CA2829669A1 - Btk and hdac inhibitors to treat non-hematologic cancers - Google Patents
Btk and hdac inhibitors to treat non-hematologic cancers Download PDFInfo
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- CA2829669A1 CA2829669A1 CA 2829669 CA2829669A CA2829669A1 CA 2829669 A1 CA2829669 A1 CA 2829669A1 CA 2829669 CA2829669 CA 2829669 CA 2829669 A CA2829669 A CA 2829669A CA 2829669 A1 CA2829669 A1 CA 2829669A1
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
- A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
Description
i ) Application number / Numero de demande:
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_________________________________________________________________ --Pages: 5115 )-11) 35) 3-q __ p-Pq ,6139 4-16 ) q) 35 Isq _ , -7-)-561--.
, (-.-attboills in cDoif)'3 ' i (-) if Unscannable items reciciec vit 1 this application (Request original documents in File Prep. Section on the 10th floor) Documents rccu avec cette dcmande lie pouvant atre balayos (Commander les documents originaux clans la section de la preparation des dossiers au I Oieme etage) MIAMI MICE eTh CONFIDENTIAL
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 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MIAMI MICE cm---\ CON F IDE NTIAL
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 Page 2 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MIAMI MICE
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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.
Page 3 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MIAMI MICE CONFIDENTIAL
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 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.
Page 4 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MIAMI MICE er) CONFIDENTIAL
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 Page 6 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MIAMI MICE r), CONFIDENTIAL
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 Page 8 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MIAMI MICE CONFIDENTIAL
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 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 Page 9 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 tY.,01 r CONFIDENTIAL
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 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.
Page 11 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MIAMI w 167.
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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 Page 12 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 Li e - =
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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.
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_________________________________________________________________ --Pages: 5115 )-11) 35) 3-q __ p-Pq ,6139 4-16 ) q) 35 Isq _ , -7-)-561--.
, (-.-attboills in cDoif)'3 ' i (-) if Unscannable items reciciec vit 1 this application (Request original documents in File Prep. Section on the 10th floor) Documents rccu avec cette dcmande lie pouvant atre balayos (Commander les documents originaux clans la section de la preparation des dossiers au I Oieme etage) MIAMI MICE eTh CONFIDENTIAL
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 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MIAMI MICE cm---\ CON F IDE NTIAL
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 Page 2 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MIAMI MICE
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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 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 Page 6 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MIAMI MICE r), CONFIDENTIAL
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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= agg CONFIDENTIAL
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 Page 8 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MIAMI MICE CONFIDENTIAL
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 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 Page 9 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 tY.,01 r CONFIDENTIAL
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 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 Page 12 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 Li e - =
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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 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.
15 10- Iljill 11'111 m 5-z .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
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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MIAMI =6µ CONFIDENTIAL
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 from Tcf/Lef in Wnt-mediated transcription activation. Nature structural &
molecular biology 12, 364-371, doi:10.1038/nsmb912 (2005).
8 Orian, A. et al. A Myc-Groucho complex integrates EGF and Notch signaling to regulate neural development. Proceedings of the National Academy of Sciences of the United States of America 104, 15771-15776, doi:10.1073/pnas.0707418104 (2007).
9 Chen, G., Fernandez, J., Mische, S. & Courey, A. J. A functional interaction between the histone deacetylase Rpd3 and the corepressor groucho in Drosophila development.
Genes & development 13, 2218-2230 (1999).
Stifani, S., Blaumueller, C. M., Redhead, N. J., Hill, R. E. & Artavanis-Tsakonas, S.
Human homologs of a Drosophila Enhancer of split gene product define a novel family of nuclear proteins. Nature genetics 2,343, doi:10.1038/ng1292-343a (1992).
Page 23 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel. 416-712-3215 CONFIDENTIAL
11 Pickles, L. M., Roe, S. M., Hemingway, E. J., Stifani, S. & Pearl, L. H.
Crystal structure of the C-terminal WD40 repeat domain of the human Groucho/TLE1 transcriptional corepressor. Structure 10, 751-761 (2002).
Page 13 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MIAMI MI c CONFIDENTIAL
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 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.
15 10- Iljill 11'111 m 5-z .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.
Page 17 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 " ev"Th CONFIDENTIAL
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
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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MIAMI =6µ CONFIDENTIAL
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.
Page 22 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 ME CONFIDENTIAL
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 from Tcf/Lef in Wnt-mediated transcription activation. Nature structural &
molecular biology 12, 364-371, doi:10.1038/nsmb912 (2005).
8 Orian, A. et al. A Myc-Groucho complex integrates EGF and Notch signaling to regulate neural development. Proceedings of the National Academy of Sciences of the United States of America 104, 15771-15776, doi:10.1073/pnas.0707418104 (2007).
9 Chen, G., Fernandez, J., Mische, S. & Courey, A. J. A functional interaction between the histone deacetylase Rpd3 and the corepressor groucho in Drosophila development.
Genes & development 13, 2218-2230 (1999).
Stifani, S., Blaumueller, C. M., Redhead, N. J., Hill, R. E. & Artavanis-Tsakonas, S.
Human homologs of a Drosophila Enhancer of split gene product define a novel family of nuclear proteins. Nature genetics 2,343, doi:10.1038/ng1292-343a (1992).
Page 23 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel. 416-712-3215 CONFIDENTIAL
11 Pickles, L. M., Roe, S. M., Hemingway, E. J., Stifani, S. & Pearl, L. H.
Crystal structure of the C-terminal WD40 repeat domain of the human Groucho/TLE1 transcriptional corepressor. Structure 10, 751-761 (2002).
12 Mallo, M., Franco del Amo, F. & Gridley, T. Cloning and developmental expression of Grg, a mouse gene related to the groucho transcript of the Drosophila Enhancer of split complex. Mechanisms of development 42, 67-76 (1993).
13 Miyasaka, H., Choudhury, B. K., Hou, E. W. & Li, S. S. Molecular cloning and expression of mouse and human cDNA encoding AES and ESG proteins with strong similarity to Drosophila enhancer of split groucho protein. European journal of biochemistry / FEBS 216, 343-352 (1993).
14 Marcal, N. et al. Antagonistic effects of Grg6 and Groucho/TLE on the transcription repression activity of brain factor 1/FoxG1 and cortical neuron differentiation.
Molecular and cellular biology 25, 10916-10929, doi:10.1128/MCB.25.24.10916-10929.2005 (2005).
Molecular and cellular biology 25, 10916-10929, doi:10.1128/MCB.25.24.10916-10929.2005 (2005).
15 Chen, P. C. et aL Novel roles for MLH3 deficiency and TLE6-like amplification in DNA
mismatch repair-deficient gastrointestinal tumorigenesis and progression. PLoS
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mismatch repair-deficient gastrointestinal tumorigenesis and progression. PLoS
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16 Schrons, H., Knust, E. & Campos-Ortega, J. A. The Enhancer of split complex and adjacent genes in the 96F region of Drosophila melanogaster are required for segregation of neural and epidermal progenitor cells. Genetics 132, 481-503 (1992).
17 Eberhard, D., Jimenez, G., Heavey, B. & Busslinger, M. Transcriptional repression by Pax5 (BSAP) through interaction with corepressors of the Grouch() family. The EMBO
journal 19, 2292-2303, doi:10.1093/emboj/19.10.2292 (2000).
journal 19, 2292-2303, doi:10.1093/emboj/19.10.2292 (2000).
18 Hasson, P. et aL EGFR signaling attenuates Groucho-dependent repression to antagonize Notch transcriptional output. Nature genetics 37, 101-105, doi:10.1038/ng1486 (2005).
19 Courey, A. J. & Jia, S. Transcriptional repression: the long and the short of it. Genes &
development 15, 2786-2796, doi:10.1101/gad.939601 (2001).
development 15, 2786-2796, doi:10.1101/gad.939601 (2001).
20 Fisher, A. L. & Caudy, M. Groucho proteins: transcriptional corepressors for specific subsets of DNA-binding transcription factors in vertebrates and invertebrates.
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development 12, 1931-1940 (1998).
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development 12, 1931-1940 (1998).
21 Flores-Saaib, R. D. & Courey, A. J. Analysis of Groucho-histone interactions suggests mechanistic similarities between Groucho- and Tupl-mediated repression.
Nucleic acids research 28, 4189-4196 (2000).
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Nucleic acids research 28, 4189-4196 (2000).
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22 Sekiya, T. & Zaret, K. S. Repression by Groucho/TLE/Grg proteins: genomic site recruitment generates compacted chromatin in vitro and impairs activator binding in vivo. Molecular cell 28, 291-303, doi:10.1016/j.molce1.2007.10.002 (2007).
23 Allen, T. et al. Grgl acts as a lung-specific oncogene in a transgenic mouse model.
Cancer research 66, 1294-1301, doi:66/3/1294 [pH] 10.1158/0008-5472.CAN-05-(2006).
Cancer research 66, 1294-1301, doi:66/3/1294 [pH] 10.1158/0008-5472.CAN-05-(2006).
24 Sadoul, K., Boyault, C., Pabion, M. & Khochbin, S. Regulation of protein turnover by acetyltransferases and deacetylases. Biochimie 90, 306-312, doi:10.1016/j.biochi.2007.06.009 (2008).
25 Beier, U. H., Akimova, T., Liu, Y., Wang, L. & Hancock, W. W.
Histone/protein deacetylases control Foxp3 expression and the heat shock response of T-regulatory cells. Current opinion in immunology 23, 670-678, doi:10.1016/j.coi.2011.07.002 (2011).
Histone/protein deacetylases control Foxp3 expression and the heat shock response of T-regulatory cells. Current opinion in immunology 23, 670-678, doi:10.1016/j.coi.2011.07.002 (2011).
26 Rorive, S. et al. Exploring the distinctive biological characteristics of pilocytic and low-grade diffuse astrocytomas using microarray gene expression profiles. Journal of neuropathology and experimental neurology 65, 794-807, doi:10.1097/01.jnen.0000228203.12292.al (2006).
27 Cuevas, 1. C. et a/. Meningioma transcript profiles reveal deregulated Notch signaling pathway. Cancer research 65, 5070-5075, doi:10.1158/0008-5472.CAN-05-0240 (2005).
28 Moreno, C. S. et al. Novel molecular signaling and classification of human clinically nonfunctional pituitary adenomas identified by gene expression profiling and proteomic analyses. Cancer research 65, 10214-10222, doi:10.1158/0008-5472.CAN-0884 (2005).
29 Ruebel, K. H. et al. Patterns of gene expression in pituitary carcinomas and adenomas analyzed by high-density oligonucleotide arrays, reverse transcriptase-quantitative PCR, and protein expression. Endocrine 29, 435-444, doi:10.1385/ENDO:29:3:435 (2006).
30 Terry, J. et al. TLE1 as a diagnostic immunohistochemical marker for synovial sarcoma emerging from gene expression profiling studies. The American journal of surgical pathology 31, 240-246, doi:10.1097/01.pas.0000213330.71745.39 (2007).
31 Lobe, C. G. et aL Z/AP, a double reporter for cre-mediated recombination.
Developmental biology 208, 281-292, doi:S0012-1606(99)99209-6 [pill 10.1006/dbio.1999.9209 (1999).
Page 25 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel. 416-712-3215 MIAMI MICE C ' CONFIDENTIAL
Developmental biology 208, 281-292, doi:S0012-1606(99)99209-6 [pill 10.1006/dbio.1999.9209 (1999).
Page 25 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel. 416-712-3215 MIAMI MICE C ' CONFIDENTIAL
32 Novak, A., Guo, C., Yang, W., Nagy, A. & Lobe, C. G. Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision.
Genesis 28, 147-155, doi:10.1002/1526-968X(200011/12)28:3/4<147::AID-GENE90>3Ø00;2-G [pH] (2000).
Genesis 28, 147-155, doi:10.1002/1526-968X(200011/12)28:3/4<147::AID-GENE90>3Ø00;2-G [pH] (2000).
33 Liu, J. & Lobe, C. G. Cre-conditional expression of constitutively active Notchl in transgenic mice. Genesis 45, 259-265, doi:10.1002/dvg.20282 (2007).
34 Guo, C., Yang, W. & Lobe, C. G. A Cre recombinase transgene with mosaic, widespread tamoxifen-inducible action. Genesis 32, 8-18, doi:10.1002/gene.10021 Ipiil (2002).
35 Brooks, C. L. & Gu, W. Ubiquitination, phosphorylation and acetylation: the molecular basis for p53 regulation. Current opinion in cell biology 15, 164-171 (2003).
36 Ito, A. et aL MDM2-HDAC1-mediated deacetylation of p53 is required for its degradation. The EMBO journal 21, 6236-6245 (2002).
37 Nakamura, S., Roth, J. A. & Mukhopadhyay, T. Multiple lysine mutations in the C-terminal domain of p53 interfere with MDM2-dependent protein degradation and ubiquitination. Molecular and cellular biology 20, 9391-9398 (2000).
38 Brantjes, H., Roose, J., van De Wetering, M. & Clevers, H. All Tcf HMG box transcription factors interact with Groucho-related co-repressors. Nucleic acids research 29, 1410-1419 (2001).
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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 Page 27 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MIAMI MICE e)--- CONFIDENTIAL
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..
.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- , -- - ...
Page 28 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 =
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 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).
Page 31 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 1 MIC=E er- CONFIDENTIAL
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 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 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MIAMI MICE en_. CONFIDENTIAL
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 Page 36 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MI MICE e(-- CONFIDENTIAL
= 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, .0 10 T IN Ibrutinib E MN Abex/Ibrut 2 10. = *
=
=
500t . . . .
it of cf,b e to"
=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 Page 38 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M50 1L7 Tel: 416-712-3215 eTh CONFIDENTIAL
= 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 Page 39 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215
Page 26 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MIAMI MICE e7_, CONFIDENTIAL
=-..
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 Page 27 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MIAMI MICE e)--- CONFIDENTIAL
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..
.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- , -- - ...
Page 28 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 =
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).
Page 29 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 4t: CONFIDENTIAL
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 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).
Page 31 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 1 MIC=E er- CONFIDENTIAL
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.
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E o z i 1 -5-0 i illill if i Lo. 1.
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cje .4,o+ cse v-e) cf' 4 if + 4' =.0 ir= .0 ii-1,-Treatment Treatment Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MIAMI MICE en_. CONFIDENTIAL
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 Page 36 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215 MI MICE e(-- CONFIDENTIAL
= 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, .0 10 T IN Ibrutinib E MN Abex/Ibrut 2 10. = *
=
=
500t . . . .
it of cf,b e to"
=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 Page 38 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M50 1L7 Tel: 416-712-3215 eTh CONFIDENTIAL
= 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 Page 39 of 39 Miami Mice Research Corp MaRS Centre, 101 College Street, Suite120-E, Toronto, Ontario M5G 1L7 Tel: 416-712-3215
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015188738A1 (en) * | 2014-06-11 | 2015-12-17 | 中国科学院合肥物质科学研究院 | New use of ibrutinib |
| US20170175125A1 (en) * | 2014-03-28 | 2017-06-22 | Board Of Regents, The University Of Texas System | Inhibition of Bruton's Tyrosine Kinase (Btk) in the Lung to Treat Severe Lung Inflammation and Lung Injury |
| CN109305944A (en) * | 2017-07-28 | 2019-02-05 | 深圳睿熙生物科技有限公司 | The inhibitor of bruton's tyrosine kinase |
| CN115554301A (en) * | 2022-10-24 | 2023-01-03 | 徐诺药业(南京)有限公司 | Use of an HDAC inhibitor and ibrutinib for the preparation of a medicament for the prevention or treatment of mantle cell lymphoma |
-
2013
- 2013-10-07 CA CA 2829669 patent/CA2829669A1/en not_active Abandoned
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170175125A1 (en) * | 2014-03-28 | 2017-06-22 | Board Of Regents, The University Of Texas System | Inhibition of Bruton's Tyrosine Kinase (Btk) in the Lung to Treat Severe Lung Inflammation and Lung Injury |
| US9982265B2 (en) * | 2014-03-28 | 2018-05-29 | Board Of Regents, The University Of Texas System | Inhibition of Bruton's tyrosine kinase (Btk) in the lung to treat severe lung inflammation and lung injury |
| WO2015188738A1 (en) * | 2014-06-11 | 2015-12-17 | 中国科学院合肥物质科学研究院 | New use of ibrutinib |
| CN109305944A (en) * | 2017-07-28 | 2019-02-05 | 深圳睿熙生物科技有限公司 | The inhibitor of bruton's tyrosine kinase |
| CN109305944B (en) * | 2017-07-28 | 2022-09-02 | 深圳睿熙生物科技有限公司 | Inhibitors of bruton's tyrosine kinase |
| CN115554301A (en) * | 2022-10-24 | 2023-01-03 | 徐诺药业(南京)有限公司 | Use of an HDAC inhibitor and ibrutinib for the preparation of a medicament for the prevention or treatment of mantle cell lymphoma |
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