-ATF5 peptide comprises the sequence
TABLE-US-00013 LARENEELLEKEAEELEQENAELEGECQGL EARNRELRERAES,
where the underlined sequence is the dominant-negative sequence and
the remainder of the sequence is the ATF5 leucine zipper. In
certain embodiments the d/n-ATF5 is encoded by a nucleic acid
comprising the sequence CTGGCGCGCGAAAACGAAGAAC
TGCTGGAAAAAGAAGCGGAAGAACTGGAACAGGAAAACGCGGAACTGGAAG
GCGAATGCCAGGGCCTGGAAGCGCGCAACCGCGAACTGCGCGAACGCGCGG AAAGCTAA. In
certain embodiments, the d/n-ATF5 peptide comprises the
sequence
TABLE-US-00014
LEQRAEELARENEELLEKEAEELEQENAELEGECQGLEARNRELRERAES,
where the underlined sequence is the dominant-negative sequence and
the remainder of the sequence is the ATF5 leucine zipper. In
certain embodiments the d/n-ATF5 is encoded by a nucleic acid
comprising the sequence CTGGAACAGCGCGCGGAAGAACTGGCGCGCGAAAACGAA
GAACTGCTGGAAAAAGAAGCGGAAGAACTGGAACAGGAAAACGCGGAACTG
GAAGGCGAATGCCAGGGCCTGGAAGCGCGCAACCGCGAACTGCGCGAACGC GCGGAAAGCTAA.
In certain embodiments, the d/n-ATF5 peptide comprises the
sequence
TABLE-US-00015
LEQRAEELARENEELLEKEAEELEQENAELEGECQGLEARNRELRERAESV,
where the underlined sequence is the dominant-negative sequence and
the remainder of the sequence is the ATF5 leucine zipper. In
certain embodiments the d/n-ATF5 is encoded by a nucleic acid
comprising the sequence CTGGAACAGCGCGCGGAAGAACTGGCGCGCGAAAACGAAGA
ACTGCTGGAAAAAGAAGCGGAAGAACTGGAACAGGAAAACGCGGAACTGGA
AGGCGAATGCCAGGGCCTGGAAGCGCGCAACCGCGAACTGCGCGAACGCGC GGAAAGCGTGTAA.
In certain embodiments, a d/n-ATF5 comprising the ATF5 leucin
zipper sequence LEGECQGLEARNRELRERAESV, will further comprise, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, or 24 additional C-terminal ATF5 leucine zipper
residues of the sequence EREIQYVKDLLIEVYKARSQRTRS. In certain
embodiments, the d/n-ATF5 peptide consists of one of the foregoing
peptide sequences.
[0044] As used herein, a "cell-penetrating peptide" or "CP" is a
peptide that comprises a short amino acid sequence (e.g., in
certain embodiments, about 12-30 residues) or functional motif that
confers the energy-independent (i.e., non-endocytotic)
translocation properties associated with transport of the
membrane-permeable complex across the plasma and/or nuclear
membranes of a cell. Representative amino acid motifs conferring
such properties are listed in U.S. Pat. No. 6,348,185, the contents
of which are expressly incorporated herein by reference. The
cell-penetrating peptides of the present invention preferably
include, but are not limited to, penetratin1, transportan, pIs1,
TAT(48-60), pVEC, MTS, and MAP.
[0045] The cell-penetrating peptides of the present invention
include those sequences that retain certain structural and
functional features of the identified cell-penetrating peptides,
yet differ from the identified peptides' amino acid sequences at
one or more positions. Such polypeptide variants can be prepared by
substituting, deleting, or adding amino acid residues from the
original sequences via methods known in the art.
[0046] In certain embodiments, such substantially similar sequences
include sequences that incorporate conservative amino acid
substitutions, as described above in connection with polypeptide
apoptotic target inhibitors. In certain embodiments, a
cell-penetrating peptide of the present invention is at least about
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% homologous to the amino acid sequence of the
identified peptide and is capable of mediating cell penetration.
The effect of the amino acid substitutions on the ability of the
synthesized peptide to mediate cell penetration can be tested using
the methods disclosed in Examples section, below.
[0047] In certain embodiments of the present invention, the
cell-penetrating peptide is penetratin1, comprising the peptide
sequence RQIKIWFQNRRMKWKK, or a conservative variant thereof. As
used herein, a "conservative variant" is a peptide having one or
more amino acid substitutions, wherein the substitutions do not
adversely affect the shape--or, therefore, the biological activity
(i.e., transport activity) or membrane toxicity--of the
cell-penetrating peptide.
[0048] Penetratin1 is a 16-amino-acid polypeptide derived from the
third alpha-helix of the homeodomain of Drosophila antennapedia.
Its structure and function have been well studied and
characterized: Derossi et al., Trends Cell Biol., 8(2):84-87, 1998;
Dunican et al., Biopolymers, 60(1):45-60, 2001; Hallbrink et al.,
Biochim. Biophys. Acta, 1515(2):101-09, 2001; Bolton et al., Eur.
J. Neurosci., 12(8):2847-55, 2000; Kilk et al., Bioconjug. Chem.,
12(6):911-16, 2001; Bellet-Amalric et al., Biochim. Biophys. Acta,
1467(1):131-43, 2000; Fischer et al., J. Pept. Res., 55(2): 163-72,
2000; Thoren et al., FEBS Lett., 482(3):265-68, 2000.
[0049] It has been shown that penetratin1 efficiently carries
avidin, a 63-kDa protein, into human Bowes melanoma cells (Kilk et
al., Bioconjug. Chem., 12(6):911-16, 2001). Additionally, it has
been shown that the transportation of penetratin1 and its cargo is
non-endocytotic and energy-independent, and does not depend upon
receptor molecules or transporter molecules. Furthermore, it is
known that penetratin1 is able to cross a pure lipid bilayer
(Thoren et al., FEBS Lett., 482(3):265-68, 2000). This feature
enables penetratin1 to transport its cargo, free from the
limitation of cell-surface-receptor/-transporter availability. The
delivery vector previously has been shown to enter all cell types
(Derossi et al., Trends Cell Biol., 8(2):84-87, 1998), and
effectively to deliver peptides (Troy et al., Proc. Natl. Acad.
Sci. USA, 93:5635-40, 1996) or antisense oligonucleotides (Troy et
al., J. Neurosci., 16:253-61, 1996; Troy et al., J. Neurosci.,
17:1911-18, 1997).
[0050] In certain embodiments, the CP-d/n-ATF5 is a peptide
comprising a Penetratin sequence operably linked to a rat d/n-ATF5
sequence. In certain embodiments the CP-d/n-ATF5 peptide sequence
is
TABLE-US-00016 MGSSHHHHHHSSGLVPRGSHMRQIKIWFQNRRMKWKKDYKDDDDK
MASMTGGQQMGRDPD L EGECQGLEARNRELRERAESV,
where the underlined residues are a 6.times.His-tag leader
sequence, the bold residues are a Penetratin sequence, the
italicized residues are a Flag tag, the residues with no font
modification are spacer amino acids, the bold and italicized
residues are a d/n sequence, and the bold and underlined residues
are an ATF5 leucine zipper truncated after its first Valine. In
certain embodiments the CP-d/n-ATF5 is encoded by a nucleic acid
comprising the sequence
TABLE-US-00017 ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTG
CCGCGCGGCAGCCATATGCGTCAAATTAAAATTTGGTTTCAAAAT
CGTCGTATGAAATGGAAAAAAGACTACAAGGACGATGATGACAAA
ATGGCATCTATGACTGGAGGACAACAAATGGGAAGAGACCCAGAC
CTCGAACAAAGAGCAGAAGAACTAGCAAGAGAAAACGAAGAACTA
CTAGAAAAAGAAGCAGAAGAACTAGAACAAGAAAATGCAGAGCTA
GAGGGCGAGTGCCAAGGGCTAGAGGCGCGGAATCGGGAGCTGAGG
GAGAGGGCAGAGTCAGTGTAG.
[0051] Other non-limiting embodiments of the present invention
involve the use of the following exemplary cell permeant molecules:
RL16 (H-RRLRRLLRRLLRRLRR-OH), a sequence derived from Penetratin1
with slightly different physical properties (Biochim Biophys Acta.
2008 July-August; 1780(7-8):948-59); and RVGRRRRRRRRR, a rabies
virus sequence which targets neurons see P. Kumar, H. Wu, J. L.
McBride, K. E. Jung, M. H. Kim, B. L. Davidson, S. K. Lee, P.
Shankar and N. Manjunath, Transvascular delivery of small
interfering RNA to the central nervous system, Nature 448 (2007),
pp. 39-43.
[0052] In certain alternative non-limiting embodiments of the
present invention, the cell-penetrating peptide is a
cell-penetrating peptides selected from the group consisting of:
transportan, pIS1, Tat(48-60), pVEC, MAP, and MTS. Transportan is a
27-amino-acid long peptide containing 12 functional amino acids
from the amino terminus of the neuropeptide galanin, and the
14-residue sequence of mastoparan in the carboxyl terminus,
connected by a lysine (Pooga et al., FASEB J., 12(1):67-77, 1998).
It comprises the amino acid sequence GWTLNSAGYLLGKINLKALAALAKKIL,
or a conservative variant thereof.
[0053] pIs1 is derived from the third helix of the homeodomain of
the rat insulin 1 gene enhancer protein (Magzoub et al., Biochim.
Biophys. Acta, 1512(1):77-89, 2001; Kilk et al., Bioconjug. Chem.,
12(6):911-16, 2001). pIs1 comprises the amino acid sequence PVIRVW
FQNKRCKDKK, or a conservative variant thereof.
[0054] Tat is a transcription activating factor, of 86-102 amino
acids, that allows translocation across the plasma membrane of an
HIV-infected cell, to transactivate the viral genome (Hallbrink et
al., Biochem. Biophys. Acta., 1515(2):101-09, 2001; Suzuki et al.,
J. Biol. Chem., 277(4):2437-43, 2002; Futaki et al., J. Biol.
Chem., 276(8):5836-40, 2001). A small Tat fragment, extending from
residues 48-60, has been determined to be responsible for nuclear
import (Vives et al., J. Biol. Chem., 272(25):16010-017, 1997); it
comprises the amino acid sequence: YGRKKRRQRRR; GRKKRRQRRRPPQ; or a
conservative variant thereof.
[0055] In certain embodiments, the CP-d/n-ATF5 is a peptide
comprising a TAT sequence operably linked to a rat d/n-ATF5
sequence. In certain embodiments the CP-d/n-ATF5 peptide sequence
is
TABLE-US-00018 MGSSHHHHHHSSGLVPRGSHMLEYGRKKRRQRRRYPYDVPDYAMA
SMTGGQQMGRDPD LEG ECQGLEARNRELRERAESV,
where the underlined residues are a 6.times.His-tag leader
sequence, the bold residues are a TAT sequence, the italicized
residues are an HA tag, the residues with no font modification are
spacer amino acids, the bold and italicized residues are a d/n
sequence, and the bold and underlined residues are an ATF5 leucine
zipper truncated after its first Valine. In certain embodiments the
CP-d/n-ATF5 is encoded by a nucleic acid comprising the
sequence
TABLE-US-00019 ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTG
CCGCGCGGCAGCCATATGCTCGAGTACGGCCGCAAGAAACGCCGC
CAGCGCCGCCGCTATCCATATGACGTCCCAGACTATGCTATGGCA
TCTATGACTGGAGGACAACAAATGGGAAGAGACCCAGACCTCGAA
CAAAGAGCAGAAGAACTAGCAAGAGAAAACGAAGAACTACTAGAA
AAAGAAGCAGAAGAACTAGAACAAGAAAATGCAGAGCTAGAGGGC
GAGTGCCAAGGGCTAGAGGCGCGGAATCGGGAGCTGAGGGAGAGG GCAGAGTCAGTGTAG.
[0056] pVEC is an 18-amino-acid-long peptide derived from the
murine sequence of the cell-adhesion molecule, vascular endothelial
cadherin, extending from amino acid 615-632 (Elmquist et al., Exp.
Cell Res., 269(2):237-44, 2001). pVEC comprises the amino acid
sequence LLIILRRRIRKQAHAH, or a conservative variant thereof.
[0057] MTSs, or membrane translocating sequences, are those
portions of certain peptides which are recognized by the acceptor
proteins that are responsible for directing nascent translation
products into the appropriate cellular organelles for further
processing (Lindgren et al., Trends in Pharmacological Sciences,
21(3):99-103, 2000; Brodsky, J. L., Int. Rev. Cyt., 178:277-328,
1998; Zhao et al., J. Immunol. Methods, 254(1-2):137-45, 2001). An
MTS of particular relevance is MPS peptide, a chimera of the
hydrophobic terminal domain of the viral gp41 protein and the
nuclear localization signal from simian virus 40 large antigen; it
represents one combination of a nuclear localization signal and a
membrane translocation sequence that is internalized independent of
temperature, and functions as a carrier for oligonucleotides
(Lindgren et al., Trends in Pharmacological Sciences, 21(3):99-103,
2000; Morris et al., Nucleic Acids Res., 25:2730-36, 1997). MPS
comprises the amino acid sequence GALFLGWLGAAGSTMGAWSQPKKKRKV, or a
conservative variant thereof.
[0058] Model amphipathic peptides, or MAPs, form a group of
peptides that have, as their essential features, helical
amphipathicity and a length of at least four complete helical turns
(Scheller et al., J. Peptide Science, 5(4):185-94, 1999; Hallbrink
et al., Biochim. Biophys. Acta., 1515(2):101-09, 2001). An
exemplary MAP comprises the amino acid sequence
KLALKLALKALKAALKLA-amide, or a conservative variant thereof.
[0059] In certain embodiments, the cell-penetrating peptides
described above are covalently bound to the d/n-ATF5, e.g., via a
peptide bond. In certain embodiments the cell-penetrating peptide
is operably linked to a d/n-ATF5 via recombinant DNA technology.
For example, the d/n-ATF5 can be introduced either upstream (for
linkage to the amino terminus of the cell-penetrating peptide) or
downstream (for linkage to the carboxy terminus of the
cell-penetrating peptide), or both, of a nucleic acid sequence
encoding the cell-penetrating peptide of interest. Such fusion
sequences comprising both the d/n-ATF5 encoding nucleic acid
sequence and the cell-penetrating peptide encoding nucleic acid
sequence can be expressed using techniques well known in the
art.
[0060] In certain embodiments the d/n-ATF5 can be operably linked
to the cell-penetrating peptide via a non-covalent linkage. In
certain embodiments such non-covalent linkage is mediated by ionic
interactions, hydrophobic interactions, hydrogen bonds, or van der
Waals forces.
[0061] In certain embodiments the d/n-ATF5 is operably linked to
the cell penetrating peptide via a chemical linker. Examples of
such linkages typically incorporate 1-30 nonhydrogen atoms selected
from the group consisting of C, N, O, S and P. Exemplary linkers
include, but are not limited to, a substituted alkyl or a
substituted cycloalkyl. Alternately, the heterologous moiety may be
directly attached (where the linker is a single bond) to the amino
or carboxy terminus of the cell-penetrating peptide. When the
linker is not a single covalent bond, the linker may be any
combination of stable chemical bonds, optionally including, single,
double, triple or aromatic carbon-carbon bonds, as well as
carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen
bonds, sulfur-sulfur bonds, carbon-sulfur bonds, phosphorus-oxygen
bonds, phosphorus-nitrogen bonds, and nitrogen-platinum bonds. In
certain embodiments, the linker incorporates less than 20
nonhydrogen atoms and are composed of any combination of ether,
thioether, urea, thiourea, amine, ester, carboxamide, sulfonamide,
hydrazide bonds and aromatic or heteroaromatic bonds. In certain
embodiments, the linker is a combination of single carbon-carbon
bonds and carboxamide, sulfonamide or thioether bonds.
[0062] A general strategy for conjugation involves preparing the
cell-penetrating peptide and the d/n-ATF5 components separately,
wherein each is modified or derivatized with appropriate reactive
groups to allow for linkage between the two. The modified d/n-ATF5
is then incubated together with a cell-penetrating peptide that is
prepared for linkage, for a sufficient time (and under such
appropriate conditions of temperature, pH, molar ratio, etc.) as to
generate a covalent bond between the cell-penetrating peptide and
the d/n-ATF5.
[0063] The present invention contemplates the use of proteins and
protein analogues generated by synthesis of polypeptides in vitro,
e.g., by chemical means or in vitro translation of mRNA. For
example, ATF5 and inhibitors thereof may be synthesized by methods
commonly known to one skilled in the art (Modern Techniques of
Peptide and Amino Acid Analysis (New York: John Wiley & Sons,
1981); Bodansky, M., Principles of Peptide Synthesis (New York:
Springer-Verlag New York, Inc., 1984). Examples of methods that may
be employed in the synthesis of the amino acid sequences, and
analogues of these sequences, include, but are not limited to,
solid-phase peptide synthesis, solution-method peptide synthesis,
and synthesis using any of the commercially-available peptide
synthesizers. The amino acid sequences of the present invention may
contain coupling agents and protecting groups, which are used in
the synthesis of protein sequences, and which are well known to one
of skill in the art.
[0064] As used herein, "amino acid residue," "amino acid," or
"residue," includes genetically encoded amino acid residues and
non-genetically encoded amino acid residues, e.g., non-genetically
encoded amino acid residues or non-natural amino acids include, but
are not limited to D-eantiomers of naturally occurring chiral amino
acids, .beta.-alanine (.beta.-Ala); 2,3-diaminopropionic acid
(Dpr); nipecotic acid (Nip); pipecolic acid (Pip); ornithine (Orn);
citrulline (Cit); t-butylalanine (t-BuA); 2-t-butylglycine (t-BuG);
N-methylisoleucine (MeIle); phenylglycine (PhG); cyclohexylalanine
(ChA); norleucine (Nle); naphthylalanine (Nal);
4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine
(Phe(2-F)); 3-fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine
(Phe(4-F)); penicillamine (Pen);
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic);
.beta.-2-thienylalanine (Thi); methionine sulfoxide (MSO);
homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric
acid (Dbu); 2,3-diaminobutyric acid (Dab); p-aminophenylalanine
(Phe (pNH2)); N-methyl valine (MeVal); homocysteine (hCys),
homophenylalanine (hPhe); homoserine (hSer); hydroxyproline (Hyp);
homoproline (hPro); and the corresponding D-enantiomer of each of
the foregoing, e.g., D-.beta.-Ala, D-Dpr, D-Nip, D-Orn, D-Cit,
D-t-BuA, D-t-BuG, D-MeIle, D-PhG, D-ChA, D-Nle, D-NaI, D-Phe(4-C1),
D-Phe(2-F), D-Phe(3-F), D-Phe(4-F), D-Pen, D-Tic, D-Thi, D-MSO,
D-hArg, D-AcLys, D-Dbu, D-Dab, D-Phe(pNH2), D-MeVal, D-hCys,
D-hPhe, D-hSer, D-Hyp, and D-hPro. Additional non-genetically
encoded amino acid residues include 3-aminopropionic acid;
4-aminobutyric acid; isonipecotic acid (Inp); aza-pipecolic acid
(azPip); aza-proline (azPro); .alpha.-aminoisobutyric acid (Aib);
.epsilon.-aminohexanoic acid (Aha); .delta.-aminovaleric acid
(Ava); N-methylglycine (MeGly).
[0065] In certain embodiments, the cell-penetrating
dominant-negative ATF5 comprises a sequence selected from the group
consisting of: (1)
TABLE-US-00020 MGSSHHHHHHSSGLVPRGSHMRQIKIWFQNRRMKWKKDYKDDDDKMA
SMTGGQQMGRDPD LEGECQGLEARNRELRERAESV,
where the underlined residues (MG-HM) are a 6.times.His-tag leader
sequence, the bold residues (RQ-KK) are a Penetratin sequence, the
italicized residues (DY-DK) are a Flag tag, the residues with no
font modification (MA-PD) are spacer amino acids, the bold and
italicized residues (LE-AE) are a d/n sequence, and the bold and
underlined residues (LE-SV) are an ATF5 leucine zipper truncated
after its first Valine; (2)
TABLE-US-00021 MGSSHHHHHHSSGLVPRGSHMLEYGRKKRRQRRRYPYDVPDYAMA
SMTGGQQMGRDPD LEGECQGLEARNRELRERAESV,
where the underlined residues (MG-LE) are a 6.times.His-tag leader
sequence, the bold residues (YG-RR) are a TAT sequence, the
italicized residues (YP-YA) are an HA tag, the residues with no
font modification (MA-PD) are spacer amino acids, the bold and
italicized residues (LE-AE) are a d/n sequence, and the bold and
underlined residues (LE-SV) are an ATF5 leucine zipper truncated
after its first Valine; (3)
TABLE-US-00022 MGSSHHHHHHSSGLVPRGSHMRQIKIWFQNRRMKWKKLEQRAEELAR
ENEELLEKEAEELEQENAELEGECQGLEARNRELKERAESV
where the where the underlined residues (MG-HM) are a
6.times.His-tag leader sequence, the bold residues (RQ-KK) are a
Penetratin sequence, the italicized residues (LE-AE) are a d/n
sequence, and the bold and underlined residues (LE-SV) are an ATF5
leucine zipper truncated after its first Valine; and (4)
TABLE-US-00023 RQIKIWFQNRRMKWKKLEQRAEELARENEELLEKEAEELEQENAELE
GECQGLEARNRELKERAESV
where the bold residues (RQ-KK) are a Penetratin sequence, the
italicized residues (LE-AE) are a d/n sequence, and the bold and
underlined residues (LE-SV) are an ATF5 leucine zipper truncated
after its first Valine. In certain embodiments, the
cell-penetrating dominant-negative ATF5 is chemically
synthesized.
[0066] 4.2 Use of D/N-ATF5 Compositions
[0067] In accordance with methods described herein, ATF5 can be
inhibited in a cell by disabling, disrupting, or inactivating the
function or activity of ATF5 in the cell. For example, the function
or activity of ATF5 in a cell may be inhibited by providing a
dominant negative-ATF5 molecule capable of inhibiting the function
or activity of native ATF5 in the cell. In certain embodiments, the
d/n-ATF5 is a CP-d/n-ATF5.
[0068] In certain embodiments, function or activity of the ATF5 in
the cell is inhibited by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%,
or greater (inclusive of intermediate ranges between those
explicitly recited, e.g., 5-10%, 10-20%, 20-30%, 40-50%, or greater
than 50% including 50%-100%). In certain embodiments, function or
activity of the ATF5 is decreased by inhibiting expression of ATF5.
Such expression can be inhibited by at least 1%, 5%, 10%, 20%, 30%,
40%, 50%, or greater (inclusive of intermediate ranges between
those explicitly recited, e.g., 5-10%, 10-20%, 20-30%, 40-50%, or
greater than 50% including 50%-100%). In certain embodiments,
expression is decreased by 60%, 80%, or 90%, as outlined in FIG.
11.
[0069] In certain embodiments, the present invention provides
methods for treating or preventing a tumor in a subject in need of
treatment, comprising administering to the subject a pharmaceutical
composition comprising a CP-d/n-ATF5 and, optionally, a
pharmaceutically-acceptable carrier. The CP-d/n-ATF5 is provided in
an amount that is effective to treat the tumor in a subject to whom
the composition is administered. As used herein, the phrase
"effective" means effective to ameliorate or minimize the clinical
impairment or symptoms of the tumor. For example, the clinical
impairment or symptoms of the tumor may be ameliorated or minimized
by diminishing any pain or discomfort suffered by the subject; by
extending the survival of the subject beyond that which would
otherwise be expected in the absence of such treatment; by
inhibiting or preventing the development or spread of the tumor; or
by limiting, suspending, terminating, or otherwise controlling the
maturation and proliferation of cells in the tumor. The amount of
CP-d/n-ATF5 effective to treat a tumor in a subject in need of
treatment will vary depending upon the particular factors of each
case, including the type of tumor, the stage of the tumor, the
subject's weight, the severity of the subject's condition, and the
method of administration. This amount can be readily determined by
the skilled artisan.
[0070] As used herein, the term "tumor" refers to a pathologic
proliferation of cells, and includes a neoplasia. The term
"neoplasia", and related terms as further used herein, refers to
the uncontrolled and progressive multiplication of tumor cells
under conditions that would not elicit, or would cause cessation
of, multiplication of normal cells. Neoplasia results in the
formation of a "neoplasm", which is defined herein to mean any new
and abnormal growth, particularly a new growth of tissue, in which
the growth of cells is uncontrolled and progressive. As used
herein, neoplasms include, without limitation, morphological
irregularities in cells in tissue of a subject, as well as
pathologic proliferation of cells in tissue of a subject, as
compared with normal proliferation in the same type of tissue.
Additionally, neoplasms include benign tumors and malignant tumors.
Malignant neoplasms are distinguished from benign in that the
former show a greater degree of anaplasia, or loss of
differentiation and orientation of cells, and have the properties
of invasion and metastasis. Thus, neoplasia includes "cancer"
including hematological cancers, which herein refers to a
proliferation of tumor cells having the unique trait of loss of
normal controls, resulting in unregulated growth, lack of
differentiation, local tissue invasion, and metastasis.
[0071] Additionally, as used herein, the term "neural tumor" refers
to a tumorigenic form of neural cells (i.e., transformed neural
cells), and includes astrocytoma cells (i.e., cells of all
astrocytomas, including, without limitation, Grades I-IV
astrocytomas, anaplastic astrocytoma, astroblastoma, astrocytoma
fibrillare, astrocytoma protoplasmaticum, gemistocytic astrocytoma,
and glioblastoma multiforme), gliomas, medulloblastomas,
neuroblastomas, and other brain tumors. Brain tumors invade and
destroy normal tissue, producing such effects as impaired
sensorimotor and cognitive function, increased intracranial
pressure, cerebral edema, and compression of brain tissue, cranial
nerves, and cerebral vessels. Metastases may involve the skull or
any intracranial structure. The size, location, rate of growth, and
histologic grade of malignancy determine the seriousness of brain
tumors. Nonmalignant tumors grow slowly, with few mitoses, no
necrosis, and no vascular proliferation. Malignant tumors grow more
rapidly, and invade other tissues. However, they rarely spread
beyond the CNS, because they cause death by local growth.
[0072] Brain tumors may be classified by site (e.g., brain stem,
cerebellum, cerebrum, cranial nerves, ependyma, meninges,
neuroglia, pineal region, pituitary gland, and skull) or by
histologic type (e.g., meningioma, primary CNS lymphoma, or
astrocytoma). Common primary childhood tumors are cerebellar
astrocytomas and medulloblastomas, ependymomas, gliomas of the
brain stem, neuroblastomas, and congenital tumors. In adults,
primary tumors include meningiomas, schwannomas, and gliomas of the
cerebral hemispheres (particularly the malignant glioblastoma
multiforme and anaplastic astrocytoma, and the more benign
astrocytoma and oligodendroglioma). Overall incidence of
intracranial neoplasms is essentially equal in males and females,
but cerebellar medulloblastoma and glioblastoma multiforme are more
common in males.
[0073] Gliomas are tumors composed of tissue representing neuroglia
in any one of its stages of development. They account for 45% of
intracranial tumors. Gliomas can encompass all of the primary
intrinsic neoplasms of the brain and spinal cord, including
astrocytomas, ependymomas, and neurocytomas. Astrocytomas are
tumors composed of transformed astrocytes, or astrocytic tumor
cells. Such tumors have been classified in order of increasing
malignancy: Grade I consists of fibrillary or protoplasmic
astrocytes; Grade II is an astroblastoma, consisting of cells with
abundant cytoplasm and two or three nuclei; and Grades III and IV
are forms of glioblastoma multiforme, a rapidly growing tumor that
is usually confined to the cerebral hemispheres and composed of a
mixture of astrocytes, spongioblasts, astroblasts, and other
astrocytic tumor cells. Astrocytoma, a primary CNS tumor, is
frequently found in the brain stem, cerebellum, and cerebrum.
Anaplastic astrocytoma and glioblastoma multiforme are commonly
located in the cerebrum. The present invention additionally
provides methods for promoting apoptosis in a neoplastic cell
comprising contacting the neoplastic cell with an ATF5 inhibitor.
The neoplastic cell can be selected from the group consisting of:
breast, ovary, endometrium, gastric, colon, liver, pancreas,
kidney, bladder, prostate, testis, skin, esophagus, tongue, mouth,
parotid, larynx, pharynx, lymph node, lung, and brain. In one
embodiment, the neoplastic cell is selected from the group
consisting of glioblastoma, astrocytoma, glioma, medulloblastoma
and neuroblastoma.
[0074] For example, but not by way of limitation, cell lines shown
through testing to be susceptible to TAT-d/n-ATF5 (1-3 .mu.M range)
include: U87 (human glioblastoma); U373 (human glioblastoma); LN229
(human glioblastoma); C6 (rat glioblastoma); Me1501 (human
melanoma); H2452 (human mesothelioma); MDA-MB-468 (human breast
cancer). In addition, a non-limiting list of cell lines shown
through testing to be susceptible to PEN-d/n-ATF5 (3 .mu.M)
include: Panc-1 (human pancreatic cancer); SH-SY5Y (human
neuroblastoma cells); and HCT-116 (colon-carcinoma cancer). The
method of the present invention can be performed in vitro as well
as in vivo in a subject. As used herein, "apoptosis" refers to cell
death which is wholly or partially genetically controlled.
[0075] As outlined in the examples below, certain CP-d/n-ATF5
compositions are effective anti-neoplatic agents across species,
e.g., rat/mouse CP-d/n-ATF5 is effective against human cancers.
Thus, in certain embodiments the CP-d/n-ATF5 can comprise a rat or
mouse d/n-ATF5 peptide sequence and the subject may be any animal,
including, but not limited to a mammal (e.g., a human, domestic
animal, or commercial animal). In certain embodiments, the
CP-d/n-ATF5 can comprise a rat or mouse d/n-ATF5 peptide sequence
and the subject is a human.
[0076] In accordance with the method of the present invention,
CP-d/n-ATF5 can be administered to a human or animal subject by
known procedures, including, without limitation, oral
administration, parenteral administration, intranasal
administration and transdermal administration. Preferably, the
inhibitors or factors are administered parenterally, by
intracranial, intraspinal, intrathecal, or subcutaneous
injection.
[0077] 4.3 D/N-ATF5 Pharmaceutical Compositions
[0078] For oral administration, CP-d/n-ATF5 can be formulated as
capsules, tablets, powders, granules, or as a suspension. The
CP-d/n-ATF5 formulation may have conventional additives, such as
lactose, mannitol, corn starch, or potato starch. The CP-d/n-ATF5
formulation also may be presented with binders, such as crystalline
cellulose, cellulose derivatives, acacia, corn starch, or gelatins.
Additionally, the CP-d/n-ATF5 formulation may be presented with
disintegrators, such as corn starch, potato starch, or sodium
carboxymethylcellulose. The CP-d/n-ATF5 formulation also may be
presented with dibasic calcium phosphate anhydrous or sodium starch
glycolate. Finally, the CP-d/n-ATF5 formulation may be presented
with lubricants, such as talc or magnesium stearate.
[0079] For parenteral administration (i.e., administration by
injection through a route other than the alimentary canal),
CP-d/n-ATF5 can be combined with a sterile aqueous solution that is
preferably isotonic with the blood of the subject. Such a
CP-d/n-ATF5 formulation can be prepared by dissolving a solid
active ingredient in water containing physiologically-compatible
substances, such as sodium chloride, glycine, and the like, and
having a buffered pH compatible with physiological conditions, so
as to produce an aqueous solution, then rendering said solution
sterile. The CP-d/n-ATF5 formulation can be presented in unit or
multi-dose containers, such as sealed ampoules or vials. The
CP-d/n-ATF5 formulation can be delivered by any mode of injection,
including, without limitation, epifascial, intracapsular,
intracranial, intracutaneous, intrathecal, intramuscular,
intraorbital, intraperitoneal, intraspinal, intrasternal,
intravascular, intravenous, parenchymatous, subcutaneous, or
sublingual.
[0080] In certain embodiments, the CP-d/n-ATF5 formulation is
prepared for intranasal delivery. For nasal administration,
solutions or suspensions comprising the CP-d/n-ATF5 formulation can
be prepared for direct application to the nasal cavity by
conventional means, for example with a dropper, pipette or spray.
Other means for delivering the nasal spray composition, such as
inhalation via a metered dose inhaler (MDI), may also be used
according to the present invention. Several types of MDIs are
regularly used for administration by inhalation. These types of
devices can include breath-actuated MDI, dry powder inhaler (DPI),
spacer/holding chambers in combination with MDI, and nebulizers.
The term "MDI" as used herein refers to an inhalation delivery
system comprising, for example, a canister containing an active
agent dissolved or suspended in a propellant optionally with one or
more excipients, a metered dose valve, an actuator, and a
mouthpiece. The canister is usually filled with a solution or
suspension of an active agent, such as the nasal spray composition,
and a propellant, such as one or more hydrofluoroalkanes. When the
actuator is depressed a metered dose of the solution is aerosolized
for inhalation. Particles comprising the active agent are propelled
toward the mouthpiece where they may then be inhaled by a subject.
The formulations may be provided in single or multidose form. For
example, in the case of a dropper or pipette, this may be achieved
by the patient administering an appropriate, predetermined volume
of the solution or suspension. In the case of a spray, this may be
achieved for example by means of a metering atomising spray pump.
To improve nasal delivery and retention the components according to
the invention may be encapsulated with cyclodextrins, or formulated
with agents expected to enhance delivery and retention in the nasal
mucosa.
[0081] Commercially available administration devices that are used
or can be adapted for nasal administration of a composition of the
invention include the AERONEB.TM. (Aerogen, San Francisco, Calif.),
AERONEB GO.TM. (Aerogen); PARI LC PLUS.TM., PARI BOY.TM. N,
PARI.TM. eflow (a nebulizer disclosed in U.S. Pat. No. 6,962,151),
PARI LC SINUS.TM., PARI SINUSTAR.TM., PARI SINUNEB.TM., VibrENT.TM.
and PARI DURANEB.TM. (PARI Respiratory Equipment, Inc., Monterey,
Calif. or Munich, Germany); MICROAIR.TM. (Omron Healthcare, Inc,
Vernon Hills, Ill.), HALOLITE.TM. (Profile Therapeutics Inc,
Boston, Mass.), RESPIMAT.TM. (Boehringer Ingelheim, Germany),
AERODOSE.TM. (Aerogen, Inc, Mountain View, Calif.), OMRON ELITE.TM.
(Omron Healthcare, Inc, Vernon Hills, Ill.), OMRON MICROAIR.TM.
(Omron Healthcare, Inc, Vernon Hills, Ill.), MABISMIST.TM. II
(Mabis Healthcare, Inc, Lake Forest, Ill.), LUMISCOPE.TM. 6610,
(The Lumiscope Company, Inc, East Brunswick, N.J.), AIRSEP
MYSTIQUE.TM., (AirSep Corporation, Buffalo, N.Y.), ACORN-1.TM. and
ACORN-II.TM. (Vital Signs, Inc, Totowa, N.J.), AQUATOWER.TM.
(Medical Industries America, Adel, Iowa), AVA-NEB.TM. (Hudson
Respiratory Care Incorporated, Temecula, Calif.), AEROCURRENT.TM.
utilizing the AEROCELL.TM. disposable cartridge (AerovectRx
Corporation, Atlanta, Ga.), CIRRUS.TM. (Intersurgical Incorporated,
Liverpool, N.Y.), DART.TM. (Professional Medical Products,
Greenwood, S.C.), DEVILBISS.TM. PULMO AIDE (DeVilbiss Corp;
Somerset, Pa.), DOWNDRAFT.TM. (Marquest, Englewood, Colo.), FAN
JET.TM. (Marquest, Englewood, Colo.), MB-5.TM. (Mefar, Bovezzo,
Italy), MISTY NEB.TM. (Baxter, Valencia, Calif.), SALTER 8900.TM.
(Salter Labs, Arvin, Calif.), SIDESTREAM.TM. (Medic-Aid, Sussex,
UK), UPDRAFT-II.TM. (Hudson Respiratory Care; Temecula, Calif.),
WHISPER JET.TM. (Marquest Medical Products, Englewood, Colo.),
AIOLOS.TM. (Aiolos Medicnnsk Teknik, Karlstad, Sweden),
INSPIRON.TM. (Intertech Resources, Inc., Bannockburn, Ill.),
OPTIMIST.TM. (Unomedical Inc., McAllen, Tex.), PRODOMO.TM.,
SPIRA.TM. (Respiratory Care Center, Hameenlinna, Finland), AERx.TM.
Essence.TM. and Ultra.TM., (Aradigm Corporation, Hayward, Calif.),
SONIK.TM. LDI Nebulizer (Evit Labs, Sacramento, Calif.),
ACCUSPRAY.TM. (BD Medical, Franklin Lake, N.J.), ViaNase ID.TM.
(electronic atomizer; Kurve, Bothell, Wash.), OptiMist.TM. device
or OPTINOSE.TM. (Oslo, Norway), MAD Nasal.TM. (Wolfe Tory Medical,
Inc., Salt Lake City, Utah), Freepod.TM. (Valois, Marly le Roi,
France), Dolphin.TM. (Valois), Monopowder.TM. (Valois), Equadel.TM.
(Valois), VP3.TM. and VP7.TM. (Valois), VP6 Pump.TM. (Valois),
Standard Systems Pumps.TM. (Ing. Erich Pfeiffer, Radolfzell,
Germany), AmPump.TM. (Ing. Erich Pfeiffer), Counting Pump.TM. (Ing.
Erich Pfeiffer), Advanced Preservative Free System.TM. (Ing. Erich
Pfeiffer), Unit Dose System.TM. (Ing. Erich Pfeiffer), Bidose
System.TM. (Ing. Erich Pfeiffer), Bidose Powder System.TM. (Ing.
Erich Pfeiffer), Sinus Science.TM. (Aerosol Science Laboratories,
Inc., Camarillo, Calif.), ChiSys.TM. (Archimedes, Reading, UK),
Fit-Lizer.TM. (Bioactis, Ltd, a SNBL subsidiary (Tokyo, J P),
Swordfish V.TM. (Mystic Pharmaceuticals, Austin, Tex.),
DirectHaler.TM. Nasal (DirectHaler, Copenhagen, Denmark) and
SWIRLER.TM. Radioaerosol System (AMICI, Inc., Spring City,
Pa.).
[0082] For transdermal administration, CP-d/n-ATF5 can be combined
with skin penetration enhancers, such as propylene glycol,
polyethylene glycol, isopropanol, ethanol, oleic acid,
N-methylpyrrolidone, and the like, which increase the permeability
of the skin to the CP-d/n-ATF5, and permit the CP-d/n-ATF5 to
penetrate through the skin and into the bloodstream. The
CP-d/n-ATF5 compositions also may be further combined with a
polymeric substance, such as ethylcellulose, hydroxypropyl
cellulose, ethylene/vinylacetate, polyvinyl pyrrolidone, and the
like, to provide the composition in gel form, which may be
dissolved in solvent, such as methylene chloride, evaporated to the
desired viscosity, and then applied to backing material to provide
a patch.
[0083] The present invention also provides therapeutic
compositions, comprising a CP-d/n-ATF5 and, optionally, a
pharmaceutically-acceptable carrier. The
pharmaceutically-acceptable carrier must be "acceptable" in the
sense of being compatible with the other ingredients of the
composition, and not deleterious to the recipient thereof. The
pharmaceutically-acceptable carrier employed herein is selected
from various organic or inorganic materials that are used as
materials for pharmaceutical formulations, and which may be
incorporated as analgesic agents, buffers, binders, disintegrants,
diluents, emulsifiers, excipients, extenders, glidants,
solubilizers, stabilizers, suspending agents, tonicity agents,
vehicles, and viscosity-increasing agents. If necessary,
pharmaceutical additives, such as antioxidants, aromatics,
colorants, flavor-improving agents, preservatives, and sweeteners,
may also be added. Examples of acceptable pharmaceutical carriers
include carboxymethyl cellulose, crystalline cellulose, glycerin,
gum arabic, lactose, magnesium stearate, methyl cellulose, powders,
saline, sodium alginate, sucrose, starch, talc, and water, among
others.
[0084] The CP-d/n-ATF5 formulations of the present invention can be
prepared by methods well-known in the pharmaceutical arts. For
example, the CP-d/n-ATF5 can be brought into association with a
carrier or diluent, as a suspension or solution. Optionally, one or
more accessory ingredients (e.g., buffers, flavoring agents,
surface active agents, and the like) also can be added. The choice
of carrier will depend upon the route of administration. The
pharmaceutical composition would be useful for administering the
CP-d/n-ATF5 to a subject to treat a tumor and/or neoplastic cell,
as discussed herein. The CP-d/n-ATF5 is provided in an amount that
is effective to treat the tumor and/or neoplastic cell in a subject
to whom the pharmaceutical composition is administered. That amount
may be readily determined by the skilled artisan, as described
above.
[0085] Compositions of the present disclosure can further include
other therapeutic agents. For example, they can include any one or
more anti-cancer agents. In certain embodiments, the one or more
anti-cancer agent will be selected from the group consisting of:
alkylating agents; anti-metabolites; anti-microtubule agents;
topoisomerase inhibitors, antibiotics, and antibodies/antibody-drug
conjugates. The amounts of those anti-cancer agents in compositions
of the present disclosure can, in certain embodiments, be reduced
as compared to normal doses of such agents administered in a
similar fashion.
[0086] The present invention also provides kits for use in treating
and/or preventing tumors and/or neoplastic cells. In certain
embodiments the kits comprise a CP-d/n-ATF5 molecule and a
pharmaceutically acceptable carrier. In certain embodiments the
kits further comprise a means for administration, for example, but
not limited to, a pre-filled syringe, pen, pump, or other
pre-filled device for parenteral administration of the CP-d/n-ATF5.
In certain embodiments, the kits will comprise a CP-d/n-ATF5
formulated for intranasal delivery and a means for intranasal
administration, such as, but not limited to, a metered dose inhaler
or other commercially available administration device which can be
used or can be adapted for nasal administration of a composition as
described herein.
[0087] The present invention is described in the following
Examples, which are set forth to aid in the understanding of the
invention, and should not be construed to limit in any way the
scope of the invention as defined in the claims which follow
thereafter.
5. EXAMPLES
5.1 CP-d/n-ATFS Example
[0088] 5.1.1 Materials & Methods
[0089] Truncation of d/n ATF-5. Using pQC eGFP-d/n-ATF5 plasmid
(see, Angelastro et al., J Neurosci 2003; 23(11):4590-600) as
template, PCR using upstream primer 5'-TCC GCG GCC GCA CCG GTC
GCC-3' and downstream primer 5'-CTC GAG GAT ATC TCA GTT ATC TAC ACT
GAC TCT GCC CTC TCC CTC AG-3' truncated 75 base pairs from the 3'
plasmid. Electrophoretically purified eGFP-d/n ATF5-tr
(tr=truncated) cDNA was ligated into pGEM-T Easy Cloning vector
(Promega), transformed into DH5.alpha. cells, and plated onto LB
agar-Ampicillin plates with blue-white selection. Selected colonies
were amplified overnight in LB plus ampicillin. Plasmids isolated
from the culture (mini-prep, Invitrogen) were digested with AgeI
and EcoRV followed by agarose gel electrophoresis to verify
d/n-ATF5-tr insertion and inserts underwent DNA sequencing for
verification. Agel/EcoRV digested eGFP-d/n-ATF5-tr cDNA was ligated
into AgeI/EcoRV-digested purified pQCXIX (Clontech) expression
vector. The ligation mixture was used to transform DH5a bacteria
and the product was verified by AgeI/EcoRV digestion and gel
electrophoresis from minipreps of bacterial cultures and DNA
sequencing of uncut plasmid. The pQC-eGFP-d/n-ATF5-tr plasmid was
grown in Maxiprep (Invitrogen).
[0090] CP-6.times.His-Pen-Flag-tagged-d/n-ATF5 protein production
and bioassay. To create
Cell-Penetrating-6.times.His-Penetratin-Flag-tagged-d/n-ATF5-tr
(CP-6.times.His-Pen-Flag-tagged-d/n-ATF5-tr) cDNA, PCR was first
employed using upstream primer 5'-TTA ATT AAG CCG CCA TGG ATG CGT
CAA ATT AAA ATT TGG TTT CAA AAT CGT CGT ATG AAA TGG AAA AAA ATG GAC
TAC AAG GAC GAT GAT-3' and downstream primer 5'-CTC GAG GGA TCC TCA
GTT ATC TAC ACT GAC TCT GCC CTC TCC CTC AG-3' and
pQC-Flag-d/n-ATF5-tr as template. The product was purified after
gel electrophoresis and ligated into pGEM-T Easy cloning vector.
This was transformed into DH5a cells and for white colony
selection. Miniprep clones were digested with EcoRV followed by gel
electrophoresis and sequencing of uncut plasmid to verify the
insert. To insert a 6.times.His tag at the N-terminus,
Pen-d/n-ATF5-RP-tr was cloned into the pET-15b expression vector
(Novagen). Both pET-15b and pGEMT-Pen-d/n-ATF5-RP-tr vectors were
digested with Nde-1 and BamH1 and the cut Pen-d/n-ATF5-RP-tr was
separated from pGEMT by gel electrophoresis. Likewise, cut pET-15b
was separated from the insert by gel electrophoresis. Both pET-15b
and Pen-d/n-ATF5-RP-tr were excised from the gel and purified and
then ligated using T4 DNA ligase. The ligated material was used to
transform DH5a cells and colonies selected. Mini-prepped constructs
were digested with Xba-1 and EcoRV to verify the presence of vector
and Pen-d/n-ATF5-RP insert using gel electrophoresis. The
pET-15b-Pen-d/n-ATF5-RP was verified by DNA sequencing for correct
orientation and sequence.
[0091] To generate CP-6.times.His-Pen-Flag-tagged-d/n-ATF5 protein,
the expression construct was transformed into BL21 DE3 pLysS cells
(Novagen). Colonies were selected and amplified in LB. Peptide
production was induced with 1 mM IPTG and verified by SDS-PAGE.
Once protein induction was verified, extractions were accomplished
with detergent-based BugBuster master mix system (Novagen).
Isolation and purification of the Pen-Flag-tagged-d/n-ATF5 peptide
was accomplished using its N-terminal 6.times.HIS-tag and cobalt
spin column system (HisPur; Thermo Fisher). Purified peptide was
desalted and buffer-exchanged to PBS using Zeba de-salt spin
columns (Thermo Fisher) or G-25 Sephadex (GE Health Care). Desalted
protein was sterile-filtered using 0.20 .mu.m polyethersulfone
membrane syringe filters (Sarstedt). Lastly, the peptide was
concentrated to 1-2 mg/ml using Amicon Ultra-4 centrifugal filter
devices (3000 MWCutoff).
[0092] A control peptide (Pen-Flag-tagged-Control) was created and
produced using the same methodology by eliminating the d/n-ATF5
portion of the construct using PCR upstream primer
5'-CCCGGGCATATGCGTCAAATTAAAATTTGGTTT-3' and downstream primer
5'-CTCGAGGGATCCTCAGTTATCTAGTCTGGGTCTCTTCC-3'.
[0093] Mass Spectroscopy. Linear MALDI-TOF analysis for nominal
molecular mass measurement: Matrix-assisted laser
desorption/ionization (MALDI) measurements were acquired on a
MALDI-TOF/TOF mass spectrometer (4700 Proteomics Analyzer, AB
Sciex) equipped with a 200 Hz ND-YAG laser source (355 nm). Samples
were spotted onto the MALDI plate with an equivolume of MALDI
matrix (sinapinic acid in 50% ACN/0.1% FA, Fluka) and air dried.
The instrument was operated at an accelerating voltage of 20 kV.
Spectra were taken from signal averaging of 4,000 laser shots. Mass
Spectra analyses were performed in positive ion linear mode with a
mass range of 10,000-60,000 m/z. Data were further analyzed by Data
Explorer 4.5 (AB Sciex).
[0094] LC-MS analysis: Samples were injected onto an Aeris Widepore
XB-C8 column (3.6.mu., 2.10.times.50 mm). A standard reverse phase
gradient was run over 8 minutes at flow rate of 250 .mu.l/min and
the eluent monitored by a LTQ-OrbitrapXL mass spectrometer (Thermo
Fisher) in profile mode. Ion Max Source (Thermo Fisher) was used as
the electrospray ionization source and source parameters were 5 kV
spray voltage, capillary temperature of 275.degree. C. and sheath
gas setting of 20. Spectral data were acquired at a resolution
setting of 15,000 FWHM with the lockmass feature.
[0095] Bioactivity of the pQC-eGFP-d/n ATF-5tr product
(C-terminally truncated d/n-ATF5). Purified pQC-eGFP-d/n-ATF5-tr,
full-length pQC-eGFP-d/n-ATF 5 positive control or pQC-eGFP
negative control plasmids were transfected into rat C6 glial cells
in 24 well plates using Lipofectamine 2000 (Invitrogen). After 48
hours, cells were stained with DAPI and 10 random fields were
viewed under fluorescent microscopy at 40.times.. Cells displaying
fragmented, condensed chromatin were scored as apoptotic and
quantified relative to total cells (n=3 independent
experiments).
[0096] Cell penetrating (CP)-6.times.His-Pen-Flag-tagged-d/n-ATF5
bioassay. For peptide bioassays, rat C6 glioblastoma cells were
maintained in serum-free DMEM for 2 hours, and then in DMEM/0.5%
FBS without or with 3 .mu.M Penetratin (Pen)-d/n-ATF5-RP peptide or
(Penetratin) Pen-control-RP. After 5 days, cells were stained with
DAPI and percent of apoptotic cells determined as described
above.
[0097] Imaging of internalized Pen-d/n-ATF5-RP (Recombinant
Protein). Rat C6 cells (from Jeff Bruce; Columbia University, New
York; authenticated 2004 by grafting into Rat brain Angelastro et
al., Oncogene 2006; 25(6):907-16) and U87 cells (purchased and
authenticated from the ATCC) were plated on fibronectin-coated
confocal microscopy coverslips and maintained overnight. 3 .mu.M
each of Pen-d/n-ATF5-RP or Pen-Control-RP were added to wells and
incubated for 1, 2, 4, or 24 hours. Cells were washed 3.times. with
PBS to remove extracellular peptide and stained with primary mouse
anti-FLAG antibody (Sigma-Aldrich) overnight followed by incubation
for two hours with secondary anti-mouse Alexa-568 (Invitrogen).
Microscopy used a Carl Zeiss Axiovert 200 with Axiocam video
capture or Delta Vision Deconvolution microscope at 0.1-.mu.m
optical sections enhanced by Huygens Deconvolution Software. Images
of xy and yz planes confirmed co-localization of Pen-d/n-ATF5-RP
and DAPI staining.
[0098] Retrovirus-induced mouse glioblastoma model and treatment
with Pen-d/n-ATF5-RP. As described previously (Arias et al.,
Oncogene 2012; 31(6):739-51), adult mice were anesthetized and
underwent stereotaxic injection of retrovirus expressing PDGF-B and
p53-shRNA to generate malignant gliomas. Analgesics were given
immediately after surgery. Injected mice were monitored
post-surgically and throughout the study period, which ranged from
52 to 438 days. Pen-d/n-ATF5-RP or Pen-Control-RP was administered
to tumor-bearing animals in treatments of four subcutaneous or
intraperitoneal injections, spaced 1-2 hours apart. The doses were
1 mg/kg (200 .mu.l, 0.9% saline) for each injection. In some
experiments as indicated, dosing was repeated 5 days later. Animals
injected with 0.9% saline at the same dosing schedule and volume
served as controls.
[0099] Brain sectioning and staining. As previously described,
(Arias et al., Oncogene 2012; 31(6):739-51), mice were euthanized
by deep isoflurane anesthesia followed by trans-cardial perfusion
with 10% formalin. Brains were fixed in 4% paraformaldehyde,
incubated overnight in 30% sucrose and were mounted in OCT medium,
frozen and cut into 14-.mu.m coronal sections. In other cases as
indicated, brains of perfused mice were incubated in 10%
formalin/PBS for 4-7 days and then paraffin-embedded. Paraffin
sections were subjected to antigen retrieval as described (Schrot
et al., J Neurooncol 2007; 85(2):149-57). Sections were stained
with DAPI and the following: Anti-Flag M2 (1:200; Sigma-Aldrich),
rabbit anti-Flag (1: 1000, Cell Signaling), rabbit anti-HA (4
.mu.g/ml; sc-805 Santa Cruz Biotechnology), or TUNEL (Roche) and
Anti-Flag M2. Sections were visualized with a DAPI filter and
immunofluorescence (Alexa 488/568; Invitrogen) or colorimetrically
with diaminobenzidine or fast red (Mach2; Biocare Medical) and
photographed on a Carl Zeiss Axiovert 200 with Axiocam video.
[0100] MRI analysis. Anesthetized (isoflurane and oxygen) mice were
fitted intravenously with a 30 gauge catheter, and positioned head
first, prone on the scanner bed. MRI acquisitions were performed on
a Bruker Biospec 7 Tesla magnet operating Paravision v5.1 and
outfitted with a 116-mm diameter gradient with integrated shim
control. Maximum gradient strength was 450 mT/m. A cross coil
configuration was used for imaging brains and a 72-mm ID linear
coil was used for RF transmission and a 4 channel phased array coil
for RF reception. Pre-contrast and 1 minute post contrast images
were acquired with FLASH 3Dslab. Gadolinium was injected
intravenously at a dose of 1 .mu.l/g body weight.
[0101] 5.1.2 Results
[0102] Generation of a Cell-Penetrating Form of d/n-ATF5.
[0103] A modified cell-penetrating form of d/n-ATF5 that could be
delivered systemically was prepared as outlined herein. This has a
potential advantage of rapid biodistribution, reduced immune
response, passage through the blood brain barrier, entry into
cells, and the capacity reaching widely dispersed tumor cells. The
original d/n-ATF5 is an N-terminally truncated form of ATF5 that
includes the wild-type leucine zipper domain with an amphipathic
.alpha.-helical sequence with leucine repeats at every seventh
residue replacing the DNA binding domain [Angelastro et al., J
Neurosci 2003; 23(10:4590-600]. The resulting protein is capable of
interacting with ATF5 and its binding partners via the enhanced
leucine zipper region, but not with DNA, and consequently acts as
an effective d/n suppressor of ATF5 actions [Angelastro et al., J
Neurosci 2003; 23(11):4590-600; Vinson et al., Genes Dev 1993;
7(6):1047-58]. Deletion of the N-terminal domain substantially
stabilizes d/n-ATF5 against degradation [Lee et al., Developmental
Neurobiology 2012; 72(6):789-804; Uekusa et al., Biochem Biophys
Res Commun 2009; 380(3):673-8]. To design a deliverable form of
d/n-ATF5, the last 25 amino acids of the protein were first
truncated, which includes the C-terminal two valine/valine heptad
repeats. Transfection of this deleted construct into C6
glioblastoma cells showed equal effectiveness as the full length
d/n-ATF5 in promoting apoptosis (FIG. 1).
[0104] To generate a cell-penetrating form of the C-terminally
truncated d/n-ATF5 (d/n-ATF5-tr), an N-terminally Flag-tagged
d/n-ATF5-tr construct N-terminally fused to a 6.times. histidine
repeat followed by a penetratin sequence was designed (FIG. 2A).
Penetratin sequence is a 16-amino acid motif from the Antennapedia
homeodomain protein permitting passage of fused cargos through
biological membranes into cells [Dupont et al., Methods in
molecular biology 2011; 683:21-9.]. Milligram quantities of the
protein (designated Pen-d/n-ATF5-Recombinant Protein (RP)) were
generated by expression in bacteria followed by purification by
cobalt resin affinity chromatography using the 6.times.His
sequence. SDS-PAGE showed the purified preparations were more than
95% homogeneous with minor species including what appeared to be
aggregated protein multimers. Calculated Mr of Pen-d/n-ATF5-RP with
normal bacterial removal of the N-formylmethionine is 12,949.18 Da,
but the major purified product shows an apparent molecular mass
between 25-28 KDa by SDS-PAGE (FIG. 2A). Wild type ATF5 and the
ATF5 leucine zipper can migrate anomalously when subjected to
SDS-PAGE and so high resolution LC-HRMS was employed to verify the
correct molecular weight of Pen-d/n-ATF5-RP as well as its solution
state. The deconvoluted spectra revealed the most abundant form to
be the predicted 12,948.7 Da monomer, with a low amount of dimer at
25,897.5 Da (FIG. 2B). Prior studies have also shown that
recombinant wild type full-length ATF5 or the bzip domain of ATF5
can form dimers in vitro. Finally, as a control for
Pen-d/n-ATF5-RP, a peptide (Pen-Control-RP) was generated by
similar means that lacks the d/n-ATF5-tr sequence (FIG. 2A). The
purified recombinant control (with a calculated molecular mass of
7,099.98 Da) migrated at an apparent MW of 7,100 Da by SDS-PAGE
(FIG. 2A).
[0105] Because Pen-d/n-ATF5-RP is designed for systemic
administration, stability in presence of human serum at 37.degree.
C. was shown with no significant degradation at 8 h and a mean loss
of 28% of full-length protein by 48 h (FIG. 2C).
[0106] Pen-d/n-ATF5-RP Rapidly Enters and Causes Apoptosis of
Cultured Glioblastoma Cells.
[0107] Before carrying out animal experiments, the ability of
Pen-d/n-ATF5-RP to enter and kill glioblastoma cells in culture was
verified. When added to serum-containing cultures of rat C6 and
human U87 glioblastoma cells, both Pen-control-RP and
Pen-d/n-ATF5-RP were readily detectable in the cells within 2-4 h
and remained detectable for at least 24 h (FIG. 3A,B). Confocal
microscopy revealed that the peptides were present in both the
cytoplasmic and nuclear compartments (FIG. 3A).
[0108] C6 cultures exposed to Pen-control-RP and Pen-d/n-ATF5-RP
were also assessed for apoptotic cell death. The Pen-Control-RP
treated cultures showed background levels of apoptotic death
similar to that in non-treated cultures, whereas cultures treated
with Pen-d/n-ATF5-RP showed greatly increased numbers of dying
cells (FIG. 4). These actions are similar to what has been
previously reported for multiple rodent and human glioblastoma
cells transfected with d/n-ATF5 constructs or exposed to ATF5 siRNA
[Angelastro et al., Oncogene 2006; 25(6):907-16; Arias et al.,
Oncogene 2012; 31(6):739-51; Sheng et al., Nat Med 2010;
16(6):671-7; Dluzen et al., The Journal of biological chemistry
2011; 286(9):7705-13].
[0109] Systemically-Delivered Pen-d/n-ATF5-RP Crosses the Blood
Brain Barrier, Enters Cells and Selectively Triggers Rapid,
Selective Apoptotic Death of Glioma Cells.
[0110] To test the capacity of Pen-d/n-ATF5-RP to reach and treat
primary brain tumors, a model in which gliomas are generated by
stereotactic injection of PDGF-B-HA/shRNA-p53 retrovirus into the
adult mouse brain was used. The tumors are presumably derived from
endogenous dividing progenitor cells and closely resemble
infiltrative human gliomas ranging from stages II-IV. The tumors
were detectable as early as 52 days post-injection by MRI (see
below) and were histologically identifiable by the presence of the
HA tag as well as by high cellularity, hyperchromatic nuclei, and
elevated Ki67 staining.
[0111] In an initial set of experiments, Pen-d/n-ATF5-RP, saline or
Pen-Control-RP was delivered intraperitoneally to tumor-bearing
mice in a single set of four injections each of 1 mg/kg at
intervals of 1-2 h. The mice were sacrificed 16-64 hours after the
last injection and the fixed brains were stained with anti-Flag
antibody to detect Pen-d/n-ATF5-RP or with anti-HA to mark
PDGF-B-HA expressing tumor cells, and for TUNEL to identify dying
cells. At 16 h, both tumor and normal brain cells (in the
contralateral hemisphere from the tumor) showed Flag staining
indicative of extensive uptake of Pen-d/n-ATF5-RP; there was no
signal with saline injection (FIG. 5A-C). Flag staining was still
evident at 40 h after treatment and was detectable, though at
reduced levels at 64 h (FIG. 6). While normal brain tissue showed
no TUNEL staining (FIG. 5B), there was extensive TUNEL staining
within the tumors one day after treatment with Pen-d/n-ATF5-RP
(FIG. 5A). Little or no TUNEL signal was observed in tumors of
animals treated with saline (FIG. 5C). Co-localized TUNEL and
PDGF-B-HA+ tumor marker staining continued to be evident at 64 h
after Pen-d/n-ATF5-RP treatment, but the signals indicated cell
degeneration and fragmentation (FIG. 5E) compared with cells
treated with this peptide for 16 h (FIG. 5A) or with Pen-Control-RP
peptide (FIG. 5D).
[0112] To enhance the potential long-term therapeutic efficacy of
Pen-d/n-ATF5-RP administration, a treatment protocol was devised in
which tumor-bearing animals received two sets of subcutaneous
injections, 5 days apart, each as described above. Tumors of mice
assessed two days after the second treatment (7 days after initial
treatment) showed patterns of HA and TUNEL staining, that, like 64
h after a single set of treatments, indicated cell degeneration and
fragmentation (FIG. 5F).
[0113] Full body necropsy of non-tumor bearing animals one (n=2) or
two days (n=2) after completion of the above dual treatment regimen
revealed no evident pathological lesions to internal organs and no
evident abnormalities of the cerebrum or cerebellum (FIG. 7 and
Table 1). In addition, a liver-kidney serum chemistry panel carried
out 1 day after the second set of Pen-d/n-ATF5-RP injections
indicated no damage to either organ (Table 1; n=2)
TABLE-US-00024 TABLE 1 Results from gross necropsy of organs,
H&E staining of tissue sections and liver-kidney function blood
panel of mice treated with Pen-d/n-ATF5-RP. H&E slides 6 Month
6 Month 1 day after 1 day after 2 day after 2 day after Post-tumor
Post-tumor Pen-d/n- Pen-d/n- Pen-d/n- Pen-d/n- treatment treatment
ATF5-RP ATF5-RP ATF5-RP ATF5-RP with Pen-d/n- with Pen-d/n- Gross
Necropsy treatment #1 treatment #2 Control treatment #1* treatment
#2* ATF5-RP #1 ATF5-RP #2 Cerebrum, No gross No gross No gross No
gross No gross No gross No gross cerebellum, nasal lesion/No
lesion/No lesion/No lesion/No lesion/No lesion/No lesion/No cavity,
liver, significant significant significant significant significant
significant significant kidneys, spleen, changes of changes of
changes of changes of changes of changes of changes of pancreas,
heart, pathological pathological pathological pathological
pathological pathological pathological lungs, trachea, significance
significance significance significance significance significance
significance esophagus, thymus, salivary glands, GI tract, hind
limb muscles, urinary bladder, reproductive tract Alkaline Alanine
Asparate Blood Urea Total Total Albumin Phosphatase Transaminase
Transaminase Nitrogen Creatinine Bilirubin Protein Mouse g/dL U/L
U/L U/L mg/dL mg/dL mg/dL g/dL Lipemia Hemolysis Male, one day 3.95
52.9 55.4 128.7 20.4 0.072 0.089 5.87 None None after Pen-d/n-
ATF5-RP treatment #1 Male, one day 3.35 46.0 21.6 66.6 19.7 0.061
0.115 5.21 None None after Pen-d/n- ATF5-RP treatment #2 JAX
database 3.77 .+-. 78.3 .+-. 52.7 .+-. 152 .+-. 23.7 .+-. 0.167
.+-. 0.695 .+-. 6.10 .+-. Not Not strain range 0.247 32.6 19.6 92.6
3.47 0.258 0.167 0.396 Listed Listed (males) The indicated organs
were collected from mice sacrificed at 1 day, 2 days and >6
months (190 days and 183 days, corresponding to mice with
eradicated tumors in FIGS. 3A-3B, and S9, respectively) after the
second of two sets of subcutaneous treatments with Pen-d/n-ATF5-RP
as described in the text. The >6 month animals had MRI-detected
tumors before treatment and no histologically detectable tumors at
the time of sacrifice. All other animals were not tumor-bearing.
The control mouse was untreated. The organs were evaluated for
gross pathological changes and then fixed, paraffin embedded and
used for preparation of slide-mounted 5 .mu.m sections. The slides
were stained with H&E and examined microscopically for possible
pathological changes. Gross pathological analysis and evaluation of
sections were carried out by the Comparative Pathology Laboratory
at the UC Davis School of Veterinary Medicine. *Regional
coagulative necrosis in the liver and focal linear pneumonia of the
lung were observed due to inadvertent needle penetration during the
injections.
[0114] For liver-kidney function panel, blood samples were obtained
1 day after the second of two sets of subcutaneous treatments with
Pen-d/n-ATF5-RP as described in the text. The animals were not
tumor-bearing. The analysis was carried out by the Comparative
Pathology Laboratory at the UC Davis School of Veterinary Medicine.
The data for the strain (C57BL/6J) range was obtained from the
Mouse Phenome Database at the Jackson Laboratory
(http://phenome.jax.org/db/q?rtn=meas/catlister&req=Dblood-clinical
%20chemistryqqq44&reqstrainid=7).
[0115] Systemically delivered Pen-d/n-ATF5-RP promotes rapid
regression of mouse gliomas without recurrence as indicated by MRI
and histology. Whether systemic administration of Pen-d/n-ATF5-RP
promoted prolonged regression of gliomas in a mouse model was
assessed. To achieve this MRI (post-contrast enhanced 3D FLASH T1
weighted) was used to assess tumors before and at various times
after treatment with Pen-d/n-ATF5-RP, Pen-control-RP or no
treatment. In many cases, the tumors were either multifocal or
present in both hemispheres prior to treatment (FIGS. 8, 9, 10, and
11). The peptides were injected subcutaneously using the two
treatment protocol described above. Treatments commenced only after
the presence of tumors was verified by MRI and were randomly
assigned.
[0116] As anticipated, in no case was tumor regression observed as
assessed by MRI in untreated animals (n=5) or animals treated with
Pen-control-RP (n=4). A typical example for an animal treated with
control peptide is shown in FIG. 8. Tumor presence was verified by
histology on brains of animals those either died or were sacrificed
after exhibiting moribund behavior or that survived beyond the
study endpoint (6 months after MRI tumor detection). The tumors
were HA+(FIGS. 8E and 10), indicating the presence of the tagged
PDGF-B and exhibited hyperchromatic nuclei (FIG. 8D) and elevated
Ki67 staining typical of gliomas (FIG. 8F). The infiltrative tumor
boundaries matched those in the MRI images (FIG. 8).
[0117] For mice treated with Pen-d/n-ATF5-RP, MRI revealed
significant reduction (2/5; FIG. 9 and FIG. 11) or un-detectability
(3/5) of tumor signals at 8 days after treatment (the earliest time
monitored) and full loss of detectable tumor signal within 3 weeks
(n=7/7). When assessed by MRI at 176-225 days after peptide
treatment, 7/7 mice assessed were tumor-free (see for example, FIG.
8, FIG. 11 and FIG. 12B). Thus, Pen-d/n-ATF5-RP treatment appeared
to rapidly clear gliomas without MRI-detectable recurrence for at
least 6-7 months.
[0118] Postmortem histology (n=6; 183-259 days after treatment;
190-305 days after tumor detection) corroborated the MRI findings
of tumor regression/eradication (FIGS. 8, 11, and 12C). As in the
rest of the brain, areas that initially had been tumor positive by
MRI, showed an absence of hyperchromatic nuclei or high cellularity
or elevated Ki67 staining (FIG. 8 and FIG. 11). There was also no
staining (other than scarce scattered single cells) for
PDGF-B-HA+(FIG. 8 and FIG. 11). There were however, foci of GFAP+
cells, suggesting glial activation and scarring in the areas where
tumors had been present (FIG. 8 and FIG. 11).
[0119] Systemically delivered Pen-d/n-ATF5-RP promotes long-term
survival while maintaining normal brain and tissue integrity. All
eight tumor-bearing mice treated with Pen-d/n-ATF5-RP survived to
the nominal 180 day endpoint of the study after detection of tumors
(FIG. 12A). In contrast, 6/9 control mice died within this time. In
a past study 40% (n=16) of mice died within 180 days of tumor
initiation [Arias et al., Oncogene 2012; 31(6):739-51].
[0120] In addition to the 6 mice that were sacrificed for histology
at 6-8 months after Pen-d/n-ATF5-RP treatment, 2 animals have been
maintained for a 12-month post-treatment time.
[0121] Other than the absence of tumors and the presence of glial
scarring in areas of prior tumor localization, H&E staining of
the brains of the animals sacrificed 6-8 months after
Pen-d/n-ATF5-RP treatment indicated no evident abnormalities and
both the subventricular and hippocampal subgranular zones appeared
normal (FIG. 7). Additionally, the weights of the treated mice
prior to sacrifice were either within (4/6) or greater than (2/6)
one standard deviation of the mean weight of age-matched controls
given in the Mouse Phenome Database at the Jackson Laboratory
(http://phenome.jax.org/db/q?rtn=strains/details&strainid=7).
Two mice were also subjected to full body necropsy at >6 months
of treatment (190 days and 183 days, corresponding to mice with
eradicated tumors in FIG. 8 and FIG. 11, respectively). No
pathological changes were seen in any of the organs surveyed (Table
1).
[0122] 5.1.3 Discussion
[0123] The findings presented herein show that Pen-d/n-ATF5-RP
enters and promotes apoptotic activity in cultured GBM cells and
that when systemically administered to animals, crosses the blood
brain barrier, enters brain and tumor cells and causes massive
tumor cell death and long-term tumor regression/eradication without
apparent harm to normal tissues.
[0124] Another feature of the study presented herein was that the
treated tumor-bearing animals survived for at least 6-12 months. By
contrast, 2/3 of control animals died or showed morbidity within
189 days of tumor detection and all were tumor positive at death or
at the 6 month point. Taken together, the results presented herein
provide proof that a cell penetrating form of d/n-ATF5 can be used
to treat malignant gliomas.
[0125] A model in which malignant gliomas were induced in adult
mice by retrovirally expressed PDGF-B and p53 shRNA, presumably by
transformation of PDGF-.alpha.-receptor+ neural progenitors and
oligodendrocyte precursors, was used in the instant study. Such
tumors resemble high grade human glioma [Arias et al., Oncogene
2012; 31(6):739-51] and, like the latter, are highly diffuse and
relatively large and can invade both hemispheres. Given the wide
expression of ATF5 in human GBMs and lower grade gliomas and the
variety of human and rodent-derived GBM cell lines (with and
without compromised p53 and PTEN) that express and require ATF5 for
survival [Arias et al., Oncogene 2012; 31(6):739-51], it is
expected that, based on the data presented herein, a range of
malignant glioma cell types will be susceptible to treatment with
cell-penetrating d/n-ATF5. Furthermore, although malignant gliomas
are the focus of this study, it is significant to note that ATF5 is
expressed by a wide variety of carcinomas [Sheng et al., Oncotarget
2010; 1(6):457-60; Chen A et al., International journal of
gynecological pathology 2012; 31(6):532-7; Fernandez et al.,
Oncogene 2004; 23(29):5084-91; Kong et al., Experimental and
therapeutic medicine 2011; 2(5):827-831; Monaco et al., Int J
Cancer 2007; 120(9):1883-90; and Hu et al., Anticancer research
2012; 32(10):4385-94], and that culture studies have shown
apoptotic actions of d/n-ATF5 or ATF5 siRNA on tumor cells from a
diverse range of tissues. [Sheng et al., Oncotarget 2010;
1(6):457-60; Chen A et al., International journal of gynecological
pathology 2012; 31(6):532-7; Monaco et al., Int J Cancer 2007;
120(9):1883-90; and Hu et al., Anticancer research 2012;
32(10):4385-94]. Thus, based on the data presented herein, a
diverse range of cancers will be susceptible to treatment with
cell-penetrating d/n-ATF5.
[0126] An important aspect of the instant study was that although
Pen-d/n-ATF5-RP promoted regression/eradication of tumors, it had
no apparent adverse effects on normal tissue. It is significant
that treated animals survived without apparent effect for at least
6-12 months and that no evident acute or long term tissue damage
was observed. In addition, any potential negative effects of
Pen-d/n-ATF5-RP may be mitigated by the limited duration of
treatment.
5.2 Addition Cell Lines and CP-d/n-ATF5 Compositions
[0127] 5.2.1 TAT-d/n-ATF5 Promotes Apoptotic Death of Cultured
Melanoma MEL501 Cells
[0128] TAT-linked dominant-negative ATF5 peptide was added to
medium of MEL501 melanoma cells at the concentrations (in .mu.M)
indicated in FIG. 13. Four days later the cells were stained with
Hoescht dye and the cells were stained for proportion with
apoptotic nuclei. As illustrated in FIG. 10, TAT-d/n/ATF5 promoted
apoptosis in a dose-dependent fashion.
[0129] 5.2.2 TAT-d/n-ATF5 Reduces Expression of Endogenous ATF5 in
Cultured U373 Glioblastoma Cells
[0130] TAT-linked dominant-negative ATF5 peptide was added to
medium of U373 glioblastoma cells at the concentrations (in .mu.M)
indicated in FIG. 14 for 17 hrs day and the cells were then
harvested and analyzed by Western immunoblotting for levels of
endogenous ATF5. Note that the TAT-d/n-ATF5 greatly reduces
expression of endogenous ATF5. As previous studies have shown that
tumor cells require endogenous ATF5 to survive, but without being
bound by theory, the mechanism of action by which the
cell-penetrating TAT-ZIP peptide kills may be by causing loss of
the endogenous ATF5 protein. Note also the smear above the
endogenous ATF5 when the TAT-ZIP peptide is present. This suggests
that TAT-ZIP reduces endogenous ATF5 by causing its ubiquitination
and proteasomal degradation.
[0131] 5.2.3 TAT-d/n-ATF5 induces Expression of the Pro-Death Gene
DDIT3
[0132] TAT-d/n-ATF5 (TAT-ZIP) peptide induces expression of the
pro-death gene DDIT3 (CHOP) in various tumor cell lines. Cells were
treated with TAT-d/n-ATF5 for the times and doses (in .mu.M)
indicated in FIG. 15 and then harvested and analyzed by Western
immunoblotting for expression of CHOP and other non-responsive
proteins. Note the elevation of CHOP in all cases. Since CHOP may
promote cell death, these data indicate that induction of CHOP
protein may be one mechanism by which TAT-d/n-ATF5 kills tumor
cells.
[0133] 5.2.4 Silencing of CHOP Protein with siRNA Partially
Protects U87 Cells from TAT-d/n-ATF5
[0134] Silencing of CHOP protein with siRNA (top Western immunoblot
of FIG. 16) partially protects U87 cells from death caused by
TAT-d/n-ATF5 peptide. Cells were treated with siCHOP to silence
CHOP expression (top Western immunoblot of FIG. 16) or with control
siRNA. They were then exposed to TAT-d/n-ATF5 for 2 days and
assessed for proportion of cells with apoptotic nuclei. The data
indicate that part of the mechanism by which TAT-d/n-ATF5 kills
tumor cells is by increasing their expression of CHOP which in turn
mediates death.
[0135] 5.2. TAT-D/N-ATF5 Down-Regulates BCL2 Survival Protein
[0136] TAT-D/N-ATF5 down-regulates BCL2 survival protein. As
outlined in FIG. 17, cultured U87 human glioblastoma cells were
treated with the indicated concentrations of TATZIP (TAT-d/n-ATF5
peptide) (in .mu.M) for 30 hrs. The cells were then harvested and
assessed by Western immunoblotting for expression of the survival
protein BCL2. These findings indicate that in addition to elevating
pro-death CHOP, TAT-d/n-ATF5 may also kill tumor cells by reducing
their levels of the BCL2 survival protein.
[0137] 5.9 TAT-D/N-ATF5 Synergizes with Temozolomide to Kill
Cultured U87 Glioblastoma Cells
[0138] TAT-D/N-ATF5 synergizes with temozolomide (TMZ) to kill
cultured U87 glioblastoma cells. As outlined in FIG. 18, cells were
cultured for one day with sub-lethal levels of TAT-d/n-ATF5 (TZIP 1
.mu.M) and TMZ (50 .mu.M) either separately or in combination, and
then assessed for proportion of cells with apoptotic nuclei. TMZ is
presently the first-line treatment for human GBM. The data reveal
that TAT-d/n-ATF5 not only functions in presence of TMZ, but that
the two drugs act in synergy to kill GBM cells. This indicates that
TAT-d/n-ATF5 can be administered to patients who are taking
TMZ.
[0139] 5.10 TAT-D/N-ATF5 Decreases Viability of U87, U373, and MSG
Cells
[0140] As outlined in FIG. 19, recombinant TAT-d/n-ATF5 (3 .mu.M)
treatment for 3-5 days decreases viability of two human and one
mouse GMB cell line as detected using an MTA assay.
[0141] 5.11 Synthetic PEN-D/N-ATF5 Decreases Viability of U87
Cells
[0142] As outlined in FIG. 20, synthetic PEN-d/n-ATF5 decreases
cell viability of cultured U87 human glioblastoma cells. 5 days
treatment at indicated concentrations (.mu.M), as detected using an
MTA assay.
[0143] 5.12 TAT-D/N-ATF5 Promotes Cell Death of U87 Cells
[0144] As outlined in FIG. 21, Recombinant TAT-d/n-ATF5 promotes
death of cultured U87 human glioblastoma cells as indicated by
Annexin V/PI staining and flow cytometry. Proportions of viable
cells are shown in lower left quadrant (88% control vs 58%
treated). Dying cell proportions are in the lower right and upper
right quadrants (9% in controls vs 36% in treated).
[0145] 5.13 Synthetic PEN-D/N-ATF5 Promotes Apoptosis of GS9-6
Cells
[0146] As outlined in FIG. 22, Synthetic PEN-d/n-ATF5 promotes
apoptotic death of primary GS9-6 human glioblastoma stem cells
growing in culture as spheres. Data reflects 6 days of treatment.
Data determined by Annexin V/PI staining and flow cytometry.
[0147] 5.13 Recombinant PEN-D/N-ATF5 Promotes Apoptosis of GS9-6
Cells
[0148] As outlined in FIG. 23, Recombinant PEN-d/n-ATF5 promotes
apoptotic death of primary GS9-6 human glioblastoma stem cells
growing in culture as spheres. 5 days treatment. Data determined by
Annexin V/PI staining and flow cytometry.
[0149] Various publications are cited herein, the contents of which
are hereby incorporated by reference in their entireties.
Sequence CWU 1
1
5117PRTUnknownDescription of Unknown ATF5 peptide sequence 1Leu Glu
Gln Glu Asn Ala Glu 1 5 214PRTUnknownDescription of Unknown ATF5
peptide sequence 2Leu Glu Lys Glu Ala Glu Glu Leu Glu Gln Glu Asn
Ala Glu 1 5 10 322PRTUnknownDescription of Unknown ATF5 peptide
sequence 3Leu Ala Arg Glu Asn Glu Glu Leu Leu Glu Lys Glu Ala Glu
Glu Leu 1 5 10 15 Glu Gln Glu Asn Ala Glu 20
429PRTUnknownDescription of Unknown ATF5 peptide sequence 4Leu Glu
Gln Arg Ala Glu Glu Leu Ala Arg Glu Asn Glu Glu Leu Leu 1 5 10 15
Glu Lys Glu Ala Glu Glu Leu Glu Gln Glu Asn Ala Glu 20 25
528PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 5Leu Glu Gln Glu Asn Ala Glu Leu Glu Gly Glu Cys
Gln Gly Leu Glu 1 5 10 15 Ala Arg Asn Arg Glu Leu Lys Glu Arg Ala
Glu Ser 20 25 635PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 6Leu Glu Lys Glu Ala Glu Glu Leu Glu
Gln Glu Asn Ala Glu Leu Glu 1 5 10 15 Gly Glu Cys Gln Gly Leu Glu
Ala Arg Asn Arg Glu Leu Lys Glu Arg 20 25 30 Ala Glu Ser 35
743PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 7Leu Ala Arg Glu Asn Glu Glu Leu Leu Glu Lys
Glu Ala Glu Glu Leu 1 5 10 15 Glu Gln Glu Asn Ala Glu Leu Glu Gly
Glu Cys Gln Gly Leu Glu Ala 20 25 30 Arg Asn Arg Glu Leu Lys Glu
Arg Ala Glu Ser 35 40 849PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 8Leu Glu Gln Arg Ala Glu
Glu Leu Ala Arg Asn Glu Glu Leu Leu Glu 1 5 10 15 Lys Glu Ala Glu
Glu Leu Glu Gln Glu Asn Ala Glu Leu Glu Gly Glu 20 25 30 Cys Gln
Gly Leu Glu Ala Arg Asn Arg Glu Leu Lys Glu Arg Ala Glu 35 40 45
Ser 951PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 9Leu Glu Gln Arg Ala Glu Glu Leu Ala Arg Glu
Asn Glu Glu Leu Leu 1 5 10 15 Glu Lys Glu Ala Glu Glu Leu Glu Gln
Glu Asn Ala Glu Leu Glu Gly 20 25 30 Glu Cys Gln Gly Leu Glu Ala
Arg Asn Arg Glu Leu Lys Glu Arg Ala 35 40 45 Glu Ser Val 50
1028PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Leu Glu Gln Glu Asn Ala Glu Leu Glu Gly Glu Cys
Gln Gly Leu Glu 1 5 10 15 Ala Arg Asn Arg Glu Leu Arg Glu Arg Ala
Glu Ser 20 25 1135PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 11Leu Glu Lys Glu Ala Glu Glu Leu
Glu Gln Glu Asn Ala Glu Leu Glu 1 5 10 15 Gly Glu Cys Gln Gly Leu
Glu Ala Arg Asn Arg Glu Leu Arg Glu Arg 20 25 30 Ala Glu Ser 35
1243PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 12Leu Ala Arg Glu Asn Glu Glu Leu Leu Glu Lys
Glu Ala Glu Glu Leu 1 5 10 15 Glu Gln Glu Asn Ala Glu Leu Glu Gly
Glu Cys Gln Gly Leu Glu Ala 20 25 30 Arg Asn Arg Glu Leu Arg Glu
Arg Ala Glu Ser 35 40 1350PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 13Leu Glu Gln Arg Ala Glu
Glu Leu Ala Arg Glu Asn Glu Glu Leu Leu 1 5 10 15 Glu Lys Glu Ala
Glu Glu Leu Glu Gln Glu Asn Ala Glu Leu Glu Gly 20 25 30 Glu Cys
Gln Gly Leu Glu Ala Arg Asn Arg Glu Leu Arg Glu Arg Ala 35 40 45
Glu Ser 50 1451PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 14Leu Glu Gln Arg Ala Glu Glu Leu
Ala Arg Glu Asn Glu Glu Leu Leu 1 5 10 15 Glu Lys Glu Ala Glu Glu
Leu Glu Gln Glu Asn Ala Glu Leu Glu Gly 20 25 30 Glu Cys Gln Gly
Leu Glu Ala Arg Asn Arg Glu Leu Arg Glu Arg Ala 35 40 45 Glu Ser
Val 50 15111PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 15Met Gly Ser Ser His His His His
His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Arg
Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg 20 25 30 Met Lys Trp Lys
Lys Asp Tyr Lys Asp Asp Asp Asp Lys Met Ala Ser 35 40 45 Met Thr
Gly Gly Gln Gln Met Gly Arg Asp Pro Asp Leu Glu Gln Arg 50 55 60
Ala Glu Glu Leu Ala Arg Glu Asn Glu Glu Leu Leu Glu Lys Glu Ala 65
70 75 80 Glu Glu Leu Glu Gln Glu Asn Ala Glu Leu Glu Gly Glu Cys
Gln Gly 85 90 95 Leu Glu Ala Arg Asn Arg Glu Leu Arg Glu Arg Ala
Glu Ser Val 100 105 110 166PRTArtificial SequenceDescription of
Artificial Sequence Synthetic 6xHis tag 16His His His His His His 1
5 17109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 17Met Gly Ser Ser His His His His His His Ser
Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Leu Glu Tyr Gly
Arg Lys Lys Arg Arg Gln Arg 20 25 30 Arg Arg Tyr Pro Tyr Asp Val
Pro Asp Tyr Ala Met Ala Ser Met Thr 35 40 45 Gly Gly Gln Gln Met
Gly Arg Asp Pro Asp Leu Glu Gln Arg Ala Glu 50 55 60 Glu Leu Ala
Arg Glu Asn Glu Glu Leu Leu Glu Lys Glu Ala Glu Glu 65 70 75 80 Leu
Glu Gln Glu Asn Ala Glu Leu Glu Gly Glu Cys Gln Gly Leu Glu 85 90
95 Ala Arg Asn Arg Glu Leu Arg Glu Arg Ala Glu Ser Val 100 105
1888PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 18Met Gly Ser Ser His His His His His His Ser
Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Arg Gln Ile Lys
Ile Trp Phe Gln Asn Arg Arg 20 25 30 Met Lys Trp Lys Lys Leu Glu
Gln Arg Ala Glu Glu Leu Ala Arg Glu 35 40 45 Asn Glu Glu Leu Leu
Glu Lys Glu Ala Glu Glu Leu Glu Gln Glu Asn 50 55 60 Ala Glu Leu
Glu Gly Glu Cys Gln Gly Leu Glu Ala Arg Asn Arg Glu 65 70 75 80 Leu
Lys Glu Arg Ala Glu Ser Val 85 1967PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
19Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1
5 10 15 Leu Glu Gln Arg Ala Glu Glu Leu Ala Arg Glu Asn Glu Glu Leu
Leu 20 25 30 Glu Lys Glu Ala Glu Glu Leu Glu Gln Glu Asn Ala Glu
Leu Glu Gly 35 40 45 Glu Cys Gln Gly Leu Glu Ala Arg Asn Arg Glu
Leu Lys Glu Arg Ala 50 55 60 Glu Ser Val 65 2087DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 20ctggaacagg aaaacgcgga actggaaggc gaatgccagg
gcctggaagc gcgcaaccgc 60gaactgaaag aacgcgcgga aagctaa
8721108DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 21ctggaaaaag aagcggaaga actggaacag
gaaaacgcgg aactggaagg cgaatgccag 60ggcctggaag cgcgcaaccg cgaactgaaa
gaacgcgcgg aaagctaa 10822132DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 22ctggcgcgcg
aaaacgaaga actgctggaa aaagaagcgg aagaactgga acaggaaaac 60gcggaactgg
aaggcgaatg ccagggcctg gaagcgcgca accgcgaact gaaagaacgc
120gcggaaagct aa 1322350PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 23Leu Glu Gln Arg Ala Glu
Glu Leu Ala Arg Glu Asn Glu Glu Leu Leu 1 5 10 15 Glu Lys Glu Ala
Glu Glu Leu Glu Gln Glu Asn Ala Glu Leu Glu Gly 20 25 30 Glu Cys
Gln Gly Leu Glu Ala Arg Asn Arg Glu Leu Lys Glu Arg Ala 35 40 45
Glu Ser 50 24153DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 24ctggaacagc gcgcggaaga
actggcgcgc gaaaacgaag aactgctgga aaaagaagcg 60gaagaactgg aacaggaaaa
cgcggaactg gaaggcgaat gccagggcct ggaagcgcgc 120aaccgcgaac
tgaaagaacg cgcggaaagc taa 15325156DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 25ctggaacagc
gcgcggaaga actggcgcgc gaaaacgaag aactgctgga aaaagaagcg 60gaagaactgg
aacaggaaaa cgcggaactg gaaggcgaat gccagggcct ggaagcgcgc
120aaccgcgaac tgaaagaacg cgcggaaagc gtgtaa 1562622PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 26Leu
Glu Gly Glu Cys Gln Gly Leu Glu Ala Arg Asn Arg Glu Leu Lys 1 5 10
15 Glu Arg Ala Glu Ser Val 20 2787DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 27ctggaacagg
aaaacgcgga actggaaggc gaatgccagg gcctggaagc gcgcaaccgc 60gaactgcgcg
aacgcgcgga aagctaa 8728108DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 28ctggaaaaag
aagcggaaga actggaacag gaaaacgcgg aactggaagg cgaatgccag 60ggcctggaag
cgcgcaaccg cgaactgcgc gaacgcgcgg aaagctaa 10829132DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
29ctggcgcgcg aaaacgaaga actgctggaa aaagaagcgg aagaactgga acaggaaaac
60gcggaactgg aaggcgaatg ccagggcctg gaagcgcgca accgcgaact gcgcgaacgc
120gcggaaagct aa 13230153DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 30ctggaacagc
gcgcggaaga actggcgcgc gaaaacgaag aactgctgga aaaagaagcg 60gaagaactgg
aacaggaaaa cgcggaactg gaaggcgaat gccagggcct ggaagcgcgc
120aaccgcgaac tgcgcgaacg cgcggaaagc taa 15331156DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
31ctggaacagc gcgcggaaga actggcgcgc gaaaacgaag aactgctgga aaaagaagcg
60gaagaactgg aacaggaaaa cgcggaactg gaaggcgaat gccagggcct ggaagcgcgc
120aaccgcgaac tgcgcgaacg cgcggaaagc gtgtaa 1563222PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 32Leu
Glu Gly Glu Cys Gln Gly Leu Glu Ala Arg Asn Arg Glu Leu Arg 1 5 10
15 Glu Arg Ala Glu Ser Val 20 3324PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 33Glu Arg Glu Ile Gln Tyr
Val Lys Asp Leu Leu Ile Glu Val Tyr Lys 1 5 10 15 Ala Arg Ser Gln
Arg Thr Arg Ser 20 3416PRTDrosophila sp. 34Arg Gln Ile Lys Ile Trp
Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15
35336DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 35atgggcagca gccatcatca tcatcatcac
agcagcggcc tggtgccgcg cggcagccat 60atgcgtcaaa ttaaaatttg gtttcaaaat
cgtcgtatga aatggaaaaa agactacaag 120gacgatgatg acaaaatggc
atctatgact ggaggacaac aaatgggaag agacccagac 180ctcgaacaaa
gagcagaaga actagcaaga gaaaacgaag aactactaga aaaagaagca
240gaagaactag aacaagaaaa tgcagagcta gagggcgagt gccaagggct
agaggcgcgg 300aatcgggagc tgagggagag ggcagagtca gtgtag
3363616PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 36Arg Arg Leu Arg Arg Leu Leu Arg Arg Leu Leu Arg
Arg Leu Arg Arg 1 5 10 15 3712PRTRabies virus 37Arg Val Gly Arg Arg
Arg Arg Arg Arg Arg Arg Arg 1 5 10 3827PRTUnknownDescription of
Unknown Transportan peptide 38Gly Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Lys Ile Asn Leu 1 5 10 15 Lys Ala Leu Ala Ala Leu Ala
Lys Lys Ile Leu 20 25 3916PRTRattus sp. 39Pro Val Ile Arg Val Trp
Phe Gln Asn Lys Arg Cys Lys Asp Lys Lys 1 5 10 15 4011PRTHuman
immunodeficiency virus 40Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg
Arg 1 5 10 4113PRTHuman immunodeficiency virus 41Gly Arg Lys Lys
Arg Arg Gln Arg Arg Arg Pro Pro Gln 1 5 10 42330DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
42atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat
60atgctcgagt acggccgcaa gaaacgccgc cagcgccgcc gctatccata tgacgtccca
120gactatgcta tggcatctat gactggagga caacaaatgg gaagagaccc
agacctcgaa 180caaagagcag aagaactagc aagagaaaac gaagaactac
tagaaaaaga agcagaagaa 240ctagaacaag aaaatgcaga gctagagggc
gagtgccaag ggctagaggc gcggaatcgg 300gagctgaggg agagggcaga
gtcagtgtag 3304316PRTMus sp. 43Leu Leu Ile Ile Leu Arg Arg Arg Ile
Arg Lys Gln Ala His Ala His 1 5 10 15 4427PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 44Gly
Ala Leu Phe Leu Gly Trp Leu Gly Ala Ala Gly Ser Thr Met Gly 1 5 10
15 Ala Trp Ser Gln Pro Lys Lys Lys Arg Lys Val 20 25
4518PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 45Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys
Ala Ala Leu Lys 1 5 10 15 Leu Ala 4621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
46tccgcggccg caccggtcgc c 214747DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 47ctcgaggata tctcagttat
ctacactgac tctgccctct ccctcag 474890DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
48ttaattaagc cgccatggat gcgtcaaatt aaaatttggt ttcaaaatcg tcgtatgaaa
60tggaaaaaaa tggactacaa ggacgatgat 904947DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
49ctcgagggat cctcagttat ctacactgac tctgccctct ccctcag
475033DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 50cccgggcata tgcgtcaaat taaaatttgg ttt
335138DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 51ctcgagggat cctcagttat ctagtctggg tctcttcc 38
* * * * *
References