U.S. patent application number 15/101577 was filed with the patent office on 2016-10-20 for combination therapy for treating cancer.
The applicant listed for this patent is Epizyme, Inc.. Invention is credited to Heike Keilhack, Sarah K. Knutson, Kevin W. Kuntz.
Application Number | 20160303135 15/101577 |
Document ID | / |
Family ID | 53274220 |
Filed Date | 2016-10-20 |
United States Patent
Application |
20160303135 |
Kind Code |
A1 |
Keilhack; Heike ; et
al. |
October 20, 2016 |
COMBINATION THERAPY FOR TREATING CANCER
Abstract
The present invention relates to compositions comprising
inhibitors of human histone methyltransferase EZH2 and one or more
other therapeutic agents, particularly anticancer agents such as
prednisone, and methods of combination therapy for administering to
subjects in need thereof for the treatment of cancer.
Inventors: |
Keilhack; Heike; (Belmont,
MA) ; Knutson; Sarah K.; (Cambridge, MA) ;
Kuntz; Kevin W.; (Woburn, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Epizyme, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
53274220 |
Appl. No.: |
15/101577 |
Filed: |
December 8, 2014 |
PCT Filed: |
December 8, 2014 |
PCT NO: |
PCT/US2014/069167 |
371 Date: |
June 3, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61913063 |
Dec 6, 2013 |
|
|
|
61934388 |
Jan 31, 2014 |
|
|
|
61992881 |
May 13, 2014 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/573 20130101;
A61K 31/4965 20130101; A61K 39/3955 20130101; A61K 31/496 20130101;
A61K 31/436 20130101; A61P 35/02 20180101; A61K 31/404 20130101;
A61K 31/519 20130101; A61P 43/00 20180101; A61K 2039/505 20130101;
A61K 45/06 20130101; A61K 31/5377 20130101; A61K 31/635 20130101;
A61K 31/52 20130101; A61K 31/675 20130101; A61P 35/00 20180101;
A61K 31/69 20130101; A61K 31/4375 20130101; C07K 16/2887 20130101;
A61K 31/5377 20130101; A61K 2300/00 20130101; A61K 31/496 20130101;
A61K 2300/00 20130101; A61K 31/404 20130101; A61K 2300/00 20130101;
A61K 31/4965 20130101; A61K 2300/00 20130101; A61K 31/436 20130101;
A61K 2300/00 20130101; A61K 31/519 20130101; A61K 2300/00 20130101;
A61K 31/4375 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 31/5377 20060101
A61K031/5377; A61K 31/573 20060101 A61K031/573; A61K 31/404
20060101 A61K031/404; A61K 31/519 20060101 A61K031/519; C07K 16/28
20060101 C07K016/28; A61K 31/436 20060101 A61K031/436; A61K 31/675
20060101 A61K031/675; A61K 31/4375 20060101 A61K031/4375; A61K
31/69 20060101 A61K031/69; A61K 39/395 20060101 A61K039/395; A61K
31/635 20060101 A61K031/635; A61K 31/52 20060101 A61K031/52 |
Claims
1. A method for treating cancer in a patient in need thereof
comprising administering (i) a therapeutically effective amount of
an EZH2 inhibitor and a therapeutically effective amount of a
standard of care agent, (ii) a therapeutically effective amount of
a combination comprising an EZH2 inhibitor and a standard of care
agent, or (iii) a therapeutically effective amount of a composition
comprising an EZH2 inhibitor and a standard of care agent.
2-3. (canceled)
4. The method of claim 1, wherein the cancer is a Non-Hodgkin's
lymphoma.
5. The method of claim 4, wherein the Non-Hodgkin lymphoma is DLBCL
(diffuse large B-cell lymphoma) or GCB (germinal center
B-cell-like) lymphoma.
6. The method of claim 1, wherein (i) the cancer is an EZH2 wild
type cancer, or (ii) the cancer is characterized by increased
trimethylation at H3K27, or (iii) the cancer is an EZH2 inhibitor
resistant or refractory cancer.
7. (canceled)
8. The method of claim 5, wherein the lymphoma is an EZH2 mutant
lymphoma.
9. The method of claim 8, wherein the EZH2 mutant lymphoma has an
Y646, A682 or A692 mutation.
10. (canceled)
11. The method of claim 1, wherein the standard of care agent is
one or more compounds selected from the group consisting of an
R-CHOP component, a BCL inhibitor, or a BCR inhibitor.
12. The method of claim 11, wherein the R-CHOP is a
glucocorticosteroid receptor agonist.
13. The method of claim 12, wherein the glucocorticosteroid
receptor agonist is prednisolone or dexamethasone.
14. The method of claim 11, wherein doxorubicin is omitted from
R-CHOP.
15. The method of claim 11, wherein the BCL inhibitor is
navitoclax, obatoclax or ABT-199.
16. The method of claim 11, wherein the BCR inhibitor is a
PI3K/Akt/mTOR signaling cascade inhibitor.
17. The method of claim 11, wherein the BCR inhibitor is rituximab,
MK-2206, idelalisib, trametinib, tamatanib, everolimus, VELCADE, or
ibrutinib.
18. The method of claim 1, wherein the EZH2 inhibitor is Compound
44 having the following formula: ##STR00003## or a pharmaceutically
acceptable salt thereof.
19. The method of claim 18, wherein the EZH2 inhibitor and the
standard of care agent are administered simultaneously or
sequentially.
20. The method of claim 18, wherein the EZH2 inhibitor is
administered prior to administration of the standard of care
agent.
21. The method of claim 18, wherein at least one gene is
upregulated in the patient.
22. The method of claim 21, wherein the gene is selected from the
group consisting of Sestrin, TNF, and GILZ, or the gene is a
glucocorticoid target gene.
23. (canceled)
24. The method of claim 21, wherein the upregulation of a gene is
used to determine or adjust the therapeutically effective amount of
the EZH2 inhibitor of claim 1, or the upregulation of a gene is
used to determine or adjust the therapeutically effective amount of
the standard of care agent of claim 1.
25. (canceled)
26. A method of selecting a patient for a method of treatment
according to claim 1, wherein the patient is selected based on the
expression profile of one or more genes selected from the group
consisting of Sestrin, TNF and GILZ.
27. A method of treatment according to claim 1 wherein the patient
has upregulated expression of Sestrin, TNF or GILZ.
Description
RELATED APPLICATIONS
[0001] This application claims priority to, and the benefit of,
U.S. provisional application No. 61/913,063, filed Dec. 6, 2013,
61/934,388, filed Jan. 31, 2014, and 61/922,881, filed May 13,
2014, the contents of each of which are incorporated herein by
reference in their entireties.
FIELD OF THE INVENTION
[0002] This invention relates to compositions comprising inhibitors
of human histone methyltransferase EZH2, the catalytic subunit of
the PRC2 complex which catalyzes the mono- through tri-methylation
of lysine 27 on histone H3 (H3-K27), and one or more other
therapeutic agents, particularly anticancer agents, and methods of
combination therapy for treating cancer.
BACKGROUND OF THE INVENTION
[0003] Combination-therapy treatments for cancer have become more
common, in part due to the perceived advantage of attacking the
disease via multiple avenues. Although many effective
combination-therapy treatments have been identified over the past
few decades; in view of the continuing high number of deaths each
year resulting from cancer, a continuing need exists to identify
effective therapeutic regimens for use in anticancer treatment.
SUMMARY OF THE INVENTION
[0004] The instant invention is based at least in part on the
discovery that an EZH2 inhibitor such as Compound 44 (also known as
EPZ-6438, E7438)
##STR00001##
in combination with a variety of agents, including the current
standard of care, is very active in the treatment of certain
cancers regardless of EZH2 mutation status. In a certain embodiment
the cancer is a lymphoma. In a certain embodiment the cancer is a
Non-Hodgkin's Lymphoma (NHL) or Diffuse Large B-cell Lymphoma
(DLBCL) of germinal center B cell (GCB) origin. In certain
embodiments the lymphoma is an EZH2 mutant lymphoma. In certain
embodiments the lymphoma is an EZH2 non-mutant or EZH2 wild-type
lymphoma. The instant invention is also based upon the discovery
that EZH2 inhibitors, such as Compound 44 and glucocorticoid
receptor agonists (GRags), such as Prednisone, Prednisolone or
Dexamethasone, cooperate to dramatically enhance therapeutic
activity in cancer. The combination of Compound 44 and prednisolone
extends the range of cells that are sensitive to EZH2 inhibition,
from mutant-bearing only to all GCB NHL cells.
[0005] In one aspect, the present invention is directed to a method
for treating cancer in a patient in need thereof comprising
administering a therapeutically effective amount of an EZH2
inhibitor and a therapeutically effective amount of a standard of
care agent.
[0006] In another aspect, the present invention is directed to a
method for treating cancer in a patient in need thereof comprising
administering a therapeutically effective amount of a combination
comprising an EZH2 inhibitor and a standard of care agent.
[0007] Another aspect of the present invention is directed to a
method for treating cancer in a patient in need thereof comprising
administering a therapeutically effective amount of a composition
comprising an EZH2 inhibitor and a standard of care agent.
[0008] In some embodiment, the EZH2 mutant lymphoma is an Y646,
A682, or A692 mutation.
[0009] In some embodiments, the standard of care agent is one or
more compounds selected from the group consisting of an R-CHOP
component, a BCL inhibitor, and a BCR inhibitor.
[0010] In some embodiments, the R-CHOP is a GRag component of CHOP,
prednisolone or dexamethasone.
[0011] In some embodiments, R-CHOP is a glucocorticosteroid
receptor agonist. In certain embodiments, the glucocorticosteroid
receptor agonist is prednisolone or dexamethasone.
[0012] In some embodiments, doxorubicin is omitted from R-CHOP.
[0013] In some embodiments, the BCL inhibitor is navitoclax,
obatoclax or ABT-19.
[0014] In some embodiments, the BCR inhibitor is rituximab, the AKT
inhibitor MK-2206, idelalisib, trametinib, tamatanib, everolimus or
ibrutinib.
[0015] In some embodiments, the BCR inhibitor is PI3K/Akt/mTOR
signaling cascade inhibitor.
[0016] In some embodiments, the BCR inhibitor is rituximab,
MK-2206, idelalisib, trametinib, tamatanib, everolimus, VELCADE, or
ibrutinib.
[0017] In some embodiments, the EZH2 inhibitor and the standard of
care agent are administered simultaneously or sequentially. In
other embodiments, the EZH2 inhibitor is administered prior to
administration of the standard of care agent.
[0018] In some embodiments, at least one gene is upregulated in the
patient. In certain embodiments, the gene that is upregulated is
selected from the group consisting of Sestrin, TNF, and GILZ. In
other embodiments, the gene the gene that is upregulated is a
glucocorticoid target gene.
[0019] In some embodiments, the upregulation of a gene is used to
determine or adjust the therapeutically effective amount of the
EZH2 inhibitor and the standard of care agent.
[0020] In another aspect, the present invention is directed to a
method of selecting a patient for treatment wherein the patient is
selected based on the expression profile of one or more genes
selected from the group consisting of Sestrin, TNF and GILZ.
[0021] In one aspect, the present invention is directed to a method
for treating cancer in a patient in need thereof comprising
administering a therapeutically effective amount of an EZH2
inhibitor and a therapeutically effective amount of a standard of
care agent wherein the patient has upregulated expression of
Sestrin, TNF or GILZ.
[0022] In some embodiments, the cancer is an EZH2 inhibitor
resistant or refractory cancer.
[0023] In some embodiments, the cancer is characterized by
increased trimethylation at H3K27.
[0024] One aspect of the invention is directed to the combination
of the EZH2 inhibitor and the GRag reverses the insensitivity in
EZH2-inhibitor resistant or refractory mutant cells, including EZH2
mutation bearing cells.
[0025] In certain embodiments, the EZH2 inhibitor is Compound 44,
or a pharmaceutically acceptable salt or solvate thereof and one or
more other therapeutic agents.
[0026] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF FIGURES
[0027] FIGS. 1A-1F are a series of Fa-CI plots demonstrating
combination benefit with CHOP components and Compound 44 (Cpd 44)
in mutant EZH2 germinal center B-cell lymphoma cell lines. Compound
44 and doxorubicin act synergistically in the WSU-DLCL2 cells (FIG.
1A) and produce an additive effect in SU-DHL-10 cells (FIG. 1D).
Combination benefit is observed with mafosfamide in WSU-DLCL2 cells
(FIG. 1C) and SU-DHL-10 cells (FIG. 1F). Combination benefit is
also observed with vincristine in both EZH2 Y646 mutant cell lines:
WSU-DLCL2 cells (FIG. 1B) and SU-DHL-10 cells (FIG. 1E). In
WSU-DLCL2 doses ranged from 0.16-20 nM for doxorubicin, 0.04-5 nM
for vincristine, 0.156-10 .mu.M for mafosfamide, and 15-1000 nM for
Compound 44. In SU-DHL-10 cells doses ranged from 0.5-60 nM for
doxorubicin, 0.016-2 nM for vincristine, 0.156-10 .mu.M for
mafosfamide, and 1.56-100 nM for Compound 44. Cells were treated
according to pretreatment model A, and data analyzed with the
Calcusyn software.
[0028] FIGS. 2A-2D are a series of plots demonstrating that
glucocorticoid agonists enhance potency of Compound 44 (Cpd 44) in
EZH2 mutant lymphoma lines. Potency of Compound 44 is dramatically
increased when combined with glucocorticoid agonists. The addition
of prednisolone (FIG. 2A, 2C) or dexamethasone (FIG. 2B, 2D) in 2
EZH2 Y646F mutant DLBCL lines according to pre-treatment model A
produces a dose dependent shift in the IC.sub.50 of Compound 44.
Doses ranged from 15 nM-1000 nM for prednisolone and 1.5 nM-100 nM
for dexamethasone in both cell lines. Doses of Compound 44 ranged
from 15-1000 nM in WSU-DLCL2 cells and 1.5-100 nM in SU-DHL-10
cells.
[0029] FIGS. 3A-3D are a series of dose response plots
demonstrating the benefits of combinations of Compound 44 (Cpd 44)
with prednisolone or dexamethasone in WSU-DLCL2 EZH2 mutant (FIG.
3A, 3B) and DOHH2 EZH2 wild-type (FIG. 3C, 3D) GCB lymphoma cell
lines, respectively. Doses of Compound 44 ranged from 15.6-1000 nM,
doses of prednisolone ranged from 7.8-1000 nM, and doses of
dexamethasone ranged from 0.8-100 nM. (FIGS. 3A and 3B). Potency of
Compound 44 was increased with prednisolone or dexamethasone in
EZH2 mutant WSU-DLCL2 cells (FIGS. 3C and 3D). Compound 44 showed
no anti-proliferative effect as a single agent in DOHH2 EZH2
wild-type cells, therefore the potency shift of prednisolone or
dexamethasone was measured. The potency of prednisolone or
dexamethasone was increased with addition of Compound 44 in DOHH2
cells.
[0030] FIG. 4 is a summary table showing that Compound 44 (Cpd
44)/glucocorticoid agonist combination overcomes EZH2 inhibitors
(EZH2i) insensitivity in cell lines resistant to EZH2 inhibitors.
Overall, a combination of prednisolone and Compound 44 leads to
greater sensitivity in all GCB cell lines tested, not just EZH2i
sensitive cell lines. Except for RL cells, where sequence of drug
addition is crucial as preincubation with prednisolone, followed by
Compound 44, is not effective.
[0031] FIGS. 5A and 5B are two plots showing the very strong
synergy observed in the EZH2 mutant lymphoma cell line with the
combination of Compound 44 (Cpd 44) and other targeted therapies.
Very strong synergy is observed when Compound 44 is combined with
the BCL2 inhibitor navitoclax (in FIG. 5A), as well as with the
mTOR inhibitor everolimus (in FIG. 5B). Dose ranges for navitoclax
are 0.16-10 .mu.M, 0.04-5 nM for everolimus, and 31-2000 nM for
Compound 44. These data were generated in the pretreatment model A
and data analyzed with Calcusyn software.
[0032] FIG. 6 is a summary table of the results from combinations
of various drugs and/or drug therapies with Compound 44 (Cpd 44).
Combination benefit with Compound 44 was achieved with all drugs
tested in EZH2 mutant lymphoma lines. Glucocorticoid agonists
demonstrated combination benefit with EZH2 WT and mutant GCB
lymphoma lines.
[0033] FIGS. 7A-7C are a series of plots demonstrating that
Compound 44 (Cpd 44)-CHOP combinations show enhanced anti-tumor
activity compared to single agents in several EZH2 mutant lymphoma
xenograft models. WSU-DLCL2 (EZH2 Y646F) xenografts were treated
with Compound 44, CHOP, or the combination for 28 days, as
specified in the methods (FIG. 7A). Mean tumor volumes+/-SEM are
plotted. Both doses of Compound 44 at 150 mg/kg TID and 225 mg/kg
BID were statistically more significant in tumor growth inhibition
than vehicle alone (*p value<0.05). Treatment with Compound 44
at 225 mg/kg BID plus CHOP resulted in greater tumor regression
than with any single agent alone (***p value<0.001 versus
vehicle). Statistics calculated by repeated measures ANOVA. SU-DHL6
(EZH2 Y646N) xenografts were treated with Compound 44, CHOP, or the
combination for 28 days, as specified in the methods (FIG. 7B).
Mean tumor volumes+/-SEM are plotted in top panel. CHOP or single
agent Compound 44 alone had no effect on tumor growth, but
treatment with Compound 44 at 225 mg/kg BID plus CHOP resulted in
tumor growth regression during the treatment period of 28 days,
while also maintaining tumor growth delay after 32 days of dosing
cessation (*p value<0.0001). Survival curves (bottom panel) out
to 60 days demonstrate significant tumor growth delay in animals
treated with a combination of Compound 44 and CHOP (**p
value<0.05). Statistics calculated by two-tailed t-test.
SUDHL-10 (EZH2 Y646F) xenografts were treated with Compound 44, COP
(SOC without the doxorubicin component), or the combination for 28
days, as specified in the methods (FIG. 7C). Mean tumor
volumes+/-SEM are plotted in top panel. Percent survival out to 60
days in a tumor growth delay study is plotted in the middle panel
(Note: 500 mg/kg and 250 mg/kg+COP survival curves are
overlapping). Mean tumor weights are compared in the bottom panel,
demonstrating the significant differences in tumor weight between
groups (*p value<0.05, **p value<0.01, ****p
value<0.0001).
[0034] FIGS. 8A-8C are panels showing the change in expression
levels of glucocorticoid target genes Sestrin 1 (SESN1, FIG. 8A),
TNF (FIG. 8B) and GILZ (FIG. 8C) when various cell lines are
treated with Compound 44, prednisolone, a combination of Compound
44 and prednisolone, or DMSO. As shown in FIGS. 8A-8C, an increase
in the expression levels of Sestrin 1, TNF, and GILZ was observed
after co-treatment compared to Compound 44 or prednisolone
alone.
[0035] FIGS. 9A-9D are panels showing that global H3K27 acetylation
and trimethylation are unaffected by prednisolone or combination
treatment. Cells were treated for 4 days with increasing doses of
prednisolone, Compound 44 (Cpd 44), or a combination of Compound 44
with a constant dose of prednisolone. Acid extracted histones were
analyzed by ELISA for H3K27Me3 levels (FIG. 9A) (prednisolone
alone, left panel; Compound 44/prednisolone combination, right
panel, with IC.sub.50 values as insets of each graph). For
prednisolone treatment, H3K27Me3 values are represented as a bar
graph as there were no dose dependent changes observed with this
compound. WSU-DLCL2 (FIG. 9B), OCI-LY19 (FIG. 9C) or RL cells (FIG.
9D) were treated for 4 days with increasing doses of prednisolone,
Compound 44, or a combination of Compound 44 with a constant dose
of prednisolone. Acid extracted histones were analyzed by western
blot for H3K27 acetylation levels.
[0036] FIG. 10 is a western blot showing that single agent
treatment with Compound 44 or prednisolone has no effect on SMARCB1
protein levels.
[0037] FIGS. 11A and 11D are Fa-CI plots demonstrating the
combination benefit of Compound 44 and everolimus. FIGS. 11B and
11E are panels showing apoptosis in WSU-DLCL2 and SU-DHL-5 cells
treated with, Compound 44, everolimus, a combination of Compound 44
and everolimus, or DMSO. FIGS. 11C and 11F are plots showing the
changes in the G1 phase of cell cycle observed after co-treatment
compared to Compound 44 alone in both WSU-DLCL2 and SU-DHL-5 cells.
Strong synergistic effects were observed for a combination of
Compound 44 and everolimus in both WSU-DLCL2 cells and SU-DHL-5
(FIG. 11A, 11D).
[0038] FIGS. 12A and 12D are Fa-CI plots demonstrating the
combination benefit of Compound 44 and ibrutinib. FIGS. 12B and 12E
are panels showing apoptosis in WSU-DLCL2 and SU-DHL-5 cells
treated with Compound 44, ibrutinib, a combination of Compound 44
and ibrutinib, or DMSO. FIGS. 12C and 12F are plots showing the
changes in the G1 phase of cell cycle observed after co-treatment
compared to Compound 44 alone in both WSU-DLCL2 and SU-DHL-5 cells.
Strong synergistic effects were observed for a combination of
Compound 44 and ibrutinib in both WSU-DLCL2 cells and SU-DHL-5
(FIGS. 12A, 12D).
[0039] FIGS. 13A, 13D, and 13G are Fa-CI plots demonstrating the
combination benefit of Compound 44 and MK-2206 in WSU-DLCL2,
SU-DHL-5, and OCI-LY19 cells. FIGS. 13B, 13E, and 13H are panels
showing apoptosis in WSU-DLCL2, SU-DHL-5, and OCI-LY19 cells
treated with Compound 44, MK-2206, a combination of Compound 44 and
MK-2206, or DMSO. FIGS. 13C, 13F, and 13I are plots showing the
changes in the G1 phase of cell cycle observed after co-treatment
compared to Compound 44 alone in the three cell lines. Strong
synergistic effects were observed for a combination of Compound 44
and MK-2206 in WSU-DLCL2 cells, SU-DHL-5, and OCI-LY19 cells (FIGS.
13A, 13D and 13G).
[0040] FIGS. 14A-14C are bar graphs showing change in gene
expression of EGR1, FOS, TCL1, AICDA, and GJA1 when WSU-DLCL2 and
SU-DHL-5 cells were treated with Compound 44, ibrutinib, MK-2206, a
combination of Compound 44 and ibrutinib, or a combination of
Compound 44 and MK-2206. Downregulation of EGR1 (40 fold) and FOS
(4 fold) and upregulation of AICDA (3 fold), TCL1A (5 fold), and
GJA1 (3 fold) was observed with a combination of Compound 44 and a
second agent than was observed with treatment of single agents
alone (FIGS. 14A-14C).
[0041] FIG. 15 is a diagram of the signaling pathways implicated in
Diffuse Large B-cell Lymphoma (DLBCL) biology and the targets of
various chemotherapeutic agents within the signaling pathway.
[0042] FIGS. 16A and 16D are plots showing the changes in the G1
phase of cell cycle observed after treatment of WSU-DLCL2 and
SU-DHL-5 cells with Compound 44, everolimus, a combination of
Compound 44 and everolimus, and DMSO. FIGS. 16B and 16E are plots
showing the changes in the S phase of cell cycle observed after
treatment of WSU-DLCL2 and SU-DHL-5 cells with Compound 44,
everolimus, a combination of Compound 44 and everolimus, and DMSO.
FIGS. 16C and 16F are plots showing the changes in G2/M phases of
the cell cycle observed after treatment of WSU-DLCL2 and SU-DHL-5
cells with Compound 44, everolimus, a combination of Compound 44
and everolimus, and DMSO. Synergistic decrease of cells in G1, S,
and G2/M phases of the cell cycle, respectively, is seen 48 hours
after co-treatment on SU-DHL-5 cells (FIGS. 16D-16F). No change in
sub-G1 phase of the cell cycle was observed when WSU-DLCL2 cells
are treated with single agents or in combination (FIG. 16A).
Synergistic time-dependent decrease of cells in S phase and G2/M
phase of the cell cycle, respectively, was observed when WSU-DLCL2
cells were treated with the combination (FIG. 16B, 16C).
[0043] FIGS. 17 A and 17D are plots showing the changes in the G1
phase of cell cycle observed after treatment of WSU-DLCL2 and
SU-DHL-5 cells with Compound 44, ibrutinib a combination of
Compound 44 and ibrutinib and DMSO. FIGS. 17B and 17E are plots
showing the changes in the S phase of cell cycle observed after
treatment of WSU-DLCL2 and SU-DHL-5 cells with Compound 44,
ibrutinib, a combination of Compound 44 and ibrutinib, and DMSO.
FIGS. 17C and 17F are plots showing the changes in G2/M phases of
the cell cycle observed after treatment of WSU-DLCL2 and SU-DHL-5
cells with Compound 44, ibrutinib, a combination of Compound 44 and
ibrutinib, and DMSO. FIGS. 17A-17F show a synergistic decrease of
cells in G1, S, and G2/M phases of the cell cycle, respectively, 24
hours after co-treatment of WSU-DLCL2 cells and SU-DHL-5 cells
compared to Compound 44 or ibrutinib as single agents.
[0044] FIGS. 18A, 18D, and 18G are plots showing the changes in the
G1 phase of cell cycle observed after treatment of WSU-DLCL2,
SU-DHL-5, and OCI-LY19 cells with Compound 44, MK-2206, a
combination of Compound 44 and MK-2206, and DMSO. FIGS. 18B, 18E,
and 18H are plots showing the changes in the S phase of cell cycle
observed after treatment of WSU-DLCL2, SU-DHL-5, and OCI-LY19 cells
with Compound 44, MK-2206, a combination of Compound 44 and
MK-2206, and DMSO. FIGS. 18C, 18F, and 18I are plots showing the
changes in G2/M phases of the cell cycle observed after treatment
of WSU-DLCL2, SU-DHL-5, and OCI-LY19 cells with Compound 44,
MK-2206, a combination of Compound 44 and MK-2206, and DMSO. FIGS.
18A-18I show a synergistic decrease of cells in G1, S, and G2/M
phases of the cell cycle, respectively after co-treatment of
WSU-DLCL2 cells and SU-DHL-5 cells compared to Compound 44 or
MK-2206 as single agents.
[0045] FIG. 19 is a bar graph showing the change in expression
levels of the glucocorticoid receptor, normalized to DMSO controls,
for EZH2 wild-type (OCI-LY19, DOHH2), EZH2 Y646-sensitive
(WSU-DLCL2, SUDHL10), and EZH2 Y646-resistant (RL, SUDHL) cell
lines treated with the Compound 44, prednisolone, a combination of
Compound 44 and prednisolone, or DMSO. Fold change values were
quantified using the .DELTA..DELTA.Ct method and ACTB, B2M and
GAPDH as reference genes. As the results show, the expression
levels of glucocorticoid receptors were not commonly affected among
cell lines in the combination.
[0046] FIGS. 20A-20C show the effects of omitting one or all
chemotherapy components from the CHOP regime in xenograft-bearing
mice. FIG. 20A is a plot showing the change in tumor weight in
SUDHL10 (EZH2 Y646F) xenograft-bearing mice treated with Compound
44, COP (chemotherapy without the Doxorubicin component), or their
combination for 28 days. FIG. 20B is a is a plot showing the change
in tumor volume in SUDHL10 (EZH2 Y646F) xenograft-bearing mice
treated for 28 days with two doses of Compound 44, Prednisone, or
their combination. FIG. 20C is a plot showing the change in body
weight in SUDHL10 (EZH2 Y646F) xenograft-bearing mice treated with
Compound 44, Prednisone, or their combination (See FIG. 20B). Mice
dosed with the maximal tolerated dose of Compound 44 or with the
Compound 44/COP combination showed 100% survival on day 60, the
combination group showed the smallest day 28 tumor weights from all
other treatment groups, including the maximal tolerated dose for
Compound 44 (FIG. 20A). Prednisone dosing alone did not induce any
significant anti-tumor effect (FIG. 20B). In line with the previous
study, dosing of Compound 44 generated only a partial response, but
co-dosing of Compound 44 with Prednisone, but not with the 2 cycle
Prednisone regimen, induced the maximal possible regression
achieved with higher doses of Compound 44 alone.
DETAILED DESCRIPTION OF THE INVENTION
[0047] The instant invention is based at least in part on the
discovery that Compound 44 in combination with a variety of agents,
including the current standard of care, is active in the treatment
of certain cancers regardless of EZH2 mutation status. In a certain
embodiment the cancer is a lymphoma. In a certain embodiment the
cancer is a Non-Hodgkin's Lymphoma (NHL) or Diffuse Large B-cell
Lymphoma (DLBCL) of germinal center B cell (GCB) origin. In certain
embodiments the lymphoma is an EZH2 mutant lymphoma. In certain
embodiments the lymphoma is an EZH2 non-mutant or EZH2 wild-type
lymphoma.
[0048] In certain aspects of the invention, the EZH2 inhibitor is
Compound 44 (also known as EPZ-6438, E7438) having the following
formula:
##STR00002##
or a pharmaceutically acceptable salt thereof.
[0049] The present invention is based upon the discovery that EZH2
histone methyltransferase inhibitors and other anti-cancer agents
can be used in combination to treat certain tumors with superior
results than those achieved by treating tumors with EZH2 histone
methyltransferase inhibitors and the anti-cancer agents alone.
Accordingly, the present invention provides a composition
comprising an EZH2 histone methyltransferase inhibitor and one or
more other therapeutic agents, and methods for their use to treat
diseases the course of which can be influenced by modulating the
methylation status of histones or other proteins, e.g., cancer. In
a certain embodiment, the present invention features a composition
comprising Compound 44 and prednisone. The present invention also
includes methods for combination therapies comprising EZH2 histone
methyltransferase inhibitor and one or more therapeutic agents,
such as a Compound 44 and prednisone, to treat cancer, e.g.,
follicular lymphoma (FL) and diffuse cell large B-cell lymphoma
(DCLBL). Specifically, the methods of the present invention are
useful for treating or preventing cancer or inhibiting cancer cell
proliferation.
[0050] An aspect of the present invention relates to methods for
treating or alleviating a symptom of cancer or precancerous
condition in a subject by administering to a subject expressing a
mutant EZH2 a therapeutically effective amount of an EZH2 inhibitor
and one or more other therapeutic agents. The mutant EZH2 of the
present invention refers to a mutant EZH2 polypeptide or a nucleic
acid sequence encoding a mutant EZH2 polypeptide. In certain
embodiments the mutant EZH2 comprises one or more mutations in its
substrate pocket domain.
[0051] Another aspect of the present invention relates to methods
for treating or alleviating a symptom of cancer or precancerous
condition in a subject by administering to a subject expressing a
mutant EZH2 or a wild-type EZH2 a therapeutically effective amount
of an EZH2 inhibitor and one or more other therapeutic agents. The
mutant EZH2 of the present invention refers to a mutant EZH2
polypeptide or a nucleic acid sequence encoding a mutant EZH2
polypeptide. In certain embodiments the mutant EZH2 comprises one
or more mutations in its substrate pocket domain.
[0052] In another aspect, the present invention relates to methods
for treating or alleviating a symptom of cancer or precancerous
condition in a subject by administering to a subject expressing a
mutant EZH2 or a wild-type EZH2 a therapeutically effective amount
of an EZH2 inhibitor, e.g., Compound 44 and one or more
glucocorticoid receptor agonists (GRags), e.g., Prednisone,
Prednisolone or Dexamethasone. The mutant EZH2 of the present
invention refers to a mutant EZH2 polypeptide or a nucleic acid
sequence encoding a mutant EZH2 polypeptide. In certain embodiments
the mutant EZH2 comprises one or more mutations in its substrate
pocket domain.
[0053] Human EZH2 nucleic acids and polypeptides have previously
been described. See, e.g., Chen et al. (1996) Genomics 38:30-7 [746
amino acids]; Swiss-Prot Accession No. Q15910 [746 amino acids];
GenBank Accession Nos. NM_004456 and NP_004447 (isoform a [751
amino acids]); and GenBank Accession Nos. NM_152998 and NP_694543
(isoform b [707 amino acids]), each of which is incorporated herein
by reference in its entirety.
[0054] For purposes of this application, amino acid residue Y641 of
human EZH2 is to be understood to refer to the tyrosine residue
that is or corresponds to Y641 in Swiss-Prot Accession No.
Q15910.
[0055] Also for purposes of this application, a Y641 mutant of
human EZH2, and, equivalently, a Y641 mutant of EZH2, is to be
understood to refer to a human EZH2 in which the amino acid residue
corresponding to Y641 of wild-type human EZH2 is substituted by an
amino acid residue other than tyrosine.
[0056] In certain embodiments the R-CHOP is a GRag component of
CHOP, prednisolone or dexamethasone. In certain embodiments the
B-cell receptor (BCR) signaling pathways inhibitor is rituximab,
the AKT inhibitor MK-2206, idelalisib, trametinib, tamatanib,
everolimus or ibrutinib.
[0057] The invention is based, in part, on the discovery that
inhibitors of the PI3K-AKT-mTOR BCR signaling pathway, e.g.,
idelalisib, MK-2206 and everolimus, induced very strong synergy in
the WSU-DLCL2 and SU-DHL-10 cell lines when combined with Compound
44. The invention is also based, in part, on the discovery that the
combination of Compound 44 and inhibitors of the B-cell receptor
pathway, e.g., ibrutinib and tamatanib displayed very strong
synergy in both mutant cell lines. In certain embodiments, the BCL
receptor inhibitor is navoticlax or ABT-199.
[0058] In some embodiments, the cancer is a Non-Hodgkin's Lymphoma,
Diffuse Large B-cell Lymphoma, or Non-Hodgkin's Lymphoma germinal
center B cell.
[0059] In some embodiments, the standard of care agent is one or
more compounds selected from the group consisting of R-CHOP, a BCL
inhibitor, and a BCR inhibitor.
[0060] In some embodiments, the R-CHOP is a GRag component of CHOP,
prednisolone or dexamethasone.
[0061] In some embodiments, the BCR inhibitor is rituximab, the AKT
inhibitor MK-2206, idelalisib, trametinib, tamatanib, everolimus or
ibrutinib.
[0062] In some embodiments, the cancer is an EZH2 mutant
cancer.
[0063] In some embodiments, the cancer is an EZH2 inhibitor
resistant or refractory cancer.
[0064] In one embodiment the amino acid sequence of a Y641 mutant
of EZH2 differs from the amino acid sequence of wild-type human
EZH2 only by substitution of a single amino acid residue
corresponding to Y641 of wild-type human EZH2 by an amino acid
residue other than tyrosine.
[0065] In one embodiment the amino acid sequence of a Y641 mutant
of EZH2 differs from the amino acid sequence of wild-type human
EZH2 only by substitution of phenylalanine (F) for the single amino
acid residue corresponding to Y641 of wild-type human EZH2. The
Y641 mutant of EZH2 according to this embodiment is referred to
herein as a Y641F mutant or, equivalently, Y641F.
[0066] In one embodiment the amino acid sequence of a Y641 mutant
of EZH2 differs from the amino acid sequence of wild-type human
EZH2 only by substitution of histidine (H) for the single amino
acid residue corresponding to Y641 of wild-type human EZH2. The
Y641 mutant of EZH2 according to this embodiment is referred to
herein as a Y641H mutant or, equivalently, Y641H.
[0067] In one embodiment the amino acid sequence of a Y641 mutant
of EZH2 differs from the amino acid sequence of wild-type human
EZH2 only by substitution of asparagine (N) for the single amino
acid residue corresponding to Y641 of wild-type human EZH2. The
Y641 mutant of EZH2 according to this embodiment is referred to
herein as a Y641N mutant or, equivalently, Y641N.
[0068] In one embodiment the amino acid sequence of a Y641 mutant
of EZH2 differs from the amino acid sequence of wild-type human
EZH2 only by substitution of serine (S) for the single amino acid
residue corresponding to Y641 of wild-type human EZH2. The Y641
mutant of EZH2 according to this embodiment is referred to herein
as a Y641S mutant or, equivalently, Y641 S.
[0069] In one embodiment the amino acid sequence of a Y641 mutant
of EZH2 differs from the amino acid sequence of wild-type human
EZH2 only by substitution of cysteine (C) for the single amino acid
residue corresponding to Y641 of wild-type human EZH2. The Y641
mutant of EZH2 according to this embodiment is referred to herein
as a Y641C mutant or, equivalently, Y641C.
[0070] In one embodiment the amino acid sequence of a A677 mutant
of EZH2 differs from the amino acid sequence of wild-type human
EZH2 only by substitution of a non-alanine amino acid, preferably
glycine (G) for the single amino acid residue corresponding to A677
of wild-type human EZH2. The A677 mutant of EZH2 according to this
embodiment is referred to herein as an A677 mutant, and preferably
an A677G mutant or, equivalently, A677G. A677 is also referred to
as A682.
[0071] In one embodiment the amino acid sequence of a A687 mutant
of EZH2 differs from the amino acid sequence of wild-type human
EZH2 only by substitution of a non-alanine amino acid, preferably
valine (V) for the single amino acid residue corresponding to A687
of wild-type human EZH2. The A687 mutant of EZH2 according to this
embodiment is referred to herein as an A687 mutant and preferably
an A687V mutant or, equivalently, A687V. A687 is also referred to
as A692.
[0072] In one embodiment the amino acid sequence of a mutant of
EZH2 differs from the amino acid sequence of wild-type human EZH2
in one or more amino acid residues in its substrate pocket domain.
The mutant of EZH2 according to this embodiment is referred to
herein as an EZH2 mutant.
[0073] Other exemplary substitution amino acid mutation includes a
substitution at amino acid position 677, 687, or 641, such as, but
is not limited to a substitution of glycine (G) for the wild type
residue alanine (A) at amino acid position 677 (A677G); a
substitution of valine (V) for the wild type residue alanine (A) at
amino acid position 687 (A687V); a substitution of phenylalanine
(F) for the wild type residue tyrosine (Y) at amino acid position
641 (Y641F); a substitution of histidine (H) for the wild type
residue tyrosine (Y) at amino acid position 641 (Y641H); a
substitution of asparagine (N) for the wild type residue tyrosine
(Y) at amino acid position 641 of (Y641N); a substitution of serine
(S) for the wild type residue tyrosine (Y) at amino acid position
641 of (Y641 S); or a substitution of cysteine (C) for the wild
type residue tyrosine (Y) at amino acid position 641 (Y641C). Y641
is also referred to as Y646.
[0074] Cells heterozygous for EZH2 would be expected to display a
malignant phenotype due to the efficient formation of H3-K27me1 by
the WT enzyme and the efficient, subsequent transition of this
progenitor species to H3-K27me2, and, especially, H3-K27me3, by the
mutant enzyme form(s).
[0075] Another aspect of the invention is a method for inhibiting
in a subject conversion of H3-K27 to trimethylated H3-K27. The
inhibition can involve inhibiting in a subject conversion of
unmethylated H3-K27 to monomethylated H3-K27, conversion of
monomethylated H3-K27 to dimethylated H3-K27, conversion of
dimethylated H3-K27 to trimethylated H3-K27, or any combination
thereof, including, for example, conversion of monomethylated
H3-K27 to dimethylated H3-K27 and conversion of dimethylated H3-K27
to trimethylated H3-K27. As used herein, unmethylated H3-K27 refers
to histone H3 with no methyl group covalently linked to the amino
group of lysine 27. As used herein, monomethylated H3-K27 refers to
histone H3 with a single methyl group covalently linked to the
amino group of lysine 27. Monomethylated H3-K27 is also referred to
herein as H3-K27me1. As used herein, dimethylated H3-K27 refers to
histone H3 with two methyl groups covalently linked to the amino
group of lysine 27. Dimethylated H3-K27 is also referred to herein
as H3-K27me2. As used herein, trimethylated H3-K27 refers to
histone H3 with three methyl groups covalently linked to the amino
group of lysine 27. Trimethylated H3-K27 is also referred to herein
as H3-K27me3.
A composition of the present invention comprises Compound 44 and
one or more other therapeutic agents. The compounds and
combinations of the invention are suitable for administration as
part of a combination therapy with one or more other therapeutic
agents or treatment modality, suitable to be administered together,
sequentially, or in alternation. Other compounds suitable for the
methods of the invention are described in U.S. Publication
20120264734, the contents of which are hereby incorporated by
reference in their entireties.
[0076] In certain aspects of the invention an inhibitor of EZH2
"selectively inhibits" histone methyltransferase activity of the
mutant EZH2 when it inhibits histone methyltransferase activity of
the mutant EZH2 more effectively than it inhibits histone
methyltransferase activity of wild-type EZH2. For example, in one
embodiment the selective inhibitor has an IC50 for the mutant EZH2
that is at least 40 percent lower than the IC50 for wild-type EZH2.
In one embodiment the selective inhibitor has an IC50 for the
mutant EZH2 that is at least 50 percent lower than the IC50 for
wild-type EZH2. In one embodiment the selective inhibitor has an
IC50 for the mutant EZH2 that is at least 60 percent lower than the
IC50 for wild-type EZH2. In one embodiment the selective inhibitor
has an IC50 for the mutant EZH2 that is at least 70 percent lower
than the IC50 for wild-type EZH2. In one embodiment the selective
inhibitor has an IC50 for the mutant EZH2 that is at least 80
percent lower than the IC50 for wild-type EZH2. In one embodiment
the selective inhibitor has an IC50 for the mutant EZH2 that is at
least 90 percent lower than the IC50 for wild-type EZH2.
[0077] In certain aspects of the invention the inhibitor inhibits
conversion of H3-K27me2 to H3-K27me3. In one embodiment the
inhibitor is said to inhibit trimethylation of H3-K27. Since
conversion of H3-K27me1 to H3-K27me2 precedes conversion of
H3-K27me2 to H3-K27me3, an inhibitor of conversion of H3-K27me1 to
H3-K27me2 naturally also inhibits conversion of H3-K27me2 to
H3-K27me3, i.e., it inhibits trimethylation of H3-K27. It is also
possible to inhibit conversion of H3-K27me2 to H3-K27me3 without
inhibition of conversion of H3-K27me1 to H3-K27me2. Inhibition of
this type would also result in inhibition of trimethylation of
H3-K27, albeit without inhibition of dimethylation of H3-K27.
[0078] In one embodiment the inhibitor inhibits conversion of
H3-K27me1 to H3-K27me2 and the conversion of H3-K27me2 to
H3-K27me3. Such inhibitor may directly inhibit the conversion of
H3-K27me1 to H3-K27me2 alone. Alternatively, such inhibitor may
directly inhibit both the conversion of H3-K27me1 to H3-K27me2 and
the conversion of H3-K27me2 to H3-K27me3.
[0079] In certain aspects of the invention, the inhibitor compound
inhibits histone methyltransferase activity Inhibition of histone
methyltransferase activity can be detected using any suitable
method. The inhibition can be measured, for example, either in
terms of rate of histone methyltransferase activity or as product
of histone methyltransferase activity.
[0080] The inhibition is a measurable inhibition compared to a
suitable control. In one embodiment, inhibition is at least 10
percent inhibition compared to a suitable control. That is, the
rate of enzymatic activity or the amount of product with the
inhibitor is less than or equal to 90 percent of the corresponding
rate or amount made without the inhibitor. In various other
embodiments, inhibition is at least 20, 25, 30, 40, 50, 60, 70, 75,
80, 90, or 95 percent inhibition compared to a suitable control. In
one embodiment, inhibition is at least 99 percent inhibition
compared to a suitable control. That is, the rate of enzymatic
activity or the amount of product with the inhibitor is less than
or equal to 1 percent of the corresponding rate or amount made
without the inhibitor.
[0081] A composition of the present invention comprises an EZH2
inhibitor or Compound 44 or a pharmaceutically acceptable salt
thereof, and one or more other therapeutic agents, or a
pharmaceutically acceptable salt thereof. The present invention
provides for the administration of an EZH2 inhibitor or Compound 44
or a pharmaceutically acceptable salt thereof, and one or more
therapeutic agents or a pharmaceutically acceptable salt thereof,
as a co-formulation or separate formulations, wherein the
administration of formulations is simultaneous, sequential, or in
alternation. In certain embodiments, the other therapeutic agents
can be an agent that is recognized in the art as being useful to
treat the disease or condition being treated by the composition of
the present invention. In other embodiment, the other therapeutic
agent can be an agent that is not recognized in the art as being
useful to treat the disease or condition being treated by the
composition of the present invention. In one aspect, the other
therapeutic agents can be an agent that imparts a beneficial
attribute to the composition of the present invention (e.g., an
agent that affects the viscosity of the composition). The
beneficial attribute to the composition of the present invention
includes, but is not limited to, pharmacokinetic or pharmacodynamic
co-action resulting from the combination of an EZH2 inhibitor or
Compound 44 and one or more other therapeutic agents. For example,
the one or more other therapeutic agents can be anticancer agents
or chemotherapeutic agents. For example, the one or more other
therapeutic agents can be glucocorticoids. For example, the one or
more other therapeutic agents can be selected from prednisone,
prednisolone, cyclophosphamide, vincristine, doxorubicin,
mafosfamide, cisplatin, AraC, everolimus, decitabine,
dexamethasone, or functional analogs, derivatives, prodrugs, and
metabolites thereof. In another aspect, the other therapeutic agent
can be Prednisone or its active metabolite, Prednisolone.
[0082] The therapeutic agents set forth below are for illustrative
purposes and not intended to be limiting. The present invention
includes at least one other therapeutic agent selected from the
lists below. The present invention can include more than one other
therapeutic agent, e.g., two, three, four, or five other
therapeutic agents such that the composition of the present
invention can perform its intended function.
[0083] In another embodiment, the other therapeutic agent is a
chemotherapeutic agent (also referred to as an anti-neoplastic
agent or anti-proliferative agent), selected from the group
including an alkylating agent; an antibiotic; an anti-metabolite; a
detoxifying agent; an interferon; a polyclonal or monoclonal
antibody; an EGFR inhibitor; a HER2 inhibitor; a histone
deacetylase inhibitor; a hormone; a mitotic inhibitor; an MTOR
inhibitor; a multi-kinase inhibitor; a serine/threonine kinase
inhibitor; a tyrosine kinase inhibitors; a VEGF/VEGFR inhibitor; a
taxane or taxane derivative, an aromatase inhibitor, an
anthracycline, a microtubule targeting drug, a topoisomerase poison
drug, an inhibitor of a molecular target or enzyme (e.g., a kinase
or a protein methyltransferase), a cytidine analogue drug or any
chemotherapeutic, anti-neoplastic or anti-proliferative agent
listed in www.cancer.org/docroot/cdg/cdg_0.asp.
[0084] The present invention provides methods for combination
therapy in which a composition comprising an EZH2 inhibitor or
Compound 44 or a pharmaceutically acceptable salt thereof, and one
or more other therapeutic agents are administered to a subject in
need for treatment of a disease or cancer. The combination therapy
can also be administered to cancer cells to inhibit proliferation
or induce cell death. In one aspect Compound 44 or a
pharmaceutically acceptable salt thereof is administered subsequent
to administration of the composition of the present invention
comprising Compound 44 or a pharmaceutically acceptable salt
thereof, and one or more other therapeutic agents. In one aspect,
Compound 44 or a pharmaceutically acceptable salt thereof is
administered prior to administration of the composition of the
present invention comprising Compound 44 or a pharmaceutically
acceptable salt thereof, and one or more other therapeutic agents.
In one aspect, Compound 44 or a pharmaceutically acceptable salt
thereof is administered subsequent to administration of one or more
therapeutic agents, such that the other therapeutic agents are
administered either in a single composition or in two or more
compositions, e.g. administered simultaneously, sequentially, or in
alternation. In one aspect, Compound 44 or a pharmaceutically
acceptable salt thereof is administered prior to administration of
one or more therapeutic agents, such that the other therapeutic
agents are administered either in a single composition or in two or
more compositions, e.g. administered simultaneously, sequentially,
or in alternation.
[0085] In one embodiment, a composition of the present invention
includes Compound 44 or a pharmaceutically acceptable salt thereof,
and one or more anticancer agents, e.g., CHOP (cyclophosphamide,
hydroxydaunorubicin, oncovin, and prednisone or prednisolone) or
R-CHOP (rituximab, cyclophosphamide, hydroxydaunorubicin, oncovin,
prednisone or prednisolone). In one embodiment, a composition of
the present invention includes Compound 44 or a pharmaceutically
acceptable salt thereof, and prednisone or prednisolone. Methods of
the present invention include the combination therapy of
administering a compound of Compound 44 or a pharmaceutically
acceptable salt thereof, and anticancer agents, wherein the
anticancer agents are CHOP, R-CHOP, prednisone, or
prednisolone.
[0086] In certain embodiments, "combination comprising an EZH2
inhibitor and a standard of care agent" is intended to embrace
administration of therapeutic agents that are not
co-formulated.
[0087] In certain embodiments, "combination therapy" is intended to
embrace administration of these therapeutic agents in a sequential
manner, wherein each therapeutic agent is administered at a
different time, as well as administration of these therapeutic
agents, or at least two of the therapeutic agents concurrently, or
in a substantially simultaneous manner. Simultaneous administration
can be accomplished, for example, by administering to the subject a
single capsule having a fixed ratio of each therapeutic agent or in
multiple, single capsules for each of the therapeutic agents.
Sequential or substantially simultaneous administration of each
therapeutic agent can be effected by any appropriate route
including, but not limited to, oral routes, intravenous routes,
intramuscular routes, and direct absorption through mucous membrane
tissues. The therapeutic agents can be administered by the same
route or by different routes. For example, a first therapeutic
agent of the combination selected may be administered by
intravenous injection while the other therapeutic agents of the
combination may be administered orally. Alternatively, for example,
all therapeutic agents may be administered orally or all
therapeutic agents may be administered by intravenous injection.
Therapeutic agents may also be administered in alternation.
[0088] In certain aspects of the invention, the combination
therapies featured in the present invention can result in a
synergistic effect in the treatment of a disease or cancer. A
"synergistic effect" is defined as where the efficacy of a
combination of therapeutic agents is greater than the sum of the
effects of any of the agents given alone. A synergistic effect may
also be an effect that cannot be achieved by administration of any
of the compounds or other therapeutic agents as single agents. The
synergistic effect may include, but is not limited to, an effect of
treating cancer by reducing tumor size, inhibiting tumor growth, or
increasing survival of the subject. The synergistic effect may also
include reducing cancer cell viability, inducing cancer cell death,
and inhibiting or delaying cancer cell growth.
[0089] In certain aspects of the invention "combination therapy"
also embraces the administration of the therapeutic agents as
described above in further combination with other biologically
active ingredients and non-drug therapies (e.g., surgery or
radiation treatment). Where the combination therapy further
comprises a non-drug treatment, the non-drug treatment may be
conducted at any suitable time so long as a beneficial effect from
the co-action of the combination of the therapeutic agents and
non-drug treatment is achieved. For example, in appropriate cases,
the beneficial effect is still achieved when the non-drug treatment
is temporally removed from the administration of the therapeutic
agents, perhaps by days or even weeks.
[0090] In another aspect, a composition of the present invention,
or a pharmaceutically acceptable salt or solvate thereof, may be
administered in combination with radiation therapy. Radiation
therapy can also be administered in combination with a composition
of the present invention and another chemotherapeutic agent
described herein as part of a multiple agent therapy.
[0091] Combination therapy can be achieved by administering two or
more agents, e.g., a Compound 44 and one or more other therapeutic
agents, each of which is formulated and administered separately, or
by administering two or more agents in a single formulation. Other
combinations are also encompassed by combination therapy. For
example, two agents can be formulated together and administered in
conjunction with a separate formulation containing a third agent.
While the two or more agents in the combination therapy can be
administered simultaneously, they need not be. For example,
administration of a first agent (or combination of agents) can
precede administration of a second agent (or combination of agents)
by minutes, hours, days, or weeks. Thus, the two or more agents can
be administered within minutes of each other or within 1, 2, 3, 6,
9, 12, 15, 18, or 24 hours of each other or within 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 12, 14 days of each other or within 2, 3, 4, 5, 6,
7, 8, 9, or 10 weeks of each other. In some cases even longer
intervals are possible. While in many cases it is desirable that
the two or more agents used in a combination therapy be present in
within the patient's body at the same time, this need not be
so.
[0092] The present invention also provides pharmaceutical
compositions comprising Compound 44 or pharmaceutically acceptable
salts thereof, and one or more other therapeutic agents disclosed
herein, mixed with pharmaceutically suitable carriers or
excipient(s) at doses to treat or prevent a disease or condition as
described herein. The pharmaceutical compositions of the present
invention can also be administered in combination with other
therapeutic agents or therapeutic modalities simultaneously,
sequentially, or in alternation.
[0093] Mixtures of compositions of the present invention can also
be administered to the patient as a simple mixture or in suitable
formulated pharmaceutical compositions. For example, one aspect of
the invention relates to a pharmaceutical composition comprising a
therapeutically effective dose of an EZH2 inhibitor or Compound 44,
or a pharmaceutically acceptable salt, hydrate, enantiomer or
stereoisomer thereof; one or more other therapeutic agents, and a
pharmaceutically acceptable diluent or carrier.
[0094] A "pharmaceutical composition" is a formulation containing
the compounds of the present invention in a form suitable for
administration to a subject. Compound 44 and one or more other
therapeutic agents described herein each can be formulated
individually or in multiple pharmaceutical compositions in any
combinations of the active ingredients. Accordingly, one or more
administration routes can be properly elected based on the dosage
form of each pharmaceutical composition. Alternatively, Compound 44
and one or more other therapeutic agents described herein can be
formulated as one pharmaceutical composition.
[0095] In one embodiment, the pharmaceutical composition is in bulk
or in unit dosage form. The unit dosage form is any of a variety of
forms, including, for example, a capsule, an IV bag, a tablet, a
single pump on an aerosol inhaler, or a vial. The quantity of
active ingredient (e.g., a formulation of the disclosed compound or
salt, hydrate, solvate or isomer thereof) in a unit dose of
composition is an effective amount and is varied according to the
particular treatment involved. One skilled in the art will
appreciate that it is sometimes necessary to make routine
variations to the dosage depending on the age and condition of the
patient. The dosage will also depend on the route of
administration. A variety of routes are contemplated, including
oral, pulmonary, rectal, parenteral, transdermal, subcutaneous,
intravenous, intramuscular, intraperitoneal, inhalational, buccal,
sublingual, intrapleural, intrathecal, intranasal, and the like.
Dosage forms for the topical or transdermal administration of a
compound of this invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches and inhalants. In
one embodiment, the active compound is mixed under sterile
conditions with a pharmaceutically acceptable carrier, and with any
preservatives, buffers, or propellants that are required.
[0096] As used herein, the phrase "pharmaceutically acceptable"
refers to those compounds, anions, cations, materials,
compositions, carriers, and/or dosage forms which are, within the
scope of sound medical judgment, suitable for use in contact with
the tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio.
[0097] "Pharmaceutically acceptable excipient" means an excipient
that is useful in preparing a pharmaceutical composition that is
generally safe, non-toxic and neither biologically nor otherwise
undesirable, and includes excipient that is acceptable for
veterinary use as well as human pharmaceutical use. A
"pharmaceutically acceptable excipient" as used in the
specification and claims includes both one and more than one such
excipient.
[0098] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), and transmucosal administration. Solutions
or suspensions used for parenteral, intradermal, or subcutaneous
application can include the following components: a sterile diluent
such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates, and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
The pH can be adjusted with acids or bases, such as hydrochloric
acid or sodium hydroxide. The parenteral preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic.
[0099] A composition of the invention can be administered to a
subject in many of the well-known methods currently used for
chemotherapeutic treatment. For example, for treatment of cancers,
a compound of the invention may be injected directly into tumors,
injected into the blood stream or body cavities or taken orally or
applied through the skin with patches. The dose chosen should be
sufficient to constitute effective treatment but not so high as to
cause unacceptable side effects. The state of the disease condition
(e.g., cancer, precancer, and the like) and the health of the
patient should preferably be closely monitored during and for a
reasonable period after treatment.
[0100] The term "therapeutically effective amount", as used herein,
refers to an amount of a pharmaceutical agent to treat, ameliorate,
or prevent an identified disease or condition, or to exhibit a
detectable therapeutic or inhibitory effect. The effect can be
detected by any assay method known in the art. The precise
effective amount for a subject will depend upon the subject's body
weight, size, and health; the nature and extent of the condition;
and the therapeutic or combination of therapeutics selected for
administration. Therapeutically effective amounts for a given
situation can be determined by routine experimentation that is
within the skill and judgment of the clinician. In a preferred
aspect, the disease or condition to be treated is cancer. In
another aspect, the disease or condition to be treated is a cell
proliferative disorder.
[0101] In certain embodiments the therapeutically effective amount
of each pharmaceutical agent used in combination will be lower when
used in combination in comparison to monotherapy with each agent
alone. Such lower therapeutically effective amount could afford for
lower toxicity of the therapeutic regimen.
[0102] For any compound, the therapeutically effective amount can
be estimated initially either in cell culture assays, e.g., of
neoplastic cells, or in animal models, usually rats, mice, rabbits,
dogs, or pigs. The animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans. Therapeutic/prophylactic efficacy and
toxicity may be determined by standard pharmaceutical procedures in
cell cultures or experimental animals, e.g., ED.sub.50 (the dose
therapeutically effective in 50% of the population) and LD.sub.50
(the dose lethal to 50% of the population). The dose ratio between
toxic and therapeutic effects is the therapeutic index, and it can
be expressed as the ratio, LD.sub.50/ED.sub.50. Pharmaceutical
compositions that exhibit large therapeutic indices are preferred.
The dosage may vary within this range depending upon the dosage
form employed, sensitivity of the patient, and the route of
administration.
[0103] Dosage and administration are adjusted to provide sufficient
levels of the active agent(s) or to maintain the desired effect.
Factors which may be taken into account include the severity of the
disease state, general health of the subject, age, weight, and
gender of the subject, diet, time and frequency of administration,
drug combination(s), reaction sensitivities, and tolerance/response
to therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular
formulation.
[0104] The pharmaceutical compositions containing active compounds
of the present invention may be manufactured in a manner that is
generally known, e.g., by means of conventional mixing, dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating,
entrapping, or lyophilizing processes. Pharmaceutical compositions
may be formulated in a conventional manner using one or more
pharmaceutically acceptable carriers comprising excipients and/or
auxiliaries that facilitate processing of the active compounds into
preparations that can be used pharmaceutically. Of course, the
appropriate formulation is dependent upon the route of
administration chosen.
[0105] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. 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. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol and sorbitol, and sodium chloride in
the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, for example, aluminum
monostearate and gelatin.
[0106] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, methods of preparation are vacuum
drying and freeze-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0107] Oral compositions generally include an inert diluent or an
edible pharmaceutically acceptable carrier. They can be enclosed in
gelatin capsules or compressed into tablets. For the purpose of
oral therapeutic administration, the active compound can be
incorporated with excipients and used in the form of tablets,
troches, or capsules. Oral compositions can also be prepared using
a fluid carrier for use as a mouthwash, wherein the compound in the
fluid carrier is applied orally and swished and expectorated or
swallowed. Pharmaceutically compatible binding agents, and/or
adjuvant materials can be included as part of the composition. The
tablets, pills, capsules, troches and the like can contain any of
the following ingredients, or compounds of a similar nature: a
binder such as microcrystalline cellulose, gum tragacanth or
gelatin; an excipient such as starch or lactose, a disintegrating
agent such as alginic acid, Primogel, or corn starch; a lubricant
such as magnesium stearate or Sterotes; a glidant such as colloidal
silicon dioxide; a sweetening agent such as sucrose or saccharin;
or a flavoring agent such as peppermint, methyl salicylate, or
orange flavoring.
[0108] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser, which contains a suitable propellant, e.g., a gas
such as carbon dioxide, or a nebulizer.
[0109] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0110] The active compounds can be prepared with pharmaceutically
acceptable carriers that will protect the compound against rapid
elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811.
[0111] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved.
[0112] In therapeutic applications, the dosages of the EZH2
inhibitor compounds described herein, other therapeutic agents
described herein, compositions comprising Compound 44 and one or
more other therapeutic agents, or the pharmaceutical compositions
used in accordance with the invention vary depending on the agent,
the age, weight, and clinical condition of the recipient patient,
and the experience and judgment of the clinician or practitioner
administering the therapy, among other factors affecting the
selected dosage. Generally, the dose should be sufficient to result
in slowing, and preferably regressing, the growth of the tumors and
also preferably causing complete regression of the cancer. Dosages
can range from about 0.01 mg/kg per day to about 5000 mg/kg per
day. In preferred aspects, dosages can range from about 1 mg/kg per
day to about 1000 mg/kg per day. In an aspect, the dose will be in
the range of about 0.1 mg/day to about 50 g/day; about 0.1 mg/day
to about 25 g/day; about 0.1 mg/day to about 10 g/day; about 0.1 mg
to about 3 g/day; or about 0.1 mg to about 1 g/day, in single,
divided, or continuous doses (which dose may be adjusted for the
patient's weight in kg, body surface area in m.sup.2, and age in
years). An effective amount of a pharmaceutical agent is that which
provides an objectively identifiable improvement as noted by the
clinician or other qualified observer. For example, regression of a
tumor in a patient may be measured with reference to the diameter
of a tumor. Decrease in the diameter of a tumor indicates
regression. Regression is also indicated by failure of tumors to
reoccur after treatment has stopped. As used herein, the term
"dosage effective manner" refers to amount of an active compound to
produce the desired biological effect in a subject or cell.
[0113] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0114] The composition of the present invention is capable of
further forming salts. The composition of the present invention is
capable of forming more than one salt per molecule, e.g., mono-,
di-, tri-. All of these forms are also contemplated within the
scope of the claimed invention.
[0115] As used herein, "pharmaceutically acceptable salts" refer to
derivatives of the compounds of the present invention wherein the
parent compound is modified by making acid or base salts thereof.
Examples of pharmaceutically acceptable salts include, but are not
limited to, mineral or organic acid salts of basic residues such as
amines, alkali or organic salts of acidic residues such as
carboxylic acids, and the like. The pharmaceutically acceptable
salts include the conventional non-toxic salts or the quaternary
ammonium salts of the parent compound formed, for example, from
non-toxic inorganic or organic acids. For example, such
conventional non-toxic salts include, but are not limited to, those
derived from inorganic and organic acids selected from
2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic,
benzene sulfonic, benzoic, bicarbonic, carbonic, citric, edetic,
ethane disulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic,
gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic,
hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxymaleic,
hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulfonic,
maleic, malic, mandelic, methane sulfonic, napsylic, nitric,
oxalic, pamoic, pantothenic, phenylacetic, phosphoric,
polygalacturonic, propionic, salicyclic, stearic, subacetic,
succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluene
sulfonic, and the commonly occurring amine acids, e.g., glycine,
alanine, phenylalanine, arginine, etc.
[0116] Other examples of pharmaceutically acceptable salts include
hexanoic acid, cyclopentane propionic acid, pyruvic acid, malonic
acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid,
4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,
4-toluenesulfonic acid, camphorsulfonic acid,
4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylic acid,
3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic
acid, muconic acid, and the like. The present invention also
encompasses salts formed when an acidic proton present in the
parent compound either is replaced by a metal ion, e.g., an alkali
metal ion, an alkaline earth ion, or an aluminum ion; or
coordinates with an organic base such as ethanolamine,
diethanolamine, triethanolamine, tromethamine, N-methylglucamine,
and the like.
[0117] It should be understood that all references to
pharmaceutically acceptable salts include solvent addition forms
(solvates), of the same salt.
[0118] The composition, or pharmaceutically acceptable salts or
solvates thereof, are administered orally, nasally, transdermally,
pulmonary, inhalationally, buccally, sublingually,
intraperintoneally, subcutaneously, intramuscularly, intravenously,
rectally, intrapleurally, intrathecally and parenterally. In one
embodiment, the compound is administered orally. One skilled in the
art will recognize the advantages of certain routes of
administration.
[0119] The dosage regimen utilizing the compounds is selected in
accordance with a variety of factors including type, species, age,
weight, sex and medical condition of the patient; the severity of
the condition to be treated; the route of administration; the renal
and hepatic function of the patient; and the particular compound or
salt thereof employed. An ordinarily skilled physician or
veterinarian can readily determine and prescribe the effective
amount of the drug required to prevent, counter, or arrest the
progress of the condition.
[0120] Techniques for formulation and administration of the
disclosed compounds of the invention can be found in Remington: the
Science and Practice of Pharmacy, 19.sup.th edition, Mack
Publishing Co., Easton, Pa. (1995). In an embodiment, the compounds
described herein, and the pharmaceutically acceptable salts
thereof, are used in pharmaceutical preparations in combination
with a pharmaceutically acceptable carrier or diluent. Suitable
pharmaceutically acceptable carriers include inert solid fillers or
diluents and sterile aqueous or organic solutions. The compounds
will be present in such pharmaceutical compositions in amounts
sufficient to provide the desired dosage amount in the range
described herein.
[0121] All percentages and ratios used herein, unless otherwise
indicated, are by weight. Other features and advantages of the
present invention are apparent from the different examples. The
provided examples illustrate different components and methodology
useful in practicing the present invention. The examples do not
limit the claimed invention. Based on the present disclosure the
skilled artisan can identify and employ other components and
methodology useful for practicing the present invention.
[0122] The present invention provides compositions and methods for
treating conditions and diseases the course of which can be
influenced by modulating the methylation status of histones or
other proteins, wherein said methylation status is mediated at
least in part by the activity of EZH2. Modulation of the
methylation status of histones can in turn influence the level of
expression of target genes activated by methylation, and/or target
genes suppressed by methylation. The method includes administering
to a subject in need of such treatment, a therapeutically effective
amount of a composition of the present invention or a
pharmaceutically acceptable salt or solvate thereof, to a subject
in need of such treatment.
[0123] Based at least on the fact that abnormal histone methylation
has been found to be associated with certain cancers and
precancerous conditions, a method for treating cancer or a
precancerous condition with a mutant EZH2 in a subject comprises
administering to the subject in need thereof a therapeutically
effective amount of a compound that inhibits methylation. In one
embodiment a method for treating cancer or a precancerous condition
in a subject comprises administering to the subject in need thereof
a therapeutically effective amount of a compound that inhibits
conversion of unmethylated H3-K27 to monomethylated H3-K27
(H3-K27me1). In one embodiment a method for treating cancer or a
precancerous condition in a subject comprises administering to the
subject in need thereof a therapeutically effective amount of a
compound that inhibits conversion of monomethylated H3-K27
(H3-K27me1) to dimethylated H3-K27 (H3-K27me2). In one embodiment a
method for treating cancer or a precancerous condition in a subject
comprises administering to the subject in need thereof a
therapeutically effective amount of a compound that inhibits
conversion of H3-K27me2 to trimethylated H3-K27 (H3-K27me3). In one
embodiment a method for treating cancer or a precancerous condition
in a subject comprises administering to the subject in need thereof
a therapeutically effective amount of a compound that inhibits both
conversion of H3-K27me1 to H3-K27me2 and conversion of H3-K27me2 to
H3-K27me3. It is important to note that disease-specific increase
in methylation can occur at chromatin in key genomic loci in the
absence of a global increase in cellular levels of histone or
protein methylation. For example, it is possible for aberrant
hypermethylation at key disease-relevant genes to occur against a
backdrop of global histone or protein hypomethylation.
[0124] Modulators of methylation can be used for modulating cell
proliferation, generally. For example, in some cases excessive
proliferation may be reduced with agents that decrease methylation,
whereas insufficient proliferation may be stimulated with agents
that increase methylation. Accordingly, diseases that may be
treated include hyperproliferative diseases, such as benign cell
growth and malignant cell growth (cancer).
[0125] The disorder in which EZH2-mediated protein methylation
plays a part can be cancer, a cell proliferative disorder, or a
precancerous condition. The present invention further provides the
use of a composition of the present invention, or a
pharmaceutically acceptable salt or solvate thereof, to a subject
in need of such treatment, for the preparation of a medicament
useful for the treatment of cancer. Exemplary cancers that may be
treated include lymphomas, including non-Hodgkin lymphoma,
follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL),
including GCB lymphoma.
[0126] In general, compounds that are methylation modulators can be
used for modulating cell proliferation, generally. For example, in
some cases excessive proliferation may be reduced with agents that
decrease methylation, whereas insufficient proliferation may be
stimulated with agents that increase methylation. Accordingly,
diseases that may be treated by the compounds of the invention
include hyperproliferative diseases, such as benign cell growth and
malignant cell growth.
[0127] As used herein, a "subject in need thereof" is a subject
having a disorder in which EZH2-mediated protein methylation plays
a part, or a subject having an increased risk of developing such
disorder relative to the population at large. A subject in need
thereof can have a precancerous condition. Preferably, a subject in
need thereof has cancer. A "subject" includes a mammal. The mammal
can be e.g., any mammal, e.g., a human, primate, bird, mouse, rat,
dog, cat, cow, horse, goat, camel, sheep or a pig. Preferably, the
mammal is a human.
[0128] The subject of the present invention includes any human
subject who has been diagnosed with, has symptoms of, or is at risk
of developing a cancer or a precancerous condition. The subject of
the present invention includes any human subject expressing a
mutant EZH2. For example, a mutant EZH2 comprises one or more
mutations, wherein the mutation is a substitution, a point
mutation, a nonsense mutation, a missense mutation, a deletion, or
an insertion or any other EZH2 mutation described herein.
[0129] A subject in need thereof may have refractory or resistant
cancer. "Refractory or resistant cancer" means cancer that does not
respond to treatment. The cancer may be resistant at the beginning
of treatment or it may become resistant during treatment. In some
embodiments, the subject in need thereof has cancer recurrence
following remission on most recent therapy. In some embodiments,
the subject in need thereof received and failed all known effective
therapies for cancer treatment. In some embodiments, the subject in
need thereof received at least one prior therapy. In certain
embodiments the prior therapy is monotherapy. In certain
embodiments the prior therapy is combination therapy.
[0130] In some embodiments, a subject in need thereof may have a
secondary cancer as a result of a previous therapy. "Secondary
cancer" means cancer that arises due to or as a result from
previous carcinogenic therapies, such as chemotherapy.
[0131] The subject may also exhibit resistance to EZH2 histone
methyltransferase inhibitors or any other therapeutic agent.
[0132] The invention also features a method of selecting a
combination therapy for a subject having cancer. The method
includes the steps of: detecting one or more EZH2 mutations
described herein in a sample from the subject; and selecting, based
on the presence of the one or more EZH2 mutations, a combination
therapy for treating cancer. In one embodiment, the therapy
includes administering to the subject a composition of the
invention. In one embodiment, the method further includes
administrating to the subject a therapeutically effective amount of
a composition of the invention. An EZH2 mutation can be detected
using any suitable method known in the art. More methods are
described in U.S. patent publication US 20130040906, which is
incorporated herein by reference in their entireties.
[0133] The methods and uses described herein may include steps of
detecting one or more EZH2 mutations described herein in a sample
from a subject in need thereof prior to and/or after the
administration of a composition of the invention (e.g., a
composition comprising a Compound 44) or pharmaceutically
acceptable salts thereof, and one or more therapeutic agents) to
the subject. The presence of the one or more EZH2 mutations
described herein in the tested sample indicates the subject is
responsive to the combination therapy of the invention.
[0134] The present invention provides personalized medicine,
treatment and/or cancer management for a subject by genetic
screening of one or more EZH2 mutations described herein in the
subject. For example, the present invention provides methods for
treating or alleviating a symptom of cancer or a precancerous
condition in a subject in need thereof by determining
responsiveness of the subject to a combination therapy and when the
subject is responsive to the combination therapy, administering to
the subject a composition of the m invention. The responsiveness is
determined by obtaining a sample from the subject and detecting one
or more EZH2 mutations described herein, and the presence of such
one or more EZH2 mutations described herein indicates that the
subject is responsive to the composition of the invention. Once the
responsiveness of a subject is determined, a therapeutically
effective amount of a composition, for example, a composition
comprising Compound 44 or pharmaceutically acceptable salts
thereof, and one or more therapeutic agents, can be administered.
The therapeutically effective amount of a composition can be
determined by one of ordinary skill in the art.
[0135] As used herein, the term "responsiveness" is interchangeable
with terms "responsive", "sensitive", and "sensitivity", and it is
meant that a subject is showing therapeutic responses when
administered a composition of the invention, e.g., tumor cells or
tumor tissues of the subject undergo apoptosis and/or necrosis,
and/or display reduced growing, dividing, or proliferation. This
term is also meant that a subject will or has a higher probability,
relative to the population at large, of showing therapeutic
responses when administered a composition of the invention, e.g.,
tumor cells or tumor tissues of the subject undergo apoptosis
and/or necrosis, and/or display reduced growing, dividing, or
proliferation.
[0136] By "sample" it means any biological sample derived from the
subject, includes but is not limited to, cells, tissues samples,
body fluids (including, but not limited to, mucus, blood, plasma,
serum, urine, saliva, and semen), tumor cells, and tumor tissues.
Preferably, the sample is selected from bone marrow, peripheral
blood cells, blood, plasma and serum. Samples can be provided by
the subject under treatment or testing. Alternatively samples can
be obtained by the physician according to routine practice in the
art.
[0137] As used herein, the term "cell proliferative disorder"
refers to conditions in which unregulated or abnormal growth, or
both, of cells can lead to the development of an unwanted condition
or disease, which may or may not be cancerous. Exemplary cell
proliferative disorders of the invention encompass a variety of
conditions wherein cell division is deregulated. Exemplary cell
proliferative disorder include, but are not limited to, neoplasms,
benign tumors, malignant tumors, pre-cancerous conditions, in situ
tumors, encapsulated tumors, metastatic tumors, liquid tumors,
solid tumors, immunological tumors, hematological tumors, cancers,
carcinomas, leukemias, lymphomas, sarcomas, and rapidly dividing
cells. The term "rapidly dividing cell" as used herein is defined
as any cell that divides at a rate that exceeds or is greater than
what is expected or observed among neighboring or juxtaposed cells
within the same tissue. A cell proliferative disorder includes a
precancer or a precancerous condition. A cell proliferative
disorder includes cancer. Preferably, the methods provided herein
are used to treat or alleviate a symptom of cancer. The term
"cancer" includes solid tumors, as well as, hematologic tumors
and/or malignancies. A "precancer cell" or "precancerous cell" is a
cell manifesting a cell proliferative disorder that is a precancer
or a precancerous condition. A "cancer cell" or "cancerous cell" is
a cell manifesting a cell proliferative disorder that is a cancer.
Any reproducible means of measurement may be used to identify
cancer cells or precancerous cells. Cancer cells or precancerous
cells can be identified by histological typing or grading of a
tissue sample (e.g., a biopsy sample). Cancer cells or precancerous
cells can be identified through the use of appropriate molecular
markers.
[0138] A cancer that is to be treated can be evaluated by DNA
cytometry, flow cytometry, or image cytometry. A cancer that is to
be treated can be typed as having 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, or 90% of cells in the synthesis stage of cell division
(e.g., in S phase of cell division). A cancer that is to be treated
can be typed as having a low S-phase fraction or a high S-phase
fraction.
[0139] As used herein, a "normal cell" is a cell that cannot be
classified as part of a "cell proliferative disorder". A normal
cell lacks unregulated or abnormal growth, or both, that can lead
to the development of an unwanted condition or disease. Preferably,
a normal cell possesses normally functioning cell cycle checkpoint
control mechanisms.
[0140] As used herein, "contacting a cell" refers to a condition in
which a compound or other composition of matter is in direct
contact with a cell, or is close enough to induce a desired
biological effect in a cell.
[0141] As used herein, "candidate compound" refers to a compound of
the present invention, or a pharmaceutically acceptable salt or
solvate thereof, that has been or will be tested in one or more in
vitro or in vivo biological assays, in order to determine if that
compound is likely to elicit a desired biological or medical
response in a cell, tissue, system, animal or human that is being
sought by a researcher or clinician. A candidate compound is a
compound of the present invention, or a pharmaceutically acceptable
salt or solvate thereof. The biological or medical response can be
the treatment of cancer. The biological or medical response can be
treatment or prevention of a cell proliferative disorder. In vitro
or in vivo biological assays can include, but are not limited to,
enzymatic activity assays, electrophoretic mobility shift assays,
reporter gene assays, in vitro cell viability assays, and the
assays described herein.
[0142] As used herein, "treating" or "treat" describes the
management and care of a patient for the purpose of combating a
disease, condition, or disorder and includes the administration of
a compound of the present invention, or a pharmaceutically
acceptable salt or solvate thereof, to alleviate the symptoms or
complications of a disease, condition or disorder, or to eliminate
the disease, condition or disorder.
[0143] A composition of the present invention, or a
pharmaceutically acceptable salt or solvate thereof, can also be
used to prevent a disease, condition or disorder. As used herein,
"preventing" or "prevent" describes reducing or eliminating the
onset of the symptoms or complications of the disease, condition or
disorder.
[0144] As used herein, the term "alleviate" is meant to describe a
process by which the severity of a sign or symptom of a disorder is
decreased. Importantly, a sign or symptom can be alleviated without
being eliminated. In a preferred embodiment, the administration of
pharmaceutical compositions of the invention leads to the
elimination of a sign or symptom, however, elimination is not
required. Effective dosages are expected to decrease the severity
of a sign or symptom. For instance, a sign or symptom of a disorder
such as cancer, which can occur in multiple locations, is
alleviated if the severity of the cancer is decreased within at
least one of multiple locations.
[0145] As used herein, the term "severity" is meant to describe the
potential of cancer to transform from a precancerous, or benign,
state into a malignant state. Alternatively, or in addition,
severity is meant to describe a cancer stage, for example,
according to the TNM system (accepted by the International Union
Against Cancer (UICC) and the American Joint Committee on Cancer
(AJCC)) or by other art-recognized methods. Cancer stage refers to
the extent or severity of the cancer, based on factors such as the
location of the primary tumor, tumor size, number of tumors, and
lymph node involvement (spread of cancer into lymph nodes).
Alternatively, or in addition, severity is meant to describe the
tumor grade by art-recognized methods (see, National Cancer
Institute, www.cancer.gov). Tumor grade is a system used to
classify cancer cells in terms of how abnormal they look under a
microscope and how quickly the tumor is likely to grow and spread.
Many factors are considered when determining tumor grade, including
the structure and growth pattern of the cells. The specific factors
used to determine tumor grade vary with each type of cancer.
Severity also describes a histologic grade, also called
differentiation, which refers to how much the tumor cells resemble
normal cells of the same tissue type (see, National Cancer
Institute, www.cancer.gov). Furthermore, severity describes a
nuclear grade, which refers to the size and shape of the nucleus in
tumor cells and the percentage of tumor cells that are dividing
(see, National Cancer Institute, www.cancer.gov).
[0146] In another aspect of the invention, severity describes the
degree to which a tumor has secreted growth factors, degraded the
extracellular matrix, become vascularized, lost adhesion to
juxtaposed tissues, or metastasized. Moreover, severity describes
the number of locations to which a primary tumor has metastasized.
Finally, severity includes the difficulty of treating tumors of
varying types and locations. For example, inoperable tumors, those
cancers which have greater access to multiple body systems
(hematological and immunological tumors), and those which are the
most resistant to traditional treatments are considered most
severe. In these situations, prolonging the life expectancy of the
subject and/or reducing pain, decreasing the proportion of
cancerous cells or restricting cells to one system, and improving
cancer stage/tumor grade/histological grade/nuclear grade are
considered alleviating a sign or symptom of the cancer.
[0147] As used herein the term "symptom" is defined as an
indication of disease, illness, injury, or that something is not
right in the body. Symptoms are felt or noticed by the individual
experiencing the symptom, but may not easily be noticed by others.
Others are defined as non-health-care professionals.
[0148] As used herein the term "sign" is also defined as an
indication that something is not right in the body. But signs are
defined as things that can be seen by a doctor, nurse, or other
health care professional.
[0149] Cancer is a group of diseases that may cause almost any sign
or symptom. The signs and symptoms will depend on where the cancer
is, the size of the cancer, and how much it affects the nearby
organs or structures. If a cancer spreads (metastasizes), then
symptoms may appear in different parts of the body.
[0150] Treating cancer can result in a reduction in size of a
tumor. A reduction in size of a tumor may also be referred to as
"tumor regression". Preferably, after treatment, tumor size is
reduced by 5% or greater relative to its size prior to treatment;
more preferably, tumor size is reduced by 10% or greater; more
preferably, reduced by 20% or greater; more preferably, reduced by
30% or greater; more preferably, reduced by 40% or greater; even
more preferably, reduced by 50% or greater; and most preferably,
reduced by greater than 75% or greater. Size of a tumor may be
measured by any reproducible means of measurement. The size of a
tumor may be measured as a diameter of the tumor.
[0151] Treating cancer can result in a reduction in tumor volume.
Preferably, after treatment, tumor volume is reduced by 5% or
greater relative to its size prior to treatment; more preferably,
tumor volume is reduced by 10% or greater; more preferably, reduced
by 20% or greater; more preferably, reduced by 30% or greater; more
preferably, reduced by 40% or greater; even more preferably,
reduced by 50% or greater; and most preferably, reduced by greater
than 75% or greater. Tumor volume may be measured by any
reproducible means of measurement.
[0152] Treating cancer results in a decrease in number of tumors.
Preferably, after treatment, tumor number is reduced by 5% or
greater relative to number prior to treatment; more preferably,
tumor number is reduced by 10% or greater; more preferably, reduced
by 20% or greater; more preferably, reduced by 30% or greater; more
preferably, reduced by 40% or greater; even more preferably,
reduced by 50% or greater; and most preferably, reduced by greater
than 75%. Number of tumors may be measured by any reproducible
means of measurement. The number of tumors may be measured by
counting tumors visible to the naked eye or at a specified
magnification. Preferably, the specified magnification is 2.times.,
3.times., 4.times., 5.times., 10.times., or 50.times..
[0153] Treating cancer can result in a decrease in number of
metastatic lesions in other tissues or organs distant from the
primary tumor site. Preferably, after treatment, the number of
metastatic lesions is reduced by 5% or greater relative to number
prior to treatment; more preferably, the number of metastatic
lesions is reduced by 10% or greater; more preferably, reduced by
20% or greater; more preferably, reduced by 30% or greater; more
preferably, reduced by 40% or greater; even more preferably,
reduced by 50% or greater; and most preferably, reduced by greater
than 75%. The number of metastatic lesions may be measured by any
reproducible means of measurement. The number of metastatic lesions
may be measured by counting metastatic lesions visible to the naked
eye or at a specified magnification. Preferably, the specified
magnification is 2.times., 3.times., 4.times., 5.times., 10.times.,
or 50.times..
[0154] Treating cancer can result in an increase in average
survival time of a population of treated subjects in comparison to
a population receiving carrier alone. Preferably, the average
survival time is increased by more than 30 days; more preferably,
by more than 60 days; more preferably, by more than 90 days; and
most preferably, by more than 120 days. An increase in average
survival time of a population may be measured by any reproducible
means. An increase in average survival time of a population may be
measured, for example, by calculating for a population the average
length of survival following initiation of treatment with an active
compound. An increase in average survival time of a population may
also be measured, for example, by calculating for a population the
average length of survival following completion of a first round of
treatment with an active compound.
[0155] Treating cancer can result in an increase in average
survival time of a population of treated subjects in comparison to
a population of untreated subjects. Preferably, the average
survival time is increased by more than 30 days; more preferably,
by more than 60 days; more preferably, by more than 90 days; and
most preferably, by more than 120 days. An increase in average
survival time of a population may be measured by any reproducible
means. An increase in average survival time of a population may be
measured, for example, by calculating for a population the average
length of survival following initiation of treatment with an active
compound. An increase in average survival time of a population may
also be measured, for example, by calculating for a population the
average length of survival following completion of a first round of
treatment with an active compound.
[0156] Treating cancer can result in increase in average survival
time of a population of treated subjects in comparison to a
population receiving monotherapy with a drug that is not a compound
of the present invention, or a pharmaceutically acceptable salt or
solvate thereof. Preferably, the average survival time is increased
by more than 30 days; more preferably, by more than 60 days; more
preferably, by more than 90 days; and most preferably, by more than
120 days. An increase in average survival time of a population may
be measured by any reproducible means. An increase in average
survival time of a population may be measured, for example, by
calculating for a population the average length of survival
following initiation of treatment with an active compound. An
increase in average survival time of a population may also be
measured, for example, by calculating for a population the average
length of survival following completion of a first round of
treatment with an active compound.
[0157] Treating cancer can result in a decrease in the mortality
rate of a population of treated subjects in comparison to a
population receiving carrier alone. Treating cancer can result in a
decrease in the mortality rate of a population of treated subjects
in comparison to an untreated population. Treating cancer can
result in a decrease in the mortality rate of a population of
treated subjects in comparison to a population receiving
monotherapy with a drug that is not a compound of the present
invention, or a pharmaceutically acceptable salt or solvate
thereof. Preferably, the mortality rate is decreased by more than
2%; more preferably, by more than 5%; more preferably, by more than
10%; and most preferably, by more than 25%. A decrease in the
mortality rate of a population of treated subjects may be measured
by any reproducible means. A decrease in the mortality rate of a
population may be measured, for example, by calculating for a
population the average number of disease-related deaths per unit
time following initiation of treatment with an active compound. A
decrease in the mortality rate of a population may also be
measured, for example, by calculating for a population the average
number of disease-related deaths per unit time following completion
of a first round of treatment with an active compound.
[0158] Treating cancer can result in a decrease in tumor growth
rate. Preferably, after treatment, tumor growth rate is reduced by
at least 5% relative to number prior to treatment; more preferably,
tumor growth rate is reduced by at least 10%; more preferably,
reduced by at least 20%; more preferably, reduced by at least 30%;
more preferably, reduced by at least 40%; more preferably, reduced
by at least 50%; even more preferably, reduced by at least 50%; and
most preferably, reduced by at least 75%. Tumor growth rate may be
measured by any reproducible means of measurement. Tumor growth
rate can be measured according to a change in tumor diameter per
unit time.
[0159] Treating cancer can result in a decrease in tumor regrowth.
Preferably, after treatment, tumor regrowth is less than 5%; more
preferably, tumor regrowth is less than 10%; more preferably, less
than 20%; more preferably, less than 30%; more preferably, less
than 40%; more preferably, less than 50%; even more preferably,
less than 50%; and most preferably, less than 75%. Tumor regrowth
may be measured by any reproducible means of measurement. Tumor
regrowth is measured, for example, by measuring an increase in the
diameter of a tumor after a prior tumor shrinkage that followed
treatment. A decrease in tumor regrowth is indicated by failure of
tumors to reoccur after treatment has stopped.
[0160] Treating or preventing a cell proliferative disorder can
result in a reduction in the rate of cellular proliferation.
Preferably, after treatment, the rate of cellular proliferation is
reduced by at least 5%; more preferably, by at least 10%; more
preferably, by at least 20%; more preferably, by at least 30%; more
preferably, by at least 40%; more preferably, by at least 50%; even
more preferably, by at least 50%; and most preferably, by at least
75%. The rate of cellular proliferation may be measured by any
reproducible means of measurement. The rate of cellular
proliferation is measured, for example, by measuring the number of
dividing cells in a tissue sample per unit time.
[0161] Treating or preventing a cell proliferative disorder can
result in a reduction in the proportion of proliferating cells.
Preferably, after treatment, the proportion of proliferating cells
is reduced by at least 5%; more preferably, by at least 10%; more
preferably, by at least 20%; more preferably, by at least 30%; more
preferably, by at least 40%; more preferably, by at least 50%; even
more preferably, by at least 50%; and most preferably, by at least
75%. The proportion of proliferating cells may be measured by any
reproducible means of measurement. Preferably, the proportion of
proliferating cells is measured, for example, by quantifying the
number of dividing cells relative to the number of nondividing
cells in a tissue sample. The proportion of proliferating cells can
be equivalent to the mitotic index.
[0162] Treating or preventing a cell proliferative disorder can
result in a decrease in size of an area or zone of cellular
proliferation. Preferably, after treatment, size of an area or zone
of cellular proliferation is reduced by at least 5% relative to its
size prior to treatment; more preferably, reduced by at least 10%;
more preferably, reduced by at least 20%; more preferably, reduced
by at least 30%; more preferably, reduced by at least 40%; more
preferably, reduced by at least 50%; even more preferably, reduced
by at least 50%; and most preferably, reduced by at least 75%. Size
of an area or zone of cellular proliferation may be measured by any
reproducible means of measurement. The size of an area or zone of
cellular proliferation may be measured as a diameter or width of an
area or zone of cellular proliferation.
[0163] Treating or preventing a cell proliferative disorder can
result in a decrease in the number or proportion of cells having an
abnormal appearance or morphology. Preferably, after treatment, the
number of cells having an abnormal morphology is reduced by at
least 5% relative to its size prior to treatment; more preferably,
reduced by at least 10%; more preferably, reduced by at least 20%;
more preferably, reduced by at least 30%; more preferably, reduced
by at least 40%; more preferably, reduced by at least 50%; even
more preferably, reduced by at least 50%; and most preferably,
reduced by at least 75%. An abnormal cellular appearance or
morphology may be measured by any reproducible means of
measurement. An abnormal cellular morphology can be measured by
microscopy, e.g., using an inverted tissue culture microscope. An
abnormal cellular morphology can take the form of nuclear
pleiomorphism.
[0164] As used herein, the term "selectively" means tending to
occur at a higher frequency in one population than in another
population. The compared populations can be cell populations.
Preferably, a compound of the present invention, or a
pharmaceutically acceptable salt or solvate thereof, acts
selectively on a cancer or precancerous cell but not on a normal
cell. Preferably, a compound of the present invention, or a
pharmaceutically acceptable salt or solvate thereof, acts
selectively to modulate one molecular target (e.g., a target
protein methyltransferase) but does not significantly modulate
another molecular target (e.g., a non-target protein
methyltransferase). The invention also provides a method for
selectively inhibiting the activity of an enzyme, such as a protein
methyltransferase. Preferably, an event occurs selectively in
population A relative to population B if it occurs greater than two
times more frequently in population A as compared to population B.
An event occurs selectively if it occurs greater than five times
more frequently in population A. An event occurs selectively if it
occurs greater than ten times more frequently in population A; more
preferably, greater than fifty times; even more preferably, greater
than 100 times; and most preferably, greater than 1000 times more
frequently in population A as compared to population B. For
example, cell death would be said to occur selectively in cancer
cells if it occurred greater than twice as frequently in cancer
cells as compared to normal cells.
[0165] A composition of the present invention, e.g., Compound 44 or
pharmaceutically acceptable salt thereof, and one or more other
therapeutic agents, such as prednisone, can modulate the activity
of a molecular target (e.g., a target protein methyltransferase).
Modulating refers to stimulating or inhibiting an activity of a
molecular target. Preferably, a compound of the present invention,
or a pharmaceutically acceptable salt or solvate thereof, modulates
the activity of a molecular target if it stimulates or inhibits the
activity of the molecular target by at least 2-fold relative to the
activity of the molecular target under the same conditions but
lacking only the presence of said compound. More preferably, a
compound of the present invention, or a pharmaceutically acceptable
salt or solvate thereof, modulates the activity of a molecular
target if it stimulates or inhibits the activity of the molecular
target by at least 5-fold, at least 10-fold, at least 20-fold, at
least 50-fold, at least 100-fold relative to the activity of the
molecular target under the same conditions but lacking only the
presence of said compound. The activity of a molecular target may
be measured by any reproducible means. The activity of a molecular
target may be measured in vitro or in vivo. For example, the
activity of a molecular target may be measured in vitro by an
enzymatic activity assay or a DNA binding assay, or the activity of
a molecular target may be measured in vivo by assaying for
expression of a reporter gene.
[0166] A composition of the present invention does not
significantly modulate the activity of a molecular target if the
addition of the compound does not stimulate or inhibit the activity
of the molecular target by greater than 10% relative to the
activity of the molecular target under the same conditions but
lacking only the presence of said compound.
[0167] As used herein, the term "isozyme selective" means
preferential inhibition or stimulation of a first isoform of an
enzyme in comparison to a second isoform of an enzyme (e.g.,
preferential inhibition or stimulation of a protein
methyltransferase isozyme alpha in comparison to a protein
methyltransferase isozyme beta). Preferably, a compound of the
present invention, or a pharmaceutically acceptable salt or solvate
thereof, demonstrates a minimum of a fourfold differential,
preferably a tenfold differential, more preferably a fifty fold
differential, in the dosage required to achieve a biological
effect. Preferably, a compound of the present invention, or a
pharmaceutically acceptable salt or solvate thereof, demonstrates
this differential across the range of inhibition, and the
differential is exemplified at the IC.sub.50, i.e., a 50%
inhibition, for a molecular target of interest.
[0168] Administering a composition of the present invention to a
cell or a subject in need thereof can result in modulation (i.e.,
stimulation or inhibition) of an activity of a protein
methyltransferase of interest.
[0169] Administering a compound of the present invention, e.g., a
composition comprising Compound 44 or pharmaceutically acceptable
salt thereof, and one or more other therapeutic agents, such as
prednisone, to a cell or a subject in need thereof results in
modulation (i.e., stimulation or inhibition) of an activity of an
intracellular target (e.g., substrate). Several intracellular
targets can be modulated with the compounds of the present
invention, including, but not limited to, protein
methyltransferase.
[0170] Activating refers to placing a composition of matter (e.g.,
protein or nucleic acid) in a state suitable for carrying out a
desired biological function. A composition of matter capable of
being activated also has an unactivated state. An activated
composition of matter may have an inhibitory or stimulatory
biological function, or both. Elevation refers to an increase in a
desired biological activity of a composition of matter (e.g., a
protein or a nucleic acid). Elevation may occur through an increase
in concentration of a composition of matter.
[0171] As used herein, "a cell cycle checkpoint pathway" refers to
a biochemical pathway that is involved in modulation of a cell
cycle checkpoint. A cell cycle checkpoint pathway may have
stimulatory or inhibitory effects, or both, on one or more
functions comprising a cell cycle checkpoint. A cell cycle
checkpoint pathway is comprised of at least two compositions of
matter, preferably proteins, both of which contribute to modulation
of a cell cycle checkpoint. A cell cycle checkpoint pathway may be
activated through an activation of one or more members of the cell
cycle checkpoint pathway. Preferably, a cell cycle checkpoint
pathway is a biochemical signaling pathway.
[0172] As used herein, "cell cycle checkpoint regulator" refers to
a composition of matter that can function, at least in part, in
modulation of a cell cycle checkpoint. A cell cycle checkpoint
regulator may have stimulatory or inhibitory effects, or both, on
one or more functions comprising a cell cycle checkpoint. A cell
cycle checkpoint regulator can be a protein or not a protein.
[0173] Treating cancer or a cell proliferative disorder can result
in cell death, and preferably, cell death results in a decrease of
at least 10% in number of cells in a population. More preferably,
cell death means a decrease of at least 20%; more preferably, a
decrease of at least 30%; more preferably, a decrease of at least
40%; more preferably, a decrease of at least 50%; most preferably,
a decrease of at least 75%. Number of cells in a population may be
measured by any reproducible means. A number of cells in a
population can be measured by fluorescence activated cell sorting
(FACS), immunofluorescence microscopy and light microscopy. Methods
of measuring cell death are as shown in Li et al., Proc. Natl.
Acad. Sci. USA. 100(5): 2674-8, 2003. In an aspect, cell death
occurs by apoptosis.
[0174] Preferably, an effective amount of a composition of the
present invention, or a pharmaceutically acceptable salt or solvate
thereof, is not significantly cytotoxic to normal cells. A
therapeutically effective amount of a compound is not significantly
cytotoxic to normal cells if administration of the compound in a
therapeutically effective amount does not induce cell death in
greater than 10% of normal cells. A therapeutically effective
amount of a compound does not significantly affect the viability of
normal cells if administration of the compound in a therapeutically
effective amount does not induce cell death in greater than 10% of
normal cells. In an aspect, cell death occurs by apoptosis.
[0175] Contacting a cell with a composition of the present
invention, or a pharmaceutically acceptable salt or solvate
thereof, can induce or activate cell death selectively in cancer
cells. Administering to a subject in need thereof a compound of the
present invention, or a pharmaceutically acceptable salt or solvate
thereof, can induce or activate cell death selectively in cancer
cells. Contacting a cell with a composition of the present
invention, or a pharmaceutically acceptable salt or solvate
thereof, can induce cell death selectively in one or more cells
affected by a cell proliferative disorder. Preferably,
administering to a subject in need thereof a composition of the
present invention, or a pharmaceutically acceptable salt or solvate
thereof, induces cell death selectively in one or more cells
affected by a cell proliferative disorder.
[0176] The present invention relates to a method of treating or
preventing cancer by administering a composition of the present
invention, or a pharmaceutically acceptable salt or solvate
thereof, to a subject in need thereof, where administration of the
composition of the present invention, or a pharmaceutically
acceptable salt or solvate thereof, results in one or more of the
following: prevention of cancer cell proliferation by accumulation
of cells in one or more phases of the cell cycle (e.g. G1, G1/S,
G2/M), or induction of cell senescence, or promotion of tumor cell
differentiation; promotion of cell death in cancer cells via
cytotoxicity, necrosis or apoptosis, without a significant amount
of cell death in normal cells, antitumor activity in animals with a
therapeutic index of at least 2. As used herein, "therapeutic
index" is the maximum tolerated dose divided by the efficacious
dose.
[0177] One skilled in the art may refer to general reference texts
for detailed descriptions of known techniques discussed herein or
equivalent techniques. These texts include Ausubel et al., Current
Protocols in Molecular Biology, John Wiley and Sons, Inc. (2005);
Sambrook et al., Molecular Cloning, A Laboratory Manual (3.sup.rd
edition), Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
(2000); Coligan et al., Current Protocols in Immunology, John Wiley
& Sons, N.Y.; Enna et al., Current Protocols in Pharmacology,
John Wiley & Sons, N.Y.; Fingl et al., The Pharmacological
Basis of Therapeutics (1975), Remington's Pharmaceutical Sciences,
Mack Publishing Co., Easton, Pa., 18.sup.th edition (1990). These
texts can, of course, also be referred to in making or using an
aspect of the invention.
Example 1
Synergistic Anti-Tumor Activity of EZH2 Inhibitors and
Glucocorticoid
[0178] Compound 44 was synthesized as described in U.S. Pat. No.
8,410,088 which is incorporated herein by reference in its
entirety.
[0179] Dramatic synergy was observed when Compound 44 (Cpd 44) is
combined just with the glucocorticoid receptor agonist (GRag)
prednisolone of CHOP or with other GRag, such as dexamethasone.
When combined with CHOP, the antiproliferative effects of Compound
44 were greatly enhanced and most of this synergy can be ascribed
to the GRag component of CHOP, prednisolone (the active metabolite
of prednisone). Remarkably, the combination of Compound 44 and
prednisolone extends the range of cells that are sensitive to EZH2
inhibition, from mutant-bearing only to all GCB NHL cells.
[0180] Two EZH2 mutant cell lines, WSU-DLCL2 and SU-DHL10, were
pre-treated with Compound 44 for 4 days and then co-treated with
the combination of Compound 44 plus individual CHOP components for
3 additional days (4+3 model). Mafosfamide (an analog of
cyclophosphamide), doxorubicin, and vincristine, all showed
concentration-dependent growth inhibition in the mutant cell lines
by themselves. Hence, combination indices (CI, calculated using
Calcusyn software) were obtained for these drugs in combination
with Compound 44. These cell lines, however, showed no sensitivity
to prednisolone (the active metabolite of prednisone) by itself.
Thus, in this case a CI could not be determined and instead an
enhancement of potency was calculated based on the shift in
IC.sub.50 of Compound 44 seen with a concentration-response curve
of prednisolone.
[0181] The combination of Compound 44 and mafosfamide led to an
overall additive combination benefit in both EZH2 mutant cell lines
(FIG. 1C, 1F). In WSU-DLCL2 cells, the combination of Compound 44
and doxorubicin acted synergistically in the 4+3 model (FIG. 1A),
while this combination was additive in SU-DHL10 cells (FIG. 1D).
The combination of Compound 44 and vincristine also demonstrated
additivity in both EZH2 mutant cell lines (FIG. 1B, 1E). When
WSU-DLCL2 cells were treated with the combination of prednisolone
and Compound 44, a 9-fold shift to greater potency was observed for
Compound 44. Treatment with a different GRag, dexamethasone,
resulted in an even greater shift in the IC.sub.50 of Compound 44
of 17-fold (FIG. 2A, 2B). A similar trend in potency shift for
Compound 44 was observed in SU-DHL10 cells (FIG. 2C, 2D).
[0182] Whether the combination effect of Compound 44 and CHOP could
render WT EZH2 lymphoma cell lines, sensitive to Compound 44 was
investigated. Since Compound 44 treatment alone does not induce
growth inhibition in EZH2 WT lymphoma lines, shifts in potency were
calculated based on the concentration-response curves of the
individual CHOP components. Of the four CHOP components tested,
only the combination of GRag and Compound 44 led to a potency shift
in a WT GCB lymphoma cell line.
[0183] Whether the combination effect of Compound 44 and CHOP could
render EZH2 mutant and wild-type cell lines, WSU-DLCL2 EZH2 mutant
(FIG. 3A, 3B) and DOHH2 EZH2 wild-type (FIG. 3C, 3D) GCB lymphoma
cell lines, sensitive to Compound 44 was investigated next.
Treatment of WSU-DLCL2 cells with a combination of Prednisolone and
Compound 44 caused an enhancement of Compound 44 activity (FIG.
3A), with a maximum 24-fold reduction in Compound 44 IC.sub.50.
Treatment with a different GRag, Dexamethasone, resulted in an even
greater 30-fold reduction in the IC.sub.50 of Compound 44 (FIG.
3B). At biologically relevant concentrations of 1 .mu.M for
Prednisolone and 100 nM for Dexamethasone the potency enhancements
were 7 and 15-fold, respectively. Compound 44 showed no
anti-proliferative effect as a single agent in DOHH2 EZH2 wild-type
cells (FIG. 3C, 3D), therefore the potency shift of Prednisolone or
Dexamethasone was measured. Interestingly, when compound 44 was
tested in a wild-type GCB lymphoma cell line (DOHH2), only the GRag
component of CHOP demonstrated enhanced potency in the presence of
Compound 44 (FIG. 3C, 3D). The potency of Prednisolone or
Dexamethasone was increased with addition of Compound 44 in DOHH2
cells (FIG. 3C, 3D).
[0184] Given that only the GRag and EZH2i combination induced
dramatically enhanced antiproliferative effects, compared to either
single agent, in EZH2 WT and mutant GCB lymphoma cell lines,
whether duration of treatment and/or sequence of addition of
compounds affected sensitivity was determined. The cell line panel
was also extended to include EZH2 WT, EZH2 mutant, Compound 44
sensitive, and EZH2 mutant, Compound 44 insensitive cell line
(previously reported by McCabe et al, and unpublished internal
data). In the previous 4+3 model, the potency shift was based on
either Compound 44 (in EZH2 Y646 (also known as Y641) sensitive
cell lines) or prednisolone (in EZH2 WT cell lines) exposure. For
this set of experiments, the Compound 44 IC.sub.50 shift at a fixed
concentration of prednisolone was used to determine the combination
benefit in cell lines treated with either the 4+3 model, 4 day or 7
day co-treatment, or 4 day prednisolone pre-treatment plus 3 days
of co-treatment. When EZH2 mutant, Compound 44 sensitive cell lines
were co-treated for 4 days, a 30-60 fold lower IC.sub.50 of
Compound 44 was observed, demonstrating similar trends to that of
the 4+3 treatment schedule (Table 1). Similar results were observed
with 7 day co-treatment, and the 4+3 model (Table 1). In EZH2 WT
GCB cell lines, despite yielding no measureable Compound 44
IC.sub.50 after 4 days, both cell lines exhibited decreased
proliferation and a measurable Compound 44 IC.sub.50 after 4 days
of co-treatment with prednisolone (Table 1). EZH2 WT GCB cells also
responded to the 4+3 model and/or 7 day co-treatment schedules
(Table 1). Strikingly, EZH2 mutant, Compound 44 insensitive cell
lines, which also exhibit no measurable Compound 44 IC.sub.50 after
4 day treatment, demonstrated decreased proliferation with 4 day
co-treatment, with even greater response to the combination with
the 4+3 treatment schedule as well as with 7 day co-treatment
(Table 1). Only one of the cell lines demonstrated a combination
benefit when cells were pre-treated with prednisolone, then
co-treated with Compound 44 and prednisolone, suggesting that the
order of drug addition is important for the synergy effect (Table
1).
TABLE-US-00001 TABLE 1 Compound 44/GRag Combination Increases EZH2i
Sensitivity in EZH2 Y646 (Y641) Cell Lines and Overcomes EZH2i
Insensitivity in Cell Lines Resistant to EZH2i 4 Day Cpd44
IC.sub.50 (uM) 7 Day Cpd44 IC.sub.50 (uM) Cpd44 Cpd44 4 d Cpd44
Pre/ 4 d Pred Pre/ 7 d Cell Line Alone Co-treatment 3 d Co-treat 3
d Co-treat Co-treatment WSU 0.53 +/- 0.014 0.020 +/- 0.021 0.011
+/- 0.0062 >1 0.014 +/- 0.0049 (Y646-Sens) SU-DHL10 0.64 +/-
0.26 0.0092 +/- 0.0044 0.0027 +/- 0.0013 0.52, >1 0.020 +/-
0.0057 (Y646-Sens) RL >1 0.0096 +/- 0.0066 <<0.004 0.38
<0.004 (Y646-Res) SU-DHL4 >1 >1, 0.2, >1 0.035 +/-
0.043 >1 0.51 +/- 0.35 (Y646-Res) DOHH2 >1 0.20 +/- 0.25
>1, 0.03, >1 >1 0.34 +/- 0.078 (WT) OCI-Ly19 >1 0.19
+/- 0.11 0.0055 +/- 0.0047 >1 0.026, <0.004 (WT)
[0185] To evaluate potential mechanisms responsible for the
observed combination benefits of Compound 44 and GRag in these cell
lines, we determined whether Prednisolone treatment affected global
methylation and acetylation of H3K27 following a four day treatment
either alone or in combination with Compound 44 in WSU-DLCL2,
OCI-LY19, and RL cells (two independent experiments). Single agent
Prednisolone had no effect on H3K27Me3 levels in WSU-DLCL2 or RL
cells, but did increase H3K27Me3 levels at higher doses in OCI-LY19
cells (FIG. 9A). Due to the high sensitivity of OCI-LY19 cells to
Prednisolone, in contrast to the Prednisolone-insensitive EZH2
mutant lines, a lower Prednisolone dose was necessary for the
treatment of OCY-LY19 cells. The inclusion of Prednisolone did not
alter the Compound 44 IC.sub.50 for H3K27Me3 inhibition in any cell
line (FIG. 9A). Likewise, global H3K27 acetylation levels were not
affected by Prednisolone alone or the combination of Compound 44
and prednisolone (FIGS. 9B, 9C & 9D).
[0186] Having found that global levels of H3K27 acetylation or
trimethylation were unaffected, transcriptional regulation of GR
signaling pathways was studied. WSU-DLCL2, SU-DHL10, RL, SU-DHL4,
OCI-LY19, and DOHH2 cells were treated with a single concentration
of Compound 44, prednisolone, or the combination for 4 days, and
gene expression was analyzed using a glucocorticoid signaling PCR
array (Table 4). Overall, a larger number of genes were
down-regulated with both prednisolone and combination treatments in
all cell lines, pointing to a role of GR as both activator and
repressor of gene expression. Here, the activating function of GR
was focused on and 3 genes which have a synergistic up-regulation
in the panel of cell lines with combination treatment were
described. Sestrin (SESN1), a putative tumor suppressor that
inhibits mTOR signaling (ref), was identified as a gene commonly
up-regulated among the 4 EZH2 mutant cell lines in a synergistic
manner with combination treatment, but not in EZH2 WT cell lines
(FIG. 8A and Table 2). TNF expression was synergistically
up-regulated only in one of the two EZH2 mutant, Compound 44
insensitive cell lines (SUDHL4), with a trend for the other EZH2
mutant, Compound 44 insensitive cell line (RL) showing the same
result (FIG. 8B and Table 2). Expression of TSC22D3/GILZ, while
up-regulated in all cell lines by prednisolone, is only
synergistically enhanced by combination treatment in EZH2 mutant,
Compound 44 sensitive cells (FIG. 8C and Table 2).
TABLE-US-00002 TABLE 2 Statistical Analysis of Gene Expression Data
Presented in FIG. 8 Sestrin TNF GILZ P Value P Value P Value Cell
Line Comparison P Value Summary P Value Summary P Value Summary
OCI-LY19 DMSO vs 0.9164 ns 0.0071 ** 0.0075 ** Combo OCI-LY19
EPZ-6438 vs 0.3232 ns 0.1553 ns 0.0326 * Combo OCI-LY19
Prednisolone 0.1486 ns 0.5050 ns 0.6353 ns vs Combo DOHH2 DMSO vs
0.0063 ** 0.0589 ns 0.0056 ** Combo DOHH2 EPZ-6438 vs 0.0186 *
0.1401 ns 0.0071 ** Combo DOHH2 Prednisolone 0.557 ns 0.1000 ns
0.2828 ns vs Combo WSU-DLCL2 DMSO vs <0.0001 **** 0.0001 ***
<0.0001 **** Combo WSU-DLCL2 EPZ-6438 vs <0.0001 **** 0.3813
ns <0.0001 **** Combo WSU-DLCL2 Prednisolone <0.0001 ****
0.9483 ns 0.0001 *** vs Combo SUDHL10 DMSO vs 0.0073 ** 0.0058 **
0.0102 * Combo SUDHL10 EPZ-6438 vs 0.0081 ** 0.0050 ** 0.0076 **
Combo SUDHL10 Prednisolone 0.0126 * 0.1159 ns 0.0236 * vs Combo RL
DMSO vs 0.0449 * 0.0529 ns 0.0623 ns Combo RL EPZ-6438 vs 0.0484 *
0.0639 ns 0.0635 ns Combo RL Prednisolone 0.2329 ns 0.0997 ns
0.5716 ns vs Combo SUDHL4 DMSO vs 0.0033 ** 0.0043 ** 0.0275 *
Combo SUDHL4 EPZ-6438 vs 0.0045 ** 0.0059 ** 0.0196 * Combo SUDHL4
Prednisolone 0.010 * 0.0205 * 0.0107 ns vs Combo Pairwise
statistical comparisons were performed by two-tailed t test. ns:
not significant; * p < 0.05; ** p < 0.01; *** p < 0.001;
**** p < 0.0001
[0187] Expression levels of glucocorticoid receptor, normalized to
DMSO controls, for EZH2 wild-type (i.e., OCI-LY19, DOHH2), EZH2
Y646-sensitive (i.e., WSU-DLCL2, SUDHL10), and EZH2 Y646 resistant
(i.e., RL, SUDHL4) cell lines were measured after treatment with
the indicated Compound 44, Prednisolone, the combination of
Compound 44 and prednisolone, or DMSO (2 biological replicates, see
methods materials and methods section 5 for details). As the
results show, the expression levels of glucocorticoid receptors
were not commonly affected among cell lines in the combination.
(FIG. 19) Fold change values were quantified using the
.DELTA..DELTA.Ct method and ACTB, B2M and GAPDH as reference
genes.
[0188] The effects of omitting one or all chemotherapy components
from the CHOP regime in two additional xenograft studies were then
examined. SUDHL10 (EZH2 Y646F) xenograft-bearing mice were treated
with Compound 44, COP (chemotherapy without the Doxorubicin
component), or their combination for 28 days (FIG. 20A). Mean tumor
weights from 8/16 mice, euthanized on day 28, were compared,
demonstrating the significant differences in tumor weight between
groups (*p<0.05, **p<0.01, ****p<0.0001; two-tailed t
test). Mice dosed with the maximal tolerated dose of Compound 44 or
with the Compound 44/COP combination showed 100% survival on day
60, the combination group showed the smallest day 28 tumor weights,
statistically different (p<0.05) from all other treatment
groups, including the maximal tolerated dose for Compound 44 (FIG.
20A).
[0189] Then, we investigated combination dosing of Compound 44 with
Prednisone for 28 days in the SUDHL10 xenograft model with two
doses of Compound 44 or Prednisone at two different schedules
(Pred-1=Prednisone at 0.15 mg/kg BID.times.5 on days 1-5 and 22-26;
Pred-2=Prednisone 0.15 mg/kg BID.times.28). As suggested by the in
vitro data, Prednisone dosing alone did not induce any significant
anti-tumor effect (FIG. 20B). In line with the previous study, 125
mg/kg BID (twice daily) dosing of Compound 44 generated only a
partial response, but co-dosing of Compound 44 with Prednisone at
0.15 mg/kg BID, but not with the 2 cycle Prednisone regimen,
induced the maximal possible regression achieved with higher doses
of Compound 44 alone. Body weight for all mice dosed is shown in
FIG. 20C.
[0190] SUDHL10 (EZH2 Y646F) xenograft-bearing mice were treated
with Compound 44, COP (chemotherapy without the Doxorubicin
component), or their combination for 28 days, as specified in the
methods. Mean tumor weights from 8/16 mice, euthanized on day 28,
are compared, demonstrating the significant differences in tumor
weight between groups (*p<0.05, **p<0.01, ****p<0.0001;
two-tailed t test). B) SUDHL10 (EZH2 Y646F) xenograft-bearing mice
were treated for 28 days with two doses of Compound 44 or
Prednisone at two different schedules (Pred-1=Prednisone at 0.15
mg/kg BID.times.5 on days 1-5 and 22-26; Pred-2=Prednisone 0.15
mg/kg BID.times.28). Both compounds were also administered in
combination as indicated. Mean tumor volumes.+-.SEM (n=10) are
plotted in top panel. All groups administered EPZ-6438 show
statistically significant reduction in tumor growth (p<0.01 at
least, vs. vehicle or Prednisone single agent at both schedules;
repeated measures ANOVA, Dunnett's post test), while Prednisone
single agent did not elicit any significant anti-tumor effect
compared to vehicle.
TABLE-US-00003 TABLE 3 Summary of Combinations with Compound 44
Cell Lines EZH2 Mutant GCB EZH2 WT GCB WSU- SU- SU- SU- OCI- DLCL2
DHL10 DHL6 DOHH2 DHL5 LY-19 Toledo C Mafosfanide Additive Additive
Additive No effect -- -- No effect H Doxorubicin Synergy Additive
Additive No effect -- -- No effect O Vincristine Additive Additive
Additive No effect -- -- No effect P Prednisolone Synergy Synergy
Synergy Synergy Synergy Synergy No effect Dexamethasone Synergy
Synergy Synergy Synergy Synergy Synergy No effect
[0191] Finally, tumor growth inhibition was assessed in 3 different
EZH2 mutant lymphoma xenograft models. SCID or nude mice bearing
subcutaneous lymphoma xenografts were co-dosed with Compound 44 and
chemotherapy, either CHOP or COP (CHOP without doxorubicine), and
compared to single agent treatments. In WSU-DLCL2 xenograft bearing
mice, tumor growth inhibition was achieved at all Compound 44 doses
and schedules employed, and was better than CHOP chemotherapy alone
(FIG. 7A). Moreover, the combination therapy of Compound 44 and
CHOP induced a robust anti-tumor response and significantly
(p<0.001) better tumor growth inhibition (93%) than with either
single agent alone (45% and 71%, for CHOP and Compound 44,
respectively). All single treatments were tolerated; there was
minor body weight loss (11.3%) in the Compound 44/CHOP combo group
after the first cycle after which the mice recovered before the
next cycle of treatment.
[0192] In a SU-DHL6 xenograft model, significant tumor growth
inhibition was not observed with CHOP alone, or with Compound 44
(FIG. 7B, top panel), in contrast to results previously published
by Beguelin et al. using the EZH2 inhibitor GSK503. Strikingly, the
combination of Compound 44/CHOP resulted in tumor regression. When
dosing was stopped at day 28 and mice were observed out to day 60
for tumor growth delay, this combination resulted in tumor free
survival in 58% of the mice (FIG. 7B, bottom panel).
[0193] The doxorubicin component of CHOP has a lifetime cumulative
dosing limit of <550 mg/m.sup.2 due to its cardiotoxicity.
Therefore, the combination benefit of a Compound 44/chemotherapy
regimen that eliminated this component was investigated. In a third
study, SU-DHL10 xenograft bearing mice were treated for 28 days
with either increasing doses of Compound 44 (BID), doxorubicin-free
chemotherapy regimen (COP), or a combination of COP and Compound 44
Tumor growth inhibition was observed at all Compound 44 doses as
well as with COP (FIG. 7C, top panel). The 266 mg/kg, 532 mg/kg and
COP/Compound 44 combination treatments resulted in regressions that
were statistically different from vehicle (p>0.001) as assessed
by repeated measures ANOVA and Dunnett's post test, with the
Compound 44/COP combination group demonstrating the best overall
response. After the 28 day dosing, a sub-group of mice with the
smallest tumor burden (8 mice per group) were kept alive without
further dosing for a tumor growth delay endpoint. There was a clear
dose dependent tumor growth delay benefit for mice treated with
Compound 44, while COP treated tumors progressed faster than those
treated with Compound 44 (FIG. 7C, middle panel). While mice
treated with the maximal tolerated dose of Compound 44 or with the
Compound 44/COP combination showed 100% survival on Day 60, the
combination group showed the smallest terminal tumor weights,
statistically different (p>0.05) from all other treatment
groups, including the maximal tolerated dose for Compound 44 (FIG.
7C, bottom panel).
[0194] Standard treatments for B-cell NHL are combination
chemotherapy regimens composed of cyclophosphamide, doxorubicin,
vincristine and prednisolone. While complete response rates of
40-50% can be achieved, a substantial proportion of patients
relapse, with 3-year overall survival rates of only about 30%.
Relapsed lymphomas can exhibit resistance to a wide range of
anticancer drugs, which poses a severe challenge in the clinic to
manage these aggressive malignancies. Acquisition of drug
resistance in lymphoma is partly driven by the genetic
heterogeneity and instability of the tumor cells. Successful
treatment of chemoresistant NHL will thus require rational
combinations of drugs targeting multiple pathways specific to the
different subtypes of B-cell NHL. For instance, in lymphomas of the
activated B cell type, constitutive activation of the NFkB pathway
has been implicated in therapy resistance, and several novel
targeted therapies have shown promise in this subtype.
[0195] Epigenetic effectors, such as polycomb, have also been
implicated in cancer cell chemo-resistance. EZH2, the catalytic
subunit of polycomb repressive complex 2 (PRC2) is a critical
oncogenic driver in germinal center derived B-cell lymphomas. These
more primitive B-cell malignancies, especially variants expressing
EZH2 mutants with altered catalytic activity, require EZH2 for
proliferation and survival. Results from preclinical studies
forecast great promise for EZH2 catalytic inhibitors for the
treatment of such genetically defined cancers, and EZH2 inhibitors
may also mitigate chemotherapy resistance. The data presented
herein show that Compound 44, a clinical stage EZH2 inhibitor,
shows various degrees of combination benefit, ranging from
additivity to synergy, with the components of CHOP. Those
combination effects were specifically found in lymphomas of the
germinal center origin, and, in the case of cyclophosphamide,
doxorubicine and vincristine, were restricted to EZH2
mutant-bearing cells. Significant synergy in lymphoma cell killing
was also found when Compound 44 was co-dosed with CHOP in vivo.
This was especially true in the SU-DHL6 xenograft model where
neither single agent showed any significant antitumor activity, but
the combination induced durable regressions in >50% of mice.
This reiterates the potential importance of overactive EZH2 in
chemoresistance of EZH2 mutant lymphoma. Among the CHOP components,
Compound 44 combinations with prednisone induced the strongest
antiproliferative activity, and this combination could also render
insensitive GCB lymphoma cell lines sensitive to EZH2 inhibition,
regardless of the EZH2 mutational status. Additionally, this
combination benefit is more apparent when Compound 44 and
prednisolone are either dosed together or in a sequence specific
manner; thus, priming cells with an EZH2 inhibitor, followed by
treatment with GR agonists proved particularly effective. This
surprising finding has potentially important implications for the
application of EZH2 inhibitors in the clinic. First, the widely
used GRag are frequently co-administrated with anticancer drugs to
prevent drug-induced allergic reactions and to relieve pain,
nausea, and emesis, and are pivotal in the treatment of
hematopoietic malignancies owing to their ability to induce
apoptosis in these cancers. Compared to the other CHOP components,
GRag induces the least severe adverse effects. Further, the
opportunity to eliminate doxorubicin from the CHOP regime while
preserving a combination benefit with Compound 44, as suggested by
the data in the SU-DHL10 xenograft model, could spare patients from
the dose-limiting cardiotoxic side effects of doxorubicin. Finally,
preclinical studies have shown that single agent EZH2 inhibitors
induce significant cell killing only in EZH2 mutant-bearing
lymphomas, which represent a fraction (20%) of GCB lymphoma
patients with high unmet clinical need. The results here
demonstrate that GRag/EZH2 inhibitor combinations may have clinical
utility in all germinal center derived B cell lymphomas.
[0196] Glucocorticoid bound GR molecules move to the nucleus and
can act as either transcriptional activator or repressor, depending
on the cellular environment. It has been suggested that GR
constantly samples the nucleosome for a productive interaction, and
the purpose of chromatin-modifying enzymes is to provide regulated
access of GR, its cofactors and the basal transcription machinery
to DNA. Other studies show that GR often binds to preexisting
regions of open chromatin, and the chromatin architecture in a
given cell type is organized such that GR can act in a tissue
specific manner. Accessibility to GR binding sites can further be
enhanced by ATP-dependent chromatin remodeling, and the SWI/SNF
complex plays a key role in this activity. Not wishing to be bound
by a particular theory or a specific mechanism of action, it is
conceivable that aberrant chromatin repression, induced by EZH2
mediated hypertrimethylation of H3K27, can block some of the
otherwise accessible GR binding sites, interfering with normal GR
mediated gene induction or repression. Indeed, all EZH2 mutant
lymphoma cell lines are insensitive to GRag treatment, while
concentration-dependent cell killing is observed in EZH2 WT cells.
The observation that pretreatment with prednisolone, followed by
Compound 44 treatment, cannot induce synergy in almost all cell
lines tested, points towards the possibility of EZH2 inhibitor
induced chromatin remodeling being the rate limiting step for the
enhanced action of GR. Also, PRC2 is known to antagonize with
SWI/SNF function and the down-regulation of core subunits of the
SWI/SNF complex--SMARCA4, ARID1A, and INI1--have been associated
with resistance to prednisolone in acute lymphoblastic T-cell
leukemia. Since the relationship of INI1 loss and EZH2
over-activation has been established in rhabdoid tumors, whether
global INI1 protein levels would increase in various lymphoma cells
exposed to Compound 44 or prednisolone, potentially allowing
greater accessibility of GR to its binding sites after increased
SWI/SNF function, was investigated.
[0197] GR pathway gene expression arrays revealed both increased
and decreased gene expression after treatment of several GCB
lymphoma cells (both EZH2 WT and mutant) with Compound 44,
prednisolone or their combination, confirming the dual function of
GR. The only gene that was synergistically up-regulated with the
combination in all EZH2 mutant lymphoma cells was SESN1, a TP53
tumor suppressor with functions in cellular response to DNA damage
and oxidative stress. Sestrins inhibit cell growth by activating
AMP-activated protein kinase, resulting in the inhibition of the
mTOR pathway. Hence SESN1 mediated mTOR pathway inhibition may be
an important mechanism of reintroducing GRag sensitivity in EZH2
mutant lymphoma cells after Compound 44 treatment.
[0198] Conversely, GRag/Compound 44 combination treatment could
also induce cell killing in those EZH2 mutant lymphoma cell lines
that have been reported as refractory to EZH2 inhibitor treatment
(RL, SU-DHL4). SESN1 was induced with combination treatment in
those cell lines as well, but an additional synergistic
up-regulation of TNF, a potent inflammatory cytokine, was observed
specifically in RL and SU-DHL4 cells. This observation seems
surprising as TNF and glucocorticoids usually act antagonistically.
TNF, through its receptor TNFR-1, can induce apoptosis, but also
has the ability to transduce survival signals, mainly through the
NFkB pathway. It is thus possible that increased TNF expression,
induced by the Compound 44/prednisolone combination, may shift TNF
action towards apoptosis in the context of GR agonist repression of
NFkB-mediated transcription. It is unclear, however, why this
mechanism would result in synergistic cell killing in Compound 44
insensitive EZH2 mutant cells. The potential importance of aberrant
repression of negative regulators of the NFkB pathway in GRag
resistance and the potential role of EZH2 mediating that is further
supported by our observation that GILZ is synergistically
up-regulated in 2 out of 6 cells lines with the combination.
Methods
Medium Throughput Assay
[0199] Lymphoma cells were seeded into flasks (50,000 cells/mL for
WSU-DLCL2, and DOHH2, 10,000 cells/mL for SU-DHL10, and 100,000
cells/mL for Toledo) and pretreated with 7 doses of Compound 44 or
DMSO for 4 days or 6 days for Toledo assays. Cells were then split
back to 50,000 cells/mL for WSU-DLCL2 and DOHH2 or 30,000 cells/mL
for SU-DHL-10 and co-treated with Compound 44 and compound of
interest using the HP D300 digital dispenser (Tecan). Both drugs
were serially diluted two-fold and combined in a matrix with
constant ratios diagonally across the plate with a final DMSO
content of 0.11% (v/v). After 3 days of co-treatment (5 days for
Toledo assays), cell viability was measured via ATP content using
CellTiter-Glo.RTM. (Promega) and luminescence was detected using a
SpectraMax M5 microplate reader (Molecular Devices).
[0200] Synergy quantification is performed using the Chou-Talalay
method for drug combination (Ref 1). The Combination Index (CI)
equation offers a quantitative definition for additivity (CI=1),
synergism (CI<1), and antagonism (CI>1). This equation used
fractional effect (Fa) values from a constant ratio of drug
combination to determine CI values. The resulting plot (Fa-CI) plot
shows the resultant CI values bracketed by 95% confidence
intervals. These Fa-CI plots are generated using the Calcusyn for
Windows software (Ref 2). CI values <1 with confidence interval
lines also below 1 indicate statistically significant
synergism.
[0201] For drug combinations where only one drug showed more than
50% inhibition, Potency shifts were determined. Dose responses were
plotted using Graphpad Prism and either 50% or 60% inhibitory
concentrations were interpolated from the dose response curves.
Potency shifts were considered significant when confidence
intervals for dose responses did not overlap.
Cell Lines, Compounds, and Treatment Outline
[0202] WSU-DLCL2, SU-DHL10, RL, SU-DHL4, OCI-LY19, and DOHH2 were
previously described (NatChemBio 2012). For combination studies, a
modified version of our proliferation assay in suspension cells was
used, as previously described (Daigle et al, Cancel Cell, Vol. 20,
1. Pg. 53-65 (2011); Daigle et al., Blood, 121, 13, 2533-2541
(2013)). Briefly, on day 0, cells were plated in triplicate in
96-well plates at initial densities to ensure linear log phase
growth over 4 days. Cells were treated with either a dose curve of
Compound 44 (starting at a top dose of 1 .mu.M), a single dose of
prednisolone (Catalog # and Manufacturer) at a concentration
10-fold lower than the 4-day IC50 of the drug, or a combination of
Compound 44 and prednisolone. On day 4, cells were counted using
Viacount reagent in the guava easyCyte flow cytometer, and the
viable cell number was used to replate cells at the original
densities for 3 additional days. Cells that were pre-treated with
Compound 44 either received continuous Compound 44 alone, or the
combination of Compound 44 and prednisolone (constant dose); cells
pre-treated with prednisolone either received continuous
prednisolone, or the combination of prednisolone and Compound 44;
cells co-treated for 4 days continued to receive co-treatment
through 7 days.
Xenograft Studies
[0203] All the procedures related to animal handling, care and the
treatment in this study were performed according to the guidelines
approved by the Institutional Animal Care and Use Committee (IACUC)
of CRL Piedmont and Shanghai ChemPartner following the guidance of
the Association for Assessment and Accreditation of Laboratory
Animal Care (AAALAC). WSU-DLCL2, SU-DHL6, or SU-DHL10 cells were
harvested during mid-log phase growth, and re-suspended in PBS with
50% Matrigel.TM. (BD Biosciences), and injected into
immune-compromised mice. Each mouse received 1.times.107 cells (0.2
mL cell suspension) subcutaneously in the right flank, and once
tumors reached a predetermined size, mice were orally dosed with
different doses of Compound 44 at various schedules for up to 28
days and/or CHOP/COP on the following schedules: Cyclophosphamide
was administered intraperitoneally (i.p.), and doxorubicin and
vincristine were each administered via bolus tail vein injections
(i.v.); each was given once daily on Days 1 and 8 in the SU-DHL6
study, and on Days 1 and 22 in the WSU-DLCL2 and SU-DHL10 studies.
Prednisone was administered p.o. on two cycles of five daily doses,
starting on Days 1 and 8 ((qd.times.5).times.2, Days 1, 8) in the
SU-DHL6 study, and on Days 1 and 22 ((qd.times.5).times.2, Days 1,
22) in the WSU-DLCL2 and SU-DHL10 studies. Each dose was delivered
in a volume of 0.2 mL/20 g mouse (10 mL/kg), and adjusted for the
last recorded weight of individual animals. Tumor measurements and
body weights were collected twice-weekly for 28 days for all
studies. To determine tumor growth delay in the SU-DHL10 and
SU-DHL6 studies, each test animal was euthanized when its neoplasm
reached the endpoint volume of 2000 mm3 or on the last day of the
study (day 60), whichever came first.
Quantitative PCR
[0204] WSU-DLCL2, SU-DHL10, RL, SU-DHL4, OCI-LY19, and DOHH2 cells
were treated in parallel with DMSO, 1 .mu.M of Compound 44
(SU-DHL10 treated with 100 nM Compound 44), a dose of prednisolone
at a concentration 10-fold lower than the 4-day IC.sub.50, or the
combination of drugs for 4 days. Cells were harvested and total
mRNA was extracted from cell pellets using the RNeasy Plus Mini Kit
(Qiagen; 74134). For the RT2 Glucocorticoid Signaling PCR array
(Qiagen; PAHS-154ZE-4), cDNA was made by RT2 First Strand Kit
(Qiagen; 330401). Array RT-PCR was performed using ViiA 7 Real-Time
PCR Systems [Applied Biosystems (AB)] with RT2 SYBR Green ROX qPCR
Mastermix (Qiagen; 330521). Gene expression was normalized to
array's B2M and fold change compared to DMSO was calculated using
the .DELTA..DELTA.Ct method. To validate array data, TaqMan probe
based qPCR was carried out using TaqMan Fast Advanced Master Mix
(AB; 4444964) and TaqMan primer/probe sets for Sestrin (AB;
Hs00902787_m1) and TNF (AB; Hs01113624_m1). Fold change was
calculated as above, normalizing to RPLPO (AB; 4333761F).
ELISA
[0205] Histones were extracted from tumor samples as described
above. Histones were prepared in equivalent concentrations in
coating buffer (PBS+0.05% BSA) yielding 0.5 ng/.mu.l of sample, and
100 .mu.l of sample or standard was added in duplicate to 2 96-well
ELISA plates (Thermo Labsystems, Immulon 4HBX #3885). The plates
were sealed and incubated overnight at 4.degree. C. The following
day, plates were washed 3.times. with 300 .mu.l/well PBST
(PBS+0.05% Tween 20; 10.times.PBST, KPL #51-14-02) on a Bio Tek
plate washer. Plates were blocked with 300 .mu.l/well of diluent
(PBS+2% BSA+0.05% Tween 20), incubated at RT for 2 hours, and
washed 3.times. with PBST. All antibodies were diluted in diluent.
100 .mu.l/well of anti-H3K27me3 (CST #9733, 50% glycerol stock
1:1,000) or anti-total H3 (Abcam ab1791, 50% glycerol 1:10,000) was
added to each plate. Plates were incubated for 90 min at RT and
washed 3.times. with PBST. 100 .mu.l/well of anti-Rb-IgG-HRP (Cell
Signaling Technology, 7074) was added 1:2,000 to the H3K27Me3 plate
and 1:6,000 to the H3 plate and incubated for 90 min at RT. Plates
were washed 4.times. with PBST. For detection, 100 .mu.l/well of
TMB substrate (BioFx Laboratories, #TMBS) was added and plates
incubated in the dark at RT for 5 min. Reaction was stopped with
100 .mu.l/well 1N H.sub.2SO.sub.4. Absorbance at 450 nm was read on
SpectaMax M5 Microplate reader.
TABLE-US-00004 TABLE 4a Ct values and fold changes from the RT2
Glucocorticoid signaling PCR array analysis for OCI cell line. Ct
Values .DELTA.CT (B2M) Gene DMSO Cpd44 Pred Combo DMSO Cpd44 Pred
Combo ADARB1 24.373 23.799 24.946 24.323 7.368 6.580 7.319 7.177
AFF1 21.574 21.780 21.892 21.613 4.589 4.561 4.265 4.467 AK2 20.300
20.497 20.859 20.656 3.295 3.278 3.232 3.510 AMPD3 27.424 26.984
27.937 27.892 10.419 9.765 10.310 10.746 ANGPTL4 30.465 30.374
30.333 29.769 13.460 13.155 12.706 12.623 ANXA4 23.319 23.379
24.130 23.394 6.314 6.160 6.503 6.248 AQP1 Undetermined 31.992
Undetermined Undetermined #VALUE! 14.773 #VALUE! #VALUE! ARID58
22.092 22.537 22.635 22.538 5.087 5.318 5.008 5.392 ASPH 27.926
27.556 28.894 27.701 10.921 10.337 11.267 10.555 ATF4 18.500 18.838
19.578 19.368 1.495 1.619 1.951 2.222 BCL6 27.421 26.240 28.282
26.459 10.416 9.021 10.655 9.313 BMPER Undetermined 34.674
Undetermined 32.290 #VALUE! 17.455 #VALUE! 15.144 CALCR
Undetermined Undetermined Undetermined Undetermined #VALUE! #VALUE!
#VALUE! #VALUE! CEBPA 30.199 27.522 30.852 28.731 13.194 10.303
13.225 11.585 CEBPB 23.119 23.723 24.427 24.675 6.114 6.504 5.800
7.532 COL4A2 32.777 33.300 35.000 32.293 15.772 16.081 17.373
15.147 CREB1 22.477 22.697 23.159 22.702 5.472 5.478 5.532 5.556
CREB3 24.708 24.979 25.174 24.863 7.703 7.760 7.547 7.717 CREB3L4
24.162 24.000 24.936 24.497 7.157 6.781 7.309 7.351 CTGF 21.557
21.719 21.099 20.311 4.552 4.500 9.472 3.165 CYB561 Undetermined
33.134 Undetermined 32.534 #VALUE! 15.315 #VALUE! 15.388 DDIT4
24.102 23.567 23.551 23.195 7.097 5.348 5.924 6.049 DIRAS2
Undetermined Undetermined Undetermined Undetermined #VALUE! #VALUE!
#VALUE! #VALUE! DUSP1 20.981 20.800 21.767 21.200 3.976 3.581 4.140
4.054 EDN1 Undetermined Undetermined 33.433 32.487 #VALUE! #VALUE!
15.806 15.341 EHD3 28.984 28.117 28.833 27.236 11.979 10.898 11.206
10.090 ERRFI1 Undetermined Undetermined 32.824 Undetermined #VALUE!
#VALUE! 15.197 #VALUE! FKBP5 22.604 22.499 22.353 21.699 5.599
5.280 4.726 4.553 FOSL2 26.226 26.214 26.368 25.547 9.221 8.995
8.741 8.401 GDPD1 26.444 26.633 27.196 26.808 9.439 9.419 9.569
9.662 GHRHR 37.467 33.641 35.486 36.113 20.462 18.422 17.859 18.967
GLUL 22.916 22.385 23.448 22.402 5.911 5.166 5.821 5.256 GOT1
23.034 23.224 23.810 23.450 6.089 6.005 6.183 6.304 H6PD 26.842
26.141 26.981 26.440 9.837 8.922 9.354 9.294 HAS2 Undetermined
Undetermined Undetermined Undetermined #VALUE! #VALUE! #VALUE!
#VALUE! HNRPLL 29.840 29.708 30.306 29.439 12.835 12.489 12.679
12.293 IL10 Undetermined Undetermined 34.155 Undetermined #VALUE!
#VALUE! 16.528 #VALUE! IL1RN 33.932 32.902 Undetermined
Undetermined 16.927 15.683 #VALUE! #VALUE! IL6 Undetermined
Undetermined Undetermined 32.602 #VALUE! #VALUE! #VALUE! 15.456
IL6R Undetermined Undetermined Undetermined Undetermined #VALUE!
#VALUE! #VALUE! #VALUE! KLF13 23.416 23.178 23.963 23.145 6.411
5.953 6.336 5.999 KLF9 29.545 28.545 28.597 27.791 12.541 11.326
10.970 10.545 LOX 33.344 32.825 32.787 31.904 16.339 15.606 15.160
14.758 MERTK 29.340 28.749 29.685 28.885 12.335 11.530 12.058
11.739 MT1E Undetermined Undetermined Undetermined Undetermined
#VALUE! #VALUE! #VALUE! #VALUE! MT2A 22.845 22.941 23.298 22.515
5.840 5.722 5.671 5.369 NFKBIA 21.672 21.905 22.337 21.755 4.667
4.686 4.710 4.609 NR3C1 20.940 21.151 21.276 20.605 3.935 3.912
3.649 3.459 PDCD7 23.121 23.359 28.314 23.491 6.116 6.140 10.687
6.345 PDGFRB 32.160 33.308 30.540 30.932 15.155 16.089 12.913
13.786 PDP1 26.092 25.788 26.292 25.520 9.087 8.569 8.665 8.374
PER1 24.615 25.503 25.500 26.016 7.610 8.284 7.873 8.870 PER2
Undetermined 23.177 23.707 23.482 #VALUE! 5.958 6.080 6.336 PIK3R1
23.175 23.115 23.678 23.317 6.170 5.896 6.051 6.171 PLD1
Undetermined Undetermined Undetermined 33.540 #VALUE! #VALUE!
#VALUE! 16.394 PLEKHF1 30.216 29.694 30.977 30.285 13.211 12.475
13.350 13.139 POU2F1 24.562 24.656 25.232 24.555 7.557 7.437 7.605
7.409 POU2F2 31.495 31.740 31.543 31.643 14.490 14.521 13.916
14.497 RASA3 23.112 23.251 23.743 23.462 6.107 6.032 6.115 6.316
RGS2 28.455 27.701 29.467 28.122 11.450 10.482 11.840 10.976 RHOB
22.108 20.944 20.967 19.659 5.103 3.725 3.340 2.513 RHOJ
Undetermined Undetermined Undetermined Undetermined #VALUE! #VALUE!
#VALUE! #VALUE! SESN1 22.463 22.424 23.126 22.491 5.458 5.205 5.499
5.345 SGK1 26.351 26.107 25.819 24.816 9.346 8.888 8.192 7.670
SLC10A6 31.409 30.432 31.656 32.746 14.398 13.213 14.029 15.600
SLC19A2 24.878 24.881 25.910 25.274 7.873 7.662 3.283 8.128 SLC22A5
29.254 29.101 30.112 29.115 12.249 11.882 12.485 11.969 SNTA1
28.151 27.457 28.892 28.483 11.146 10.238 11.265 11.337 SPHK1
28.555 28.787 29.199 29.124 11.550 11.568 11.572 11.978 SPSB1
27.338 27.455 28.347 28.097 10.333 10.236 10.720 10.951 STAT5A
22.115 22.442 22.673 22.391 5.110 5.223 5.046 5.245 STAT5B 22.886
22.979 23.838 23.297 5.881 5.760 6.211 6.151 TBL1XR1 21.317 21.488
21.705 21.430 4.312 4.269 4.078 4.284 TNF 24.765 24.377 24.612
23.620 7.758 7.158 6.985 6.474 TNFAIP3 22.296 22.827 23.168 23.327
5.291 5.608 5.541 6.181 TSC22D3 25.692 25.235 24.619 24.219 3.687
8.016 6.992 7.073 USP2 33.949 31.341 33.986 32.493 16.944 14.122
16.359 15.347 USP54 24.856 25.235 25.764 24.989 7.851 8.016 8.137
7.843 VDR 25.099 24.754 24.985 24.651 8.088 7.535 7.355 7.505 VLDLR
28.968 28.902 29.671 29.488 11.963 11.683 12.044 12.342 XDH
Undetermined Undetermined Undetermined Undetermined #VALUE! #VALUE!
#VALUE! #VALUE! ZFP36 24.550 25.191 Undetermined 25.349 7.545 7.972
#VALUE! 8.203 ZHX3 24.941 24.761 24.833 24.322 7.936 7.542 7.206
7.176 ZNF2B1 22.504 23.249 23.997 23.695 5.499 6.030 6.370 6.549
ACTB 15.098 14.892 16.093 14.987 -1.907 -2.327 -1.534 -2.159 B2M
17.005 17.219 17.627 17.146 0.000 0.000 0.000 0.000 GAPDH 15.880
16.149 16.519 16.647 -1.125 -1.070 -1.108 -0.499 HPRT1 21.462
21.828 22.125 21.813 4.457 4.609 4.498 4.667 RPLP0 14.351 14.350
15.011 14.197 -2.654 -2.869 -2.616 -2.949 HGDC Undetermined
Undetermined Undetermined Undetermined #VALUE! #VALUE! #VALUE!
#VALUE! RTC 22.174 21.961 21.962 22.251 5.169 4.742 4.335 5.105 RTC
22.089 21.953 22.140 22.008 5.084 4.734 4.515 4.862 RTC 22.195
21.961 22.167 21.993 5.190 4.742 4.540 4.847 PPC 18.397 18.266
18.432 18.371 1.392 1.049 0.805 1.225 PPC 18.426 18.330 18.320
18.347 1.421 1.111 0.693 1.201 PPC 18.301 17.672 18.372 18.378
1.296 0.453 0.745 1.232 Cpd44 Pred Combo Gene .DELTA..DELTA.CT Fold
Change .DELTA..DELTA.CT Fold Change .DELTA..DELTA.CT Fold Change
ADARB1 -0.788 1.727 -0.049 1.035 -0.191 1.142 AFF1 -0.008 1.006
-0.304 1.235 -0.102 1.073 AK2 -0.017 1.012 -0.063 1.045 0.215 0.862
AMPD3 -0.654 1.574 -0.109 1.078 0.327 0.797 ANGPTL4 -0.305 1.235
-0.754 1.686 -0.837 1.786 ANXA4 -0.154 1.113 0.189 0.877 -0.066
1.047 AQP1 #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! ARID58
0.231 0.852 -0.079 1.056 0.305 0.809 ASPH -0.584 1.499 0.346 0.737
-0.366 1.289 ATF4 0.124 0.918 0.456 0.729 0.727 0.604 BCL6 -1.395
2.630 0.239 0.847 -1.103 2.148 BMPER #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! CALCR #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! CEBPA -2.891 7.418 0.031 0.979 -1.609 3.050 CEBPB
0.390 0.763 0.686 0.622 1.418 0.374 COL4A2 0.309 0.807 1.601 0.330
-0.625 1.542 CREB1 0.006 0.996 0.060 0.959 0.084 0.943 CREB3 0.057
0.961 -0.156 1.114 0.014 0.990 CREB3L4 -0.376 1.298 0.152 0.900
0.194 0.874 CTGF -0.052 1.037 -1.080 2.114 -1.387 2.615 CYB561
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! DDIT4 -0.749 1.681
-1.173 2.255 -1.048 2.068 DIRAS2 #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! DUSP1 -0.395 1.315 0.164 0.893 0.078 0.947 EDN1
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! EHD3 -1.081 2.116
-0.773 1.709 -1.889 3.704 ERRFI1 #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! FKBP5 -0.319 1.247 -0.873 1.831 -1.046 2.065 FOSL2
-0.226 1.170 -0.480 1.395 -0.820 1.765 GDPD1 -0.020 1.014 0.130
0.914 0.223 0.857 GHRHR -4.040 16.450 -2.603 6.075 -1.495 2.819
GLUL -0.745 1.675 -0.090 1.064 -0.655 1.575 GOT1 -0.084 1.060 0.094
0.937 0.215 0.862 H6PD -0.915 1.886 -0.483 1.398 -0.543 1.457 HAS2
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! HNRPLL -0.346 1.271
-0.156 1.114 -0.542 1.456 IL10 #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! IL1RN -1.244 2.369 #VALUE! #VALUE! #VALUE! #VALUE!
IL6 #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! IL6R #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! KLF13 -0.452 1.368 -0.075
1.053 -0.412 1.331 KLF9 -1.215 2.321 -1.571 2.971 -1.896 3.722 LOX
-0.733 1.662 -1.179 2.264 -1.581 2.992 MERTK -0.805 1.747 -0.277
1.212 -0.596 1.512 MT1E #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! MT2A -0.118 1.085 -0.169 1.124 -0.471 1.386 NFKBIA 0.019
0.987 0.043 0.971 -0.058 1.041 NR3C1 -0.023 1.016 -0.286 1.219
-0.476 1.391 PDCD7 0.024 0.984 4.571 0.042 0.229 0.853 PDGFRB 0.934
0.523 -2.242 4.731 -1.369 2.593 PDP1 -0.518 1.432 -0.422 1.340
-0.713 1.639 PER1 0.674 0.627 0.263 0.833 1.250 0.418 PER2 #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! PIK3R1 -0.274 1.209 -0.119
1.086 0.001 0.999 PLD1 #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! PLEKHF1 -0.736 1.666 0.139 0.908 -0.072 1.051 POU2F1 -0.120
1.087 0.048 0.967 -0.148 1.108 POU2F2 0.331 0.979 -0.574 1.489
0.007 0.995 RASA3 -0.075 1.053 0.009 0.994 0.209 0.865 RGS2 -0.968
1.956 0.390 0.763 -0.474 1.389 RHOB -1.378 2.599 -1.763 3.394
-2.590 6.021 RHOJ #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
SESN1 -0.253 1.192 0.041 0.372 -0.113 1.081 SGK1 -0.458 1.374
-1.154 2.225 -1.676 3.195 SLC10A6 -1.185 2.274 -0.369 1.291 1.202
0.435 SLC19A2 -0.211 1.157 0.410 0.753 0.255 0.838 SLC22A5 -0.367
1.290 0.236 0.849 -0.280 1.214 SNTA1 -0.908 1.876 0.119 0.921 0.191
0.876 SPHK1 0.018 0.988 0.022 0.985 0.428 0.743 SPSB1 -0.097 1.070
0.387 0.765 0.618 0.552 STAT5A 0.113 0.925 -0.064 1.045 0.135 0.911
STAT5B -0.121 1.087 0.330 0.796 0.270 0.829 TBL1XR1 -0.043 1.030
-0.234 1.176 -0.028 1.020 TNF -0.600 1.516 -0.773 1.709 -1.284
2.435 TNFAIP3 0.317 0.803 0.250 0.841 0.890 0.540 TSC22D3 -0.671
1.592 -1.695 3.238 -1.614 3.061 USP2 -2.822 7.071 -0.585 1.500
-1.597 3.025 USP54 0.165 0.892 0.286 0.820 -0.003 1.006 VDR -0.553
1.467 -0.730 1.559 -0.583 1.498 VLDLR -0.280 1.214 0.081 0.945
0.379 0.769 XDH #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
ZFP36 0.427 0.744 #VALUE! #VALUE! 0.658 0.634 ZHX3 -0.394 1.314
-0.730 1.559 -0.760 1.693 ZNF2B1 0.531 0.692 0.871 0.547 1.050
0.483 ACTB B2M GAPDH HPRT1 RPLP0 HGDC RTC RTC RTC PPC PPC PPC
TABLE-US-00005 TABLE 4b Ct values and fold changes from the RT2
Glucocorticoid signaling PCR array analysis for DOHH2 cell line. Ct
Values .DELTA.CT (B2M) Gene DMSO Cpd44 Pred Combo DMSO Cpd44 Pred
Combo ADARB1 31.818 31.431 33.560 30.189 12.809 12.855 14.676
12.038 AFF1 24.684 23.838 23.992 23.224 5.675 5.312 5.108 5.073 AK2
20.334 20.173 20.262 19.961 1.325 1.597 1.378 1.810 AMPD3 26.401
26.146 27.535 26.852 7.392 7.570 8.651 8.701 ANGPTL4 31.134 30.820
31.538 30.854 12.125 12.244 12.654 12.703 ANXA4 24.817 24.273
24.997 24.268 5.808 5.697 6.113 6.117 AQP1 Undetermined
Undetermined Undetermined Undetermined #VALUE! #VALUE! #VALUE!
#VALUE! ARID5B 23.881 23.782 23.885 23.886 4.872 5.206 5.001 5.735
ASPH 22.970 22.823 23.369 22.996 3.961 4.247 4.485 4.845 ATF4
19.156 19.190 19.313 18.983 0.147 0.614 0.429 0.832 BCL6 21.529
21.323 21.801 21.773 2.520 2.747 2.917 3.622 BMPER 38.037 33.092
39.378 39.656 19.028 20.516 20.494 21.505 CALCR Undetermined 33.630
Undetermined Undetermined #VALUE! 15.054 #VALUE! #VALUE! CEBPA
34.654 30.676 32.188 30.646 15.645 12.100 13.304 12.495 CEBPB
23.911 23.925 24.317 24.001 4.902 5.349 5.433 5.850 COL4A2 32.314
34.119 38.993 34.143 13.305 15.543 20.109 15.992 CREB1 22.930
22.746 22.890 22.730 3.921 4.170 4.006 4.579 CREB3 24.929 24.840
24.865 24.647 5.920 6.264 5.981 6.496 CREB3L4 24.405 24.110 24.616
24.373 5.396 5.534 5.732 6.222 CTGF 33.711 32.760 33.728 33.696
14.702 14.184 14.844 15.545 CYB561 37.790 31.945 39.582 34.331
18.781 13.369 20.698 16.180 DDIT4 23.934 23.508 24.105 22.948 4.925
4.932 5.221 4.797 DIRAS2 Undetermined Undetermined Undetermined
Undetermined #VALUE! #VALUE! #VALUE! #VALUE! DUSP1 27.604 27.132
27.866 27.262 8.595 8.556 8.982 9.111 EDN1 31.233 32.260 32.263
31.224 12.224 13.684 13.379 13.073 EHD3 32.315 28.852 31.098 28.674
13.306 10.276 12.214 10.523 ERRFI1 32.525 30.163 32.635 29.588
13.516 11.587 13.751 11.437 FKBP5 21.985 21.520 20.912 20.512 2.976
2.944 2.028 2.361 FOSL2 31.767 29.872 31.543 29.925 12.758 11.295
12.659 11.774 GDPD1 27.532 27.570 27.884 27.396 8.523 8.994 9.000
9.245 GHRHR 37.684 39.644 36.095 37.813 18.675 21.068 17.211 19.662
GLUL 36.133 36.671 34.574 36.099 17.124 18.095 15.690 17.948 GOT1
23.427 23.126 23.532 22.880 4.418 4.550 4.648 4.729 H6PD 24.717
24.377 24.969 24.453 5.708 5.801 6.085 6.302 HAS2 Undetermined
Undetermined Undetermined Undetermined #VALUE! #VALUE! #VALUE!
#VALUE! HNRPLL 30.324 29.151 33.284 31.380 11.315 10.575 14.400
13.229 IL10 Undetermined Undetermined Undetermined Undetermined
#VALUE! #VALUE! #VALUE! #VALUE! IL1RN Undetermined 32.271 33.560
31.586 #VALUE! 13.695 14.676 13.435 IL6 Undetermined Undetermined
34.758 37.608 #VALUE! #VALUE! 15.874 19.457 IL6R Undetermined
31.962 Undetermined 32.383 #VALUE! 13.386 #VALUE! 14.232 KLF13
22.951 22.420 22.546 21.765 3.942 3.844 3.662 3.614 KLF9 28.691
28.439 28.547 27.741 9.682 9.863 9.663 9.590 LOX 33.562 32.997
34.158 32.855 14.553 14.421 15.274 14.704 MERTK 32.997 32.456
32.892 31.474 13.988 13.880 14.008 13.323 MT1E 39.692 Undetermined
Undetermined Undetermined 20.683 #VALUE! #VALUE! #VALUE! MT2A
39.646 Undetermined Undetermined Undetermined 20.637 #VALUE!
#VALUE! #VALUE! NFKBIA 22.891 22.625 22.830 22.625 3.882 4.049
3.946 4.474 NR3C1 22.602 22.430 22.794 22.573 3.593 3.854 3.910
4.422 PDCD7 23.656 23.417 23.552 23.397 4.647 4.841 4.668 5.246
PDGFRB Undetermined 35.193 34.934 31.552 #VALUE! 16.617 16.050
13.401 PDP1 25.863 25.175 25.682 25.330 6.854 6.599 6.798 7.179
PER1 24.944 24.717 25.142 25.289 5.935 6.141 6.258 7.138 PER2
24.642 23.835 24.159 23.476 5.633 5.259 5.275 5.325 PIK3R1 24.177
23.712 23.850 23.610 5.168 5.136 4.966 5.459 PLD1 37.038
Undetermined 37.120 38.323 18.029 #VALUE! 18.236 20.172 PLEKHF1
29.886 28.946 29.414 28.738 10.877 10.370 10.530 10.587 POU2F1
24.378 24.003 24.648 23.667 5.369 5.427 5.764 5.516 POU2F2 22.469
22.167 22.489 21.930 3.460 3.591 3.605 3.779 RASA3 27.152 27.636
27.803 28.392 8.143 9.060 8.919 10.241 RGS2 24.790 24.861 25.514
25.639 5.781 6.285 6.630 7.488 RHOB 32.661 30.745 33.162 30.702
13.652 12.169 14.278 12.551 RHOJ Undetermined Undetermined
Undetermined Undetermined #VALUE! #VALUE! #VALUE! #VALUE! SESN1
24.226 23.848 22.839 21.993 5.217 5.272 3.955 3.842 SGK1 27.633
27.821 29.628 29.125 8.624 9.245 10.744 10.974 SLC10A6 34.483
36.435 36.176 32.738 15.474 17.859 17.292 14.587 SLC19A2 25.600
24.859 25.455 24.769 6.591 6.283 6.571 6.618 SLC22A5 28.392 27.992
28.915 27.835 9.383 9.416 10.031 9.684 SNTA1 24.884 24.550 25.124
25.000 5.575 5.974 6.240 6.849 SPHK1 30.677 28.863 29.971 28.646
11.668 10.287 11.087 10.495 SPSB1 27.110 26.652 26.911 26.621 8.101
8.076 8.027 8.470 STAT5A 24.237 23.771 23.885 23.477 5.228 5.195
5.001 5.326 STAT5B 22.503 22.328 22.632 22.414 3.434 3.752 3.748
4.263 TBL1XR1 21.397 20.994 21.304 21.133 2.388 2.418 2.420 2.982
TNF 31.328 31.849 31.956 31.194 12.319 13.273 13.072 13.043 TNFAIP3
28.260 27.520 Undetermined 30.586 9.251 8.944 #VALUE! 12.435
TSC22D3 25.176 24.752 23.310 22.374 6.157 6.176 4.426 4.223 USP2
24.104 23.684 23.501 22.971 5.095 5.108 4.617 4.820 USP54 26.599
25.892 26.683 25.856 7.590 7.316 7.799 7.705 VDR 27.406 26.426
26.847 26.577 8.397 7.850 7.963 8.426 VLDLR 27.166 27.232 28.584
27.543 8.157 8.656 9.700 9.332 XDH Undetermined Undetermined
Undetermined Undetermined #VALUE! #VALUE! #VALUE! #VALUE! ZFP36
24.170 23.980 24.270 24.094 5.161 5.404 5.386 5.943 ZHX3 25.200
24.611 24.418 23.897 6.191 6.035 5.534 5.746 ZNF2B1 24.066 23.541
23.828 23.343 5.057 4.965 4.944 5.192 ACTB 14.843 14.519 14.721
14.509 -4.166 -4.057 -4.163 -3.642 B2M 19.009 18.576 18.884 18.151
0.000 0.000 0.000 0.000 GAPDH 16.513 16.197 16.551 16.157 -2.496
-2.379 -2.333 -1.994 HPRT1 21.698 21.561 21.777 21.657 2.689 2.985
2.893 3.506 RPLP0 15.187 14.935 15.128 14.595 -3.822 -3.641 -3.756
-3.556 HGDC Undetermined Undetermined Undetermined Undetermined
#VALUE! #VALUE! #VALUE! #VALUE! RTC 21.284 21.345 21.449 21.483
2.275 2.769 2.565 3.332 RTC 21.287 21.410 21.464 21.371 2.278 2.834
2.580 3.220 RTC 21.358 21.384 21.483 21.483 2.349 2.808 2.599 3.332
PPC 18.611 18.672 18.684 18.624 -0.398 0.096 -0.200 0.473 PPC
18.638 19.142 18.699 18.587 -0.371 0.566 -0.185 0.436 PPC 18.646
18.711 19.076 18.685 -0.363 0.135 0.192 0.534 Cpd44 Pred Combo Gene
.DELTA..DELTA.CT Fold Change .DELTA..DELTA.CT Fold Change
.DELTA..DELTA.CT Fold Change ADARB1 0.046 0.969 1.867 0.274 -0.771
1.706 AFF1 -0.363 1.286 -0.567 1.481 -0.602 1.518 AK2 0.272 0.828
0.053 0.964 0.485 0.714 AMPD3 0.178 0.884 1.259 0.418 1.309 0.404
ANGPTL4 0.119 0.921 0.529 0.693 0.578 0.670 ANXA4 -0.111 1.080
0.305 0.809 0.309 0.807 AQP1 #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! ARID5B 0.334 0.793 0.129 0.914 0.863 0.550 ASPH
0.286 0.820 0.524 0.695 0.884 0.542 ATF4 0.467 0.723 0.282 0.822
0.685 0.622 BCL6 0.227 0.854 0.397 0.759 1.102 0.466 BMPER 1.488
0.357 1.466 0.362 2.477 0.180 CALCR #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! CEBPA -3.545 11.672 -2.341 5.067 -3.150 8.877 CEBPB
0.447 0.734 0.531 0.692 0.948 0.518 COL4A2 2.238 0.212 6.804 0.009
2.687 0.155 CREB1 0.249 0.841 0.085 0.943 0.658 0.634 CREB3 0.344
0.788 0.061 0.959 0.576 0.671 CREB3L4 0.138 0.909 0.336 0.792 0.826
0.564 CTGF -0.518 1.432 0.142 0.906 0.843 0.557 CYB561 -5.412
42.577 1.917 0.265 -2.601 6.067 DDIT4 0.007 0.995 0.296 0.815
-0.128 1.093 DIRAS2 #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
DUSP1 -0.039 1.027 0.387 0.765 0.516 0.699 EDN1 1.460 0.363 1.155
0.449 0.849 0.555 EHD3 -3.030 8.168 -1.092 2.132 -2.783 6.883
ERRFI1 -1.929 3.808 0.235 0.850 -2.079 4.225 FKBP5 -0.032 1.022
-0.948 1.929 -0.615 1.532 FOSL2 -1.462 2.755 -0.099 1.071 -0.984
1.978 GDPD1 0.471 0.721 0.477 0.718 0.722 0.606 GHRHR 2.393 0.190
-1.464 2.759 0.987 0.505 GLUL 0.971 0.510 -1.434 2.702 0.824 0.565
GOT1 0.132 0.913 0.230 0.853 0.311 0.806 H6PD 0.093 0.938 0.377
0.770 0.594 0.663 HAS2 #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! HNRPLL -0.740 1.670 3.085 0.118 1.914 0.265 IL10 #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! IL1RN #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! IL6 #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! IL6R #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! KLF13 -0.098 1.070 -0.280 1.214 -0.328 1.255 KLF9 0.181
0.882 -0.019 1.013 -0.092 1.066 LOX -0.132 1.096 0.721 0.607 0.151
0.901 MERTK -0.108 1.078 0.020 0.986 -0.665 1.586 MT1E #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! MT2A #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! NFKBIA 0.167 0.891 0.064 0.357
0.592 0.663 NR3C1 0.261 0.835 0.317 0.803 0.829 0.563 PDCD7 0.194
0.874 0.021 0.986 0.599 0.660 PDGFRB #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! PDP1 -0.255 1.193 -0.056 1.040 0.325 0.798
PER1 0.206 0.867 0.323 0.799 1.203 0.434 PER2 -0.374 1.296 -0.358
1.282 -0.308 1.238 PIK3R1 -0.032 1.022 -0.202 1.150 0.291 0.817
PLD1 #VALUE! #VALUE! 0.207 0.866 2.143 0.226 PLEKHF1 -0.507 1.421
-0.347 1.272 -0.290 1.223 POU2F1 0.058 0.961 0.395 0.760 0.147
0.903 POU2F2 0.131 0.913 0.145 0.904 0.319 0.802 RASA3 0.917 0.530
0.776 0.584 2.098 0.234 RGS2 0.504 0.705 0.849 0.555 1.707 0.306
RHOB -1.483 2.795 0.626 0.648 -1.101 2.145 RHOJ #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! SESN1 0.055 0.963 -1.262 2.398
-1.375 2.594 SGK1 0.621 0.650 2.120 0.230 2.350 0.196 SLC10A6 2.385
0.191 1.818 0.284 -0.887 1.849 SLC19A2 -0.308 1.238 -0.020 1.014
0.027 0.981 SLC22A5 0.033 0.977 0.648 0.638 0.301 0.812 SNTA1 0.399
0.758 0.665 0.631 1.274 0.414 SPHK1 -1.381 2.604 -0.581 1.496
-1.173 2.255 SPSB1 -0.025 1.017 -0.074 1.053 0.369 0.774 STAT5A
-0.033 1.023 -0.227 1.170 0.098 0.934 STAT5B 0.258 0.836 0.254
0.839 0.769 0.587 TBL1XR1 0.030 0.979 0.032 0.978 0.594 0.663 TNF
0.954 0.516 0.753 0.593 0.724 0.605 TNFAIP3 -0.307 1.237 #VALUE!
#VALUE! 3.184 0.110 TSC22D3 0.009 0.994 -1.741 3.343 -1.944 3.348
USP2 0.013 0.991 -0.478 1.393 -0.275 1.210 USP54 -0.274 1.209 0.209
0.865 0.115 0.923 VDR -0.547 1.461 -0.434 1.351 0.029 0.980 VLDLR
0.499 0.708 1.543 0.343 1.235 0.425 XDH #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! ZFP36 0.243 0.845 0.225 0.856 0.782 0.582
ZHX3 -0.156 1.114 -0.657 1.577 -0.445 1.361 ZNF2B1 -0.092 1.066
-0.113 1.081 0.135 0.911 ACTB B2M GAPDH HPRT1 RPLP0 HGDC RTC RTC
RTC PPC PPC PPC
TABLE-US-00006 TABLE 4c Ct values and fold changes from the RT2
Glucocorticoid signaling PCR array analysis for WSU cell line. Ct
Values .DELTA.CT (B2M) Gene DMSO Cpd44 Pred Combo DMSO Cpd44 Pred
Combo ADARB1 26.316 25.386 26.108 26.018 6.866 5.701 6.963 5.845
AFF1 28.103 27.925 27.334 26.727 8.653 8.240 8.189 6.554 AK2 20.644
21.365 20.433 22.069 1.194 1.680 1.288 1.896 AMPD3 28.467 27.162
27.943 26.347 9.017 7.477 8.798 6.674 ANGPTL4 31.444 30.487 30.810
31.510 11.994 10.802 11.665 11.337 ANXA4 27.736 24.659 27.406
25.013 8.286 4.974 8.261 4.840 AQP1 Undetermined 33.645 33.595
32.796 #VALUE! 13.960 14.450 12.623 ARID5B 26.244 26.126 26.721
27.140 6.794 6.441 7.576 8.967 ASPH 22.285 22.415 21.939 22.834
2.835 2.730 2.794 2.661 ATF4 19.874 20.470 19.659 20.871 0.424
0.785 9.514 0.698 BCL6 20.954 20.795 20.898 21.133 1.504 1.110
1.753 0.960 BMPER 39.814 Undetermined Undetermined 38.494 20.364
#VALUE! #VALUE! 18.321 CALCR Undetermined Undetermined Undetermined
Undetermined #VALUE! #VALUE! #VALUE! #VALUE! CEBPA 28.438 27.014
27.838 27.647 8.988 7.329 8.693 7.474 CEBPB 25.266 26.770 25.775
27.187 5.816 7.085 6.630 7.014 COL4A2 Undetermined Undetermined
34.328 Undetermined #VALUE! #VALUE! 15.183 #VALUE! CREB1 23.170
23.413 22.732 23.778 3.720 3.728 3.587 3.605 CREB3 25.309 25.459
24.551 25.393 5.859 5.774 5.406 5.220 CREB3L4 25.072 24.392 24.437
24.344 5.622 4.707 5.292 4.171 CTGF Undetermined Undetermined
Undetermined Undetermined #VALUE! #VALUE! #VALUE! #VALUE! CYB561
36.874 31.478 32.971 33.799 17.424 11.793 13.826 13.626 DDIT4
24.229 24.404 22.252 22.739 4.779 4.719 3.107 2.566 DIRAS2
Undetermined Undetermined Undetermined Undetermined #VALUE! #VALUE!
#VALUE! #VALUE! DUSP1 25.679 27.284 25.828 26.552 6.229 7.599 6.683
6.379 EDN1 Undetermined 26.349 30.819 26.407 #VALUE! 6.664 11.674
6.234 EHD3 29.674 24.270 27.724 24.165 10.224 4.585 8.579 3.993
ERRFI1 Undetermined 32.771 Undetermined 32.896 #VALUE! 13.086
#VALUE! 12.723 FKBP5 22.873 23.267 21.321 21.824 3.423 3.582 2.176
1.651 FOSL2 31.109 34.140 33.647 34.690 11.659 14.455 14.502 14.517
GDPD1 28.371 27.494 28.235 27.303 8.921 7.809 9.090 7.130 GHRHR
34.636 39.957 37.789 Undetermined 15.186 20.272 18.644 #VALUE! GLUL
Undetermined 28.395 31.475 30.591 #VALUE! 8.710 12.330 10.418 GOT1
22.884 23.827 22.841 24.411 3.434 4.142 3.696 4.238 H6PD 26.360
25.976 26.197 25.435 6.910 6.291 7.052 5.262 HAS2 Undetermined
Undetermined Undetermined Undetermined #VALUE! #VALUE! #VALUE!
#VALUE! HNRPLL 23.972 24.170 24.750 26.864 4.522 4.485 5.605 6.691
IL10 Undetermined 34.229 34.306 35.010 #VALUE! 14.544 15.161 14.837
IL1RN 32.606 28.388 33.599 29.393 13.156 8.703 14.454 9.220 IL6
Undetermined Undetermined Undetermined Undetermined #VALUE! #VALUE!
#VALUE! #VALUE! IL6R Undetermined 33.814 Undetermined Undetermined
#VALUE! 14.123 #VALUE! #VALUE! KLF13 24.539 23.800 23.792 23.671
5.089 4.115 4.647 3.498 KLF9 30.841 28.881 30.105 28.187 11.391
9.196 10.960 8.014 LOX 34.266 34.399 34.511 34.207 14.816 14.714
15.366 14.034 MERTK Undetermined 31.323 32.524 Undetermined #VALUE!
11.638 13.379 #VALUE! MT1E Undetermined Undetermined Undetermined
Undetermined #VALUE! #VALUE! #VALUE! #VALUE! MT2A 24.717 23.499
24.518 24.350 5.267 3.814 5.373 4.177 NFKBIA 22.371 23.807 22.895
23.454 2.921 4.122 3.750 3.281 NR3C1 23.250 23.121 23.110 23.300
3.800 3.436 3.965 3.127 PDCD7 24.179 24.740 23.874 25.248 4.729
5.055 4.729 5.075 PDGFRB Undetermined Undetermined Undetermined
Undetermined #VALUE! #VALUE! #VALUE! #VALUE! PDP1 25.371 25.226
24.957 25.536 5.921 5.541 5.812 5.363 PER1 25.109 25.820 24.989
26.651 5.659 6.135 5.844 6.478 PER2 24.451 24.837 24.218 25.563
5.001 5.152 5.073 5.390 PIK3R1 23.734 24.332 23.429 24.080 4.284
4.647 4.284 3.907 PLD1 Undetermined Undetermined 35.266
Undetermined #VALUE! #VALUE! 16.121 #VALUE! PLEKHF1 27.205 28.660
26.977 29.585 7.755 8.975 7.832 9.412 POU2F1 24.234 24.671 24.368
24.732 4.784 4.986 5.223 4.559 POU2F2 23.123 22.678 22.565 22.920
3.673 2.993 3.420 2.747 RASA3 23.952 23.208 23.454 23.293 4.502
3.523 4.309 3.120 RGS2 22.902 24.859 23.962 27.302 3.452 5.184
4.817 7.129 RHOB 29.724 27.234 28.803 27.392 10.274 7.549 9.658
7.219 RHOJ Undetermined Undetermined Undetermined Undetermined
#VALUE! #VALUE! #VALUE! #VALUE! SESN1 28.689 25.215 25.915 21.378
9.239 5.530 6.770 1.205 SGK1 25.579 28.624 27.309 30.174 6.129
8.939 8.164 10.001 SLC10A6 36.617 35.684 37.200 39.653 17.167
15.999 18.055 19.480 SLC19A2 26.638 26.125 25.887 26.101 7.188
6.440 6.742 5.928 SLC22A5 28.901 26.640 29.427 27.488 9.451 6.955
10.282 7.315 SNTA1 24.438 14.181 24.329 25.156 4.988 4.495 5.184
4.993 SPHK1 29.643 23.333 29.804 29.702 10.193 9.648 10.659 9.529
SPSB1 29.613 26.952 29.963 27.294 10.163 7.267 10.818 7.121 STAT5A
25.567 25.495 25.699 24.956 5.117 5.810 6.554 4.783 STAT5B 23.414
23.453 23.270 23.985 3.964 3.768 4.125 3.812 TBL1XR1 21.602 22.111
21.479 22.588 2.152 2.426 2.334 2.415 TNF 23.694 25.079 24.151
25.675 4.244 5.394 5.006 5.502 TNFAIP3 24.946 26.903 26.733 28.675
5.496 7.218 7.588 8.502 TSC22D3 25.514 25.390 22.481 21.679 6.064
5.705 3.336 1.506 USP2 22.646 21.903 20.884 20.640 3.196 2.218
1.739 0.467 USP54 26.549 26.807 26.453 27.306 7.099 7.122 7.308
7.133 VDR 30.102 27.293 29.232 27.319 10.652 7.608 10.087 7.146
VLDLR 28.252 32.346 30.596 Undetermined 8.802 12.661 11.451 #VALUE!
XDH Undetermined Undetermined Undetermined Undetermined #VALUE!
#VALUE! #VALUE! #VALUE! ZFP36 24.367 25.541 25.166 26.450 4.917
5.856 6.021 6.277 ZHX3 25.774 24.831 25.570 25.485 6.324 5.146
6.425 5.312 ZNF2B1 24.007 23.913 23.608 24.577 4.557 4.228 4.463
4.404 ACTB 14.801 15.450 14.572 16.143 -4.649 -4.235 -4.573 -4.030
B2M 19.450 19.685 19.145 20.173 0.000 0.000 0.000 0.000 GAPDH
16.528 17.275 16.349 17.416 -2.922 -2.410 -2.796 -2.757 HPRT1
21.509 22.793 21.361 23.732 2.059 3.108 2.216 3.559 RPLP0 15.697
15.681 15.330 15.349 -3.753 -4.004 -3.815 -4.824 HGDC Undetermined
Undetermined Undetermined Undetermined #VALUE! #VALUE! #VALUE!
#VALUE! RTC 21.199 20.891 21.392 21.368 1.749 1.206 2.247 1.195 RTC
21.219 20.949 21.293 21.425 1.769 1.264 2.148 1.252 RTC 21.216
20.945 21.237 21.320 1.766 1.260 2.092 1.147 PPC 18.984 18.798
18.835 18.992 -0.466 -0.887 -0.310 -1.181 PPC 18.832 18.877 18.870
18.841 -0.618 -0.808 -0.275 -1.332 PPC 18.869 19.007 18.873 18.835
-0.581 -0.678 -0.272 -1.338 Cpd44 Pred Combo Gene .DELTA..DELTA.CT
Fold Change .DELTA..DELTA.CT Fold Change .DELTA..DELTA.CT Fold
Change ADARB1 -1.165 2.242 0.097 0.935 -1.021 2.029 AFF1 -0.413
1.331 -0.464 1.379 -2.099 4.284 AK2 0.486 0.714 0.094 0.937 0.702
0.615 AMPD3 -1.540 2.908 -0.219 1.164 -2.343 5.074 ANGPTL4 -1.192
2.285 -0.329 1.256 -0.657 1.577 ANXA4 -3.312 9.931 -0.025 1.017
-3.446 10.898 AQP1 #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
ARID5B -0.353 1.277 0.782 0.582 0.173 0.887 ASPH -0.105 1.075
-0.041 1.029 -0.174 1.128 ATF4 0.361 0.779 0.090 0.940 0.274 0.827
BCL6 -0.394 1.314 0.249 0.841 -0.544 1.458 BMPER #VALUE! #VALUE!
#VALUE! #VALUE! -2.043 4.121 CALCR #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! CEBPA -1.659 3.158 -0.295 1.227 -1.514 2.856 CEBPB
1.269 0.415 0.814 0.569 1.198 0.436 COL4A2 #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! CREB1 0.008 0.994 -0.133 1.097 -0.115 1.083
CREB3 -0.085 1.061 -0.453 1.369 -0.639 1.557 CREB3L4 -0.915 1.886
-0.330 1.257 -1.451 2.734 CTGF #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! CYB561 -5.631 49.556 -3.598 12.109 -3.798 13.910
DDIT4 -0.060 1.042 -1.672 3.187 -2.213 4.636 DIRAS2 #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! DUSP1 1.370 0.387 0.454 0.730 0.150
0.901 EDN1 #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! EHD3
-5.639 49.832 -1.645 3.127 -6.231 75.113 ERRFI1 #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! FKBP5 0.159 0.896 -1.247 2.373
-1.772 3.415 FOSL2 2.796 0.144 2.843 0.139 2.858 0.138 GDPD1 -1.112
2.161 0.169 0.889 -1.791 3.461 GHRHR 5.086 0.029 3.458 0.091
#VALUE! #VALUE! GLUL #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! GOT1 0.708 0.612 0.262 0.834 0.804 0.573 H6PD -0.619 1.536
0.142 0.906 -1.648 3.134 HAS2 #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! HNRPLL -0.037 1.026 1.083 0.472 2.169 0.222 IL10
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! IL1RN -4.453 21.902
1.298 0.407 -3.936 15.306 IL6 #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! IL6R #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! KLF13 -0.974 1.964 -0.442 1.358 -1.591 3.013 KLF9 -2.195
4.579 -0.431 1.348 -3.377 10.389 LOX -0.102 1.073 0.550 0.683
-0.782 1.720 MERTK #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
MT1E #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! MT2A -1.453
2.738 0.106 0.929 -1.090 2.129 NFKBIA 1.201 0.435 0.829 0.563 0.360
0.779 NR3C1 -0.364 1.287 0.165 0.892 -0.673 1.594 PDCD7 0.326 0.798
0.000 1.000 0.346 0.787 PDGFRB #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! PDP1 -0.380 1.301 -0.109 1.078 -0.558 1.472 PER1
0.476 0.719 0.185 0.880 0.819 0.567 PER2 0.151 0.901 0.072 0.951
0.389 0.764 PIK3R1 0.363 0.778 0.000 1.000 -0.377 1.299 PLD1
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! PLEKHF1 1.220 0.429
0.077 0.948 1.657 0.317 POU2F1 0.202 0.869 0.439 0.738 -0.225 1.169
POU2F2 -0.680 1.502 -0.253 1.192 -0.926 1.900 RASA3 -0.979 1.971
-0.193 1.143 -1.382 2.606 RGS2 1.732 0.301 1.365 0.388 3.677 0.078
RHOB -2.725 6.612 -0.616 1.533 -3.055 8.311 RHOJ #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! SESN1 -3.709 13.077 -2.469 5.537
-8.034 262.105 SGK1 2.810 0.143 2.035 0.244 3.372 0.068 SLC10A6
-1.168 2.247 0.888 0.540 2.313 0.201 SLC19A2 -0.748 1.679 -0.446
1.362 -1.260 2.395 SLC22A5 -2.496 5.641 0.831 0.562 -2.136 4.395
SNTA1 -0.492 1.406 0.196 0.873 0.005 0.997 SPHK1 -0.545 1.459 0.466
0.724 -0.664 1.584 SPSB1 -2.896 7.444 0.655 0.635 -3.042 8.236
STAT5A -0.307 1.237 0.437 0.739 -1.334 2.521 STAT5B -0.196 1.146
0.161 0.894 -0.152 1.111 TBL1XR1 0.274 0.827 0.182 0.881 0.263
0.833 TNF 1.150 0.451 0.762 0.590 1.258 0.418 TNFAIP3 1.722 0.303
2.092 0.235 3.006 0.124 TSC22D3 -0.359 1.283 -2.728 6.625 -4.558
23.556 USP2 -0.978 1.970 -1.457 2.745 -2.729 6.630 USP54 0.023
0.984 0.209 0.865 0.034 0.977 VDR -3.044 8.248 -0.565 1.479 -3.506
11.361 VLDLR 3.859 0.069 2.649 0.159 #VALUE! #VALUE! XDH #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! ZFP36 0.939 0.522 1.104
0.465 1.360 0.390 ZHX3 -1.178 2.263 0.101 0.932 -1.012 2.017 ZNF2B1
-0.329 1.256 -0.094 1.067 -0.153 1.112 ACTB B2M GAPDH HPRT1 RPLP0
HGDC RTC RTC RTC PPC PPC PPC
TABLE-US-00007 TABLE 4d Ct values and fold changes from the RT2
Glucocorticoid signaling PCR array analysis for SUDHL10 cell line.
Ct Values .DELTA.CT (B2M) Gene DMSO Cpd44 Pred Combo DMSO Cpd44
Pred Combo ADARB1 30.421 31.215 31.883 32.846 11.995 12.068 14.229
14.012 AFF1 28.478 29.849 27.600 27.812 10.052 10.702 9.946 8.978
AK2 20.354 20.974 19.237 20.672 1.928 1.827 1.583 1.838 AMPD3
27.489 27.654 26.390 27.563 9.063 8.507 8.736 8.729 ANGPTL4 30.771
32.107 29.894 31.412 12.345 12.960 12.240 12.578 ANXA4 26.715
24.961 25.942 24.755 8.289 5.814 8.288 5.921 AQP1 Undetermined
Undetermined Undetermined Undetermined #VALUE! #VALUE! #VALUE!
#VALUE! ARID5B 26.837 28.208 27.668 27.409 8.411 9.061 10.014 8.575
ASPH 22.820 23.837 22.217 24.322 4.394 4.690 4.563 5.488 ATF4
18.149 20.607 18.947 20.429 -0.277 1.460 1.293 1.595 BCL6 21.278
22.639 21.573 23.181 2.852 3.492 3.319 4.347 BMPER Undetermined
Undetermined Undetermined Undetermined #VALUE! #VALUE! #VALUE!
#VALUE! CALCR Undetermined Undetermined Undetermined Undetermined
#VALUE! #VALUE! #VALUE! #VALUE! CEBPA 29.205 28.900 29.217 28.372
10.779 9.753 11.563 9.538 CEBPB 22.884 26.624 24.539 25.652 4.458
7.477 6.885 6.818 COL4A2 Undetermined Undetermined 35.603 34.161
#VALUE! #VALUE! 17.949 15.327 CREB1 23.139 23.309 22.395 23.638
4.713 4.662 4.741 4.804 CREB3 25.310 26.452 24.440 25.398 6.884
7.305 6.786 6.564 CREB3L4 24.612 26.139 24.960 26.248 6.186 6.992
7.306 7.414 CTGF Undetermined Undetermined Undetermined
Undetermined #VALUE! #VALUE! #VALUE! #VALUE! CYB561 38.682 37.004
38.074 38.165 20.256 17.857 20.420 19.331 DDIT4 23.944 26.109
21.960 21.759 5.518 6.962 4.306 2.925 DIRAS2 Undetermined
Undetermined Undetermined Undetermined #VALUE! #VALUE! #VALUE!
#VALUE! DUSP1 29.480 31.300 27.989 28.385 11.054 12.153 10.335
9.551 EDN1 Undetermined Undetermined Undetermined Undetermined
#VALUE! #VALUE! #VALUE! #VALUE! EHD3 26.932 26.768 25.888 25.803
8.506 7.621 8.234 6.969 ERRFI1 Undetermined Undetermined
Undetermined Undetermined #VALUE! #VALUE! #VALUE! #VALUE! FKBP5
22.120 22.883 19.749 20.675 3.694 3.736 2.095 1.841 FOSL2 32.273
32.931 29.990 30.849 13.847 13.784 12.336 12.015 GDPD1 31.627
30.943 29.917 30.011 13.201 11.796 12.263 11.177 GHRHR Undetermined
Undetermined 36.757 Undetermined #VALUE! #VALUE! 19.103 #VALUE!
GLUL 33.940 Undetermined Undetermined Undetermined 15.514 #VALUE!
#VALUE! #VALUE! GOT1 23.510 25.306 23.519 24.803 5.084 6.159 5.865
5.969 H6PD 26.184 28.126 26.256 26.915 7.758 8.979 8.602 8.081 HAS2
Undetermined Undetermined Undetermined Undetermined #VALUE! #VALUE!
#VALUE! #VALUE! HNRPLL 22.972 23.764 22.692 24.016 4.546 4.617
5.038 5.182 IL10 Undetermined 32.505 Undetermined 32.875 #VALUE!
13.358 #VALUE! 14.041 IL1RN Undetermined 32.182 Undetermined
Undetermined #VALUE! 13.035 #VALUE! #VALUE! IL6 Undetermined
Undetermined Undetermined Undetermined #VALUE! #VALUE! #VALUE!
#VALUE! IL6R Undetermined 33.807 33.801 Undetermined #VALUE! 14.660
16.147 #VALUE! KLF13 25.451 24.536 22.488 22.832 7.025 5.389 4.834
3.998 KLF9 32.931 32.525 30.255 29.691 14.505 13.378 12.601 10.857
LOX 33.500 35.385 32.223 32.465 15.074 16.238 14.569 13.631 MERTK
Undetermined 34.552 Undetermined 33.161 #VALUE! 15.505 #VALUE!
14.327 MT1E Undetermined Undetermined Undetermined 34.503 #VALUE!
#VALUE! #VALUE! 15.659 MT2A 34.844 37.225 35.909 35.849 16.418
13.078 13.255 17.015 NFKBIA 22.331 23.654 21.628 22.744 3.905 4.507
3.974 3.910 NR3C1 22.516 23.754 22.000 22.835 4.090 4.617 4.346
4.001 PDCD7 23.600 25.123 23.256 24.731 5.174 5.976 5.602 5.897
PDGFRB Undetermined Undetermined Undetermined Undetermined #VALUE!
#VALUE! #VALUE! #VALUE! PDP1 25.438 26.175 25.178 26.259 7.012
7.028 7.524 7.425 PER1 26.209 27.710 24.762 26.686 7.783 8.563
7.108 7.852 PER2 23.618 24.780 22.642 24.465 5.192 5.633 4.988
5.631 PIK3R1 23.509 24.661 22.697 23.585 5.083 5.514 5.043 4.751
PLD1 Undetermined Undetermined Undetermined Undetermined #VALUE!
#VALUE! #VALUE! #VALUE! PLEKHF1 27.789 28.979 26.691 27.331 9.363
9.832 9.037 8.497 POU2F1 25.115 25.842 24.283 24.827 6.689 6.695
6.629 5.993 POU2F2 23.953 25.098 22.977 24.098 5.527 5.951 5.323
5.264 RASA3 23.171 24.277 22.449 23.649 4.745 5.130 4.795 4.815
RGS2 24.794 25.587 25.390 26.161 6.368 6.440 7.736 7.327 RHOB
28.583 27.829 27.968 26.383 10.157 8.682 10.314 7.549 RHOJ
Undetermined 36.530 Undetermined Undetermined #VALUE! 17.383
#VALUE! #VALUE! SESN1 28.405 27.480 24.220 22.646 9.979 8.333 6.566
3.812 SGK1 22.694 25.358 22.897 24.642 4.268 6.211 5.243 5.808
SLC10A6 36.987 37.060 34.670 36.258 18.561 17.913 17.016 17.424
SLC19A2 31.019 30.597 31.940 31.354 12.593 11.450 14.286 12.520
SLC22A5 31.275 30.263 32.426 29.324 12.849 11.116 14.772 10.490
SNTA1 25.751 27.003 24.913 26.374 7.325 7.856 7.259 7.540 SPHK1
26.852 27.804 25.801 27.082 8.426 8.657 8.147 8.248 SPSB1 25.856
26.133 24.455 24.642 7.430 6.986 6.801 5.808 STAT5A 24.170 25.275
23.779 24.550 5.744 6.128 6.125 5.716 STAT5B 23.533 24.281 23.480
24.231 5.107 5.134 5.826 5.397 TBL1XR1 20.891 21.846 20.224 21.815
2.465 2.699 2.570 2.981 TNF 23.208 23.725 22.657 24.669 4.782 4.578
5.003 5.835 TNFAIP3 26.832 27.677 27.010 26.749 8.406 8.530 9.356
7.915 TSC22D3 25.441 28.871 23.160 22.809 7.015 9.724 5.506 3.975
USP2 22.643 23.434 21.579 22.360 4.217 4.287 3.925 3.526 USP54
27.132 27.789 26.401 27.379 8.706 8.642 8.747 8.545 VDR 29.507
29.514 28.490 28.525 11.081 10.367 10.836 9.691 VLDLR 27.937 32.904
31.762 32.093 9.511 13.757 14.108 13.259 XDH Undetermined
Undetermined Undetermined Undetermined #VALUE! #VALUE! #VALUE!
#VALUE! ZFP36 25.707 26.643 24.515 25.887 7.281 7.496 6.861 7.053
ZHX3 26.753 26.305 26.008 26.393 8.327 7.158 8.354 7.559 ZNF2B1
23.573 23.857 22.336 23.665 5.147 4.710 4.682 4.831 ACTB 14.330
14.828 13.138 14.548 -4.096 -4.319 -4.516 -4.286 B2M 18.426 19.147
17.654 18.834 0.000 0.000 0.000 0.000 GAPDH 16.544 17.793 15.669
16.935 -1.882 -1.354 -1.985 -1.899 HPRT1 19.452 20.615 18.679
20.706 1.026 1.468 1.025 1.872 RPLP0 15.746 16.821 15.169 15.785
-2.680 -2.326 -2.485 -3.049 HGDC Undetermined Undetermined
Undetermined Undetermined #VALUE! #VALUE! #VALUE! #VALUE! RTC
22.619 22.346 22.496 23.181 4.193 3.199 4.842 4.347 RTC 22.626
22.362 22.621 23.201 4.200 3.215 4.967 4.367 RTC 22.662 22.313
22.484 23.114 4.236 3.166 4.830 4.280 PPC 18.253 18.442 17.960
18.476 -0.173 -0.705 0.305 -0.358 PPC 18.527 18.474 18.434 18.446
0.101 -0.673 0.780 -0.388 PPC 18.410 18.623 18.515 18.482 -0.016
-0.524 0.861 -0.352 Cpd44 Pred Combo Gene .DELTA..DELTA.CT Fold
Change .DELTA..DELTA.CT Fold Change .DELTA..DELTA.CT Fold Change
ADARB1 0.073 0.951 2.234 0.213 2.017 0.247 AFF1 0.650 0.637 -0.106
1.076 -1.074 2.105 AK2 -0.101 1.073 -0.345 1.270 -0.090 1.064 AMPD3
-0.556 1.470 -0.327 1.254 -0.334 1.261 ANGPTL4 0.615 0.653 -0.105
1.075 0.233 0.851 ANXA4 -2.475 5.560 -0.001 1.001 -2.368 5.162 AQP1
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! ARID5B 0.650 0.637
1.603 0.329 0.164 0.893 ASPH 0.296 0.815 0.169 0.889 1.094 0.468
ATF4 1.737 0.300 1.570 0.337 1.872 0.273 BCL6 0.640 0.542 1.067
0.477 1.495 0.355 BMPER #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! CALCR #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! CEBPA
-1.026 2.036 0.784 0.581 -1.241 2.364 CEBPB 3.019 0.123 2.427 0.186
2.360 0.195 COL4A2 #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
CREB1 -0.051 1.036 0.028 0.981 0.091 0.939 CREB3 0.421 0.747 -0.098
1.070 -0.320 1.248 CREB3L4 0.806 0.572 1.120 0.460 1.228 0.427 CTGF
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! CYB561 -2.399 5.274
0.164 0.893 -0.925 1.899 DDIT4 1.444 0.368 -1.212 2.317 -2.593
6.034 DIRAS2 #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! DUSP1
1.099 0.467 -0.719 1.646 -1.503 2.834 EDN1 #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! EHD3 -0.885 1.847 -0.272 1.207 -1.537 2.902
ERRFI1 #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! FKBP5 0.042
0.971 -1.599 3.029 -1.853 3.613 FOSL2 -0.063 1.045 -1.511 2.850
-1.832 3.560 GDPD1 -1.405 2.648 -0.938 1.916 -2.024 4.067 GHRHR
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! GLUL #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! GOT1 1.075 0.475 0.781
0.582 0.885 0.541 H6PD 1.221 0.429 0.844 0.557 0.323 0.799 HAS2
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! HNRPLL 0.071 0.952
0.492 0.711 0.635 0.643 IL10 #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! IL1RN #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! IL6 #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! IL6R
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! KLF13 -1.636 3.108
-2.191 4.566 -3.027 8.151 KLF9 -1.127 2.184 -1.904 3.742 -3.648
12.536 LOX 1.164 0.446 -0.505 1.419 -1.443 2.719 MERTK #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! MT1E #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! MT2A 1.660 0.316 1.837 0.280 0.597
0.661 NFKBIA 0.602 0.659 0.069 0.953 0.005 0.997 NR3C1 0.527 0.694
0.256 0.837 -0.089 1.064 PDCD7 0.802 0.574 0.428 0.743 0.723 0.606
PDGFRB #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! PDP1 0.016
0.989 0.512 0.701 0.413 0.751 PER1 0.780 0.582 -0.575 1.597 0.069
0.953 PER2 0.441 0.737 -0.204 1.152 0.439 0.738 PIK3R1 0.431 0.742
-0.040 1.028 -0.332 1.259 PLD1 #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! PLEKHF1 0.469 0.722 -0.326 1.254 -0.866 1.823
POU2F1 0.006 0.996 -0.060 1.042 -0.696 1.620 POU2F2 0.424 0.745
-0.204 1.152 -0.263 1.200 RASA3 0.385 0.766 0.050 0.966 0.070 0.953
RGS2 0.072 0.951 1.368 0.387 0.959 0.514 RHOB -1.475 2.780 0.157
0.897 -2.608 6.097 RHOJ #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! SESN1 -1.646 3.130 -3.413 10.652 -6.167 71.854 SGK1 1.943
0.260 0.975 0.509 1.540 0.344 SLC10A6 -0.648 1.567 -1.545 2.918
-1.137 2.199 SLC19A2 -1.143 2.208 1.633 0.309 -0.073 1.052 SLC22A5
-1.733 3.324 1.923 0.264 -2.359 5.130 SNTA1 0.531 0.692 -0.066
1.047 0.215 0.862 SPHK1 0.231 0.852 -0.279 1.213 -0.178 1.131 SPSB1
-0.444 1.360 -0.629 1.546 -1.622 3.078 STAT5A 0.384 0.766 0.381
0.768 -0.028 1.020 STAT5B 0.027 0.981 0.719 0.608 0.290 0.818
TBL1XR1 0.234 0.850 0.105 0.930 0.516 0.699 TNF -0.204 1.152 0.221
0.858 1.053 0.482 TNFAIP3 0.124 0.918 0.950 0.518 -0.491 1.405
TSC22D3 2.709 0.153 -1.509 2.846 -3.040 8.225 USP2 0.070 0.953
-0.292 1.224 -0.691 1.614 USP54 -0.064 1.045 0.041 0.972 -0.161
1.118 VDR -0.714 1.640 -0.245 1.185 -1.390 2.621 VLDLR 4.246 0.053
4.597 0.041 3.748 0.074 XDH #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! ZFP36 0.215 0.862 -0.420 1.338 -0.228 1.171 ZHX3 -1.169
2.249 0.027 0.981 -0.768 1.703 ZNF2B1 -0.437 1.354 -0.465 1.380
-0.316 1.245 ACTB B2M GAPDH HPRT1 RPLP0 HGDC RTC RTC RTC PPC PPC
PPC
TABLE-US-00008 TABLE 4e Ct values and fold changes from the RT2
Glucocorticoid signaling PCR array analysis for RI cell line. Ct
Values .DELTA.CT (B2M) Gene DMSO Cpd44 Pred Combo DMSO Cpd44 Pred
Combo ADARB1 27.745 26.650 28.557 28.623 8.964 7.292 9.377 9.377
AFF1 28.249 26.820 27.258 26.977 9.468 7.462 8.078 7.731 AK2 19.425
20.270 20.510 21.466 0.644 0.912 1.330 2.220 AMPD3 27.499 27.191
27.354 27.238 8.718 7.833 8.174 7.992 ANGPTL4 30.178 29.820 32.245
29.596 11.397 10.462 13.065 10.350 ANXA4 24.380 24.395 24.910
24.771 5.599 5.037 5.730 5.525 AQP1 Undetermined Undetermined
Undetermined 33.328 #VALUE! #VALUE! #VALUE! 14.082 ARID5B 27.976
27.333 29.208 28.495 9.195 7.975 10.028 9.249 ASPH 22.413 23.455
23.583 24.410 3.632 4.108 4.403 5.164 ATF4 17.689 18.289 19.452
20.540 -1.092 -1.089 0.272 1.294 BCL6 19.449 20.289 20.785 20.772
0.668 0.931 1.605 1.526 BMPER Undetermined Undetermined
Undetermined Undetermined #VALUE! #VALUE! #VALUE! #VALUE! CALCR
Undetermined Undetermined Undetermined Undetermined #VALUE! #VALUE!
#VALUE! #VALUE! CEBPA Undetermined 36.511 Undetermined Undetermined
#VALUE! 17.153 #VALUE! #VALUE! CEBPB 23.192 23.765 26.229 27.211
4.411 4.407 7.049 7.965 COL4A2 31.978 31.782 Undetermined 35.212
13.197 12.424 #VALUE! 15.966 CREB1 22.435 23.217 23.317 23.470
3.654 3.859 4.137 4.224 CREB3 23.790 24.178 24.991 24.735 5.009
4.820 5.771 5.489 CREB3L4 23.683 23.500 24.211 23.370 4.902 4.142
5.031 4.624 CTGF Undetermined Undetermined Undetermined
Undetermined #VALUE! #VALUE! #VALUE! #VALUE! CYB561 39.352 38.452
38.088 37.618 20.571 19.094 18.908 18.372 DDIT4 21.641 22.679
23.471 22.583 2.860 3.321 4.291 3.337 DIRAS2 Undetermined
Undetermined Undetermined Undetermined #VALUE! #VALUE! #VALUE!
#VALUE! DUSP1 25.166 25.422 25.412 24.981 6.385 6.064 6.232 5.735
EDN1 32.446 31.815 39.440 30.700 13.665 12.457 20.260 11.454 EHD3
24.957 24.572 25.411 23.975 6.175 5.214 6.231 4.729 ERRFI1
Undetermined 31.705 32.655 31.791 #VALUE! 12.347 13.475 12.545
FKBP5 20.792 21.757 20.858 20.881 2.011 2.339 1.678 1.635 FOSL2
31.458 30.761 34.157 36.459 12.677 11.403 14.977 17.213 GDPD1
27.589 27.394 28.699 28.110 8.808 8.036 9.519 8.864 GHRHR
Undetermined 37.546 33.555 29.797 #VALUE! 18.188 14.375 10.551 GLUL
30.775 28.738 32.131 32.951 11.994 9.380 13.001 13.715 GOT1 21.489
22.584 23.355 24.551 2.708 3.226 4.175 5.305 H6PD 25.108 25.012
26.442 24.742 6.327 5.654 7.262 5.496 HAS2 Undetermined
Undetermined Undetermined Undetermined #VALUE! #VALUE! #VALUE!
#VALUE! HNRPLL 21.958 22.844 23.146 24.289 3.177 3.486 3.966 5.043
IL10 32.353 31.498 32.669 Undetermined 13.572 12.140 13.489 #VALUE!
IL1RN 29.709 28.405 29.300 29.204 10.928 9.047 10.120 9.958 IL6
Undetermined Undetermined Undetermined Undetermined #VALUE! #VALUE!
#VALUE! #VALUE! IL6R 29.184 29.245 30.468 31.321 10.403 9.888
11.288 12.075 KLF13 22.843 22.854 23.590 23.322 4.062 3.496 4.410
4.076 KLF9 34.469 Undetermined Undetermined Undetermined 15.688
#VALUE! #VALUE! #VALUE! LOK 33.918 33.915 34.185 33.948 15.137
14.557 15.005 14.702 MERTK 34.533 30.691 Undetermined 31.272 15.752
11.333 #VALUE! 12.026 MT1E Undetermined 34.215 Undetermined
Undetermined #VALUE! 14.857 #VALUE! #VALUE! MT2A 23.125 24.279
25.180 25.570 4.344 4.921 6.000 6.324 NFKBIA 22.279 23.710 23.531
23.442 3.498 4.352 4.351 4.196 NR3C1 21.972 22.616 23.323 23.237
3.191 3.258 4.143 3.991 PDCD7 23.823 24.406 24.616 24.925 5.042
5.048 5.436 5.679 PDGFRB Undetermined Undetermined Undetermined
Undetermined #VALUE! #VALUE! #VALUE! #VALUE! PDP1 25.799 25.387
26.940 25.667 7.018 6.029 7.760 6.421 PER1 24.531 25.492 27.160
26.707 5.750 6.134 7.980 7.461 PER2 24.162 24.378 24.811 24.547
5.381 5.020 5.631 5.301 PIK3R1 22.958 23.908 24.331 24.602 4.177
4.550 5.151 5.356 PLD1 Undetermined Undetermined Undetermined
Undetermined #VALUE! #VALUE! #VALUE! #VALUE! PLEKHF1 23.893 30.520
31.367 34.212 11.112 11.162 12.187 14.966 POU2F1 23.693 24.243
25.222 25.607 4.912 4.885 6.042 6.361 POU2F2 21.776 21.769 22.972
22.359 2.995 2.411 3.792 3.113 RASA3 25.711 26.279 27.655 26.571
6.930 6.921 8.473 7.325 RGS2 25.306 25.721 26.477 28.212 6.525
6.363 7.297 8.966 RHOB Undetermined Undetermined Undetermined
38.682 #VALUE! #VALUE! #VALUE! 13.436 RHOJ Undetermined
Undetermined Undetermined Undetermined #VALUE! #VALUE! #VALUE!
#VALUE! SESN1 25.545 24.425 21.882 20.973 6.764 5.067 2.702 1.727
SGK1 24.884 26.262 26.131 26.411 6.103 5.904 6.951 7.165 SLC10A6
35.730 Undetermined 34.404 35.950 16.949 #VALUE! 15.224 15.704
SLC19A2 25.756 25.536 26.202 25.342 6.975 6.178 7.022 6.096 SLC22A5
32.280 Undetermined Undetermined 38.888 13.499 #VALUE! #VALUE!
19.642 SNTA1 24.937 25.554 26.480 26.133 6.156 6.196 7.300 6.887
SPHK1 31.323 31.119 34.340 Undetermined 12.542 11.781 15.160
#VALUE! SPSB1 26.573 26.285 28.977 28.200 7.792 6.927 9.797 8.954
STAT5A 22.191 22.769 23.379 22.666 3.410 3.411 4.199 3.420 STAT5B
22.200 22.683 23.111 23.737 3.419 3.325 3.931 4.431 TBL1XR1 20.394
21.663 21.587 21.692 1.613 2.305 2.407 2.446 TNF 24.972 24.712
24.773 22.812 6.191 5.554 5.593 3.566 TNFAIP3 25.433 26.896 27.393
23.474 6.652 7.533 8.213 9.228 TSC22D3 22.534 23.300 21.502 21.179
3.753 3.942 2.322 1.933 USP2 20.982 21.420 20.720 20.616 2.201
2.062 1.540 1.370 USP54 26.748 26.364 27.638 26.992 7.967 7.006
8.458 7.746 VDR 29.317 27.736 31.463 29.503 11.036 8.378 12.283
10.262 VLDLR 35.442 Undetermined 33.973 Undetermined 16.661 #VALUE!
14.799 #VALUE! XDH Undetermined Undetermined Undetermined
Undetermined #VALUE! #VALUE! #VALUE! #VALUE! ZFP36 23.833 24.374
25.160 24.997 5.052 5.016 5.980 5.751 ZHX3 24.547 29.785 25.114
24.601 5.765 10.427 5.934 5.355 ZNF281 23.044 23.667 23.814 23.669
4.263 4.309 4.634 4.423 ACTB 14.794 15.664 15.466 15.976 -3.937
-3.694 -3.714 -3.270 B2M 18.781 19.358 19.180 19.246 0.000 0.000
0.000 0.000 GAPDH 15.388 15.720 16.234 17.444 -3.393 -3.538 -2.946
-1.802 HPRT1 21.297 22.013 21.526 22.777 2.516 2.655 2.446 3.531
RPLP0 15.092 14.837 15.334 15.962 -3.689 -4.521 -3.186 -3.284 HGDC
Undetermined Undetermined Undetermined Undetermined #VALUE! #VALUE!
#VALUE! #VALUE! RTC 20.832 21.659 20.818 20.383 2.051 2.301 1.638
1.137 RTC 20.752 21.713 20.631 20.330 1.971 2.555 1.501 1.134 RTC
20.792 21.629 20.780 20.481 2.011 2.271 1.600 1.235 PPC 18.493
13.197 13.424 18.380 -0.288 -1.161 -0.756 -0.866 PPC 18.567 18.303
18.491 18.255 -0.214 -1.055 -0.689 -0.991 PPC 18.444 13.435 13.381
13.325 -0.337 -0.923 -0.799 -0.921 Cpd44 Pred Combo Gene
.DELTA..DELTA.CT Fold Change .DELTA..DELTA.CT Fold Change
.DELTA..DELTA.CT Fold Change ADARB1 -1.672 3.187 0.413 0.751 0.413
0.751 AFF1 -2.006 4.017 -1.390 2.621 -1.737 3.333 AK2 0.268 0.830
0.686 0.622 1.575 0.335 AMPD3 -0.885 1.847 -0.544 1.458 -0.726
1.654 ANGPTL4 -0.935 1.912 1.668 0.315 -1.047 2.066 ANXA4 -0.562
1.476 0.131 0.913 -0.074 1.053 AQP1 #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! ARID5B -1.220 2.329 0.833 0.561 0.054 0.953 ASPH
0.476 0.719 0.771 0.586 1.532 0.346 ATF4 0.003 0.998 1.364 0.389
2.386 0.191 BCL6 0.263 0.833 0.937 0.522 0.858 0.552 BMPER #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! CALCR #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! CEBPA #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! CEBPB -0.004 1.003 2.638 0.161 3.554 0.085
COL4A2 -0.773 1.709 #VALUE! #VALUE! 2.769 0.147 CREB1 0.205 0.868
0.483 0.715 0.570 0.674 CREB3 -0.189 1.140 0.762 0.590 0.480 0.717
CREB3L4 -0.760 1.693 0.129 0.914 -0.278 1.213 CTGF #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! CYB561 -1.477 2.784 -1.663 3.167
-2.199 4.592 DDIT4 0.461 0.726 1.431 0.371 0.477 0.718 DIRAS2
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! DUSP1 -0.321 1.249
-0.153 1.112 -0.650 1.569 EDN1 -1.208 2.310 6.595 0.010 -2.211
4.630 EHD3 -0.962 1.948 0.055 0.963 -1.447 2.726 ERRFI1 #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! FKBP5 0.388 0.764 -0.333
1.260 -0.376 1.298 FOSL2 -1.274 2.418 2.300 0.203 4.535 0.043 GDPD1
-0.772 1.708 0.711 0.611 0.056 0.962 GHRHR #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! GLUL -2.614 6.122 1.007 0.498 1.721 0.303
GOT1 0.518 0.698 1.467 0.362 2.597 0.165 H6PD -0.673 1.594 0.935
0.523 -0.831 1.779 HAS2 #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! HNRPLL 0.309 0.807 0.789 0.579 1.866 0.274 IL10 -1.432
2.698 -0.083 1.059 #VALUE! #VALUE! IL1RN -1.881 3.683 -0.808 1.751
-0.970 1.959 IL6 #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
IL6R -0.515 1.429 0.885 0.541 1.672 0.314 KLF13 -0.566 1.480 0.348
0.786 0.014 0.990 KLF9 #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! LOK -0.580 1.495 -0.132 1.095 -0.435 1.352 MERTK -4.419
21.392 #VALUE! #VALUE! -3.726 13.232 MT1E #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! MT2A 0.577 0.670 1.656 0.317 1.980 0.253
NFKBIA 0.354 0.553 0.353 0.554 0.698 0.616 NR3C1 0.067 0.955 0.952
0.517 0.800 0.574 PDCD7 0.005 0.996 0.394 0.751 0.537 0.543 PDGFRB
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! PDP1 -0.989 1.985
0.742 0.593 -0.597 1.513 PER1 0.384 0.766 2.230 0.213 1.711 0.305
PER2 -0.361 1.284 0.250 0.841 -0.080 1.057 PIK3R1 0.373 0.772 0.974
0.509 1.179 0.442 PLD1 #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! PLEKHF1 0.050 0.966 1.075 0.475 3.854 0.069 POU2F1 -0.027
1.019 1.130 0.457 1.449 0.366 POU2F2 -0.584 1.499 0.797 0.576 0.118
0.921 RASA3 -0.009 1.006 1.543 0.343 0.395 0.760 RGS2 -0.162 1.119
0.772 0.586 2.441 0.184 RHOB #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! RHOJ #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! SESN1 -1.697 3.242 -4.062 16.703 -5.037 32.831 SGK1 0.801
0.574 0.848 0.556 1.062 0.479 SLC10A6 #VALUE! #VALUE! -1.725 3.306
-0.245 1.185 SLC19A2 -0.797 1.737 0.047 0.968 -0.879 1.839 SLC22A5
#VALUE! #VALUE! #VALUE! #VALUE! 6.143 0.014 SNTA1 0.040 0.973 1.144
0.453 0.731 0.502 SPHK1 -0.781 1.718 2.618 0.163 #VALUE! #VALUE!
SPSB1 -0.865 1.821 2.005 0.249 1.162 0.447 STAT5A 0.001 0.999 0.789
0.579 0.010 0.993 STAT5B -0.054 1.067 0.512 0.701 1.072 0.476
TBL1XR1 0.692 0.619 0.794 0.577 0.833 0.561 TNF -0.837 1.786 -0.598
1.514 -2.625 6.169 TNFAIP3 0.886 0.541 1.561 0.339 2.576 0.168
TSC22D3 0.189 0.877 -1.431 2.696 -1.820 3.531 USP2 -0.139 1.101
-0.661 1.581 -0.831 1.779 USP54 -0.961 1.947 0.491 0.712 -0.221
1.166 VDR -2.658 6.312 1.247 0.421 -0.774 1.710 VLDLR #VALUE!
#VALUE! -1.862 3.635 #VALUE! #VALUE! XDH #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! ZFP36 -0.036 1.025 0.928 0.526 0.699 0.616
ZHX3 4.661 0.040 0.168 0.890 -0.411 1.330 ZNF281 0.046 0.969 0.371
0.773 0.160 0.895 ACTB B2M GAPDH HPRT1 RPLP0 HGDC RTC RTC RTC PPC
PPC PPC
TABLE-US-00009 TABLE 4f Ct values and fold changes from the RT2
Glucocorticoid signaling PCR array analysis for SUDHL4 cell line.
Ct Values .DELTA.CT (B2M) Gene DMSO Cpd44 Pred Combo DMSO Cpd44
Pred Combo ADARB1 27.696 28.562 27.634 28.373 10.107 8.878 9.591
8.992 AFF1 26.492 25.936 25.874 25.660 7.394 7.118 6.965 7.788 AK2
19.861 20.311 20.602 20.682 2.416 1.846 1.340 1.157 AMPD3 25.234
25.553 24.780 25.739 7.473 6.024 6.582 6.530 ANGPTL4 29.764 29.825
29 326 30.167 11.901 10.570 10.854 11.060 ANXA4 26.847 28.717
26.973 28.902 10.636 8.217 9.746 8.143 AQP1 Undetermined
Undetermined 32.982 32.161 13.895 14.226 #VALUE! #VALUE! ARID5B
25.120 25.129 24.504 24.555 6.289 5.748 6.158 6.416 ASPH 22.618
23.348 22.741 23.094 4.828 3.985 4.377 3.914 ATF4 19.323 18.778
18.988 18.352 0.086 0.232 -0.193 0.619 BCL6 20.521 21.075 20.634
21.163 2.897 1.878 2.104 1.817 BMPER Undetermined Undetermined
Undetermined Undetermined #VALUE! #VALUE! #VALUE! #VALUE! CALCR
Undetermined Undetermined Undetermined Undetermined #VALUE! #VALUE!
#VALUE! #VALUE! CEBPA 28.837 31.206 28.782 30.802 12.536 10.026
12.235 10.133 CEBPB 24.507 23.911 23.944 22.678 4.412 5.188 4.940
5.803 COL4A2 Undetermined Undetermined Undetermined 33.904 15.638
#VALUE! #VALUE! #VALUE! CREB1 22.906 22.973 22.993 22.816 4.550
4.237 4.002 4.202 CREB3 24.330 24.566 24.421 24.421 6.155 5.665
5.595 5.626 CREB3L4 24.709 25.089 24.418 24.583 6.317 5.662 6.118
6.005 CTGF Undetermined Undetermined Undetermined Undetermined
#VALUE! #VALUE! #VALUE! #VALUE! CYB561 33.993 36.737 33.006 36.752
18.486 14.250 17.766 15.289 DDIT4 21.247 21.455 21.854 22.681 4.415
3.098 2.484 2.543 DIRAS2 Undetermined Undetermined 33.382
Undetermined #VALUE! 14.626 #VALUE! #VALUE! DUSP1 26.436 26.325
26.754 26.713 8.447 7.998 7.354 7.732 EDN1 32.440 33.297 32.372
Undetermined #VALUE! 13.616 14.326 13.736 EHD3 24.298 25.766 24.878
26.386 8.120 6.122 6.795 5.594 ERRFI1 Undetermined Undetermined
Undetermined Undetermined #VALUE! #VALUE! #VALUE! #VALUE! FKBP5
20.533 20.494 21.869 21.371 3.105 3.113 1.523 1.829 FOSL2 35.083
31.757 33.362 Undetermined #VALUE! 14.606 12.786 16.379 GDPD1
27.358 28.134 26.972 27.838 9.572 8.216 9.163 8.654 GHRHR 36.313
37.623 Undetermined 36.734 18.468 #VALUE! 18.652 17.609 GLUL 35.436
35.795 34.414 Undetermined #VALUE! 15.658 16.824 16.732 GOT1 22.400
22.607 22.859 22.304 4.038 4.103 3.636 3.696 H6PD 25.209 25.743
24.819 24.878 6.612 6.063 6.772 6.505 HAS2 Undetermined 35.628
31.961 Undetermined #VALUE! 13.205 16.557 #VALUE! HNRPLL 22.667
22.977 22.577 22.558 4.292 3.821 4.006 3.963 IL10 32.210 33.099
31.119 32.524 14.258 12.363 14.128 13.506 IL1RN Undetermined
Undetermined 36.259 Undetermined #VALUE! 17.503 #VALUE! #VALUE! IL6
Undetermined Undetermined Undetermined Undetermined #VALUE! #VALUE!
#VALUE! #VALUE! IL6R 32.607 33.589 31.995 33.408 15.142 13.239
14.618 13.903 KLF13 22.256 22.649 22.834 22.878 4.612 4.078 3.678
3.552 KLF9 26.816 27.456 26.769 27.872 9.306 8.013 8.485 8.112 LOX
33.947 Undetermined Undetermined Undetermined #VALUE! #VALUE!
#VALUE! 15.243 MERTK 33.257 Undetermined 33.859 31.392 13.126
15.103 #VALUE! 14.553 MT1E Undetermined Undetermined Undetermined
Undetermined #VALUE! #VALUE! #VALUE! #VALUE! MT2A 23.416 24.175
23.522 23.341 5.075 4.766 5.204 4.712 NFKBIA 22.744 22.909 23.016
22.914 4.648 4.260 3.938 4.040 NR3C1 22.602 22.803 22.781 22.525
4.259 4.025 3.832 3.898 PDCD7 23.859 24.858 24.113 23.750 5.484
5.357 5.887 5.155 PDGFRB Undetermined Undetermined 35.205
Undetermined #VALUE! 16.449 #VALUE! #VALUE! PDP1 25.255 26.112
25.507 25.928 7.662 6.751 7.141 6.551 PER1 24.612 24.926 24.973
25.007 6.741 6.217 5.955 5.908 PER2 23.794 24.371 24.403 24.767
6.501 5.647 5.400 5.090 PIK3R1 23.210 23.440 23.615 23.627 5.361
4.859 4.469 4.506 PLD1 Undetermined Undetermined Undetermined
Undetermined #VALUE! #VALUE! #VALUE! #VALUE! PLEKHF1 27.292 27.868
27.684 28.448 10.182 8.928 8.897 8.568 POU2F1 23.799 24.224 23.866
23.951 5.685 5.110 5.253 5.095 POU2F2 21.502 21.920 21.841 21.890
3.624 3.085 2.949 2.798 RASA3 22.754 23.207 22.984 23.225 4.959
4.228 4.236 4.050 RGS2 24.883 25.145 24.670 24.812 6.546 5.914
6.174 6.179 RHOB 30.760 32.584 30.084 31.155 12.889 11.328 13.613
12.056 RHOJ Undetermined Undetermined 38.493 Undetermined #VALUE!
19.737 #VALUE! #VALUE! SESN1 22.189 Undetermined 25.195 26.963
8.697 6.439 #VALUE! 3.485 SGK1 25.886 25.808 26.513 25.449 7.183
7.757 6.837 7.182 SLC10A6 37.655 34.857 34.336 38.026 19.760 15.580
15.886 18.951 SLC19A2 26.295 27.465 26.633 27.755 9.489 7.877 8.494
7.591 SLC22A5 27.847 28.544 27.725 28.010 9.744 8.969 9.573 9.143
SNTA1 24.008 24.797 24.422 24.779 6.513 5.666 5.826 5.304 SPHK1
29.372 30.619 29.007 29.583 11.317 10.251 11.648 10.668 SPSB1
25.736 26.495 25.588 25.722 7.456 6.832 7.524 7.032 STAT5A 24.652
25.174 24.761 24.858 6.592 6.005 6.203 5.948 STAT5B 21.986 22.153
21.908 21.716 3.450 3.152 3.182 3.282 TBL1XR1 20.756 20.805 20.855
20.821 2.555 2.099 1.834 2.052 TNF 27.723 29.337 29.509 31.477
13.211 10.753 10.366 9.019 TNFAIP3 28.965 28.521 27.807 26.978
8.712 9.051 9.550 10.261 TSC22D3 21.819 21.432 23.384 22.896 4.630
4.628 2.461 3.115 USP2 20.842 21.342 22.120 22.318 4.052 3.364
2.371 2.133 USP54 26.333 26.952 33.990 27.307 9.041 15.234 7.981
7.629 VDR 27.497 28.330 26.956 28.621 10.355 8.200 9.359 8.793
VLDLR 30.410 28.792 27.824 26.896 8.630 9.068 9.821 11.706 XDH
Undetermined Undetermined Undetermined Undetermined #VALUE! #VALUE!
#VALUE! #VALUE! ZFP36 24.715 24.706 24.770 24.542 6.276 6.014 5.735
6.012 ZHX3 24.009 24.719 24.325 24.882 6.616 5.569 5.748 5.305
ZNF281 23.423 23.881 23.813 23.935 5.669 5.057 4.910 4.719 ACTB
13.717 14.247 14.284 14.272 -3.994 -4.472 -4.724 -4.987 B2M 18.704
18.972 18.756 18.266 0.000 0.000 0.000 0.000 GAPDH 15.435 15.835
15.790 15.782 -2.484 -2.966 -3.136 -3.269 HPRT1 21.349 21.358
21.582 21.214 2.948 2.826 2.387 2.645 RPLP0 15.192 15.469 15.266
15.194 -3.072 -3.480 -3.502 -3.512 HGDC Undetermined Undetermined
Undetermined Undetermined #VALUE! #VALUE! #VALUE! #VALUE! RTC
21.372 21.163 21.388 21.673 3.407 2.632 2.192 2.668 RTC 21.441
21.008 21.369 21.554 3.288 2.613 2.037 2.737 RTC 21.504 21.137
21.357 21.500 3.234 2.601 2.166 2.800 PPC 18.529 18.295 18.338
18.368 0.102 -0.418 -0.676 -0.175 PPC 18.544 18.326 19.432 18.405
0.133 0.676 -0.645 -0.160 PPC 18.784 18.935 18.081 18.679 0.413
-0.675 -0.036 0.080 Cpd44 Pred Combo Gene .DELTA..DELTA.CT Fold
Change .DELTA..DELTA.CT Fold Change .DELTA..DELTA.CT Fold Change
ADARB1 -1.229 2.344 -0.516 1.430 -1.115 2.166 AFF1 -0.276 1.211
-0.429 1.346 0.394 0.761 AK2 -0.570 1.485 -1.076 2.108 -1.259 2.393
AMPD3 -1.449 2.730 -0.891 1.854 -0.943 1.923 ANGPTL4 -1.331 2.516
-1.047 2.066 -0.841 1.791 ANXA4 -2.419 5.348 -0.890 1.853 -2.493
5.629 AQP1 0.331 0.795 #VALUE! #VALUE! #VALUE! #VALUE! ARID5B
-0.541 1.455 -0.131 1.095 0.127 0.916 ASPH -0.843 1.794 -0.451
1.367 -0.914 1.884 ATF4 0.146 0.904 -0.279 1.213 0.533 0.691 BCL6
-1.019 2.027 -0.793 1.733 -1.080 2.114 BMPER #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! CALCR #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! CEBPA -2.510 5.696 -0.301 1.232 -2.403
5.289 CEBPB 0.776 0.584 0.528 0.694 1.391 0.381 COL4A2 #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! CREB1 -0.313 1.242 -0.548
1.462 -0.348 1.273 CREB3 -0.490 1.404 -0.560 1.474 -0.529 1.443
CREB3L4 -0.655 1.575 -0.199 1.148 -0.312 1.241 CTGF #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! CYB561 -4.236 18.844 -0.720 1.647
-3.197 9.170 DDIT4 -1.317 2.491 -1.931 3.813 -1.872 3.660 DIRAS2
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! DUSP1 -0.449 1.365
-1.093 2.133 -0.715 1.641 EDN1 #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! EHD3 -1.998 3.994 -1.325 2.505 -2.526 5.760 ERRFI1
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! FKBP5 0.008 0.994
-1.582 2.994 -1.276 2.422 FOSL2 #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! GDPD1 -1.356 2.560 -0.409 1.328 -0.918 1.889 GHRHR
#VALUE! #VALUE! 0.184 0.880 -0.859 1.814 GLUL #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! GOT1 0.065 0.956 -0.402 1.321
-0.342 1.268 H6PD -0.549 1.463 0.160 0.895 -0.107 1.077 HAS2
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! HNRPLL -0.471 1.386
-0.286 1.219 -0.329 1.256 IL10 -1.895 3.719 -0.130 1.094 -0.752
1.634 IL1RN #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! IL6
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! IL6R -1.903 3.740
-0.524 1.438 -1.239 2.360 KLF13 -0.534 1.448 -0.934 1.911 -1.060
2.085 KLF9 -1.293 2.450 -0.821 1.767 -1.194 2.238 LOX #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! MERTK 1.977 0.254 #VALUE!
#VALUE! 1.427 0.372 MT1E #VALUE! #VALUE! #VALUE! #VALUE! #VALUE!
#VALUE! MT2A -0.309 1.239 0.129 0.914 -0.363 1.286 NFKBIA -0.388
1.309 -0.710 1.636 -0.608 1.524 NR3C1 -0.234 1.176 -0.427 1.344
-0.361 1.284 PDCD7 -0.127 1.092 0.403 0.756 -0.329 1.256 PDGFRB
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! #VALUE! PDP1 -0.911 1.880
-0.521 1.435 -1.111 2.160 PER1 -0.524 1.438 -0.786 1.724 -0.833
1.781 PER2 -0.854 1.808 -1.101 2.145 -1.411 2.659 PIK3R1 -0.502
1.416 -0.892 1.856 -0.855 1.809 PLD1 #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! PLEKHF1 -1.254 2.385 -1.285 2.437 -1.594
3.019 POU2F1 -0.575 1.490 -0.432 1.349 -0.590 1.505 POU2F2 -0.539
1.453 -0.675 1.597 -0.826 1.773 RASA3 -0.731 1.660 -0.723 1.651
-0.909 1.878 RGS2 -0.632 1.550 -0.372 1.294 -0.367 1.290 RHOB
-1.561 2.951 0.724 0.505 -0.833 1.781 RHOJ #VALUE! #VALUE! #VALUE!
#VALUE! #VALUE! #VALUE! SESN1 -2.258 4.783 #VALUE! #VALUE! -5.212
37.065 SGK1 0.574 0.672 -0.346 1.271 -0.001 1.001 SLC10A6 -4.180
18.126 -3.874 14.662 -0.809 1.752 SLC19A2 -1.512 3.057 -0.995 1.993
-1.898 3.727 SLC22A5 -0.775 1.711 -0.171 1.126 -0.601 1.517 SNTA1
-0.847 1.799 -0.687 1.610 -1.209 2.312 SPHK1 -1.066 2.094 0.331
0.795 -0.649 1.568 SPSB1 -0.624 1.541 0.068 0.954 -0.424 1.342
STAT5A -0.587 1.502 -0.389 1.309 -0.644 1.563 STAT5B -0.298 1.229
-0.268 1.204 -0.168 1.123 TBL1XR1 -0.456 1.372 -0.721 1.648 -0.503
1.417 TNF -2.458 5.495 -2.845 7.185 -4.192 18.278 TNFAIP3 0.339
0.791 0.838 0.559 1.549 0.342 TSC22D3 -0.002 1.001 -2.169 4.497
-1.515 2.858 USP2 -0.688 1.611 -1.681 3.207 -1.914 3.769 USP54
6.193 0.014 -1.060 2.085 -1.412 2.661 VDR -2.155 4.454 -0.996 1.994
-1.562 2.953 VLDLR 0.438 0.738 1.191 0.438 3.076 0.113 XDH #VALUE!
#VALUE! #VALUE! #VALUE! #VALUE! #VALUE! ZFP36 -0.262 1.199 -0.541
1.455 -0.264 1.201 ZHX3 -1.047 2.066 -0.868 1.825 -1.311 2.481
ZNF281 -0.612 1.528 -0.759 1.692 -0.950 1.932 ACTB B2M GAPDH HPRT1
RPLP0 HGDC RTC RTC RTC PPC PPC PPC
Example 2
Compound 44 and Everolimus Act Synergistically to Enhance Cell
Cycle Arrest in G1 Phase in EZH2 Mutant WSU-DLCL2 Cells, Apoptosis
in Wild Type EZH2 SU-DH-L5 Cells
[0206] In FIG. 11 panels B and E, each point represents the mean of
percentage of gated cells in early and late apoptosis (Annexin-V
positive, mean+/-S.D., n=3). In panels C and F, points on the
progress curve represent the mean percentage of gated cells by DNA
content (PI positive, mean+/-S.D., n=2). In panel A, WSU-DLCL2
cells were treated at a 400:1 constant ratio with a combination of
Compound 44 and Everolimus. The combination was shown to induce
very strong synergy with CI values of 0.34-0.003. In panel B)
Apoptosis levels assessed in WSU-DLCL2 cells treated with Compound
44 (500 nM), Everolimus (5 nM) or in combination at the same
concentrations. No increase in apoptosis on WSU-DLCL2 cells was
seen. In panel C, A significant increase in G1 phase of cell cycle
was observed after co-treatment compared to Compound 44 alone. In
panel D, SU-DHL-5 cells were treated at a 4000:3 constant ratio in
combination. The combination was shown to induce very strong
synergy with CI values of 0.135-0.008. In panel E, A significant
increase in Annexin positive cells was measured after co-treatment
(500 nM Compound 44, 0.75 nM Everolimus), compared with Compound 44
alone (p<0.0001). In panel F, A significant increase in sub-G1
phase of cell cycle was observed after co-treatment.
Example 3
Compound 44 and Ibrutinib Act Synergistically to Enhance Apoptosis
in EZH2 Mutant WSU-DLCL2 Cells and Wild Type EZH2 SU-DH-L5
Cells
[0207] In FIG. 12 panels B and E, each point represents the mean of
percentage of gated cells in early and late apoptosis (Annexin-V
positive, mean+/-S.D., n=3). In panels C and F, points on the
progress curve represent the mean percentage of gated cells by DNA
content (PI positive, mean+/-S.D., n=2). In panel A, WSU-DLCL2
cells were treated at a 4:5 constant ratio with a combination of
Compound 44 and Ibrutinib. The combination of these agents
demonstrates strong synergy with CI values between 0.39 and 0.14.
In panel B, apoptosis levels assessed in WSU-DLCL2 cells treated
with Compound 44 (500 nM), Ibrutinib (625 nM) or in combination.
This combination revealed a synergistic time-dependent increase in
apoptosis on WSU-DLCL2 cells. In panel C, cell cycle analysis
revealed a time-dependent increase in the percentage of WSU-DLCL2
cells in G1-phase with a steep increase after combination
treatment. In panel D, SU-DHL-5 cells were treated at a 1:5
constant ratio of Compound 44: Ibrutinib. The combination induced
very strong synergy with CI values of 0.222-0.002. In panel E,
synergistic and time-dependent increase of Annexin positive
staining of SU-DHL-5 cells after cotreatment with Compound 44 (1000
nM) and ibrutinib (2500 nM) compared with Compound 44 alone
(p<0.0001). In panel F, cell cycle analysis of SU-DHL-5 cells
treated in combination revealed an increase in the cells in the
sub-G1 population after co-treatment compared with each agent
alone.
Example 4
Compound 44 and MK-2206 Act Synergistically to Enhance Apoptosis in
EZH2 Mutant WSU-DLCL2 Cells and Wild Type EZH2 (SU-DH-L5 and
OCI-LY-19) Cells
[0208] In FIG. 13 panel A, WSU-DLCL2 cells were treated at a 4:1
constant ratio with a combination of Compound 44 and MK-2206. Fa-CI
plot demonstrates very strong synergy with CI values between
0.77-0.005. In panel B, Time dependent increase in the percentage
of Annexin positive WSU-DLCL2 cells when co-treated with Compound
44 (2000 nM) and MK-2206 (400 nM). In panel C, cell cycle analysis
revealed an increase in the percentage of WSU-DLCL2 cells in
G1-phase with a steep increase after one day of co-treatment
compared with Compound 44 alone (p<0.0001). In panel D, SU-DHL-5
cells were treated at a 2:1 constant ratio for Compound 44 and
MK-2206. The combination induced very strong synergy with CI values
of 0.276-0.001. In panel E, apoptosis level assessment in SU-DHL-5
revealed an increase in Annexin positive cells after 24 hours of
co-treatment (500 nM Compound 44, 250 nM MK-2206) compared with
Compound 44 alone (p<0.0001). In panel F, cell cycle analysis of
SU-DHL-5 cells treated in combination showed an increase in the
percentage of cells in sub-G1 population compared with treatment of
the agents individually. In panel G, strong synergy in OCI-LY19
cells was observed by treatment with a combination of Compound 44
and MK-2206 with a 1/a value of 71.4. In panel H, Time-dependent
increase in apoptosis was shown when OCI-LY19 cells were treated
with the combination (1000 nM Compound 44, 2500 nM MK-2206)
compared with Compound 44 alone (p<0.0001). In panel I, cell
cycle analysis of OCI-LY19 cells treated with the combination
revealed a time-dependent increase of cells in sub-G1 phase of the
cell cycle (p<0.0001).
Example 5
Regulation of Target Genes with Combinations of Compound 44 and BCR
Pathway Inhibitors
[0209] In FIG. 14 panel A, downregulation of EGR1 (40 fold) and FOS
(4 fold) with a combination of Compound 44 and Ibrutinib compared
to single agents in WSU-DLCL2 cells. In panel B, upregulation of
AICDA (3 fold) and TCL1A (5 fold) with a combination of Compound 44
and MK-2206 is compared to single agents in WSU-DLCL2 cells. In
panel C, upregulation of GJA1 (3 fold) with a combination of
Compound 44 and Ibrutinib is compared to single agents in SU-DHL-5
cells. Value for statistical analysis are a mean of duplicate or
triplicate+/-SD. t test, *P<0.05, **P<0.01, ***P<0.001,
****P<0.0001
Example 6
Synergistic Interactions Between EZH2 Inhibition and Modulation of
the BCR Signaling Pathway, BCL2 Inhibition and GR Agonism in
Germinal Center B Cell Lines
[0210] Several synergistic combinations were uncovered in this
study with key players in the signaling pathways implicated in
DLBCL biology (see FIG. 15). Inhibitors targeting nodes of the
B-cell receptor pathway such as those of the PI3K/Akt/mTOR
signaling cascade, MEK1/2 in the MAPK cascade, SYK and BTK showed
very strong synergy when combined with EZP-6438 extending the
impact of EZH2 inhibition from mutant EZH2 bearing GCB cell lines
to those of the wild type subtype Inhibitors of BCL-2 family of
proteins, obatoclax, navitoclax and ABT-199 showed synergistic
antiproliferative activity in combination with Compound 44.
Glucorticoid receptor agonists, prednisolone and dexamethasone
display a dramatic enhancement of EZH2 inhibition in mutant cell
lines and sensitize wild type to EZH2i. Rituximab, the antibody
combined with chemotherapeutics in R-CHOP targets cd-20 to elicit
enhanced antiproliferative effects in vitro in mutant cell
lines.
Example 7
Compound 44 and Everolimus Act Synergistically to Decrease
Populations of Cells in S and G2/M Phases of Mutant WSU-DLCL2 Cells
and G1, S, and G2/M Phases in Wild Type SU-DHL-5 Cells
[0211] WSU-DLCL2, SU-DHL-5, and OCI-LY19 (data not shown) cells
were pretreated with Compound 44 (500 nM for WSU and SU-DHL-5)
followed by co-treatment with a combination of Compound 44 and
Everolimus (WSU: 5 nM, SU-DHL-5: 0.75 nM). In FIG. 16 panel A, no
change in sub-G1 phase of the cell cycle is seen when WSU-DLCL2
cells are treated with single agents or in combination. In panels B
and C, synergistic time-dependent decrease of cells in S phase and
G2/M phase of the cell cycle, respectively, is seen when WSU-DLCL2
cells were treated with the combination. In panels D, E, and F,
synergistic decrease of cells in G1, S, and G2/M phases of the cell
cycle, respectively, is seen 48 hours after co-treatment on
SU-DHL-5 cells.
Example 8
Compound 44 and Ibrutinib Act Synergistically to Decrease
Populations of Cells in G1, S and G2/M Phases of Mutant WSU-DLCL2
Cells and Wild Type SU-DHL-5 Cells
[0212] WSU-DLCL2, SU-DHL-5, and OCI-LY19 (data not shown) cells
were pretreated with Compound 44 (WSU: 500 nM, SU-DHL-5: 1000 nM)
followed by co-treatment with a combination of Compound 44 and
Ibrutinib (WSU: 625 nM, SU-DHL-5: 2500 nM). In FIG. 17 panels A, B,
and C, synergistic decrease of cells in G1, S, and G2/M phases of
the cell cycle, respectively, is seen 24 hours after co-treatment
of WSU-DLCL2 cells compared to Compound 44 or Ibrutinib as single
agents. In panels D, E, and F, synergistic time dependent decrease
of cells in G1, S, and G2/M phases of the cell cycle, respectively,
is seen after co-treatment of SU-DHL-5 cells compared to Compound
44 or Ibrutinib as single agents.
Example 9
Compound 44 and MK-2206 Act Synergistically to Decrease Populations
of Cells in G1, S and G2/M Phases of Mutant WSU-DLCL2 Cells and
Wild Type SU-DHL-5 and OCI-LY19 Cells
[0213] WSU-DLCL2, SU-DHL-5, and OCI-LY19 cells were pretreated with
Compound 44 (2000 nM, 500 nM, and 1000 nM respectively) followed by
co-treatment with a combination of Compound 44 and MK-2206 (400 nM,
250 nM, and 2500 nM respectively). In FIG. 18 panel A, a
synergistic time-dependent decrease in G1 phase of the cell cycle
is seen when WSU-DLCL2 cells were treated in combination with
MK-2206. In panels B and C, a synergistic decrease of cells in S
and G2/M phases of the cell cycle, respectively, is seen when
WSU-DLCL2 cells were treated in combination. In panels D, E, and F,
synergistic decrease of cells in G1, S, and G2/M phases of the cell
cycle, respectively, is seen 48 hours after co-treatment of
SU-DHL-5 cells compared to single agents. In panels G, H, and I,
synergistic time-dependent decrease of cells in G1, S, and G2/M
phases of the cell cycle, respectively, is seen when OCI-LY19 cells
were treated in combination.
The outcomes of proliferation studies using the combination of
Compound 44 with individual SOC, or other selected agents against
wild type and EZH2 mutant bearing DLBCL cell lines are shown in
Table 5.
TABLE-US-00010 TABLE 5 Proliferation study results. WSU-DLCL2
SU-DHL-10 SU-DHL-5 CI range CI range CI range DOHH2 OCI-LY19 Toledo
Compound CR or 1/.alpha. CR or 1/.alpha. CR or 1/.alpha. 1/.alpha.
1/.alpha. 1/.alpha. Prednisolone 9.7 4.2 7.6 9.5 4.2 No effect
Dexamethasone 17.sup.b .sup. 3.7 400:1 0.42- 4.2 7.7 No 0.076
effect ABT-199 4:3 0.27- 3:200 1.2- No 1.9.sup.b 4.20 1.9.sup.b
0.002 1.4.sup.e Effect Navitoclax 1:5 0.42- 1:100 0.90- No
1.5.sup.b 6.60 No 0.067 0.36.sup.a Effect effect Obatoclax 40:3
.sup. 1.10.sup.e 1:1 0.91- 320:1 1.26- 1.4.sup.b 1.50 1.1.sup.b
1.36.sup.e 1.61.sup.e Ibrutinib 4:5 0.39- 1:10 0.78- 1:5 0.22- 0.67
No No 0.14 0.062 0.002 effect effect Idelalisib 1:5 0.31- 3:200
0.64- 2:5 0.24- 0.59 No 1.1.sup. 0.062 0.02 0.000025 effect
Everolimus 400:1 0.34- 100:3 0.65- 4000:3 0.14- 0.83 No No 0.003
0.14 0.008 effect effect Tamatinib 1:5 0.24- 3:50 1.1.sup.e- 1:5
0.57- 0.81 No No 0.025 0.061 0.19 effect effect Trametinib 1:5
0.45- .sup. 5.6.sup.b 2:5 0.031- 1.2.sup.b No No 0.16 0.001 effect
effect MK-2206 4:1 0.77- 3:20 0.56- 2:1 0.28- 0.64 71.40 1.7.sup.b
0.005 0.04 0.001 Rituximab 2.6 Bortezomib 400:3 1.4- 15:1 1.5-
1600:1 1.00 0.96 1.67 0.96 1.3.sup.e 1.sup.e CR = combination
ratio, CI = combination index CI range above Fractional effect of
0.5 .sup.abased on 1 experiment, other experiments are IC.sub.50
shift values between top concentration of 6438 and drug alone,
because 50% inhibition was not achieved with Compound 44
.sup.bcould not calculate an alpha value so IC.sub.50 shift was
reported .sup.cDOHH2 data normalized to individual 6438
concentrations instead of DMSO d- Concentrations of Rituximab are
.mu.g/mL .sup.eThese CI values were not significantly different
from 1
The potency of compounds used in proliferation assays, and dose
ranges used in each cell line are shown in Table 6.
TABLE-US-00011 TABLE 6 Compound potency and dose ranges. GCB
sub-type EZH2 Y646F GCB sub-type WT EZH2 WSU-DLCL2 SU-DHL-10
SU-DHL-5 DOHH-2 OCI-LY19 Toledo drug drug drug drug drug drug
Potency range Potency range Potency range range range range (nM)
(nM) (nM) (nM) (nM) (nM) Potency (nM) Potency (nM) Potency (nM)
Prednisolone 90.6 7.8- >1000 7.8- 39 0.625- 133 7.8- 47 0.78-
>1000 7.8- 1000 1000 80 1000 100 1000 Dexa- >10000 0.78-
>100 0.78- 3.4 0.078- 5.6 1.56- 79 0.078- >100 0.78-
methasone 100 100 10 200 10 100 ABT-199 1942 23.4- 3037 78-
>10000 78- 77 7.8- 53 1.56- 190 4.69- 3000 10000 10000 1000 200
600 Navitoclax 3539 78- >10,000 78- >10000 78- 540 78- 131
15.6- 590 11.7- 10000 10000 10000 10000 2000 1500 Obatoclax 59 1.2-
19.5 0.78- 9.8 0.39- 51 1.56- 42 1.56- 96 1.17- 50 100 50 200 200
150 Ibrutinib 277.7 39- 1146 187- 1327 312- 956 39- >10000 78-
>10000 78- 5000 3000 5000 5000 10000 10000 Idelalisib 2046 78-
8433 1250- 2587 78- 2984 78- >10000 78- 9796 78- 10000 10000
10000 10000 10000 10000 Everolimus 0.653 0.039- 0.854 0.09- 0.72
0.09- 0.13 0.078- ND 7.8- 0.1 0.078- 5 6 3 5 1000 10 Tamatinib
3415.2 78- 2214 312.5- 3761 313- 1209 78- >10000 78- 3200 78-
10000 5000 10000 10000 10000 10000 Trametinib 8608 78- >10,000
78- >10000 78- >10000 78- ND 78- >10000 78- 10000 10000
10000 10000 (*>10,000 10000 10000 prism) MK-2206 127 7.8- 274.6
7.8- 162 7.8- 86 7.8- 304 78- 95 7.8- 500 500 1000 1000 1000 1000
Rituximab .sup. >10.sup.a 10- 0.39.sup.a Bortezomib 6.5 0.94-
8.6 5.0- 4 0.04- 6.6 0.078- 12 0.23- 4.3 0.12- 7.5 20 5 10 30 15
Compound 310 31- 73 3.1- 3300 62- >10000 31.3- >10000 125-
>10000 15.6- 44 2000 200 4000 2000 8000 1000
.sup.aConcentrations of Rituximab are in .mu.g/ml IC.sub.50 values
listed are calculated after 3 days of dosing except for Toledo
which were dosed for 5 days Compound 44 IC.sub.50s were calculated
after 7 days for all cell lines except for Toledo which was
calculated after 11 days of treatment
INCORPORATION BY REFERENCE
[0214] All publications and patent documents cited herein are
incorporated herein by reference as if each such publication or
document was specifically and individually indicated to be
incorporated herein by reference. Citation of publications and
patent documents is not intended as an admission that any is
pertinent prior art, nor does it constitute any admission as to the
contents or date of the same. The invention having now been
described by way of written description, those of skill in the art
will recognize that the invention can be practiced in a variety of
embodiments and that the foregoing description and examples below
are for purposes of illustration and not limitation of the claims
that follow.
EQUIVALENTS
[0215] The invention can be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting on the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes that come within the meaning and range of equivalency of
the claims are intended to be embraced therein.
* * * * *
References