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TABLE OF CONTENTS
1. INTRODUCTION ........................................................ 3
2. SUMMARY OF RESULTS .................................................. 4
3. BACKGROUND .......................................................... 5
a. .....................................................................
Groucho proteins 5
b. .....................................................................
Function of Groucho proteins 6
c. .....................................................................
Groucho proteins in cancer 6
d. .....................................................................
Groucho-related gene 1 (Grgl) transgenic mice 7
4. METHODS ............................................................. 11
a. Mice ................................................................ 11
b. ..................................................................... Drug
preparation and dosage 11
c. Quantitation ........................................................ 13
d. ..................................................................... Blood
samples 13
5. RESULTS ............................................................. 14
a. ..................................................................... Three
month old 14
b. ..................................................................... Six
month old 17
6. CONCLUSIONS ......................................................... 22
7. REFERENCES .......................................................... 23
APPENDIX 1:3 MONTH SAMPLES - MOUSE ID AND TUMOR NUMBERS ................ 27
APPENDIX 2:6 MONTH SAMPLES - MOUSE ID AND TUMOR NUMBERS ................ 28
TABLE OF FIGURES
Figure 1: Average number of tumors in control and treated mice ......... 4
Figure 2: Gro proteins are transcriptional co-repressors with conserved
domains 5
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Figure 3: Transgene construct in the Grgl transgenic mice ............. 8
Figure 4: Molecular changes accompanying tumor progression in Grgl mice .. 9
Figure 5: Typical molecular changes in human non-small lung cancer .... 9
Figure 6: Number of surface tumors per mouse at 3 months old .......... 14
Figure 7: Whole-mount left lung lobe at 3 months ...................... 15
Figure 8: Sections from tumors at 3 months ............................ 16
Figure 9: Number of surface tumors at 6 months old .................... 17
Figure 10A: Tumor diameter in tissue sections at 6 months old ......... 18
Figure 10B: Sum of tumor diameters in tissue sections at 6 months old .. 18
Figure 11: Number of tumors over 1 mm in 6 month samples .............. 19
Figure 12: Wholemount left lung lobe at 6 months ...................... 20
Figure 13: Sections of lung tumors at 6 months ........................ 21
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1. Introduction
The aim of the experiment was to evaluate the effect of two drugs on tumor
development in a
mouse model of non-small cell lung cancer.
The two drugs tested were Abexinostat (PCI-24781), a histone deacetylase
complex (HDAC)
inhibitor, and Ibrutinib (PCI-32765), a Bruton's tyrosine kinase (BTK)
inhibitor.
Grgl transgenic mice were used to evaluate the drugs. Grgl transgenic mice
overexpress
Groucho-related gene 1 (Grgl). The mice develop lung tumors that resemble
human non-small
cell lung cancer. Tumors initiate at 1 month of age and progress to invasive
adenocarcinoma by 8
months of age. The drugs were administered to 2 month-old mice or to 5 month-
old mice, and
mice were treated for 4 weeks. The former group is referred to as the 3 month
samples and the
latter as the 6 month samples.
Grgl functions by directly interacting with histone deacetylase to repress
transcription and alter
protein stability. HDAC inhibitors such as Abexinostat may therefore be able
to interrupt Grgl
function and alter tumor progression in the Grgl transgenic mice.
BTK inhibitors inhibit the B cell receptor signaling pathway and are being
evaluated in B cell
malignancies, however their effect on the progression of solid tumors is not
known. To evaluate
the possibility of BTK inhibitors slowing tumor growth, Ibrutinib was tested
in the Grgl
transgenic mice.
This report presents the protocol, data collection, data treatment and
results.
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2. Summary of Results
Drug treatment of 2 to 3 month old mice greatly reduced tumor number compared
to the control
group: Abexinostat reduced tumor number by 50% and Ibrutinib reduced tumor
number by 60%.
The drugs were more effective when administered together, with 75% reduction
in tumor
number.
With drug treatment from 5 to 6 months, both drugs reduced the number of large
tumors by 80%
compared to the control group. Abexinostat-treated tumors exhibited large
regions of tumor cell
death. Ibrutinib potentially altered the immune response as reflected by the
number of apoptotic
macrophages in the tumors of 6 month-old mice.
25- 2.5-
NI Control
e 20- 2.0- Abexinostat
Ibrutinib
a 15- 1.5- Abexilbrut
10- T
0.-5-
z loll ii
,40 0
41/4*
*GP =c4
Figure 1: Average number of tumors in control and treated mice
The average number of tumors for control and drug-treated mice is shown. The
bars
at the left show the number of visible surface tumors in mice treated for 4
weeks
beginning at 2 months (3 month samples) and the bars in the middle show number
of
visible surface tumors in mice treated for 4 weeks beginning at 5 months (6
month
samples). The average number of tumors over 1 mm for 6 month samples is shown
in
the bars at the right.
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proteins, TLE6/Grg6 does not repress transcription when recruited to DNA and
antagonizes
TLE1 activity.
b. Function of Groucho proteins
Gro proteins are broadly expressed and play an important role in the
regulation of patterning and
cell differentiation during development. For example, in Drosophila one
function of Gro is to act
with basic-helix-loop-helix transcription factors in the decision between a
neural or epidermal
cell fate16. In mammals, Gro proteins bind with the Pax5 transcription factor
to promote
commitment to B cell differentiation".
Gro proteins act in several signaling pathways, including the Notch,
Wingless/Wnt and
Dpp/BMP/TGF-beta pathways. The activity of Gro in the Notch signaling pathway
can be
attenuated through Epidermal Growth Factor Receptor (EGFR) signaling, by
phosphorylation in
the SP and WD40 domains by mitogen-activated protein kinase (MAPK)18.
Gro proteins act as transcriptional co-repressors. They lack intrinsic DNA-
binding activity but
are recruited to target genes by DNA-binding transcription factors, including
LEF1/TCF/FoxA,
c-Myc7'8, Hes, Runx, Engrailed, Goosecoid, Pax, Nkx and FoxD11. The
recruitment of Groucho
to specific gene regulatory sequences results in transcriptional repression.
Gro proteins also interact with histone deacetylase complex (HDAC) molecules,
and
hypoacetylated histones4'9'19'20. One possible mechanism for their
transcriptional repression is by
direct binding and modification of chromatin 21'22. Another possible mechanism
is through
indirect chromatin modification, through Gro binding to HDAC via the GP
domain9.
Groucho proteins also appear to down-regulate expression at the protein level,
by acting with
HDAC to deacetylate proteins at lysine residues, which targets proteins for
ubiquitination and
proteasomal degradation 23-25.
c. Groucho proteins in cancer
Deregulated expression of human Groucho family members (TLE proteins) occurs
in several
types of cancer.
TLE1 is overexpressed in a significant number of non-small cell lung cancer
(NSCLC) tumors23.
A tumor tissue microarray from NSCLC samples was screened for overexpression
of TLE1 by
immunohistochemical staining. Overexpression was observed in 11% of squamous
cell
carcinomas and 20% of adenocarcinomas, whereas only moderate staining was seen
for
carcinoid tumors and small cell lung cancer. Preliminary studies show it may
be possible to
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identify lung cancer patients with overexpression of TLE1 using a blood test
that measures the
protein in an ELISA assay (Lobe, unpublished).
TLE6 overexpression was observed in human colorectal cancers's. Using mice
with mutations in
the APC and DNA mismatch repair genes, it was found TLE6 was amplified and its
overexpression contributed to tumor progression in the mice15. TLE6 bound to
the RUNX3
tumor suppressor and antagonized RUNX3 target gene activation. It was
subsequently found,
using RT-PCR on human tumor samples, that TLE6 and RUNX3 are overexpressed in
a subset
of human colorectal cancer samples".
Gro proteins are also expressed in other types of cancers. TLE2 is
differentially expressed in
grade I astrocytomas26. TLE2 and TLE3 expression is induced in high-grade
menangiomas,
together with HES1 transcription factor, Notchl and Notch2 27. Overexpression
of TLE2, PITX2
and NOTCH3 was observed in pituitary adenomas28, and in another patient study
TLE-4 was
overexpressed in some pituitary adenomas29. TLE1 is expressed in cell nuclei
in synovial
sarcoma at higher levels than in normal or other mesenchymal tumors, as
measured using
immunohistochemistry 30
.
d. Groucho-related gene 1 (Grgl) transgenic mice
i. Transgene strategy
Grgl transgenic mice overexpress Grgl and develop lung adenocarcinoma. The
transgenic mice
were originally generated using a Cre-conditional expression system 23,31,32
(see Figure 3,
following page). In this system, the mice initially express p-geo (lacZlneoR)
from the transgene
in all tissues. When Cre is introduced, P-geo is excised and Grgl is expressed
instead. In
addition to Grgl, human Placental Alkaline Phosphatase (hPLAP) is co-expressed
to serve as a
histological marker of transgene expression.
Expression of Grgl in the mice can be widespread, tissue specific, or
temporally-controlled,
depending on how Cre is introduced 23'32-34. For widespread Grgl expression,
mice are crossed to
the pCX-NLS-Cre mice so that Cre excision occurs at the 4- to 8-cell embryo
stage. For
convenience, these mice are referred to here as Grgl transgenic mice.
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Before Cre Excision
- I i*-hr;-L7wYkJ,00C
kmp
toRNA
After Cre Excision
_ r
4+ tkiatt,-0-Aain Cqz1 IRrs-hPLO A100(
hPLAP
Figure 3: Transgene construct in the Grgl transgenic mice
Grgl tumor phenotype
The Grgl transgenic mice develop lung tumors, beginning at 1 month of age.
Offspring of Grgl
mice that retain the Grgl transgene but lack the Cre transgene have the same
tumor phenotype.
No other phenotype has been observed in the mice, although Grgl is
overexpressed in all tissues.
In addition, no phenotype is observed without Cre excision, when the mice
express fi-geo, and no
phenotype is observed in mice that express hPLAP without Grgl 23, thus the
phenotype is not
associated with the transgene insertion site or the co-expressed histochemical
marker.
At one month, the tumors begin as small foci of proliferative tall columnar
epithelial cells on
preexisting alveolar septae, thus resembling early noninvasive
bronchioloalveolar carcinoma. At
3 to 5 moths, tumor nodules are visible on the pleural surface, and at 5 to 6
months the number
and size of tumors visible on the surface increases substantially. By 8
months, solid tumor
masses replace large portions of the normal lung and appear as invasive
adenocarcinoma with
areas of necrosis. The tumors also display infiltration of lymphocytes and
macrophages.
Histopathological analysis is consistent with human lung mucinous
adenocarcinoma 23.
iii. Molecular changes accompanying the Grgl phenotype
The development of lung tumors in the Grg I trans genie mice is accompanied by
molecular
changes that resemble those seen in human non-small cell lung cancer. At 1
month, when small
hyperplasia are first observed in the lung, p53 protein is dramatically
reduced, hypo-
phosphorylated Rb is also reduced, and ErbB1 and Mdm2 levels are increased.
Subsequently, at
3 months Cyclin Dl and D2 is increased and Rb is decreased. After 3 months,
ErbB2 is
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increased in the lung and K-Ras protein is reduced in tumors relative to
adjacent normal lung
tissue. These changes (increased p53 and Rb, reduced ErbBl, ErbB2 and Cyclin
D1, and altered
K-Ras) are also typically observed in human NSCLC.
¨1 wk ____________ 1 mo _________ 3 mo ____________________ 6 mo¨o-
lung-
ErbB1 CyclinDl/D2 ErbB2
Mdm2
t Grgl µ,
tumor
02
hypoP-Rb Rb
Ras
Figure 4: Molecular changes accompanying tumor progression in Grgl mice
p53 mutation: ¨50%
____________________________________ K-ras mutation; ¨30%
At) pical
Normal lung adclotnatous Bronchioalveolar Adenocarcinoma
hyperplasia carcinoma
Loss:
Rh ErbBliEGFR
p53 ErbBIIIER2ineu
PHIT Cyclin DI
p27 c-Myc
TGFct
Figure 5: Typical molecular changes in human non-small lung cancer
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The reduction in p53 protein appears to be largely regulated at the post-
translational level in the
Grgl mice, since the level of p53 mRNA did not have a corresponding decrease.
A possible
mechanism for Grgl regulation of p53 is through recruitment into HDAC
1/Mdm2/p53-
containing complexes. HDAC1 and Mdm2 deacetylate and ubiquitylate p53 on
lysine residues,
which target p53 for degradation in the pi roteasome 35-37. As Groucho
proteins directly bind with
HDAC to deacetylate histone proteins 9,-38, Grgl may also be part of the HDAC1
complex that
deacetylates p53. Indeed, when Grgl and Mdm2 expression constructs were co-
transfected into
NIH 3T3 cells, they cooperatively decreased p53 protein levels 23.
iv. Grg5 transgenic mice
Grg5 transgenic mice were developed in parallel using the same Cre-conditional
expression
system as the Grgl mice 23. Grg5 is the short version of the mouse Gro
proteins, with only the Q
and GP domains. Following Cre excision, mice with widespread Grg5 expression
do not develop
lung tumors.
Although the Grg5 transgenic mice do not develop tumors, they have some of the
molecular
changes observed in the Grgl mice. Most notably they exhibit the reduction in
p53, suggesting a
shared function by long and short Groucho isoforms and demonstrating that
lower p53 levels are
not solely responsible for the lung tumor phenotype of Grgl mice.
v. Grgl mice for drug testing
The Grgl transgenic mice represent an ideal model to test the effectiveness
and molecular
mechanisms for HDAC inhibitors in anti-tumor activity. The model is relevant
to human cancer
because Grgl/TLE1 overexpression occurs in a significant number of human non-
small cell lung
cancers. As a Gro protein, Grgl exerts its activity with HDAC, therefore HDAC
inhibitors are
expected to reduce the tumor burden in the Grgl mice. The mice also have a
completely
functional immune system and the tumors develop within the lung over a period
of time,
resembling development of tumors in human cancer and unlike xenograft tumor
models.
The intact immune system of the mice and in situ development of tumors also
make the Grgl
mice an attractive model to test the potential of BTK inhibitors in treating
solid tumors. Further,
if BTK inhibitors are effective in reducing tumor burden in the Grgl mice, it
could have an
important application in treating non-small cell lung cancer, for which there
is a paucity of
effective treatments.
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4. Methods
a. Mice
The experiments were carried out at the Toronto Centre for Phenogenomics,
which is a
pathogen-free facility. Mice were housed and treated following the applicable
Standard
Operating Procedures and according to the Canadian Animal Care Committee
standards.
Mice were bred to generate double transgenic (Grgl/Cre recombinase) mice on a
CD1
background. Four groups of female double transgenic mice were established:
Group 1 ¨ Control (no treatment, injection of PBS, or water with carrier and
no drug)
Group 2 ¨ Abexinostat¨treated
Group 3 ¨ Ibrutinib¨treated
Group 4 ¨ Abexinostat + Ibrutinib¨treated
At least 8 mice in each group were treated at 2 months old and 8 mice in each
group were treated
at 5 months old. Treatments lasted 4 weeks, and mice were sacrificed at 3
months and 6 months.
Therefore samples are referred to as 3 month samples or 6 month samples.
The mouse ID numbers, drug treatment, number of tumors, and paraffin block
numbers are listed
in Appendix 1.
b. Drug preparation and dosage
Abexinostat was administered by intraperitoneal (i.p.) injection, twice per
day for five days each
week. Mice were weighed and injected with 6 !,t1 per gram body weight of a 1
mg/ml solution.
The dosage was 12 mg/kg BID.
Ibrutinib was administered by supplying it in the drinking water, for a dosage
of ¨ 22
mg/kg/day.
Mice were treated for 4 weeks. After treatment, mice were euthanized and lung
tissue was
examined.
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Drugs were prepared and administered as follows:
Abexinostat (HDACi, PCI-24781)
Formulation
= Add DMSO to powder stock in vial to make a 200mg/m1 solution. Aliquot out
into 100
!Al. This stock can be stored at -20 C.
= Every two weeks, as required, thaw a 100 pi aliquot and add 900 v,1
sterile water. Aliquot
into 20 p.1 aliquots of 20 mg/ml.
= Store this stock at 4 C.
Dosage
= 20 R1 of 20mg/m1 stock in the fridge
= inject at 1 mg/ml concentration in sterile water.
= On the day of injection, add 380 tl sterile water or PBS and pipet well
to mix thoroughly.
= inject 6 tl per g body weight TWICE DAILY.
Ibrutinib (BTKi, PCI-32765)
Formulation
= 100 ml of the 10x concentrate of Ibrutinib.
= Dilute 1 part of concentrate with 9 parts water.
= Both the 10x and lx dilutions can be stored at room temperature. PCI-
32765 is >99%
stable in this formulation after 6 weeks at room temperature (22 C).
Dosage
= Administered by drinking water
= average consumption is 4 mL/day/mouse, corresponding to a final dose of
¨22
mg/kg/day.
= Thus 4m1 x 4 mice = 16m1/day x 7 days = 140m1 per week
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c. Quantitation
For quantitation of tumors, the total visible tumors on the lung surface were
counted using a
dissecting microscope. In addition, the left lung lobe was fixed in 4% PFA,
paraffin-embedded
and 5 pm sections were taken for 100 p.m at two levels. The first level began
30 m below the
lung surface and the second level began 180 [Am below the lung surface.
Alternate slides were
H&E stained, and tumors were detected and counted using a dissecting
microscope. Tumor
diameter was measured using an inverted microscope and reticule.
The unstained sections and the tissue blocks are available for further
histological analysis.
The right lung lobes were snap-frozen and stored for DNA or protein analysis.
Statistical analysis of the data was performed using one-way ANOVA followed by
multiple
comparison tests. Error bars on graphs indicate the standard deviation from
the mean.
d. Blood samples
For 6 month-old mice, blood samples were collected in EDTA tubes (VWR
8881311149), and
peripheral blood mononuclear cells (PBMC) and plasma were prepared, frozen and
stored as
follows:
I. A minimum of 500 IAL of blood is drawn into the EDTA tube
2. Transfer the blood into 1.5 mL eppendort tubes and centrifuge at 350 x g
for 5 minutes to
separate the plasma and the cells.
3. Save the supernatant/plasma.
4. Add 1 ml of IX RBC Lysis Buffer (Sigma #R7757) to the pellet
5. Gently vortex each tube immediately after adding the lysing solution.
6. Incubate at room temperature, protected from light, for 5 minutes.
7. Centrifuge 350 x g for 2 minutes.
8. Aspirate supernatant without disturbing pellet.
9. Wash the pellet once with 1 mL of PBS to the sample and centrifuge 350 x
g for 2
minutes
10. Aspirate supernatant without disturbing the pellet.
11. Freeze the PBMC pellet at -80C
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5. Results
a. Three month old
The average number of surface tumors visible at 3 months, after 4 weeks of
treatment, is shown
in Figure 6. For the control group, the average number of tumors was 5.9
(n=10). For the
Abexinostat-treated mice, the average tumor number was 3.0 (n=10), for
Ibrutinib it was 2.6
(n=8), and for treatment with both drugs 1.4 (n=11).
la
110_1
E 5
0 IVO Mil
oi1/4.4% 41/4.
+ %
cf =O'
VP:"
Treatment
Figure 6: Number of surface tumors per mouse at 3 months old
The tumors visible on the lung surface are shown in Figure 7. Tumors in the 3
month samples
were further analyzed for four mice in each treatment group by sectioning the
left lung lobe and
H&E staining alternate slides. The tumor histology is presented in Figure 8.
The tumors are carcinoma that are less than 400 Rm. The tumor histology
appears similar in
treated and control mice. Tumors in Abexinostat-treated mice had some visible
apoptotic cells.
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b. Six month old
The average number of surface tumors visible at 6 months, after 4 weeks of
treatment, is shown
in Figure 9. For the control group, the average number of tumors was 7.8
(n=8). For the
Abexinostat-treated mice, the average tumor number was 4.6 (n=8), for
Ibrutinib it was 7.6
(n=5), and for treatment with both drugs 8.4 (n=5). Note that fewer mice are
in the Ibrutinib
group at 6 months because of a shortage of drug for one set of mice, therefore
there are 5 mice
rather than 8 in the Ibrutinib groups.
20-
15-
15 10- Iljill 11'111
m 5-
z
0
.c!6>
o
veS
Treatment
Figure 9: Number of surface tumors at 6 months old
The number of visible surface tumors in the 6 month-old mice was quite
variable and did not
show a significant difference between treatment groups.
The tumors were further analyzed by sectioning the left lung lobe and H&E
staining alternate
slides. To measure and compare the tumor load, the tumor diameter in the
tissue sections was
measured.
The tumor diameters for each mouse in each treatment group are shown in Figure
10A. Each
column in the table shows the range of tumor sizes for one mouse. The range in
tumor diameter
appears higher in the untreated mice.
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The treated mice had fewer large tumors than the control mice. The average
number of tumors
over 1 mm in diameter per mouse is shown in Figure 11. Control mice had an
average of 1 large
tumor per mouse, Abexinostat-, Ibrutinib- and Abexinostat+Ibrutinib-treated
mice had an
average of 0.2 large tumors per mouse.
E 2.
OET
0
VI I
1.0
o.
0.0 ____________________________________________________
'1/4" = C%.4;1 C4µ
%)=
CIOC%.4%
e+
s?'
Treatment
Figure 11: Number of tumors over 1 mm in 6 month samples
The average number of large tumors per mouse for each treatment group is
shown.
Images of the whole left lung lobe from control and treated mice are shown in
Figure 12 and the
tissue sections showing the tumor histology are shown in Figure 13.
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6. Conclusions
The data analysis reveals that both Abexinostat and Ibrutinib greatly reduce
tumor number in
mice treated from 2 to 3 months and the drugs appear to act synergistically.
The reduction was
50-60% for each drug alone and 75% when the drugs were used together. Although
the drugs
reduce tumor number, the tumors appear morphologically similar between treated
and control
samples at 3 months.
At 6 months, the tumor number is variable, but in treated mice the tumor load
and the number of
large tumors (over 1 mm) is much lower. Tumors in mice receiving Abexinostat
have a high
number of apoptotic cells and large areas of tumor cell death. The tumors for
control and treated
mice have lymphocyte infiltration, but tumors in mice receiving Ibrutinib lack
the high number
of apoptotic macrophages seen in the control and Abexinostat-treated mice.
The data supports that Abexinostat induces apoptosis in the tumor while
Ibrutinib inhibits
macrophage and/or other immune cell infiltration, thus delaying tumor growth.
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7. References
1 Jennings, B. H. & Ish-Horowicz, D. The Groucho/TLE/Grg family of
transcriptional
co-repressors. Genome biology 9, 205, doi:10.1186/gb-2008-9-1-205 (2008).
2 Paroush, Z. et aL Groucho is required for Drosophila neurogenesis,
segmentation, and
sex determination and interacts directly with hairy-related bHLH proteins.
Cell 79,
805-815 (1994).
3 Koop, K. E., MacDonald, L. M. & Lobe, C. G. Transcripts of Grg4, a murine
groucho-
related gene, are detected in adjacent tissues to other murine neurogenic gene
homologues during embryonic development. Mechanisms of development 59, 73-87,
doi:0925477396005825 [pH] (1996).
4 Chen, G. & Courey, A. J. Groucho/TLE family proteins and transcriptional
repression. Gene 249, 1-16 (2000).
Pinto, M. & Lobe, C. G. Products of the grg (Groucho-related gene) family can
dimerize through the amino-terminal Q domain. The Journal of biological
chemistry
271, 33026-33031 (1996).
6 Chen, G., Nguyen, P. H. & Courey, A. J. A role for Groucho
tetramerization in
transcriptional repression. Molecular and cellular biology 18, 7259-7268
(1998).
7 Daniels, D. L. & Weis, W. I. Beta-catenin directly displaces Groucho/TLE
repressors
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22 Sekiya, T. & Zaret, K. S. Repression by Groucho/TLE/Grg proteins: genomic
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32 Novak, A., Guo, C., Yang, W., Nagy, A. & Lobe, C. G. Z/EG, a double
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=-..
Appendix 1: 3 month samples - Mouse ID and tumor numbers
Treatment Cage Mouse ID surface tumors paraffin blk # # of
tumors in
sections
3 month samples
control 2374913 Z923 11 12 6
control 2374984 z907 4 15 2
control 2374984 z942 7 16 7
control 2521646 A944 8 31
control 2674290 C731 5 49
control 2674290 C737 5 50 5
control 2674290 C749 13 51
control 2674300 C741 2 52
control 2674300 C743 1 53
control 2674300 C745 3 54
.. .
Abexinostat 2405404 ' - z804 ' '1 . 23 bad
histology
Abexinostat 2405404 z806... 1 24 5
Abexinostat 2409770 z831 4 25
Abexinostat 2409770 z834 2 '' - 26
=
Abexinostat 2445448 A935 ' 5 32
Abexinostat 2445448 A937 8 33
Abexinostat 2445448 A938 1 34 3
Abexinostat 2445448 A930 2 35 1
Abexinostat 2674371 C760 2 55
- Abexinostat 2674371 C788 4 56
Ibrutinib 2408949 z813 1 27
lbrutinib 2408949 z826 3 28
-'''''-. ',7',..' 'f.A=*`1,7,-= :1,A
Ibrutinib 2408949 z827 3 29 2
Ibrutinib 2408949 z982 1 30 2
Ibrutinib 2486455 A803 5 40
Ibrutinib 2486455 A804 2 41
Ibrutinib 2488073 A826 3 42 2
Ibrutirub 2488073 A828 3 43 1 ,
Abex + !brut 2456288 A901 ' " ' 1 , ' . ' 36 , .,
,.= -;-,, ,,,,:õ ,t, ;4,::
Abex + 'brut 2456288 A903 2 37
Abex + Ibrut 2466288 A933 1 38 , - -
. = = = ..-..';''''.- '-'' r-'
Abex + Ibrut 2456288 A922 0 39
Abu + Ibrut 2507926 A818 0 44 1
'
Abex + Ibrut 2488087 A812 3 46 2
-
Abex + [brut 2683966 C769 1 57 5
Abex + !brut 2683966 C782 0 . . . ' ' 58
Abex + !brut 2683966 C750 1 59
., .
Abex + Ibrut 2684010 D902 4 ' ,Nri.:-,'=--- _.:- = ,.., 62
...-:---d, f--ke, =-,-;., =- - ¨,,,,,,,,,_,,-_,--- ,
Abex + 'brut 2684010 D903
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Appendix 2: 6 month samples - Mouse ID and tumor numbers
Treatment Cage Mouse ID surface tumors paraffin blk # *of
tumors in
sections
6 month samples
injection 2556548 , b039 ++,..-;-iihni.f.- .,-
.10 206
. . .
control
lilt. _:;,r
40.4i. .4..?....,.,.... -
injection 2556548 .-.1.-1..p7., b4(L)41 ....?..= µ.9.1.1.Jr
..;VA. si 207 3 large 1 med
control Ilili',.4:' T.*'4'7':;3441r,2k;": l'''
4_!'t4'..*4.:.1A0iyili.õ0,,,,,ci
control 2556548 ,..! b046 N.::ttl:....;1-
`,.;,..õ4`, 247 ....µ47.= *. - 208
control 255654811'111! b025õailf=!'...iil- ,,J .,,
..,,, 209 3 small
water , 2575924,1, ...,. 438 Er ,,- r:rjr:frili :47
7,07i 04-310
,-- 1 large
e=¨ 32: s4=1mal?.l
control ,,1
water 2575924 b388wt*13 41*
= '
\ oio 1T 0 O-011,11
= ¨ - '''' '
control
water
control
2575924 b389 '-'k ' .,.-4- Cft., ? .,--...-
4,,.'" f. ,='i:.2,¨.4 :, ' - '=; '. ,. -.`..,'-,. --,.;... .'.. ' '.'-
.''.p:->'.i'.7 'õ1 - '. '-= = i igv: -A '.'µ'IN, .. r--,1-..'1 4 ''
F'o',j '= 1 :-'. ' ,?...J:.-.-:V 'i . l .. -:n-r-
water 2575924 4-B088 n ft,nVt
-
,
control i
22i "ir J111,1,;11111114bi_14
. Abexinostat 2468143 A847 0 = 7.,i,16...., 2;
,-... . ,T. ",- '
, , ,
Abexinostat 2488143 A850 6 , :"$t 'AV ,- .g2:=;`4,,,
7.,õ rfi,,P ,. our
Abexinostat . 2488143 A851 1922 234.',.tf
Abexinostat .2488143 A888 . 4 ' '4- ' ;"-- -= - '
=-=t ... --- .' ...-
i= ,÷, - 4: &47-.- \ = ....:. =-=-: = -A':
Abexinostat ., 2486143 A889 .4', . --..µ 1 õ,, ei-i
2õ , z ,,- ., =
... -.
. , _.
Abexinostat , 2589771 8866 ,,-..,g, 0.,,,w,..-,..
214 1 small
..
Abextnosiator 2589771 8869 ' A ;', . 6 ,.÷ , ---,./
'',.'''r- ' - 215 1 large, 3 rned, i
. , - small
Abexinostat 2589771 8870 1 St-i:..,1E 216 '. .
Ibrutinib 2575768 b378 23 217
Ibrutinib 2575768 b373 4 221
Ibrutinib 2575768 b809 0 219 0
Ibrutinib 2575768 b812 3 220
lbrutinib 2575768 b813 8 218 1 large
' .
:.=-= :222:;,-''''',:''kl.,444.1-,ts.,',,,i,;...1t4".t1Mtf,-,
toikbex.+ lbrut 2675866 b833 5 - -,,. .,..= -
.''' M 4' . ,* .t..
4 -
.04Abex + (brut 2575866 b835 10 -.,..'s...
tAbex + 'brut 2575866 b854 3 µ,' ,:-
'. 1, .,,,,:, sf . 224 3 VI ,It4-.., k,.--et,.:
- .
Abex 4- ibrut 2575866 b355 13 -''' "' '
'`kr225 5 45141,110.1,
- Abex + Ibrut 2575866 b3594,%,. 11
-.-4k.*V1,-:4-'-"tltsz0\-,,,----;,t4*',-µ ' - --s- , -
- - ...
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=
CONFIDENTIAL
Activity of BTK and HDAC inhibitors alone and in combination in a mouse model
of non-
small cell lung cancer
Background
= The Grgl mouse model is a transgenic mouse line that overexpresses the
Groucho-related
gene (Grgl; TLE I in humans) and develops tumors that resemble human non-small
cell lung
cancer.
o The tumors initiate at 1 month of age, progress to bronchioalveolar
carcinoma, and
continue to invasive adenocarcinoma by 8 months (Figure 1).
o TLE I is over-expressed in approximately 20% of human non-small cell lung
cancers.
o Grgl/TLE I interacts with histone deacetylase complex (HDAC) molecules,
to modify
histones and inhibit transcription, and to deacetylate proteins and target
them for
degradation.
= Both the histone deacetylase inhibitor abexinostat (HDACi; PCI-24781)23
and the Bruton's
Tyrosine Kinase inhibitor ibrutinib (BTKi; PC1-32765)4's have shown promising
activity as
single agents in hematologic malignancies in the clinic.
= Using the Grgl mouse model, we tested abexinostat to determine if it was
effective against
the Grgl/HDAC function in lung tumor growth, and ibrutinib to determine if
this drug had
activity in vivo in solid tumors, even those not known to express BTK, and if
so to further
determine its mechanism of action.
Objective
= To test the effectiveness of two drugs, an HDAC inhibitor and a BTK
inhibitor, in a mouse
model for non-small cell lung cancer (NSCLC).
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analysis and the number of tumors in 5 urn sections over two levels of 100 um
was
determined. Tumors in the sections were counted and tumor diameter was
measured.
= Tumor sections were immunostained to visualize B cells using anti-B220
antibody, T cells
using anti-CD3 antibody, macrophages using anti-F4/80 antibody and apoptosis
using anti-
Caspase3 antibody.
Figure 2. Treatment protocol
Age of mice (mos): 0 1 2 3 4 S 6 7 8
Early intervention:
Measure tumors
PIIMINSMINOMMEIMMOWAIO
2 3 Histology
!HC
Late intervention: Cytokines
6
Mice were treated with abexinostat, ibrutinib, both drugs or vehicle for 4
weeks, beginning either
at 2 months of age (early intervention) or at 5 months of age (late
intervention). At the end of
treatment, tumors were quantitated and characterized.
Results
Early Intervention
= Treatment at the early time interval reduced tumor number for each drug
= In the 3-month-old cohort (Figure 3), the average number of surface
visible tumors in
untreated mice was 5.9 (n=10), BTKi-treated mice was 2.6 (n--8), HDACi-treated
mice
was 2.9 (n=9), and BTKi and HDACi-treated mice was 1.4 (n=11).
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c-'
Left lung lobe (left column) and tumor sections at 50X (center column) and
400X (right column)
are shown for control mice, abexinostat-treated mice, ibutinib-treated mice,
and abexinostat +
ibrutinib-treated mice. Small nodes on the lung pleura and in the tissue
sections are visible, as
indicated by arrows
Late Intervention
Figure 5. Late treatment reduces the number of large tumors
(a) (b)
20- E 2.
E
e
>
E o
2
z
i 1 -5-0 i illill if i Lo. 1.
o
1..
.g 0.
E
0.0
e =
4, c4` = =
0 **1 =
.)
c
i)cµ
cje .4,o+ cse
v-e) cf' 4 if
+ 4' =.0
ir= .0 ii-
1,-
Treatment Treatment
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Sections of large tumors from each drug treatment were stained for B and T
cells and caspase 3
(an apoptotic cell marker). All panels are 400X. Arrowheads indicate
macrophages lightly
stained for caspase 3 (yellow), darkly stained (orange) or dead (red), as they
progress through
apoptosis.
= Ibrutinib-treated tumors did not have a significant number of visible
macrophages in the
tumors (Figure 7).
Figure 8. Abexinostat increases tumor cell death
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= Abexinostat-treated and abexinostat+ibrutinib-treated tumors had larger
areas of cell
death and more apoptotic cells than control and ibrutinib-treated tumors
(Figure 8).
Therefore abexinostat treatment appears to induce tumor cell death.
Figure 9. Ibrutinib-treatment reduces tumor macrophage infiltration
(a) (b)
III Control Number of macrophage vs tumor size
Abexinostat 15,
2
.0 10 T IN Ibrutinib
E MN Abex/Ibrut
2 10. = *
=
=
500t . . . .
50
it of cf,b e to"
4 4 4 4
=e 0 500 1000 1500
Treatment and tUITIOr size tumor size
(a) The number of visible apoptotic macrophage in tumor sections was counted.
Tumors were
grouped by treatment and by size (small = under 1 mm, large = over 1 mm). The
average number
of macrophages for each group is shown. (b) The number of macrophages in each
tumor,
according to tumor size. As tumors increase in size, macrophage infiltration
was observed in
control and abexinostat-treated tumors, but not for ibrutinib- and
abexinostat+ibrutinib-treated
tumors.
= Large tumors in the control and abexinostat-treated group had a high
number of apoptotic
macrophage, whereas ibrutinib- and abexinostat+ibrutinib treated tumors did
not (Figure 9).
Thus ibrutinib appears to inhibit macrophage infiltration that would normally
occur in large
tumors.
Conclusions
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= NSCLC tumors and other solid tumors that over-express Grgl may respond to
treatment with
HDACi or BTKi by different mechanisms.
= The combination of abexinostat with ibrutinib is more active than either
agent alone for early
treatment.
= Interestingly, even solid tumors, such as lung cancer, may respond to
immunomodulatory
drugs such as BTK inhibitors by modulating infiltrating immune cells.
= Abexinostat and ibrutinib thus may have potential as a combination for
treating solid tumors.
References
1. Allen T, van Tuyl M, lyengar P et al. Cancer Res. 2006;66:1294-301
2. Morschhauser F, Ten-iou L, Coiffier B, et al. Abexinostat (S78454/PCI-
24781), an Oral
Pan-Histone Deacetylas (HDAC) Inhibitor in Patients with Refractory or
Relapsed
Hodgkin's Lymphoma, Non-Hodgkin Lymphoma and Chronic Lymphocytic Leukemia.
Results of a Phase I Dose-Escalation Study in 35 Patients. ASH Annual Meeting
Abstracts.2012:120:3643
3. Evens AM, Vose JM, Harb W et at: A Phase II Multicenter Study of the
Histone
Deacetylase Inhibitor (HDACi) Abexinostat (PCI-24781) in Relapsed/Refractory
Follicular Lymphoma (FL) and Mantle Cell Lymphoma (MCL). ASH Annual Meeting
Abstracts.2012;120:552012
4. Byrd JC, Furman RR, Coutre SE, et at. N Engl JMed.2013;369:32-42
5. Wang ML, Rule S, Martin P, et al. N Engl J Med. 2013;369:507-16
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