ODM-207, a novel BET-bromodomain inhibitor as a therapeutic approach

for the treatment of prostate and breast cancer Mari Björkman 1, Elina Mattila 1, Reetta Riikonen 1, Chandrasekhar Abbineni 2, Mahaboobi Jaleel 2, Sivapriya Marappan 2, Tarja Ikonen 1,

Daniel Nicorici 1, Juha Rantala 3, Susanta Samajdar 2, Murali Ramachandra2 Pekka Kallio 1, Anu Moilanen 1 1Orion Corporation Orion Pharma, Espoo, Finland, 2Aurigene Discovery Technologies Limited, Bangalore, India, 3Misvik Biology, Finland

BET (bromodomain and extraterminal) family proteins (BRD2, BRD3, BRD4, and BRDT) are epigenetic readers that bind to acetylated-lysine residues in histones and recruit protein complexes to promote transcription elongation. In many cancers, BET proteins have been shown to regulate expression of MYC and other oncogenic drivers that are important for cell proliferation and survival. Pharmacologic inhibition of the BET–histone interaction has been shown to result in transcriptional downregulation of a number of oncogenes and inhibition of tumor growth providing a novel strategy for treatment of cancer. ODM-207 is a novel, potent and highly selective BET bromodomain inhibitor with excellent efficacy in preclinical models of prostate and breast cancer as well as in patient-derived tumor cell cultures from various tumor types.

Biochemical activity: Binding of ODM-207 to BRD2 BD1, BRD3 BD1, BRD4 BD1, BRDT BD1 and BRD4 full length recombinant proteins was tested by measuring the displacement of bromodomain/acetylated peptide interaction using biotin conjugated Acetyl-Histone H4 [Lys5,8,12,16] peptide and the TR-FRET assay.

Bromodomain selectivity profiling: Bromodomain selectivity of ODM-207 against 51 bromodomain proteins was tested using the

BromoMELT thermal stability assays in duplicate at 25 mM (Reaction Biology) .

Cells and cell viability assays: VCaP, LNCaP, 22Rv1, MCF-7 and CAMA-1 cell lines were plated on 96-well plates and treated with ODM-207 for 4 days. Growth inhibitory effect of ODM-207 in cell lines was measured using WST-1 Cell Proliferation Assay (Roche). Patient-derived tumor cells were collected from pleural fluid or tumor samples and plated onto 384-well plate. Cells were treated with ODM-207 for 3 days (Misvik Biology). Growth inhibitory effect on patient-derived lymphoma cell lines was measured using CellTiter-Glo® (Promega).

Immunocytochemistry: Cellular senescence was detected by immunolabelling the cells with Anti-Histone H3 (tri methyl K9) antibody (Abcam, ab8688). Expression of Myc was detected by immunolabelling the cells with Anti-Myc antibody [Y69] (Abcam, ab32072). DAPI was used to count the cells for growth inhibitory effect on patient-derived cancer cells and to identify the cells for automated image analysis.

Gene expression analyses: VCaP cells were treated with vehicle control or 1 µM ODM-207 in triplicate for 6 h. Differentially expressed

genes were analyzed by RNA-seq 15M reads/sample (Illumina HiSeq). Enrichment of AR and MYC driven pathways was studied by Gene Set Enrichment Analysis (GSEA, Broad Institute).

Immunoblotting: Cells were treated for 24h with ODM-207. VCaP, LNCaP and 22Rv1 cell samples were immunoblotted with Myc

antibody (Covance, cat# MMS-150P) and MCF-7 and CAMA-1 cells with ERa (Abcam, ab16660) β-actin (Novus Biologicals, NB600-503AF488) antibodies.

22Rv1 xenograft: In efficacy and PK/PD studies, tumors were established by subcutaneous injection of 22Rv1 cells into male nude mice. After initial tumor growth, when the average tumor volume reached 122 mm3 (efficacy study) or 274 mm3 (PK/PD study), oral treatment with ODM-207 30 mg/kg qd, iBET-762 30 mg/kg qd, OTX-015 40 mg/kg qd, enzalutamide 20 mg/kg qd (only efficacy study) or vehicle was initiated and continued for 31 or 5 days, respectively. *, p<0,05. Myc protein expression levels of the tumors were measured using Milliplex MAP Myc Signaling Kit (Millipore Cat#48-602MAG) at the end of both studies.

ODM-207

Is a novel, potent and structurally distinct inhibitor of BET proteins

Inhibits proliferation of prostate cancer cell lines with intact AR

signaling in vitro and in vivo.

• Inhibition of BET proteins leads to induction of senescence and a dose-

dependent reduction of Myc mRNA and protein levels in vitro and in vivo.

Inhibits proliferation of estrogen sensitive breast cancer cell lines

• Inhibition of BET proteins is associated with induction of senescence and a

dose-dependent reduction of ERα protein levels in vitro.

Demonstrates dose-dependent inhibition of cell growth in patient-

derived cancer cells from various tumor types.

Background

Results

Conclusions

Methods

1. ODM-207 is a potent and selective BET bromodomain inhibitor

3. Antiproliferative effects of ODM-207 in breast cancer cell lines 5. Superior inhibition of tumor growth by ODM-207 in 22Rv1 prostate cancer xenograft

2. Antiproliferative effects of ODM-207 in prostate cancer cell lines

a) Growth inhibition of AR positive prostate cancer cells

b) Induction of cellular senescence

4. Antiproliferative effects of ODM-207 in patient-derived cancer cells

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be reproduced without written permission of the authors. These studies were sponsored by Orion Corporation Orion Pharma Poster presented at AACR 2016

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Abstract 4649

c) Downregulation of Myc protein expression

a) Biochemical activity of ODM-207

b) ODM-207 binds selectively BET bromodomain proteins

a) ODM-207 inhibits proliferation of breast cancer cell lines

b) Induction of cellular senescence with ODM-207

c) ODM-207 downregulates the expression of ERα

a) ODM-207 inhibits proliferation of patient-derived cancer cells

b) Induction of cellular senescence in with ODM-207 (BrCa pt2)

H3K9me3, phalloidin , DAPI

Bromodomain IC50 (nM)

BRD4 BD1 116

BRD4 full length 89

BRD3 BD1 86

BRD2 BD1 61

BRDT BD1 89

d) Inhibition of AR and MYC driven pathways in VCaP cells

a) ODM-207 inhibits tumor growth in AR-V7 expressing 22Rv1 xenograft

b) Tumor Myc levels and ODM-207 correlate in in vivo PK/PD study

c) ODM-207 inhibits Myc in in vivo PK/PD study

Enriched gene-set/pathway

Count of Differentially

Expressed Genes

Change

of Direction

P-Value

AR metabolism genes 13 Down 0.011

AR signature (UP) 18 Down >0.0001

AR signature 22 Down 0.001

MYC species independent target signature (51 genes)

40 Down >0.0001

MYC human common targets (80 genes)

61 Down >0.0001

MYC human core targets (73 genes)

57 Down >0.0001

ΔTm

22Rv1

VCaP

LNCaP

*

* ODM-207 vs. Control, p<0.05