U.S. patent application number 16/982979 was filed with the patent office on 2021-01-28 for use of 6-thio-dg to treat therapy-resistant telomerasepositive pediatric brain tumors.
This patent application is currently assigned to The Board of Regents of the University of Texas System. The applicant listed for this patent is The Board of Regents of the University of Texas System, CINCINNATI CHILDREN'S HOSPITAL MEDICAL CENTER. Invention is credited to Rachid DRISSI, Jerry SHAY.
Application Number | 20210023107 16/982979 |
Document ID | / |
Family ID | 1000005177147 |
Filed Date | 2021-01-28 |
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United States Patent
Application |
20210023107 |
Kind Code |
A1 |
SHAY; Jerry ; et
al. |
January 28, 2021 |
USE OF 6-THIO-dG TO TREAT THERAPY-RESISTANT TELOMERASEPOSITIVE
PEDIATRIC BRAIN TUMORS
Abstract
Brain tumors remain the leading cause of cancer-related deaths
in children and often are associated with long-term sequelae among
survivors of current therapies. Telomerase and telomeres play
important roles in cancer, representing attractive therapeutic
targets to treat children with poor-prognosis brain tumors such as
diffuse intrinsic pontine glioma (DIPG), high-grade glioma (HGG)
and high-risk medulloblastoma (MB). It has shown that DIPG, HGG and
MB frequently express telomerase activity. It is now shown that the
telomerase-dependent incorporation of 6-thio-2'deoxyguanosine
(6-thio-dG), a telomerase substrate precursor analog, into
telomeres leads to telomere dysfunction-induced foci (TIFs) along
with extensive genomic DNA damage, cell growth inhibition and cell
death of primary stem-like cells derived from patients with DIPG,
HGG and MB. Importantly, the effect of 6-thio-dG is persistent even
after drug withdrawal. Treatment with 6-thio-dG elicits a
sequential activation of ATR and ATM pathways and induces G.sub.2/M
arrest. In vivo, treatment of mice bearing MB xenografts with
6-thio-dG delays tumor growth, increases in-tumor TIFs and
apoptosis. Furthermore, 6-thio-dG crosses the blood-brain barrier
and specifically targets tumor cells in an orthotopic mouse model
of DIPG. Together, these findings suggest that 6-thio-dG is a
promising approach to treat therapy-resistant telomerase-positive
pediatric brain tumors.
Inventors: |
SHAY; Jerry; (Dallas,
TX) ; DRISSI; Rachid; (Cincinnati, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Regents of the University of Texas System
CINCINNATI CHILDREN'S HOSPITAL MEDICAL CENTER |
Austin
Cincinnati |
TX
OH |
US
US |
|
|
Assignee: |
The Board of Regents of the
University of Texas System
Austin
TX
Children's Hospital Medical Center
Cincinnati
OH
|
Family ID: |
1000005177147 |
Appl. No.: |
16/982979 |
Filed: |
March 22, 2019 |
PCT Filed: |
March 22, 2019 |
PCT NO: |
PCT/US2019/023596 |
371 Date: |
September 21, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62646820 |
Mar 22, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 31/708 20130101; A61P 35/00 20180101 |
International
Class: |
A61K 31/708 20060101
A61K031/708; A61P 35/00 20060101 A61P035/00 |
Claims
1. A method of treating a brain cancer in a pediatric subject,
comprising administering a telomerase substrate precursor analog to
a subject in need thereof, thereby treating pediatric brain
cancer.
2. The method of claim 1, wherein the subject's is age 1-21, 1-18
or 1-14.
3. The method of claim 1, wherein the pediatric brain cancer is
drug resistant.
4. The method of claim 1, wherein the pediatric brain cancer is
diffuse intrinsic pontine glioma (DIPG), high-grade glioma (HGG),
or high-risk medulloblastoma (MB).
5. The method of claim 1, wherein the brain cancer has telomerase
activity.
6. The method of claim 1, wherein the telomerase substrate
precursor analog is 6-thio-2' deoxyguanosine (6-thio-dG).
7. The method of claim 6, wherein 6-thio-dG induces in vivo
telomere dysfunction-induced foci (TIFs), apoptosis, and an
inhibition of tumor growth.
8. The method of claim 1, wherein the telomerase substrate
precursor analog is administered in combination or sequentially
with an immunotherapeutic agent, a targeted drug, an epigenetic
modifier, a chemotherapeutic agent, radiotherapy, or any
combination thereof.
9. The method of claim 1, wherein the telomerase substrate
precursor analog is administered in combination with an immune
checkpoint inhibitor, such as an anti-PD-L1 or PD-1 antibody.
10. The method of claim 1, further comprising the step of assessing
telomerase activity in a brain cancer cell from said subject.
11. A method of inducting G2/M cell cycle arrest in a cancer cell
comprising contacting the cell with a telomerase substrate
precursor analog and a telomerase inhibitor.
12. The method of claim 11, wherein the cancer cell is a brain
cancer cell.
13. The method of claim 12, wherein the brain cancer cell is drug
resistant.
14. The method of claim 11, wherein the brain cancer cells is
diffuse intrinsic pontine glioma (DIPG), high-grade glioma (HGG),
or high-risk medulloblastoma (MB).
15. The method of claim 11, wherein the cancer cell exhibits
telomerase activity.
16. The method of claim 11, wherein the telomerase substrate
precursor analog is 6-thio-2' deoxyguanosine (6-thio-dG).
17. The method of claim 16, wherein 6-thio-dG induces in vivo
telomere dysfunction-induced foci (TIFs), apoptosis, and an
inhibition of tumor growth.
18. The method of claim 11, wherein the telomerase substrate
precursor analog is administered in combination or sequentially
with an immunotherapeutic agent, a targeted drug, an epigenetic
modifier, a chemotherapeutic agent, radiotherapy, or any
combination thereof.
19. The method of claim 11, further comprising the step of
assessing telomerase activity in said cancer cell.
20. The method of claim 11, wherein the cell cycle arrest induces
the accumulation of genomic DNA damage.
Description
PRIORITY CLAIM
[0001] This application claims benefit of priority to U.S.
Provisional Application Ser. No. 62/646,820, filed Mar. 22, 2018,
the entire contents of which are hereby incorporated by
reference.
BACKGROUND
1. Field
[0002] The present disclosure relates to the fields of medicine,
pharmacology, molecular biology and oncology. More particular, the
disclosure relates to methods and compositions for treating
pediatric brain cancers, such as drug-resistant brain cancers.
2. Related Art
[0003] Telomeres are the physical ends of eukaryotic linear
chromosomes and, in mammals, are composed of several kilobases of
tandem TTAGGG repeats that are bound by the shelterin protein
complex (1). Shelterin proteins protect telomeres from ATM and
ATR-dependent DNA damage responses (DDR) (1). The inventors and
others have previously shown that natural telomere shortening
during replicative senescence or experimental telomere uncapping
elicit ATM-dependent DDR triggered by telomere dysfunction (2,3).
The hallmark of telomere dysfunction is the formation of DNA damage
foci localized at telomeres called TIFs (telomere
dysfunction-induced foci). TIFs are focal accumulations of DDR
factors such as ATM S1981-P, .gamma.H2AX, and 53BP1 at
dysfunctional telomeres (4). Telomeres are maintained by telomerase
activity in 73-90% of primary human cancers, while in most normal
somatic cells this activity is not detectable (5-7). Human
telomerase consists of two essential components, the protein
catalytic subunit (hTERT) and the RNA template (hTERC) that
contribute to the synthesis of telomeric repeats, thereby
maintaining telomeres. Telomerase activation, a feature of the vast
majority of cancers, is essential for maintaining an immortal
phenotype by conferring unlimited replicative potential.
[0004] Brain tumors are the most common solid tumors of childhood
and are the leading cause of cancer-related deaths in children (8).
Diffuse intrinsic pontine glioma (DIPG) is a particularly poor
prognosis brain tumor with a median overall survival of less than
one year (9). Hence, there is an urgent need to develop novel
therapies that not only improve outcome but mitigate long-term
complications in children with these poor-prognosis brain tumors.
The inventors have previously shown that over 73% of DIPG and 50%
of high-grade gliomas (HGG) (10) demonstrate telomerase activity.
The recently conducted molecular biology and phase II study of
imetelstat, a potent inhibitor of telomerase (11,12), estimated
inhibition of tumor telomerase activity and efficacy in children
with recurrent central nervous system (CNS) malignancies (13). The
regimen proved intolerable, because of thrombocytopenia that led to
bleeding. This toxicity prevented more frequent dosing of
imetelstat to allow sustained telomerase inhibition. Because
targeting telomerase directly, such as with imetelstat, would
result in a significant lag period from the initiation of treatment
until telomeres shortened sufficiently to reduce tumor burden,
stopping therapy with imetelstat would result in rapid telomere
regrowth. Thus, new approaches utilizing this almost universal
cancer target are needed.
SUMMARY
[0005] Thus, in accordance with the disclosure, there are provided
methods of treating a brain cancer in a pediatric subject,
comprising administering a telomerase substrate precursor analog to
a subject in need thereof, thereby treating pediatric brain cancer.
In some aspects, the subject's is age 1-21, 1-18 or 1-14. In some
aspects, the pediatric brain cancer is drug resistant. In some
aspects, the pediatric brain cancer is diffuse intrinsic pontine
glioma (DIPG), high-grade glioma (HGG), or high-risk
medulloblastoma (MB). In some aspects, the brain cancer has
telomerase activity. In some aspects, the telomerase substrate
precursor analog is 6-thio-2'deoxyguanosine (6-thio-dG). In certain
aspects, 6-thio-dG induces in vivo telomere dysfunction-induced
foci (TIFs), apoptosis, and an inhibition of tumor growth. In some
aspects, the telomerase substrate precursor analog is administered
in combination or sequentially with an immunotherapeutic agent, a
targeted drug, an epigenetic modifier, a chemotherapeutic agent,
radiotherapy, or any combination thereof. In some aspects, the
telomerase substrate precursor analog is administered in
combination with an immune checkpoint inhibitor, such as an
anti-PD-L1 or PD-1 antibody. In some aspects, the methods further
comprise the step of assessing telomerase activity in a brain
cancer cell from said subject.
[0006] In another embodiment, provided herein are methods of
inducting G.sub.2/M cell cycle arrest in a cancer cell comprising
contacting the cell with a telomerase substrate precursor analog
and a telomerase inhibitor. In some aspects, the cancer cell is a
brain cancer cell. In some aspects, the brain cancer cell is drug
resistant. In some aspects, the brain cancer cell is diffuse
intrinsic pontine glioma (DIPG), high-grade glioma (HGG), or
high-risk medulloblastoma (MB). In some aspects, the cancer cell
exhibits telomerase activity. In some aspects, the telomerase
substrate precursor analog is 6-thio-2'deoxyguanosine (6-thio-dG).
In some aspects, 6-thio-dG induces in vivo telomere
dysfunction-induced foci (TIFs), apoptosis, and an inhibition of
tumor growth. In some aspects, the telomerase substrate precursor
analog is administered in combination or sequentially with an
immunotherapeutic agent, a targeted drug, an epigenetic modifier, a
chemotherapeutic agent, radiotherapy, or any combination thereof.
In some aspects, the methods further comprise the step of assessing
telomerase activity in said cancer cell. In some aspects, the cell
cycle arrest induces the accumulation of genomic DNA damage.
[0007] Other objects, features and advantages of the present
disclosure will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating particular
embodiments of the disclosure, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the disclosure will become apparent to those skilled in
the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present disclosure. The disclosure may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0009] FIGS. 1A-C. 6-thio-dG specifically inhibits cell growth of
telomerase-positive cells. Cell images (upper panel) and
corresponding viable cell counts (lower panel). FIG. 1A,
telomerase-positive cells: HeLa and HFF+hTERT; telomerase-negative
cells: HFF, and Saos-2 (ALT-positive). FIG. 1B, telomerase-positive
patient-derived pediatric brain tumor cells: MB004, R0315-GBM,
SU-DIPGVI and CCHMC-DIPG-1. FIG. 1C, matched pair of
patient-derived medulloblastoma cells at biopsy (D-425, Primary)
and at post-therapy failure (D-458, Recurrence) treated with DMSO
or 0.5-10 .mu.M of 6-thio-dG for one week. Error bars represent the
standard deviation from triplicates. Each experiment was performed
at least twice.
[0010] FIGS. 2A-D. 6-thio-dG induces persistent G.sub.2/M cell
cycle arrest in telomerase-positive cells. FIG. 2A, cell cycle
analysis of HFF, HFF+hTERT, and HeLa cells treated with DMSO or
with 3 .mu.M of 6-thiodG for 3 days. FIG. 2B, schematic of the
experimental design of continuous and wash off treatment. FIG. 2C,
cell cycle plots of DMSO controls and 6-thio-dG treated HFF,
HFF+hTERT, and HeLa cells at days 5, 6, 7, and 8. FIG. 2D, cell
cycle plots of pre-treated HFF+hTERT and HeLa with DMSO or 3 .mu.M
of 6-thio-dG for 3 days at day 1 to 5 post-drug removal. The
percentage of cells in sub-G.sub.1, G.sub.1, S, G.sub.2/M and
>4n is indicated in each plot. The lower panels indicate the
percent distribution of cell cycle phases as a function of time.
The experiments were conducted at least twice and the results shown
are representative of the replicates.
[0011] FIGS. 3A-D. 6-thio-dG causes sustained telomere damage in
telomerase-positive cells. FIG. 3A, HFF and HFF+hTERT cells were
cultured with 3 .mu.M of 6-thio-dG for 2 days (initial treatment),
then continuously for another 3 days (continuous treatment) or 3
days post-drug removal (drug wash off). The number of TIFs per cell
was counted for each treatment (.about.100 cells per treatment).
Bottom panels show representative images of FISH-immunofluorescence
using a telomere specific PNA probe (red) and .gamma.H2AX staining
(green). White Arrows indicate co-localization of .gamma.H2AX and
telomere signals (yellow), indicative of TIFs. DAPI (blue)
indicates nucleus staining. Average values of at least three fields
per cell per condition were evaluated. P-values are indicated, **,
P<0.01; ***, P<0.001. FIG. 3B, cell cycle plots of MB004
treated with DMSO or 3 .mu.M of 6-thio-dG for 3 days. Percent
events of cell cycle phases are indicated. FIG. 3C, cell images of
neurosphere formation assay at day 3 and day 7 post-treatment with
DMSO or 3 .mu.M of 6-thio-dG. Right panel indicates the
quantification of the number of spheres at day 3 and 7. Error bars
represent the standard deviation generated from triplicates. FIG.
3D, relative growth of MB004 cells treated with 6-thio-dG to DMSO
control. The cells were initially treated with 3 .mu.M of 6-thio-dG
for 2 days followed by 3 days of continuous or discontinuous
treatment. Bottom panel shows representative IF-FISH images.
Telomeres (red), .gamma.H2AX (green), and TIFs (yellow) are
depicted. DAPI (blue) indicates nucleus staining. Error bars
represent the standard deviation obtained from triplicates.
[0012] FIGS. 4A-C. Sequential activation of ATR and ATM in response
to 6-thio-dG. FIG. 4A, Western blot analysis of ATR-T1989P, total
ATR, ATM-S1981P, and total ATM in HFF, and HFF+hTERT cells at day
1, 2, and 3 post-treatment with 3 .mu.M of 6-thio-dG. d, indicates
day. .beta.-actin served as a loading control. Irradiated HFF cells
with 5 Gy were used as a positive control for ATM and ATR
activation. Bar diagrams (bottom) show the quantification of
ATR-T1989P, and ATM-S-1981P phosphorylation levels relative to the
corresponding DMSO. O.D. (optical density) was measured by
densitometry analysis using ImageJ software. Band intensities were
normalized to total ATR or total ATM as applicable. Average values
of two independent experiments were evaluated. FIG. 4B,
quantification of IF-FISH data in HFF+hTERT cells treated or
untreated (DMSO) with 3 .mu.M of 6-thio-dG for 1 to 3 days.
Representative images of cells (bottom panel) are shown with TIFs
(yellow foci denoted by white arrows), telomeres (red),
.gamma.H2A.X (green), and nucleus (DAPI in blue). FIG. 4C,
immunoblot analyses of ATR-T1989P, ATR, ATM 51981-P, ATM, CHK2
T68-P, CHK2, CHK1 5345-P, and CHK1 in HFF+hTERT cells treated with
10 .mu.M of ATM or 50 nM ATR specific inhibitors for 2 hours prior
to and in combination with DMSO (control) or 3 .mu.M of 6-thio-dG
for 2 days. IF-FISH was performed to assess the number of TIFs in
200 to 300 cells (right). Error bars are the standard deviations of
at least three fields per condition (50 or more cells per field).
P-values are indicated, *, P<0.05; **, P<0.01; ***,
P<0.001; ****, P<0.0001.
[0013] FIGS. 5A-C. 6-thio-dG induces apoptosis in
telomerase-positive cancer cells and senescence in
telomerase-positive normal cells. FIG. 5A, representative IF images
of cleaved-caspase-3 (green) in HFF, HFF+hTERT, and HeLa cells
treated with DMSO or 3 .mu.M of 6-thio-dG for 4 days. DAPI (blue)
indicates nucleus staining. FIG. 5B, corresponding immunoblot
analysis of cleaved-caspase-3. .beta.-actin was used as a loading
control. FIG. 5C, senescence-associated .beta.-gal assay. Senescent
HFF cells (HFF-S) at 70 population doublings (PD 70) were used as
positive control for senescence (replicative senescence). Senescent
cells are stained in blue. X-gal (+) and (-), indicate X-gal was
added or not respectively. Early passage HFF (negative control) and
HFF+hTERT were treated with 3 .mu.M of 6-thio-dG or DMSO for 23
days. The plot on the right represents the quantification of the
number of cells SA-.beta.-gal-positive (senescent). Error bars
represent the standard deviation generated from triplicates.
P-value is indicated, *, P<0.05. Each experiment was performed
at least twice.
[0014] FIGS. 6A-G. 6-thio-dG treatment inhibits tumor growth in
pediatric high-risk group 3 medulloblastoma and induces TIFs in
diffuse intrinsic pontine glioma (DIPG) xenografts. FIG. 6A,
Average weight of mice treated with DMSO-PBS (vehicle) or
6-thio-dG. MB004 tumor cells were injected subcutaneously in the
mouse flank and intraperitoneally (IP) injected when tumor was
established with DMSO-PBS (vehicle) or 2.5 mg/kg of 6-thio-dG every
two days for the indicated period of time. The arrow indicates the
time of the tumor establishment and the start of the treatment.
Error bars represent the standard deviation from 6 mice per group.
FIG. 6B, tumor growth kinetics. Each line denotes tumor growth per
mouse. Blue and red lines indicate vehicle and 6-thio-dG treatments
respectively. FIG. 6C, representative immunohistochemistry images
indicating cleaved-caspase-3 staining (brown) in FFPE sections from
MB004 tumors treated with vehicle or 6-thio-dG. The plot on the
right shows percent in-tumor apoptotic cells (cleaved-caspase-3
positive). Each dot represents average percent of apoptotic cells
per individual tumor per mouse. P-value is indicated, *, P<0.05.
Circles in FIG. 6B and FIG. 6C correspond to the same tumors
showing slower growth kinetics (FIG. 6B) and higher apoptosis (FIG.
6C). FIG. 6D, quantification of in-tumor mitotic bodies (left), and
apoptotic bodies (right) from FFPE sections stained with H&E.
Average values of at least three fields per mouse were evaluated.
P-values are indicated, ***, P<0.001. FIG. 6E, the number of
in-tumor telomere damage (TIFs) were counted and expressed as
percent of cells with TIFs. Vehicle, indicates tumor from mice
treated with DMSO-PBS and 6-thio-dG, indicates tumor from mice
treated with 6-thio-dG. Error bars were generated from standard
deviation of at least three fields (50 cells or more per field) per
mouse. P-value is indicated, ***, P<0.001. On the right,
representative images of TIFs analysis in 6-thio-dG and vehicle
treated tumors. Telomeres are in red, 53BP1 (green), and DAPI
(blue). White arrows indicate TIFs (yellow). FIG. 6F, diagrammatic
workflow showing detection of CCHMC-DIPG-1 orthotopic xenograft by
luminescence imaging followed by resection and histological
staining with H&E and Ki67 of collected tumors and matched
normal tissue. Tumor and normal tissues are indicated. Circles
indicate the tumor location in the brain. FIG. 6G, representative
images of tissue IF-TIF analysis in 6-thio-dG and vehicle treated
tumor. White arrows indicate TIFs (yellow), telomeres (red), and
53BP1 (green). DAPI (blue). On the right, quantification of TIFs.
Error bars were generated from standard deviation of at least three
fields (50 cells or more per field) counted per mouse. Significance
between vehicle and 6-thio-dG treated tumors is indicated by
P-value, **, P<0.01.
[0015] FIG. 7. Telomerase activity in pediatric brain tumor cells.
MB004, R0315-GBM, SU-DIPG-VI, CCHMC-DIPG-1, D-458, D-425, in normal
diploid fibroblast cells HFF and HFF+hTERT, and in osteosarcoma
cells Saos-2 and U2OS. (-) C, (+) C,and IC indicate negative
control, positive control, and internal control respectively.
Telomerase products are indicated.
[0016] FIGS. 8A-D. FIG. 8A, Telomerase activity in HFF+hTERT cells
treated with DMSO, 6-thio-dG (3 .mu.M for 3 days), and imetelstat
(IMT; 2 .mu.M). For combination treatment, cells were pre-treated
with IMT for 3 days and then with 6-thio-dG for another 3 days. (-)
C, (+) C, and IC indicate negative control, positive control, and
internal control, respectively. Telomerase products are indicated.
FIGS. 8B-C, effect of telomerase inhibition on cell growth of HFF
(B) and HFF+hTERT (C) cells treated with Imetelstat (IMT; 2 .mu.M)
only, 6-thio-dG (3 .mu.M) only, or 6-thio-dG in combination with
Imetelstat as described in A. Error bars represent the standard
deviation generated from triplicates. P-value, **, P<0.01. FIG.
8D, quantifications showing percent cells with TIFs n FF+hTERT
cells treated with Imetelstat (IMT; 2 .mu.M) only, 6-thio-dG (3
.mu.M for 3 days), or in combination with Imetelstat as described
in FIG. S2A. Cells counted for each treatment (n=.about.50 cells).
Bottom panels show representative images showing TRF2 (green),
.gamma.H2AX (red), and TIFs (yellow) indicated by white arrows.
DAPI (blue) indicates nucleus staining.
[0017] FIGS. 9A-B. FIG. 9A, quantification of genomic damage
(.gamma.H2AX foci) in TIFs-negative (non-TIF) and TIFs-positive
(TIF) HFF+hTERT cells treated or untreated (DMSO) with 3 mM of
6-thio-dG for 3 days. Error bars represent the standard deviation
from three independent experiments. P-value is indicated,
***<0.001. FIG. 9B, quantification of .gamma.H2AX foci in
non-TIF and TIFs-positive HFF+hTERT cells treated with DMSO,
Imetelstat (IMT; 2 .mu.M), 6-thio-dG (3 .mu.M for 3 days), or in
combination as described in FIG. S2A.
[0018] FIGS. 10A-D. FIG. 10A, cell growth kinetics of HFF,
HFF+hTERT, Saos-2, and U2OS cells. FIG. 10B, relative cell growth.
FIG. 10C, percent viability of HFF+hTERT and U2OS cells treated
with 3 mM 6-thio-dG for 3 days followed by 10-days post-drug
removal. FIG. 10D, agarose gel electrophoresis (0.7%) of genomic
DNA extracted from HFF+hTERT (lanes 2,3), U2OS (lanes 4,5), and HFF
(lanes 6,7) cells treated with DMSO or 3 .mu.M 6-thio-dG treated
for 3 days. Lane 1, molecular weight marker, lambda phage
DNA/HindIII, is indicated. Gel was run two independent times.
[0019] FIG. 11. Survival probability of 6-thio-dG treated mice with
MB004 tumors at a volume of 1500 mm.sup.3. Average values of 6 mice
per group have been considered and significance between two groups
is indicated by P-value, **, P<0.01.
[0020] FIGS. 12A-B. FIG. 12A, Telomerase activity in mouse normal
brain, orthotopic PDXDIPG tumor, and patient-derived DIPG-1 cells
(CCHMC-DIPG-1), along with negative and positive controls for TRAP
assay. IC, indicates internal control. Telomerase products are
indicated. FIG. 12B, Telomere FISH-IF images of orthotopic DIPG
tumor (PDX) and the matched normal mouse brain. TIFs (yellow) are
indicated by white arrows, telomeres (TelC; red) and 53BP1 (green).
DAPI (blue) indicates nucleus staining. The two images are from the
brain of the same mouse. Of note, mouse telomeres are longer than
human, hence the strong telomere signals (in red) in mouse brain
relative to human DIPG tumor.
DETAILED DESCRIPTION
[0021] As discussed above, current approaches to treating pediatric
brain cancer that involve shortening of telomeres has proven
problematic. Given the role played by telomerase reactivation in
oncogenesis, telomeres and telomerase remain relevant therapeutic
targets in this patient population (14-16). Recently, preclinical
studies validated a telomere targeting strategy consisting of the
incorporation of 6-thio-2'-deoxyguanosine (6-thio-dG), a telomerase
substrate precursor nucleoside analog, into telomeres by telomerase
(17). Mender et al. have shown that telomerase-dependent
incorporation of 6-thiodG into telomeres is very effective and
specific at targeting telomerase-positive cancer cells but not
telomerase silent normal cells (17). Treatment with 6-thio-dG led
to telomere damage and cell death in telomerase-positive cancer
cell lines. Since this effect appears to be telomere-length
independent, the prediction using this novel approach is that
treatment with 6-thio-dG will require a shorter time period to
achieve a rapid effect on tumor growth and progression than direct
telomerase inhibition-based therapy (18). This approach could be
beneficial for patients with aggressive brain tumors such as DIPG.
In the present study, the inventors tested the in vitro and in vivo
effect of 6-thio-dG in telomerase-positive stem-like cells derived
from poor-prognosis pediatric brain tumors and addressed the
mechanistic aspect of 6-thio-dG-induced DNA damage response in
telomerase-positive cancer and normal cells. These findings suggest
that 6-thio-dG is a promising novel approach to treat
therapy-resistant pediatric brain tumors and provide a rationale
for clinical testing of 6-thio-dG in children with brain
tumors.
[0022] These and other aspects of the disclosure are described in
detail below.
I. TELOMERES, TELOMERASE AND TELOMERE DYSFUNCTION
[0023] During mitosis, cells make copies of their genetic material.
Half of the genetic material goes to each new daughter cell. To
make sure that information is successfully passed from one
generation to the next, each chromosome has a special protective
cap called a telomere located at the end of its "arms." Telomeres
are controlled by the presence of the enzyme telomerase.
[0024] A telomere is a repeating DNA sequence (for example, TTAGGG)
at the end of the body's chromosomes. The telomere can reach a
length of 15,000 base pairs. Telomeres function by preventing
chromosomes from losing base pair sequences at their ends. They
also stop chromosomes from fusing to each other. However, each time
a cell divides, some of the telomere is lost (usually 25-200 base
pairs per division). When the telomere becomes too short, the
chromosome reaches a "critical length" and can no longer replicate.
This means that a cell becomes old and dies by a process called
apoptosis or undergoes senescence. Telomere activity is controlled
by two mechanisms: erosion and addition. Erosion, as mentioned,
occurs each time a cell divides due to the failure of lagging
strand DNA synthesis to be completed all the way to the end.
Addition is determined by the activity of telomerase.
[0025] Telomerase, also called telomere terminal transferase, is an
enzyme made of protein and RNA subunits that elongates chromosomes
by adding TTAGGG sequences to the end of existing chromosomes.
Telomerase is found in fetal tissues, adult germ cells, and also
tumor cells. Telomerase activity is regulated during development
and has a very low, almost undetectable activity in somatic (body)
cells. Because these somatic cells do not regularly use telomerase,
they age. The result of aging cells is an aging body. If telomerase
is activated in a cell, the cell will continue to grow and divide.
This "immortal cell" theory is important in two areas of research:
aging and cancer.
[0026] Cellular aging, or senescence, is the process by which a
cell becomes old and stops growing or dies. It is due to the
shortening of chromosomal telomeres to the point that the
chromosome reaches a critical length. Cellular aging is analogous
to a wind-up clock. If the clock stays wound, a cell becomes
immortal and constantly produces new cells. If the clock winds
down, the cell stops producing new cells and undergoes what is
termed replicative senescence or dies. Cells are constantly aging.
Being able to make the body's cells extend their replication
ability certainly creates some exciting possibilities especially
for disease associated with genetic inheritance of short telomeres
(termed telomeropathies or telomere spectrum disorders). Telomerase
research could therefore yield important discoveries related to the
aging process.
[0027] Cancer cells have escaped the normal short telomere aging
phenomenon and become malignant cells. The malignant cells multiply
until they form a tumor that grows uncontrollably and spreads to
distant tissue throughout the human body. Telomerase has been
detected in almost all human cancer cells. This provides a
selective growth advantage to many types of tumors. If telomerase
activity was to be turned off, then telomeres in cancer cells would
progressively shorten, just like they do in normal body cells. This
would prevent the cancer cells from dividing uncontrollably in
their early stages of development. In the event that a tumor has
already thoroughly developed, it may be removed and anti-telomerase
therapy could be administered to prevent relapse. In essence,
preventing telomerase from performing its function would change
cancer cells from immortal to mortal. However, direct telomerase
inhibitors require a lag period from initiation of treatment until
tumor shrinkage occurs and have not progressed well in clinical
development due to increased toxicities. Thus, the present
invention provides methods to reduce the lag period but require
telomerase activity to be effective and potentially reduce side
effects.
II. TREATING BRAIN CANCER
[0028] In accordance with the present disclosure, 6-thio-dG can be
employed to treat a variety of cancer types. In general, brain,
melanomas, lung cancers, pancreatic cancers and ovarian cancers.
However, more generally, tumors expressing telomerase, including
those having TERT promoter mutations and enriched telomere
transcription signatures (e.g., a telomere maintenance signature
and/or a packaging of telomere ends signature). Moreover, a variety
of therapy-resistant cancers are responsive to 6-thio-dG
therapy.
[0029] A. Brain Cancer
[0030] In particular, the present disclosure focuses on brain
cancers, more particularly pediatric brain cancer, and in
particular drug-resistant pediatric brain cancers. In the United
States more than 28,000 people under 20 are estimated to have a
brain tumor. About 3,720 new cases of brain tumors are expected to
be diagnosed in those under 15 in 2019. Higher rates were reported
in 1985-1994 than in 1975-1983. There is some debate as to the
reasons; one theory is that the trend is the result of improved
diagnosis and reporting, since the jump occurred at the same time
that MRIs became available widely, and there was no coincident jump
in mortality.
[0031] The average survival rate for all primary brain cancers in
children is 74%. Brain cancers are the most common cancer in
children under 19, and result in more death in this group than
leukemia. Younger people do less well. The most common brain tumor
types in children (0-14) are: pilocytic astrocytoma, malignant
glioma, medulloblastoma, neuronal and mixed neuronal-glial tumors,
and ependymoma.
[0032] In children under 2, about 70% of brain tumors are
medulloblastomas, ependymomas, and low-grade gliomas. Less
commonly, and seen usually in infants, are teratomas and atypical
teratoid rhabdoid tumors. Germ cell tumors, including teratomas,
make up just 3% of pediatric primary brain tumors, but the
worldwide incidence varies significantly.
[0033] A brain tumor occurs when abnormal cells form within the
brain. There are two main types of tumors: malignant or cancerous
tumors and benign tumors. Cancerous tumors can be divided into
primary tumors, which start within the brain, and secondary tumors,
which have spread from elsewhere, known as brain metastasis tumors.
All types of brain tumors may produce symptoms that vary depending
on the part of the brain involved. These symptoms may include
headaches, seizures, problems with vision, vomiting and mental
changes. The headache is classically worse in the morning and goes
away with vomiting. Other symptoms may include difficulty walking,
speaking or with sensations. As the disease progresses,
unconsciousness may occur.
[0034] The cause of most brain tumors is unknown. Uncommon risk
factors include inherited neurofibromatosis, exposure to vinyl
chloride, Epstein-Barr virus and ionizing radiation. The evidence
for mobile phone exposure is not clear. The most common types of
primary tumors in adults are meningiomas (usually benign) and
astrocytomas such as glioblastomas. In children, the most common
type is a malignant medulloblastoma. Diagnosis is usually by
medical examination along with computed tomography or magnetic
resonance imaging. The result is then often confirmed by a biopsy.
Based on the findings, the tumors are divided into different grades
of severity.
[0035] Treatment may include some combination of surgery, radiation
therapy and chemotherapy. Anticonvulsant medication may be needed
if seizures occur. Dexamethasone and furosemide may be used to
decrease swelling around the tumor. Some tumors grow gradually,
requiring only monitoring and possibly needing no further
intervention. Treatments that use a person's immune system are
being studied. Outcome varies considerably depending on the type of
tumor and how far it has spread at diagnosis. Glioblastomas usually
have poor outcomes, while meningiomas usually have good outcomes.
The average five-year survival rate for all brain cancers in the
United States is 33%.
[0036] Secondary, or metastatic, brain tumors are more common than
primary brain tumors, with about half of metastases coming from
lung cancer. Primary brain tumors occur in around 250,000 people a
year globally, making up less than 2% of cancers. In children
younger than 15, brain tumors are second only to acute
lymphoblastic leukemia as the most common form of cancer. In
Australia, the average lifetime economic cost of a case of brain
cancer is $1.9 million, the greatest of any type of cancer.
[0037] The brain is divided into four lobes and each lobe or area
has its own function. A tumor in any of these lobes may affect the
area's performance. The location of the tumor is often linked to
the symptoms experienced but each person may experience something
different. [0038] Frontal lobe tumors may contribute to poor
reasoning, inappropriate social behavior, personality changes, poor
planning, lower inhibition, and decreased production of speech
(Broca's area). [0039] Temporal lobe tumors may contribute to poor
memory, loss of hearing, difficulty in language comprehension
(Wernicke's area). [0040] Parietal lobe tumors may result in poor
interpretation of languages and difficulty speaking, difficulty
writing, drawing, naming, and recognizing, and poor spatial and
visual perception. [0041] Occipital lobe tumors may result in poor
or loss of vision. [0042] Cerebellum tumors may cause poor balance,
muscle movement, and posture. [0043] Brain stem tumors can cause
seizures, induce endocrine problems, respiratory changes, visual
changes, headaches and partial paralysis. Human brains are
surrounded by a system of connective tissue membranes called
meninges that separate the brain from the skull. This three-layered
covering is composed of (from the outside in) the dura mater ("hard
mother"), arachnoid mater ("spidery mother"), and pia mater
("tender mother"). The arachnoid and pia are physically connected
and thus often considered as a single layer, the pia-arachnoid, or
leptomeninges. Between the arachnoid mater and the pia mater is the
subarachnoid space which contains cerebrospinal fluid (CSF). This
fluid circulates in the narrow spaces between cells and through the
cavities in the brain called ventricles, to nourish, support, and
protect the brain tissue. Blood vessels enter the central nervous
system through the perivascular space above the pia mater. The
cells in the blood vessel walls are joined tightly, forming the
blood-brain barrier which protects the brain from toxins that might
enter through the blood. Tumors of the meninges are meningiomas and
are often benign.
[0044] The brains of humans and other vertebrates are composed of
very soft tissue and have a gelatin-like texture. Living brain
tissue has a pink tint in color on the outside (gray matter), and
nearly complete white on the inside (white matter), with subtle
variations in color. Three separate brain areas make up most of the
brain's volume: [0045] telencephalon (cerebral hemispheres or
cerebrum) [0046] mesencephalon (midbrain) [0047] cerebellum These
areas are composed of two broad classes of cells: neurons and glia.
These two types are equally numerous in the brain as a whole,
although glial cells outnumber neurons roughly 4 to 1 in the
cerebral cortex. Glia come in several types, which perform a number
of critical functions, including structural support, metabolic
support, insulation, and guidance of development. Primary tumors of
the glial cells are called gliomas and often are malignant by the
time they are diagnosed.
[0048] The pons in the brainstem is a specific region that consists
of myelinated axons much like the spinal cord. The thalamus and
hypothalamus of the diencephalon also consist of neuron and glial
cell tissue with the hypophysis (pituitary gland) and pineal gland
(which is glandular tissue) attached at the bottom; tumors of the
pituitary and pineal gland are often benign. The medulla oblongata
is at the start of the spinal cord and is composed mainly of neuron
tissue enveloped in oligodendrocytes and meninges tissue. The
spinal cord is made up of bundles of these axons. Glial cells such
as Schwann cells in the periphery or, within the cord itself,
oligodendrocytes, wrap themselves around the axon, thus promoting
faster transmission of electrical signals and also providing for
general maintenance of the environment surrounding the cord, in
part by shuttling different compounds around in response to injury
or other stimulus.
[0049] Although there is no specific or singular symptom or sign,
the presence of a combination of symptoms and the lack of
corresponding indications of other causes can be an indicator for
investigation towards the possibility of an brain tumor. Brain
tumors have similar characteristics and obstacles when it comes to
diagnosis and therapy with tumors located elsewhere in the body.
However, they create specific issues that follow closely to the
properties of the organ they are in.
[0050] The diagnosis will often start by taking a medical history
noting medical antecedents, and current symptoms. Clinical and
laboratory investigations will serve to exclude infections as the
cause of the symptoms. Examinations in this stage may include the
eyes, otolaryngological (or ENT) and electrophysiological exams.
The use of electroencephalography (EEG) often plays a role in the
diagnosis of brain tumors.
[0051] Brain tumors, when compared to tumors in other areas of the
body, pose a challenge for diagnosis. Commonly, radioactive tracers
are uptaken in large volumes in tumors due to the high activity of
tumor cells, allowing for radioactive imaging of the tumor.
However, most of the brain is separated from the blood by the
blood-brain barrier (BBB), a membrane which exerts a strict control
over what substances are allowed to pass into the brain. Therefore,
many tracers that may reach tumors in other areas of the body
easily would be unable to reach brain tumors until there was a
disruption of the BBB by the tumor. Disruption of the BBB is well
imaged via MRI or CT scan, and is therefore regarded as the main
diagnostic indicator for malignant gliomas, meningiomas, and brain
metastases.
[0052] Swelling or obstruction of the passage of cerebrospinal
fluid (CSF) from the brain may cause (early) signs of increased
intracranial pressure which translates clinically into headaches,
vomiting, or an altered state of consciousness, and in children
changes to the diameter of the skull and bulging of the
fontanelles. More complex symptoms such as endocrine dysfunctions
should alarm doctors not to exclude brain tumors.
[0053] A bilateral temporal visual field defect (due to compression
of the optic chiasm) or dilation of the pupil, and the occurrence
of either slowly evolving or the sudden onset of focal neurologic
symptoms, such as cognitive and behavioral impairment (including
impaired judgment, memory loss, lack of recognition, spatial
orientation disorders), personality or emotional changes,
hemiparesis, hypoesthesia, aphasia, ataxia, visual field
impairment, impaired sense of smell, impaired hearing, facial
paralysis, double vision, or more severe symptoms such as tremors,
paralysis on one side of the body hemiplegia, or (epileptic)
seizures in a patient with a negative history for epilepsy, should
raise the possibility of a brain tumor.
[0054] Tumors can be benign or malignant, can occur in different
parts of the brain, and may be primary or secondary. A primary
tumor is one that has started in the brain, as opposed to a
metastatic tumor, which is something that has spread to the brain
from another part of the body. The incidence of metastatic tumors
are more prevalent than primary tumors by 4:1. Tumors may or may
not be symptomatic: some tumors are discovered because the patient
has symptoms, others show up incidentally on an imaging scan, or at
an autopsy.
[0055] The most common primary brain tumors are: [0056] Gliomas
(50.4%) [0057] Meningiomas (20.8%) [0058] Pituitary adenomas (15%)
[0059] Nerve sheath tumors (8%) Other types include Anaplastic
astrocytoma, Astrocytoma, Central neurocytoma, Choroid plexus
carcinoma, Choroid plexus papilloma, Choroid plexus tumor,
Dysembryoplastic neuroepithelial tumour, Ependymal tumor,
Fibrillary astrocytoma, Giant-cell glioblastoma, Glioblastoma
multiforme, Gliomatosis cerebri, Gliosarcoma, Hemangiopericytoma,
Medulloblastoma, Medulloepithelioma, Meningeal carcinomatosis,
Neuroblastoma, Neurocytoma, Oligoastrocytoma, Oligodendroglioma,
Optic nerve sheath meningioma, Pediatric ependymoma, Pilocytic
astrocytoma, Pinealoblastoma, Pineocytoma, Pleomorphic anaplastic
neuroblastoma, Pleomorphic xanthoastrocytoma, Primary central
nervous system lymphoma, Sphenoid wing meningioma, Subependymal
giant cell astrocytoma, Subependymoma, Trilateral
retinoblastoma.
[0060] A medical team generally assesses the treatment options and
presented to the person affect and their family. Various types of
treatment are available depending on neoplasm type and location and
may be combined to give the best chances of survival (discussed in
greater detail in Section IV on Combination Therapies): [0061]
Surgery: complete or partial resection of the tumor with the
objective of removing as many tumor cells as possible. [0062]
Radiotherapy: the most commonly used treatment for brain tumors;
the tumor is irradiated with beta, x rays or gamma rays. [0063]
Chemotherapy: is a treatment option for cancer, however, it is not
always used to treat brain tumors as the blood-brain barrier can
prevent some drugs from reaching the cancerous cells. A variety of
experimental therapies are available through clinical trials.
Survival rates in primary brain tumors depend on the type of tumor,
age, functional status of the patient, the extent of surgical tumor
removal and other factors specific to each case.
[0064] B. Telomerase Positive Cancers
[0065] Telomerase-positive cancers are far more susceptible to the
methods of the present disclosure than are telomerase-negative
cancers. Therefore, testing a biopsy to determine whether the
cancer is or is not telomerase-positive is highly useful.
[0066] The most common methods for detecting telomerase activity
are telomeric repeat amplification protocols (TRAPs), which allow
one to perform semi-quantitative and quantitative analyses, using
some of their modifications (called ddTRAP for droplet digital
TRAP). Among these modifications are the scintillation proximity
assay, hybridization protection assay, transcription amplification
assay, and the magnetic bead-based extraction assay.
[0067] The telomeric repeat amplification protocol can be
subdivided into three main stages: primer elongation, amplification
of telomerase-synthesized DNA, and finally its detection. At the
elongation stage, telomeric repeats are added to the
telomere-imitating oligonucleotide by telomerase present in the
cell extract. PCR-amplification of telomerase-synthesized DNA is
carried out with telomere-imitating and reverse primers. Different
labels can be incorporated into the telomerase-synthesized DNA.
This stage is then followed by detection (e.g., electrophoretic
separation and imaging of PCR products).
[0068] Still other methods involve the quantitative isolation of
telomerase, and the subsequent measurement of the overall activity
of the telomerase from a given cell quantity, which can be compared
to appropriate standards. A wide variety of labeling and detection
methodologies can be employed once telomerase has been isolated and
tested in vitro.
[0069] C. Drug Resistant Cancers
[0070] Antineoplastic resistance, often used interchangeably with
chemotherapy resistance, is the resistance of neoplastic
(cancerous) cells, or the ability of cancer cells to survive and
grow despite anti-cancer therapies. In some cases, cancers can
evolve resistance to multiple drugs, called multiple drug
resistance.
[0071] There are two general causes of antineoplastic therapy
failure: Inherent genetic characteristics, giving cancer cells
their resistance and acquired resistance after drug exposure, which
is rooted in the concept of cancer cell heterogeneity.
Characteristics of resistant cells include altered membrane
transport, enhanced DNA repair, apoptotic pathway defects,
alteration of target molecules, protein and pathway mechanisms,
such as enzymatic deactivation. Since cancer is a genetic disease,
two genomic events underlie acquired drug resistance: Genome
alterations (e.g., gene amplification and deletion) and epigenetic
modifications. Cancer cells are constantly using a variety of
tools, involving genes, proteins, and altered pathways, to ensure
their survival against antineoplastic drugs.
[0072] Antineoplastic resistance, synonymous with chemotherapy
resistance, is the ability of cancer cells to survive and grow
despite different anti-cancer therapies, i.e. their multiple drug
resistance. There are two general causes of antineoplastic therapy
failure: (i) inherent resistance, such as genetic characteristics,
giving cancer cells their resistance from the beginning, which is
rooted in the concept of cancer cell heterogeneity; and (ii)
acquired resistance after drug exposure.
[0073] Since cancer is a genetic disease, two genomic events
underlie these mechanisms of acquired drug resistance: Genome
alterations (e.g., gene amplification and deletion) and epigenetic
modifications.
[0074] Chromosomal rearrangement due to genome instability can
cause gene amplification and deletion. Gene amplification is the
increase in copy number of a region of a chromosome. which occur
frequently in solid tumors, and can contribute to tumor evolution
through altered gene expression.
[0075] Hamster cell research in 1993 showed that amplifications in
the DHFR gene involved in DNA synthesis began with chromosome break
in below the gene, and subsequent cycles of bridge-breakage-fusion
formations result in large intrachromosomal repeats. The over
amplification of oncogenes can occur in response to chemotherapy,
thought to be the underlying mechanism in several classes of
resistance. For example, DHFR amplification occurs in response to
methotrexate, TYMS (involved in DNA synthesis) amplification occurs
in response to 5-fluorouracil, and BCR-ABL amplification occurs in
response to imatinib mesylate. Determining areas of gene
amplification in cells from cancer patients has huge clinical
implications. Gene deletion is the opposite of gene amplification,
where a region of a chromosome is lost and drug resistance occurs
by losing tumor suppressor genes such as TP53.
[0076] Genomic instability can occur when the replication fork is
disturbed or stalled in its migration. This can occur with
replication fork barriers, proteins such as PTIP, CHD4 and PARP1,
which are normally cleared by the cell's DNA damage sensors,
surveyors, and responders BRCA1 and BRCA2.
[0077] Epigenetic modifications in antineoplastic drug resistance
play a major role in cancer development and drug resistance as they
contribute to the regulation of gene expression. Two main types of
epigenetic control are DNA methylation and histone
methylation/acetylation. DNA methylation is the process of adding
methyl groups to DNA, usually in the upstream promoter regions,
which stops DNA transcription at the region and effectively
silences individual genes. Histone modifications, such as
deacetylation, alters chromatin formation and silence large
chromosomal regions. In cancer cells, where normal regulation of
gene expression breaks down, the oncogenes are activated via
hypomethylation and tumor suppressors are silenced via
hypermethylation. Similarly, in drug resistance development, it has
been suggested that epigenetic modifications can result in the
activation and overexpression of pro-drug resistance genes.
[0078] Studies on cancer cell lines have shown that hypomethylation
(loss of methylation) of the MDR1 gene promoter caused
overexpression and the multidrug resistance.
[0079] In a methotrexate resistant breast cancer cell lines without
drug uptake and folate carrier expression, giving DAC, a DNA
methylation inhibitor, improved drug uptake and folate carrier
expression.
[0080] Acquired resistance to the alkylating drug fotemustine in
melanoma cell showed high MGMT activity related to the
hypermethylation of the MGMT gene exons.
[0081] In Imatinib resistant cell lines, silencing of the SOCS-3
gene via methylation has been shown to cause STAT3 protein
activation, which caused uncontrolled proliferation.
[0082] Cancer cells can become resistant to multiple drugs by
altered membrane transport, enhanced DNA repair, apoptotic pathway
defects, alteration of target molecules, protein and pathway
mechanisms, such as enzymatic deactivation.
[0083] Many classes of antineoplastic drugs act on intracellular
components and pathways, like DNA, nuclear components, meaning that
they need to enter the cancer cells. The p-glycoprotein (P-gp), or
the multiple drug resistance protein, is a phosphorylated and
glycosylated membrane transporter that can shuttle drugs out of the
cell, thereby decreasing or ablating drug efficacy. This
transporter protein is encoded by the MDR1 gene and is also called
the ATP-binding cassette (ABC) protein. MDR1 has promiscuous
substrate specificity, allowing it to transport many structurally
diverse compounds across the cell membrane, mainly hydrophobic
compounds. Studies have found that the MDR1 gene can be activated
and overexpressed in response to pharmaceutical drugs, thus forming
the basis for resistance to many drugs. Overexpression of the MDR1
gene in cancer cells is used to keep intracellular levels of
antineoplastic drugs below cell-killing levels.
[0084] For example, the antibiotic rifampicin has been found to
induce MDR1 expression. Experiments in different drug resistant
cell lines and patient DNA revealed gene rearrangements which had
initiated the activation or overexpression of MDR1. A C3435T
polymorphism in exon 226 of MDR1 has also been strongly correlated
with p-glycoprotein activities.
[0085] MDR1 is activated through NF-.kappa.B, a protein complex
which acts as a transcription factor. In the rat, an NF-.kappa.B
binding site is adjacent to the mdr1b gene, NF-.kappa.B can be
active in tumour cells because its mutated NF-.kappa.B gene or its
inhibitory I.kappa.B gene mutated under chemotherapy. In colorectal
cancer cells, inhibition of NF-.kappa.B or MDR1 caused increased
apoptosis in response to a chemotherapeutic agent.
[0086] Enhanced DNA repair plays an important role in the ability
for cancer cells to overcome drug-induced DNA damages.
[0087] Platinum-based chemotherapies, such as cisplatin, target
tumor cells by cross-linking their DNA strands, causing mutation
and damage. Such damage will trigger programmed cell death (e.g.,
apoptosis) in cancer cells. Cisplatin resistance occurs when cancer
cells develop an enhanced ability to reverse such damage by
removing the cisplatin from DNA and repairing any damage done. The
cisplatin-resistant cells upregulate expression of the excision
repair cross-complementing (ERCC1) gene and protein.
[0088] Some chemotherapies are alkylating agents meaning they
attach an alkyl group to DNA to stop it from being read.
06-methylguanine DNA methyltransferase (MGMT) is a DNA repair
enzyme which removes alkyl groups from DNA. MGMT expression is
upregulated in many cancer cells, which protects them from
alkylating agents. Increased MGMT expression has been found in
colon cancer, lung cancer, non-Hodgkin's lymphoma, breast cancer,
gliomas, myeloma and pancreatic cancer.
[0089] TP53 is a tumor suppressor gene encoding the p53 protein,
which responds to DNA damage either by DNA repair, cell cycle
arrest, or apoptosis. Losing TP53 via gene deletion can allow cells
to continuously replicate despite DNA damage. The tolerance of DNA
damage can grant cancer cells a method of resistance to those drugs
which normally induce apoptosis through DNA damage.
[0090] Other genes involved in the apoptotic pathway related drug
resistance include h-ras and bcl-2/bax. Oncogenic h-ras has been
found to increase expression of ERCC1, resulting in enhanced DNA
repair (see above). Inhibition of h-ras was found to increase
cisplatin sensitivity in glioblastoma cells. Upregulated expression
of Bcl-2 in leukemic cells (non-Hodgkin's lymphoma) resulted in
decreased levels of apoptosis in response to chemotherapeutic
agents, as Bcl-2 is a pro-survival oncogene.
[0091] During targeted therapy, oftentimes the target has modified
itself and decreased its expression to the point that therapy is no
longer effective. One example of this is the loss of estrogen
receptor (ER) and progesterone receptor (PR) upon anti-estrogen
treatment of breast cancer. Tumors with loss of ER and PR no longer
respond to tamoxifen or other anti-estrogen treatments, and while
cancer cells remain somewhat responsive to estrogen synthesis
inhibitors, they eventually become unresponsive to endocrine
manipulation and no longer dependent on estrogen for growth.
[0092] Another line of therapeutics used for treating breast cancer
is targeting of kinases like human epidermal growth factor receptor
2 (HER2) from the EGFR family. Mutations often occur in the HER2
gene upon treatment with an inhibitor, with about 50% of patients
with lung cancer found to have an EGFR-T790M gatekeeper
mutation.
[0093] Treatment of chronic myeloid leukemia (CML) involves a
tyrosine kinase inhibitor that targets the BCR/ABL fusion gene
called imatinib. In some people resistant to Imatinib, the BCR/ABL
gene is reactivated or amplified, or a single point mutation has
occurred on the gene. These point mutations enhance
autophosphorylation of the BCR-ABL protein, resulting in the
stabilization of the ATP-binding site into its active form, which
cannot be bound by imatinib for proper drug activation.
[0094] Topoisomerase is a lucrative target for cancer therapy due
to its critical role as an enzyme in DNA replication, and many
topoisomerase inhibitors have been made. Resistance can occur when
topoisomerase levels are decreased, or when different isoforms of
topoisomerase are differentially distributed within the cell.
Mutant enzymes have also been reported in patient leukemic cells,
as well as mutations in other cancers that confer resistance to
topoisomerase inhibitors.
[0095] One of the mechanisms of antineoplastic resistance is
over-expression of drug-metabolizing enzymes or carrier molecules.
By increasing expression of metabolic enzymes, drugs are more
rapidly converted to drug conjugates or inactive forms that can
then be excreted. For example, increased expression of glutathione
promotes drug resistance, as the electrophilic properties of
glutathione allow it to react with cytotoxic agents, inactivating
them. In some cases, decreased expression or loss of expression of
drug-metabolizing enzymes confers resistance, as the enzymes are
needed to process a drug from an inactive form to an active form.
Arabinoside, a commonly used chemotherapy for leukemia and
lymphomas, is converted into cytosine arabinoside triphosphate by
deoxycytidine kinase. Mutation of deoxycytidine kinase or loss of
expression results in resistance to arabinoside. This is a form of
enzymatic deactivation.
[0096] Growth factor expression levels can also promote resistance
to antineoplastic therapies. In breast cancer, drug resistant cells
were found to express high levels of IL-6, while sensitive cells
did not express significant levels of the growth factor. IL-6
activates the CCAAT enhancer-binding protein transcription factors
which activate MDR1 gene expression.
III. PHARMACEUTICAL FORMULATIONS AND ROUTES OF ADMINISTRATION
[0097] Where clinical applications are contemplated, pharmaceutical
compositions will be prepared in a form appropriate for the
intended application. Generally, this will entail preparing
compositions that are essentially free of pyrogens, as well as
other impurities that could be harmful to humans or animals.
[0098] One will generally desire to employ appropriate salts and
buffers to render drugs stable and allow for uptake by target
cells. Aqueous compositions of the present disclosure comprise an
effective amount of the drug dissolved or dispersed in a
pharmaceutically acceptable carrier or aqueous medium. The phrase
"pharmaceutically or pharmacologically acceptable" refer to
molecular entities and compositions that do not produce adverse,
allergic, or other untoward reactions when administered to an
animal or a human. As used herein, "pharmaceutically acceptable
carrier" includes solvents, buffers, solutions, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents and the like acceptable for use in
formulating pharmaceuticals, such as pharmaceuticals suitable for
administration to humans. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredients of the present disclosure, its use in
therapeutic compositions is contemplated. Supplementary active
ingredients also can be incorporated into the compositions,
provided they do not inactivate the agents of the compositions.
[0099] The active compositions of the present disclosure may
include classic pharmaceutical preparations. Administration of
these compositions according to the present disclosure may be via
any common route so long as the target tissue is available via that
route, but generally including systemic administration. This
includes oral, nasal, or buccal. Alternatively, administration may
be by intradermal, subcutaneous, intramuscular, intraperitoneal or
intravenous injection, or intratumoral or regional to a tumor, such
as in the tumor vasculature. Such compositions would normally be
administered as pharmaceutically acceptable compositions, as
described supra.
[0100] The active compounds may also be administered parenterally
or intraperitoneally. By way of illustration, solutions of the
active compounds as free base or pharmacologically acceptable salts
can be prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations generally contain a preservative to prevent the growth
of microorganisms.
[0101] The pharmaceutical forms suitable for injectable use
include, for example, sterile aqueous solutions or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions. Generally, these preparations
are sterile and fluid to the extent that easy injectability exists.
Preparations should be stable under the conditions of manufacture
and storage and should be preserved against the contaminating
action of microorganisms, such as bacteria and fungi. Appropriate
solvents or dispersion media may contain, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, and
liquid polyethylene glycol, and the like), suitable mixtures
thereof, and vegetable oils. The proper fluidity can be maintained,
for example, by the use of a coating, such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. The prevention of the action of
microorganisms can be brought about by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
[0102] Sterile injectable solutions may be prepared by
incorporating the active compounds in an appropriate amount into a
solvent along with any other ingredients (for example as enumerated
above) as desired, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the desired other ingredients, e.g., as
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation include vacuum-drying and freeze-drying techniques
which yield a powder of the active ingredient(s) plus any
additional desired ingredient from a previously sterile-filtered
solution thereof.
[0103] The compositions of the present disclosure generally may be
formulated in a neutral or salt form. Pharmaceutically-acceptable
salts include, for example, acid addition salts (formed with the
free amino groups of the protein) derived from inorganic acids
(e.g., hydrochloric or phosphoric acids, or from organic acids
(e.g., acetic, oxalic, tartaric, mandelic, and the like. Salts
formed with the free carboxyl groups of the protein can also be
derived from inorganic bases (e.g., sodium, potassium, ammonium,
calcium, or ferric hydroxides) or from organic bases (e.g.,
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0104] Upon formulation, solutions are preferably administered in a
manner compatible with the dosage formulation and in such amount as
is therapeutically effective. The formulations may easily be
administered in a variety of dosage forms such as injectable
solutions, drug release capsules and the like. For parenteral
administration in an aqueous solution, for example, the solution
generally is suitably buffered and the liquid diluent first
rendered isotonic for example with sufficient saline or glucose.
Such aqueous solutions may be used, for example, for intravenous,
intramuscular, subcutaneous and intraperitoneal administration.
Preferably, sterile aqueous media are employed as is known to those
of skill in the art, particularly in light of the present
disclosure. By way of illustration, a single dose may be dissolved
in 1 ml of isotonic NaCl solution and either added to 1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, "Remington's Pharmaceutical Sciences" 15th
Edition, pages 1035-1038 and 1570-1580). Some variation in dosage
will necessarily occur depending on the condition of the subject
being treated. The person responsible for administration will, in
any event, determine the appropriate dose for the individual
subject. Moreover, for human administration, preparations should
meet sterility, pyrogenicity, general safety and purity standards
as required by FDA Office of Biologics standards.
IV. COMBINED THERAPY
[0105] In the context of the present disclosure, it also is
contemplated 6-thio-dG could be used in conjunction with chemo- or
radiotherapeutic intervention, or other treatments. It also may
prove effective, in particular, to combine 6-thio-dG with other
therapies that target different aspects of cancer cell function
(such as immune checkpoint inhibitors).
[0106] To kill cells, inhibit cell growth, inhibit metastasis,
inhibit angiogenesis or otherwise reverse or reduce the malignant
phenotype of tumor cells, using the methods and compositions of the
present disclosure, one would generally contact a "target" cell
with 6-thio-dG and at least one other agent. These compositions
would be provided in a sequential or combined amount effective to
kill or inhibit proliferation of the cell. This process may involve
contacting the cells with 6-thio-dG and the other agent(s) or
factor(s) at the same time. This may be achieved by contacting the
cell with a single composition or pharmacological formulation that
includes both agents, or by contacting the cell with two distinct
compositions or formulations, at the same time, wherein one
composition includes the interferon prodrugs according to the
present disclosure and the other includes the other agent.
[0107] Alternatively, the 6-thio-dG therapy may precede or follow
the other agent treatment by intervals ranging from minutes to
weeks. In embodiments where the other agent and the interferon
prodrugs are applied separately to the cell, one would generally
ensure that a significant period of time did not expire between
each delivery, such that the agent and expression construct would
still be able to exert an advantageously combined effect on the
cell. In such instances, it is contemplated that one would contact
the cell with both modalities within about 12-24 hours of each
other and, more preferably, within about 6-12 hours of each other,
with a delay time of only about 12 hours being most preferred. In
some situations, it may be desirable to extend the time period for
treatment significantly, however, where several days (2, 3, 4, 5, 6
or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the
respective administrations. For example, pretreating with 6-thio-dG
then adding checkpoint inhibitors antibodies (e.g. PDL-1 and PD-1
inhibitors) may improve outcomes.
[0108] It also is conceivable that more than one administration of
either interferon prodrugs or the other agent will be desired.
Various combinations may be employed, where 6-thio-dG therapy is
"A" and the other therapy is "B", as exemplified below: [0109]
A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B [0110] A/A/B/B
A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A [0111] A/A/A/B
B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B Other combinations
are contemplated. Again, to achieve cell killing, both agents are
delivered to a cell in a combined amount effective to kill the
cell.
[0112] Agents or factors suitable for cancer therapy include any
chemical compound or treatment method that induces DNA damage when
applied to a cell. Such agents and factors include radiation and
waves that induce DNA damage such as, irradiation, microwaves,
electronic emissions, and the like. A variety of chemical
compounds, also described as "chemotherapeutic" or "genotoxic
agents," may be used. This may be achieved by irradiating the
localized tumor site; alternatively, the tumor cells may be
contacted with the agent by administering to the subject a
therapeutically effective amount of a pharmaceutical
composition.
[0113] Various classes of chemotherapeutic agents are contemplated
for use with the present disclosure. Imetelstat is discussed below.
Other chemotherapeutics include selective estrogen receptor
antagonists ("SERMs"), such as Tamoxifen, 4-hydroxy Tamoxifen
(Afimoxfene), Falsodex, Raloxifene, Bazedoxifene, Clomifene,
Femarelle, Lasofoxifene, Ormeloxifene, and Toremifene. The agents
camptothecin, actinomycin-D, and mitomycin C are commonly used
chemotherapeutic drugs. The disclosure also encompasses the use of
a combination of one or more DNA damaging agents, whether
radiation-based or actual compounds, such as the use of X-rays with
cisplatin or the use of cisplatin with etoposide. The agent may be
prepared and used as a combined therapeutic composition.
[0114] Heat shock protein 90 is a regulatory protein found in many
eukaryotic cells. HSP90 inhibitors have been shown to be useful in
the treatment of cancer. Such inhibitors include Geldanamycin,
17-(Allylamino)-17-demethoxygeldanamycin, PU-H71 and Rifabutin.
[0115] Agents that directly cross-link DNA or form adducts are also
envisaged. Agents such as cisplatin, and other DNA alkylating
agents may be used. Cisplatin has been widely used to treat cancer,
with efficacious doses used in clinical applications of 20
mg/m.sup.2 for 5 days every three weeks for a total of three
courses. Cisplatin is not absorbed orally and must therefore be
delivered via injection intravenously, subcutaneously,
intratumorally or intraperitoneally.
[0116] Agents that damage DNA also include compounds that interfere
with DNA replication, mitosis and chromosomal segregation. Such
chemotherapeutic compounds include adriamycin, also known as
doxorubicin, etoposide, verapamil, podophyllotoxin, and the like.
Widely used in a clinical setting for the treatment of neoplasms,
these compounds are administered through bolus injections
intravenously at doses ranging from 25-75 mg/m.sup.2 at 21-day
intervals for doxorubicin, to 35-50 mg/m.sup.2 for etoposide
intravenously or double the intravenous dose orally. Microtubule
inhibitors, such as taxanes, also are contemplated. These molecules
are diterpenes produced by the plants of the genus Taxus and
include paclitaxel and docetaxel.
[0117] Epidermal growth factor receptor inhibitors, such as Iressa,
mTOR, the mammalian target of rapamycin (also known as
FK506-binding protein 12-rapamycin associated protein 1 (FRAP1)),
is a serine/threonine protein kinase that regulates cell growth,
cell proliferation, cell motility, cell survival, protein
synthesis, and transcription. Rapamycin and analogs thereof
("rapalogs") are therefore contemplated for use in cancer therapy
in accordance with the present disclosure. Another EGFR inhibitor
of particular utility here is Gefitinib.
[0118] Another possible therapy is TNF-.alpha. (tumor necrosis
factor-alpha), a cytokine involved in systemic inflammation and a
member of a group of cytokines that stimulate the acute phase
reaction. The primary role of TNF is in the regulation of immune
cells. TNF is also able to induce apoptotic cell death, to induce
inflammation, and to inhibit tumorigenesis and viral
replication.
[0119] Agents that disrupt the synthesis and fidelity of nucleic
acid precursors and subunits also lead to DNA damage. As such a
number of nucleic acid precursors have been developed. Particularly
useful are agents that have undergone extensive testing and are
readily available. As such, agents such as 5-fluorouracil (5-FU),
are preferentially used by neoplastic tissue, making this agent
particularly useful for targeting to neoplastic cells. Although
quite toxic, 5-FU, is applicable in a wide range of carriers,
including topical, however intravenous administration with doses
ranging from 3 to 15 mg/kg/day being commonly used.
[0120] Other factors that cause DNA damage and have been used
extensively include what are commonly known as .gamma.-rays,
x-rays, and/or the directed delivery of radioisotopes to tumor
cells. Other forms of DNA damaging factors are also contemplated
such as microwaves and UV-irradiation. It is most likely that all
of these factors effect a broad range of damage DNA, on the
precursors of DNA, the replication and repair of DNA, and the
assembly and maintenance of chromosomes. Dosage ranges for x-rays
range from daily doses of 50 to 200 roentgens for prolonged periods
of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens.
Dosage ranges for radioisotopes vary widely, and depend on the
half-life of the isotope, the strength and type of radiation
emitted, and the uptake by the neoplastic cells.
[0121] In addition, it also is contemplated that immunotherapy,
hormone therapy, toxin therapy and surgery can be used.
[0122] The skilled artisan is directed to "Remington's
Pharmaceutical Sciences" 15th Edition, Chapter 33, in particular
pages 624-652. Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject. Moreover, for human
administration, preparations should meet sterility, pyrogenicity,
general safety and purity standards as required by FDA Office of
Biologics standards.
[0123] A. Telomerase Inhibitors
[0124] A variety of telomerase inhibitors are known in the art and
include antisense oligonucleotides, RNAi, dominant-negative TERT
and ribozymes.
[0125] One oligonucleotide drug that targets telomerase is
Imetelstat (GRN163L). Imetelstat was shown to be active against
both CD138-postiive and CD138-negative cancer stem cells and
eliminated the colony forming potential of both by five weeks.
Similarly, it inhibited the in vitro clonogenic growth of
CD138-negative Multiple Myeloma Cancer Stem Cells isolated from the
bone marrow aspirates of patients with multiple myeloma. On Nov. 3,
2014, the FDA removed the full clinical hold on imetelstat and
declared the company's clinical development plan as acceptable.
[0126] Two active Phase 2 trials for Imetelstat are scheduled for
completion in 2019 and 2022, one for Myelofibrosis and the other
for Myelodysplastic Syndrome. In October 2017, Imetelstat was
granted Fast Track status by the FDA for certain patients in
Myelodysplastic Syndrome. However, side-effects that cause patients
to temporarily stop therapy with Imetelstat may results in rapid
telomere re-elongation and reduce the efficacy of such
treatments.
[0127] B. Specific Brain Cancer Therapies
[0128] Surgery, radiation therapy and chemotherapy, alone or
together, are front line therapies for brain cancer. Each of these
approaches is discussed below.
[0129] Surgery is the removal of the tumor and some surrounding
healthy tissue during an operation. It is usually the first
treatment used for a brain tumor and is often the only treatment
needed for a low-grade brain tumor. Removing the tumor can improve
neurological symptoms, provide tissue for diagnosis, help make
other brain tumor treatments more effective, and, in many
instances, improve the prognosis of a person with a brain tumor.
There have been rapid advances in surgery for brain tumors,
including the use of cortical mapping, enhanced imaging, and
fluorescent dyes.
[0130] In addition to removing or reducing the size of the brain
tumor, surgery can provide a tissue sample for biopsy analysis. For
some tumor types, the results of this analysis can help determine
if chemotherapy or radiation therapy will be useful. For a
cancerous tumor, even if it cannot be cured, removing it can
relieve symptoms from the tumor pressing on the brain.
[0131] Radiation therapy is the use of high-energy x-rays or other
particles to destroy tumor cells. Doctors may use radiation therapy
to slow or stop the growth of the tumor. It is typically given
after surgery and possibly along with chemotherapy. The most common
type of radiation treatment is called external-beam radiation
therapy, which is radiation given from a machine outside the body.
When radiation treatment is given using implants, it is called
internal radiation therapy or brachytherapy. External-beam
radiation therapy can be directed at the tumor in the following
ways: [0132] Conventional radiation therapy. The treatment location
is determined based on anatomic landmarks and x-rays. [0133]
3-dimensional conformal radiation therapy (3D-CRT). Using images
from CT and MRI scans, a 3-dimensional model of the tumor and
healthy tissue surrounding the tumor is created on a computer. This
model can be used to aim the radiation beams directly at the tumor,
sparing the healthy tissue from high doses of radiation therapy.
[0134] Intensity modulated radiation therapy (IMRT). IMRT is a type
of 3D-CRT (see above) that can more directly target a tumor. It can
deliver higher doses of radiation to the tumor while giving less to
the surrounding healthy tissue. [0135] Proton therapy. Proton
therapy is a type of external-beam radiation therapy that uses
protons rather than x-rays. At high energy, protons can destroy
tumor cells. Proton beam therapy is typically used for tumors when
less radiation is needed because of the location. [0136]
Stereotactic radiosurgery. Stereotactic radiosurgery is the use of
a single, high dose of radiation given directly to the tumor and
not healthy tissue. It works best for a tumor that is only in 1
area of the brain and certain noncancerous tumors. A modified
linear accelerator is a machine that creates high-energy radiation
by using electricity to form a stream of fast-moving subatomic
particles. A gamma knife is another form of radiation therapy that
concentrates highly focused beams of gamma radiation on the tumor.
A cyber knife is a robotic device used in radiation therapy to
guide radiation to the tumor target, particularly in the brain,
head, and neck regions. [0137] Fractionated stereotactic radiation
therapy. Radiation therapy is delivered with stereotactic precision
but divided into small daily doses called fractions given over
several weeks, in contrast to the 1-day radiosurgery. Depending on
the size and location of the tumor, the radiation oncologist may
choose any or several of the above radiation techniques.
[0138] Chemotherapy is the use of drugs to destroy tumor cells,
usually by ending the cancer cells' ability to grow and divide. The
goal of chemotherapy can be to destroy tumor cells remaining after
surgery, slow a tumor's growth, or reduce symptoms.
[0139] A chemotherapy regimen, or schedule, usually consists of a
specific number of cycles given over a set period of time. A
patient may receive 1 drug at a time or combinations of different
drugs given at the same time. Some drugs are better at going
through the blood-brain barrier, and these drugs often used for a
brain tumor.
[0140] Gliadel wafers are one way to give the drug carmustine.
These wafers are placed in the area where the tumor was removed
during surgery.
[0141] For people with glioblastoma and high-grade glioma, the
latest standard of care is radiation therapy with daily low-dose
temozolomide (Temodar). This is followed by monthly doses of
temozolomide after radiation therapy for 6 months to 1 year.
[0142] A combination of 3 drugs, lomustine (Gleostine),
procarbazine (Matulane), and vincristine (Vincasar), have been used
along with radiation therapy. This approach has helped lengthen the
lives of patients with grade III oligodendroglioma with a 1p19q
co-deletion when given either before or right after radiation
therapy.
[0143] Patients are monitored with a brain MRI while receiving
active treatment. Patients often have regular MRIs to monitor their
health after treatment is finished and the tumor has not grown.
V. EXAMPLES
[0144] The following Examples section provides further details
regarding examples of various embodiments. It should be appreciated
by those of skill in the art that the techniques disclosed in the
examples that follow represent techniques and/or compositions
discovered by the inventors to function well. However, those of
skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific
embodiments which are disclosed and still obtain a like or similar
result without departing from the spirit and scope of the
disclosure. These examples are illustrations of the methods and
systems described herein and are not intended to limit the scope of
the disclosure. Non-limiting examples of such include but are not
limited to those presented below.
Example 1--Materials and Methods
[0145] Cell Lines and Primary Tumor Cell Culture.
[0146] All patient specimens were collected after obtaining written
informed consent from patients and families in accordance with
approved IRB studies. The primary DIPG neurosphere line CCHMCDIPG-1
was aseptically isolated by dissociating the brain tumor tissue
post-autopsy from a patient consented under the Pediatric Brain
Tumor Repository (PBTR) study (IRB approved protocols 2013-1245 and
2013-5947) at Cincinnati Children's Hospital Medical center
(CCHMC). Primary patient-derived neurospheres high-risk group-3 MB
(MB004) (19,20), GBM (R0315-GBM), and DIPG)SU-DIPG-VI (21) and
CCHMC-DIPG-1 (22)) were cultured in neurosphere stem cell media as
described elsewhere (22,23). Patient-derived medulloblastoma cell
lines collected from the same patient at diagnosis D-425, and at
recurrence D-458 (24,25) were cultured in RPMI-1640 (Gibco)
supplemented with 15% FBS. Primary normal human foreskin fibroblast
(HFF) strain (ATCC CRL-2091), the HeLa human cervical carcinoma
cell line, the human osteosarcoma cell lines Saos-2 (ATCC HTB-85)
and U2OS (ATCC HTB-96) were purchased from the American Type
Culture Collection. HFF cells were immortalized with hTERT
(HFF+hTERT) by viral transduction as previously described (2). The
source of other cell lines is referenced above. Commercially
available cell lines were characterized at their original sources.
All cells were expanded upon receipt or establishment for 2-3
passages and used within 1-2 months after thawing the cryopreserved
cells without additional authentication. No testing was done by the
authors for mycoplasma. However, the inventors did not observe any
evidence of their presence.
[0147] Telomerase Activity Assay.
[0148] Telomerase activity was assayed using the TRAPeze Telomerase
Detection Kit (Millipore). Cell extracts were prepared according to
the manufacturer's protocol. 50-100 ng of total protein was used to
assess the telomerase activity by performing polyacrylamide gel
(12.5%) electrophoresis.
[0149] Drug Treatment.
[0150] 6-thio-dG (Metkinen Oy) was dissolved in DMSO:water (1:1) to
prepare a 10 mM stock solution, aliquoted and stored in -20.degree.
C. For in vitro treatments, 1 mM final concentration was prepared
in plain media. For in vivo studies, 6-thio-dG was prepared in a 5%
DMSO solution. Kinase-inhibitors of ATM (KU-55933) (26) and ATR
(VE-822) (27,28) were purchased from Selleckchem, and reconstituted
in DMSO. Imetelstat (GRN163L) (29); Geron Corp. was reconstituted
(1 mg/mL) in PBS.
[0151] Cell Growth and Sphere Formation Assay.
[0152] After dissociating primary neurospheres by TrypLE express
(Gibco), single cells were seeded in respective growth media in
6-well plates and were incubated for one week at varying
concentrations (0.5 to 10 .mu.M) of 6-thio-dG or DMSO. Fresh
culture media were added every three days. Viable cell numbers were
determined by trypan blue exclusion method. For sphere formation
assay, MB004 single cells were seeded in limited dilution (10
cells/well in 96-well plate) and treated with DMSO or 6-thio-dG.
Sphere formation was monitored by microscopy.
[0153] Cell Cycle Analysis.
[0154] Following treatment with 6-thio-dG or DMSO, cells were
collected in PBS, fixed with ice-cold 70% ethanol and were kept in
-20.degree. C. for at least an hour. Cells were then washed and
stained with propidium iodide (PI) solution containing 25 .mu.g/mL
PI (Sigma), and 100 .mu.g/mL Ribonuclease-A (Sigma) for 30 minutes
in the dark. Flow cytometry was performed on BD FACS Canto II and
cells were analyzed using FlowJo v.10 (FlowJo, USA) software.
[0155] Immunofluorescence and Telomere FISH Assay.
[0156] Cells were fixed with 4% paraformaldehyde (PFA) for 15
minutes. Cells were then washed, permeabilized with 0.5%
Triton-X-100 in PBS, blocked with 5% donkey serum and 0.3% Triton
X-100 in TBS and incubated with primary antibodies against
.gamma.H2A.X (1:500, rabbit), or cleaved caspase-3 (1:400, rabbit)
(Cell Signaling); and/or TRF2 (1:200) (Mouse; NOVUS), as
applicable, for overnight followed by TBST wash (.times.3) next
day. Corresponding secondary antibodies were added (Alexa-Fluor
488- or 594-conjugated donkey anti-rabbit, or anti-mouse (1:500)
(Jackson ImmunoResearch) for 1 hour and washed with TBS (.times.3)
before mounting. For telomere-FISH, fixed and permeabilized cells
were dehydrated with a graded ethanol concentration series,
air-dried and covered with hybridization solution (70% formamide,
0.5% Blocking Reagent (Roche Diagnostics) diluted in 100 mM maleic
acid and 150 mM sodium chloride, and 10 mM Tris (pH 7.5)) with 300
ng/mL PNA(CCCTAA).sub.3-Cy3 (Biosynthesis, USA), and denatured for
6 minutes at 84.degree. C. followed by hybridization for at least
two hours at room temperature. Cells were washed three times with
70% formamide and 10 mM Tris (pH 7.5) and three times with TBS.
Finally, they were embedded with mounting media with DAPI (Vector
Laboratories H1200). Images were captured with 60.times. oil
objective on Nikon Eclipse Ti confocal microscope.
[0157] Senescence Assay.
[0158] Senescence-associated .beta.-galactosidase was detected as
previously described (2). Cells were observed under the microscope
until the development of the blue color and the reaction was
terminated. Images were captured; stained and unstained cells were
counted from multiple fields to quantitate the percent senescent
cells.
[0159] Western Blot.
[0160] Western blot was performed as described previously (22).
Antibodies used were against ATMS1981P (R&D Systems): ATM
(SIGMA): ATR-T1989P (GeneTex); and ATR, CHK1-S345P, CHK1,
CI-IK2-T68P, CHK2, Cleaved Caspase-3, and .beta.-actin (Cell
Signaling). Bands were visualized using ECL with Azurec500 imaging
system (Azure Biosystems). Band intensities were quantified using
ImageJ software (Ver. 1.49u. NIH, USA).
[0161] DNA Extraction and Agarose Gel Electrophoresis.
[0162] Genomic DNA was extracted from DMSO or 6-thio-dG treated
HFF, HFF+hTERT, and U2OS cells using Puregene Kit (Qiagen, USA)
following the manufacturer's protocol. 100 ng of each sample was
run on a 0.7% agarose gel followed by staining with GelRed Nucleic
Acid Gel Stain (Biotium). Bands were visualized under UV
illuminator.
[0163] Mouse Subcutaneous and Orthotopic Xenograft.
[0164] Athymic Ncr-nu/nu female mice (6-7 weeks old) were
subcutaneously injected with 10,000 MB004 cells. Mice were weighed
and distributed in two groups (control and 6-thio-dG). After
tumor-establishment (day 23-24 post-implantation) with an average
volume of 100-200 mm.sub.3, mice were injected intraperitoneally
(i.p) every 2 days with 6-thio-dG (2.5 mg/kg) or DMSO-PBS (vehicle)
for 3-4 weeks until euthanization. Tumors were measured by slide
calipers taking two longest tumor-diameters (length and width)
perpendicular to each other and volumes were calculated by using
the formula: (.pi./6).times.d3, where d=mean diameter. For
orthotopic xenograft, CCHMC-DIPG-1 luciferase-positive cells
(10,000) were injected in the brain of NRG (NOD.Cg-RagI.sub.tm1Mom
Il2rg.sub.tm1Wjl/SzJ) mice. Briefly, mice were injected
stereotactically with 2 .mu.l medium containing 10,000
luciferase-positive cells. The coordinates for injection were 0.8-1
mm posterior to lambda suture and 3.5 mm deep, corresponding to the
pons location in the brain. Tumors were visualized by luminescence
using IVIS Spectrum CT in vivo imaging system (PerkinElmer). All
animal procedures were approved by the Institutional Animal Care
and Use Committee (IACUC) (protocol #IACUC2015-0066, CCHMC).
[0165] Immunohistochemistry.
[0166] Formalin-fixed paraffin-embedded (FFPE) sections were
deparaffinized in xylene followed by rehydration through a graded
ethanol concentration series. Heat-induced antigen retrieval was
performed by steaming slides for 20 minutes in 10 mM Citrate buffer
(pH 6.0). Endogenous peroxidase activity was quenched with 1%
H.sub.2O.sub.2 followed by washing, blocking with 10% goat serum in
TBST for an hour, and incubating with primary antibody Ki67
(1:1500) (rabbit; Abcam), or Cleaved caspase-3 (1:1000) (rabbit;
Cell Signaling) in 2% goat serum in TBST overnight at 4.degree. C.
Slides were washed with TBST (.times.3) and were treated with
biotinylated anti-rabbit secondary antibody (1:500) and
signal-amplified using ABC Kit (Vector Laboratories). Signal was
visualized with DAB (Vector Laboratories) and counterstained with
Harris Hematoxylin (Sigma). H&E staining was performed using
hematoxylin-1 and eosin-Y (Thermo Scientific). Tissues were mounted
with Permount (Fisher Scientific) and imaged by Nikon eclipse 80i
microscope.
[0167] Tissue TIF Assay.
[0168] Tissue samples were pre-fixed with 4% PFA and were
cryo-protected in 25% sucrose/PBS solution, then embedded in OCT
freezing molds with Neg-50 (VWR) in acetone and dry ice followed by
cryo-sectioning. Heat-induced antigen retrieval and Telomere-FISH
was performed as described above. The samples were incubated in
blocking solution (5% donkey serum, 0.3% Triton X-100 in TBS) for
30 minutes and treated with anti-53BP1 (rabbit 1:500; Novus
Biologicals) for 1 hour at room temperature. After washing in TBST
(.times.3), the samples were incubated with secondary antibody
Alexa-Fluor 488-conjugated donkey anti-rabbit (1:400; Jackson
ImmunoResearch), and washed in TBS (.times.3). The samples were
embedded in mounting media with DAPI (Vector Laboratories H1200).
Images were captured with 60.times. oil objective on Nikon Eclipse
Ti confocal microscope.
[0169] Statistical Analysis.
[0170] The statistical analyses were performed by Student's t-test
or multiple-way ANOVA as required using the GraphPad Prism (version
7.02). Each experiment was repeated at least twice. Error bars
represent standard deviation of at least three replicate wells or
fields from one representative experiment considered as technical
replicates, or from independent experiments or different animals
for biological replicates. Differences were considered significant
at P<0.05.
Example 2--Results
[0171] 6-Thio-dG Selectively Inhibits Cell Growth of
Telomerase-Positive Tumor Cells.
[0172] One of the major setbacks in oncology is the ability of
certain cancers to recur after minimal or undetectable disease is
achieved with aggressive therapies. Cancer stem-like cells have
been proposed to represent a sub-population of cells within a tumor
that self-renew to promote tumor growth and recurrence. In the
present study, primary stem-like cells were derived from DIPG, HGG
and MB patients' tumor tissue and expanded in neurosphere stem cell
media. Table S1 indicate genetic features and subtypes of the cell
lines. The inventors tested 6-thio-dG in a panel of
telomerase-positive pediatric brain tumor cells, including
high-risk group-3 MB (MB004), GBM (R0315-GBM), and DIPG (SU-DIPG-VI
and CCHMC-DIPG-1) along with a panel of control cell lines,
consisting of normal primary human foreskin fibroblasts (HFF,
telomerase-negative), HFF-ectopically expressing hTERT (HFF+hTERT,
telomerase-positive), HeLa cells (telomerase-positive), and
osteosarcoma cells Saos-2 (Telomerase-negative, Alternative
Lengthening of Telomeres or ALT-positive). Telomerase activity was
verified by the gel-based TRAP assay (FIG. 7). The inventors and
others previously reported that under serum-free culture
conditions, HGG-, DIPG- and medulloblastoma neurospheres expressed
neural stem cell markers such as nestin, CD133, and olig2, and were
capable of self-renewal and differentiation in the presence of
serum (22,23). These cancer stem-like cells are thought to be
responsible for tumor recurrence (30). Moreover, the inventors
confirmed that these cells are able to establish tumors in
immunosuppressed mice. The cells were treated with 0.5 to 10 .mu.M
of 6-thio-dG every three days for one week. As expected, treatment
with 6-thio-dG inhibited cell growth in a dose-dependent manner in
all telomerase-positive cells, including brain tumor cells, with a
minor to no effect in telomerase-negative cells HFF and Saos-2
cells up to 3 .mu.M (FIGS. 1A-B). Interestingly, 6-thio-dG
effectively inhibited cell growth of both patient-derived
medulloblastoma cell lines collected from the same patient at
diagnosis, D-425 cells (biopsy of cerebellar primary tumor of
6-year old boy) and at recurrence D-458 cells (tumor cells in CSF
following failure of radio- and chemotherapy) (24,31) (FIG. 1C).
All telomerase-positive cells including brain tumor cells were
highly sensitive compared to telomerase-negative cells as evidenced
by the IC.sub.50 ranging 0.14-1.45 .mu.M (Table S2). Telomerase
dependency of 6-thio-dG was further verified by using imetelstat
(IMT). HFF and HFF+hTERT cells were treated with either 6-thio-dG
or IMT, or in combination. The inhibition of telomerase activity by
IMT treatment was confirmed by the TRAP assay (FIG. 8A). As
expected, the inhibition of cell growth by 6-thio-dG in the
combination treatment was markedly reduced pre-treated with IMT
(FIGS. 8B-C), indicating that the 6-thio-dG effect is largely
dependent on the presence of telomerase.
[0173] 6-Thio-dG Induces Sustained G.sub.2/M Cell Cycle Arrest in
Telomerase-Positive Cells.
[0174] Next, the inventors investigated the mechanistic aspect of
6-thio-dG-induced DNA damage response (DDR). Cell growth inhibition
caused by 6-thio-dG treatment prompted the inventors to check its
effect on cell cycle progression. Three days treatment with 3 .mu.M
of 6-thio-dG caused G.sub.2/M arrest in telomerase-positive cells
HFF+hTERT and HeLa. In contrast, this effect was negligible in
telomerase-negative cells HFF (FIG. 2A). Further, they assessed the
long-term effect of 6-thio-dG on the cell cycle profile as
illustrated in FIG. 2B. In continuous treatment, 6-thio-dG-induced
G.sub.2/M arrest was sustained and more pronounced in
telomerase-positive normal cells (HFF+hTERT) along with an increase
in >4n cell population compared to telomerase-negative HFF cells
(FIG. 2C). For Hela cells, continuous treatment caused a sharp
increase in G.sub.2/M, >4n and sub-G.sub.1 cell populations,
indicating aneuploidy and cell death. G.sub.2/M fraction increased
after prolonged treatment of HFF cells with 6-thio-dG, likely due
to the accumulation of genomic damage. In contrast to
telomerase-positive cells, no increase in sub-G.sub.1 and >4n
population was noticed. Interestingly, even when the drug was
removed from the media (FIG. 2B), 6-thio-dG effect on cell cycle
progression persisted several days after drug withdrawal in HeLa
cells (FIG. 2D). In HFF+hTERT cells, G.sub.2/M and >4n
accumulation decreased over time and the cell cycle profile
partially reverted to that of the vehicle control. These results
indicate that 6-thio-dG treatment has limited effect in normal
cells (HFF), and causes G.sub.2/M arrest, aneuploidy and cell death
in HeLa cells. This effect was sustained several days after drug
withdrawal in cancer cells but declined in normal
telomerase-positive cells (HFF+hTERT).
[0175] 6-Thio-dG Induces Persistent Telomere Dysfunction in
Telomerase-Positive Cells.
[0176] Previous studies have shown that 6-thio-dG treatment leads
to telomere dysfunction-induced foci (TIFs) in telomerase-positive
cells but not in telomerase-negative cells (17). The inventors
visualized TIFs by FISH combined staining using .gamma.H2AX
co-localization with a telomere specific PNA probe. As expected,
6-thio-dG caused an acute increase of the number of cells with TIFs
(.about.25%) in telomerase-positive cells, HFF+hTERT, after two
days (FIG. 3A). TIF formation was limited in telomerase-negative
cells HFF with less than 3% after 2 or 5 days treatment. This
effect was amplified over time, .about.34% of cells were
TIF-positive at 5 days continuous treatment (FIG. 3A).
Interestingly, .about.31% of cells were still TIF-positive in
telomerase-positive cells 3 days after drug withdrawal while TIFs
completely disappeared in telomerase-negative cells. Furthermore,
IMT treatment inhibited 6-thio-dG-induced TIF formation in
HFF+hTERT (FIG. 8D) confirming telomerase dependency of
6-thio-dG-induced TIFs. As observed in the previous study (17),
6-thiodG also caused a modest increase in genomic DNA damage in
telomerase-negative cells. This general damage was more evident in
telomerase-positive cells (FIG. 3A). Importantly, TIFs-negative
cells treated with 6-thio-dG displayed markedly reduced genomic DNA
damage suggesting that TIFs exacerbate genomic DNA damage (FIG.
9A). Indeed, the inhibition of telomerase using IMT reduced the
genomic DNA damage in cells treated with 6-thio-dG (FIG. 9B). Since
telomeres are only 1/6000.sup.th of the human genome, and TIF that
is observed is unlikely to be by chance alone and emphasizes the
importance of telomerase mediating the toxic effects of
6-thio-dG.
[0177] Next, the inventors evaluated the effect of 6-thio-dG
treatment in telomerase-positive primary high-risk group 3
medulloblastoma stem-like cells MB004. Treatment with 3 .mu.M of
6-thio-dG for 3 days resulted in an increase in sub-G.sub.1 and
G.sub.2/M cell populations and a total abolition of sphere
formation ability at day 7 (FIGS. 3B-C). Of note, at day 3, cells
treated with 6-thio-dG were able to form small spheres or
"spherelets" containing 4-10 cells. At day 7, these "spherelets"
completely disappeared with 6-thio-dG treatment while the DMSO
treated spherelets continued to grow. The inventors then evaluated
the effect of continuous and discontinuous exposure to 6-thio-dG
after an initial treatment of 2 days. Both treatment schemes showed
a robust growth inhibition and sustained TIF formation indicating
that the effect of 6-thio-dG treatment persists several days after
drug withdrawal (FIG. 3D). These data demonstrate the
telomerase-dependent induction of TIFs and their persistence even
after the drug is removed, providing a possible explanation for the
sustained G.sub.2/M arrest. Together these results indicate that
6-thio-dG effect on cell growth is dependent on telomerase-induced
TIFs probably in conjunction with genomic DNA damage and this
compound is active in brain tumor cells derived from
therapy-resistant patients' tumors.
[0178] Sequential Activation of ATR and ATM Pathways in Response to
6-Thio-dG-Induced Telomere Damage.
[0179] ATM and ATR-kinases are master regulators of DDR signaling.
The inventors and others have shown that telomere dysfunction
induces ATM-dependent DDR (2,3). To extend the characterization of
6-thio-dG-induced telomere damage, they investigated ATM and ATR
DDR in telomerase-positive cells HFF+hTERT and in matched
telomerase-negative cells HFF. Both ATM and ATR signaling pathways
were engaged in response to 6-thio-dG treatment in HFF+hTERT as
evidenced by the accumulation of ATR-T1989 and ATM-S1981
phosphorylation (FIG. 4A), whereas 6-thio-dG treatment in
telomerase-negative cells correlated with the activation of the ATR
pathway, probably due to genomic DNA damage. Timing-wise, the ATR
pathway was first activated then progressively inhibited (FIG. 4A).
The decrease in ATR signaling overlapped with ATM pathway
activation. While at day 1 the inventors did not observe TIF
formation, the number of TIFs per cell and the number of cells with
TIFs markedly increased from day 1 to day 3 in the HFF+hTERT cells
(FIG. 4B). This increase correlated with a sustained increase of
ATM activation from day 2 to day 3 and a decrease of ATR activation
starting at day 2. To investigate 6-thio-dG-induced DDR further,
HFF+hTERT cells were pre-treated with a specific ATM or ATR
inhibitor for two hours prior to 6-thio-dG treatment for 48 hours.
In the presence of either ATM or ATR inhibitor, the inventors
observed a reduction in the number of cells with TIFs as well as
the number of TIFs per individual cells (FIG. 4C). Compared to ATR
inhibition, ATM inhibition led to lower number of TIFs per
individual cell. The inventors interpret these results to suggest
that 6-thio-dG-induced telomere damage sequentially activates the
ATR pathway followed by ATM activation and the formation of TIFs 15
primarily induced by ATM pathway in normal cells.
[0180] 6-Thio-dG Induced Apoptosis in Telomerase-Positive Cancer
Cells and Senescence in Telomerase-Positive Normal Cells.
[0181] The inventors have previously demonstrated that ATM
activation, .gamma.H2AX-focus formation, and p53 accumulation
increased in pre-senescent cells (2). Therefore, they sought to
investigate the ultimate fate of cancer telomerase-positive cells,
HeLa, MB004, CCHMC-DIPG-1, and the primary normal human cells: HFF
(telomerase-negative) and HFF+hTERT (telomerase-positive) treated
with 6-thio-dG. The cells were treated with 3 .mu.M of 6-thio-dG
for 4 or 7 days and were evaluated for apoptosis. While HeLa (FIG.
5A), MB004 and CCHMC-DIPG-1 (data now shown) cells showed an
increase in cleaved caspase-3 signal, HFF and HFF+hTERT did not
show any evidence of apoptosis (FIG. 5A-B). The inventors further
evaluated the effect of long-term exposure of HFF and HFF+hTERT
cells to 6-thio-dG. The cells were incubated with 3 .mu.M of
6-thio-dG for 23 days Prolonged exposure to 6-thio-dG induced a
senescence phenotype in 30% of HFF+hTERT cells as assessed by
staining for senescence-associated .beta.-galactosidase activity
(SA-.beta.-gal) while only 7% of HFF cells were
SA-.beta.-gal-positive (FIG. 5C), suggesting that the senescence
observed in HFF+hTERT cells was due to 6-thio-dG-induced telomere
dysfunction. Thus, short-term treatment with 6-thio-dG causes
apoptosis in telomerase-positive cancer cells and the long-term
treatment leads to senescence in telomerase-positive normal cells.
These results are reminiscent of replicative senescence caused by
persistent telomere damage. Interestingly, unlike cancer
telomerase-positive cells, fast growing telomerase-negative cancer
cells, U2OS, treated with 6-thio-dG, predominantly did not die
(FIGS. 10A-C). After an initial growth inhibition, probably to
repair the genomic DNA damage, the cells resumed cell growth
several days post-drug removal (FIG. 10B). Finally, the inventors
did not observe any DNA fragmentation after treatment with
6-thio-dG (FIG. 10D). These data indicate that the cell death
observed in telomerase-positive cancer cells is not due to cell
growth kinetics or genomic DNA fragmentation but rather due to
telomere damage probably in combination with genomic DNA
damage.
[0182] 6-Thio-dG Treatment Inhibited Tumor Growth in Pediatric
High-Risk Group-3 Medulloblastoma Xenografts by Inducing an
Increase in in-Tumor Telomere Dysfunction, a Decrease of Tumor
Mitotic Index and Apoptosis.
[0183] To evaluate the in vivo activity of 6-thio-dG, the inventors
subcutaneously injected primary patient-derived stem-like cells
MB004 in athymic nude mice (10,000 cells/mouse). They previously
observed that these cells form aggressive and fast-growing tumors
in mice. Tumors were established at 24 days post-implantation at
which point the tumor volumes ranged from 100 to 200 mm.sup.3. A
mixture of DMSO and PBS solution was used as vehicle control in 6
mice. To monitor 6-thio-dG toxicity in mice, the inventors weighed
both treated and untreated mice. They did not observe a noticeable
weight difference between the two groups of mice, and no
dehydration or clinical symptom of sickness were observed in the
treated group, indicating that this 6-thio-dG regimen is not toxic
(FIG. 6A). Tumor growth kinetics showed the majority of treated
mice had a slower tumor growth rate compared to the untreated group
(FIG. 6B). Four out of six treated mice had reduced, or delayed
tumor growth. Two out of six treated mice showed fast tumor growth,
most likely due to the tumor aggressive nature and bigger tumor
volume at the start of 6-thio-dG treatment. Immunohistochemistry of
cleaved caspase-3 was performed in five tumors from each group to
evaluate in-tumor apoptosis. Compared to the control group, the
inventors observed a significant increase in apoptosis in the
treated group with a higher increase in tumors with slower growth
(FIGS. 6B-C). Accordingly, hematoxylin and eosin staining (H&E)
showed a significant decrease in number of mitotic figures and an
increase in apoptotic bodies in 6-thio-dG treated mice compared to
untreated group (FIG. 6D). FISH staining using a combination of a
telomeric probe and 53BP1 immunostaining, showed a marked in-tumor
increase of TIFs compared to untreated tumors (FIG. 6E). More than
21% of cells showed at least 1-3 TIFs in the 6-thio-dG group while
only .about.6% in the control group (FIG. 6E). Moreover, a
population of cells with 4-6 TIFs per cell was detected only in
treated tumors. The probability of animal survival was
significantly higher in 6-thio-dG treated mice compared to the
vehicle group upon reaching the tumor volume at a size of 1500
mm.sup.3 (>6 times of initial volume) considered to be a tumor
burden (FIG. 11). Together, these data can be interpreted to
indicate that 6-thio-dG treatment inhibits the growth of
therapy-resistant MB004 tumors by inducing telomere dysfunction,
inhibition of cell growth and apoptosis.
[0184] 6-Thio-dG Reached the Tumor Site and Induced Intratumoral
TIFs in an Orthotopic Patient-Derived Xenograft Model of Diffuse
Intrinsic Pontine Glioma.
[0185] To investigate the penetration and activity of 6-thio-dG in
patient-derived brain tumors xenografted into mice, the inventors
injected intra-cranially luciferase-positive DIPG patient-derived
primary cancer stem-like cells, CCHMC-DIPG-1, into the pons of the
mouse brain using a stereotactic device. Tumor growth was monitored
by bioluminescence imaging of the mouse brain (FIG. 6F). Upon tumor
establishment at 8-10 days post-implantation, 6-thio-dG or DMSO-PBS
administration was started by intraperitoneal injection. The mice
were treated for 7-8 days (4-5 doses in total). Mouse brains were
collected for analyses after animal euthanasia. The tumors were
highly aggressive and histopathological staining using H&E and
Ki67 indicated high cellularity and proliferation of tumor tissue
compared to matched normal brain (FIG. 6F). Telomerase activity was
retained in the xenograft relative to the patient-derived
neurosphere cells (FIG. 12A). FISH analyses of 6-thio-dG treated
orthotopic tumors revealed an increase in the number of cells with
genomic DNA damage and TIFs as well as higher number of TIFs per
cell compared to untreated tumors (FIG. 6G). TIFs and genomic DNA
damage was not observed in normal mouse brain (FIG. 12B). This
indicates that 6-thio-dG reached the tumor in the pons, induced
TIFs and genomic DNA damage with no obvious effect on normal brain
tissue. Due to the aggressiveness of the tumors, further in vivo
studies in orthotopic models of pediatric brain tumors are
warranted to evaluate the long-term effect of 6-thio-dG on tumor
growth and mouse overall survival.
Example 3--Discussion
[0186] Telomerase activity is present in most human cancers but is
undetectable in the majority of normal human somatic cells,
supporting the rationale of targeting telomerase and telomeres to
treat cancer. The inventors' previous clinical trial of imetelstat,
a potent direct inhibitor of telomerase, proved intolerable and
ineffective in children with recurrent CNS malignancies. The
inventors believe that this was due, at least in part, to
toxicities, such as thrombocytopenia, which led to CNS bleeding
that prevented more frequent dosing of imetelstat to allow
sustained inhibition of telomerase. Mender et al. reported that no
animal deaths or weight loss were observed in mice treated with
6-thio-dG. Moreover, the treatment did not cause any toxic effects
on hematologic counts, liver and kidney functions (17).
[0187] Using a lung cancer model, the previous study has
demonstrated that 6-thio-dG caused both, in vitro and in vivo
telomere damage (TIFs) and induced rapid cancer cell death, likely
due to telomerase-dependent telomere uncapping and dysfunction
caused by 6-thio-dG treatment (17). In the present study, the
inventors sought to evaluate the effect of 6-thio-dG in
telomerase-positive primary pediatric brain tumor cells derived
from patients with high-risk and treatment-resistant tumors. They
found that treatment with 6-thio-dG caused telomere dysfunction and
cell growth inhibition in a telomerase specific manner within one
week. Interestingly, both cell lines derived from the same patient
at diagnosis and at recurrence after chemo- and radiotherapy were
sensitive to 6-thio-dG, demonstrating that cells from previously
treated and recurrent tumors remain sensitive to 6-thiodG.
[0188] Mechanistically, the inventors showed that 6-thio-dG induced
G.sub.2/M cell cycle arrest in both telomerase-positive normal
(HFF+hTERT) and cancer cells (HeLa and MB004). G.sub.2/M arrest was
sustained after 6-thio-dG removal and was associated with apoptosis
in cancer cells. Long-term exposure of telomerase-positive normal
cells to 6-thio-dG induced senescence, probably due to telomere
dysfunction previously shown to be also associated with replicative
senescence (2). Interestingly, these data can be interpreted to
suggest that 6-thio-dG-induced senescence is initiated in the
G.sub.2/M phase, while senescent cells are usually in the G.sub.1
phase (32). However, recent publications also support the
initiation of senescence from G.sub.2 (33-35).
[0189] 6-thio-dG treatment-induced G.sub.2/M arrest and TIFs
formation was sustained several days after drug withdrawal
suggesting that a short exposure time in a clinical setting may be
sufficient to have a therapeutic effect. It would be informative to
investigate a combination treatment with 6-thio-dG and G.sub.2/M
checkpoint inhibitors already tested in clinical trials such as
AZD0156 (ATM inhibitor), VX-970 (ATR inhibitor) and AZD1775 (WEE1
inhibitor) (36). The expectation is that the cells will progress to
M phase causing mitotic catastrophe and massive cell death.
6-thio-dG treatment sequentially activates ATR and ATM DDR
pathways. 6-thio-dG activated the ATR but not the ATM pathway in
normal telomerase-negative cells. In contrast, 6-thio-dG activated
ATR then ATM in normal telomerase-positive cells suggesting that
the 6-thio-dG-induced genomic DNA damage activates ATR and then
6-thio-dG-induced telomere damage activates the ATM pathway.
However, TIFs could be induced by either pathway if ATR or ATM is
inhibited. Thus, it would be informative to evaluate the cell cycle
progression in the presence of 6-thio-dG and ATM or ATR inhibitors.
Importantly, 6-thio-dG treatment completely abolished neurosphere
formation ability, suggesting that self-renewal and sternness are
potential targets of 6-thio-dG. Given the extensive genomic DNA
damage in telomerase-positive cells, the inventors are not ruling
out the possibility that both genomic and telomeric damage
contribute to the ultimate fate of the cells in the context of the
presence of 6-thio-dG-induced TIFs. Of note, it is well accepted
that unlike genomic DNA damage, replicative senescence or apoptosis
due to telomere dysfunction does not necessarily depend on the
extent of the damage (number of TIFs), but rather on the telomeric
localization of the DNA damage. Future studies are required to
investigate the link between 6-thio-dG-induced telomere damage and
genomic DNA damage.
[0190] Using an orthotopic mouse model for DIPG, the inventors
showed that 6-thio-dG provided by intraperitoneal injection reached
the tumor site in the pons and induced telomere damage in the
tumor, demonstrating that 6-thio-dG crossed the
blood-brain-barrier. Importantly, they did not observe any adverse
effects of 6-thio-dG on normal mouse brain or mouse behavior. In
addition to an increase in telomere damage, they also observed an
increase in genomic DNA damage, indicating an enhancement of
general damage initiated by the 6-thio-dG-induced telomere
dysfunction as shown previously (17,37). In future studies, the
inventors will optimize the number of injected brain tumor cells in
the pons, the dose and the timing of 6-thio-dG administration to
evaluate the effect of 6-thio-dG on mouse survival. As this
compound is not currently in clinical trials, further testing in
multiple animal models is required to fully evaluate efficacy and
toxicity.
[0191] In conclusion, these findings document that 6-thio-dG is a
promising novel approach to treat therapy-resistant pediatric brain
tumors and provides a rationale for 6-thio-dG testing as a single
agent or in combination with G.sub.2/M checkpoint inhibitors
already in clinical trials to treat children with high-risk
pediatric brain tumors.
TABLE-US-00001 TABLE S1 Genetic Features and Subtypes of the Cell
Lines Used Tumor type Cell lines Subtype TP53 c-MYC References
Medulloblastoma MD004 Group 3 Mutated Amplification 19, 20 D-425
Group 3 Mutated Amplification 20, 25 D-458 Group 3 Wild type
Amplification 20 Cell lines Histone status ATRX TP53 References GBM
R0315-GBM H3K27 wild type Wild type Wild type Unpublished DIPG
SU-DIPG-M H3.3K27M Not available Mutated 21 CCHMC-DIPG-1 H3K27 wild
type Not available Mutated 22 and data not shown
TABLE-US-00002 TABLE S2 IC.sub.50 Values (.mu.M) of 6-thio-dG
Treatment for 7 Days in the Cell Lines Used Cell Lines IC.sub.50
(.mu.M) HFF 7.905 SaOS2 7.581 HFF + hTERT 0.1794 HeLa 0.1431 MB004
0.4361 R0315-GBM 1.247 SU-DIPG-VI 0.7717 CCHMC-DIPG-1 0.6547 D-425
1.377 D-458 1.452
[0192] All of the methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present
disclosure. While the compositions and methods of this disclosure
have been described in terms of particular embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the methods and in the steps or in the sequence of steps
of the method described herein without departing from the concept,
spirit and scope of the disclosure. More specifically, it will be
apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the disclosure as defined by the appended claims.
VI. REFERENCES
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forth herein, are specifically incorporated herein by reference.
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Sequence CWU 1
1
1118DNAArtificial sequenceSynthetic oligonucleotide 1ccctaaccct
aaccctaa 18
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