![BMC Genomics BioMed - core.ac.uk · FEBS J 2005, 272:5129-5148). Results: ... Mdm2, RPA, CBP/ p300 and CSN5/Jab1 among many other proteins [52]. The C-terminal domain interacts with](https://static.fdocuments.us/doc/165x107/5b40482f7f8b9a2f138d15bb/bmc-genomics-biomed-coreacuk-febs-j-2005-2725129-5148-results-mdm2.jpg)
Abstract # 3991 Novel small molecule inhibitor of p300/CBP ... · Novel small molecule inhibitor of...
1
Novel small molecule inhibitor of p300/CBP down-regulates androgen receptor (AR) and c-Myc for the treatment of prostate cancer and beyond Neil Pegg 1 , Jenny Worthington 2 , Barbara Young 3 , Amy Prosser 3 , Luke Gaughan 4 , Gary Spencer 5 , Tim Somervaille 5 , Julie Burns 6 , Margaret Knowles 6 , Nigel Brooks 1 . 1 CellCentric Ltd, Cambridge UK; 2 Axis Bioservices, Coleraine, UK; 3 Sygnature Discovery, Nottingham UK; 4 Northern Institute for Cancer Research, Newcastle, UK; 5 CRUK Manchester Institute, Manchester, UK; 6 The University of Leeds, UK Abstract # 3991 Cell Line AR status Model CCS1477 Proliferation IC50 mM LNCaP AR-FL Hormone responsive 0.230 LNCaP-AR AR-FL over-expressed CRPC 0.150 VCaP AR-FL, AR-SV CRPC 0.049 22Rv1 AR-FL, AR-SV CRPC 0.096 DU145 AR negative Hormone independent 1.280 PC3 AR negative Hormone independent 1.490 1. CCS1477: Inhibits prostate cancer cell proliferation Conclusions CellCentric Proliferation was measured with a cell viability assay (CyQuant Direct Cell Proliferation or CellTiter Glo) in prostate cancer cells (maintained in 10% FCS) after compound treatment for 72h. Proliferation was measured with a cell viability assay (CellTiter Glo) in panel of 277 cancer cell lines after compound treatment for 7 days. 2. CCS1477 degrades AR-FL, AR-SV & c-Myc protein in 22Rv1 cells: Including downstream genes a) Representative Western analysis of AR-FL, AR-SV (V7) and c-Myc protein in 22Rv1 cells after 24h treatment with CCS1477 6. Protein biomarkers are reduced in 22Rv1 tumour bearing animals treated with CCS1477 for 7 and 28 days 22Rv1 tumour bearing athymic nude mice were treated with CCS1477 by oral gavage, once daily (10/20mg/kg) or once every other day (30mg/kg). Vehicle (5% DMSO:95% methylcellulose [0.5%w/v]) was dosed once daily. 9. CCS1477 inhibits the proliferation of AML cells mediated by G1 cell cycle arrest Introduction 7. Breadth of efficacy screening with CCS1477 demonstrates subsets of cell lines which are sensitive: including prostate cancer and beyond Western analysis of AR-FL, AR-SV, c-Myc in 22Rv1 tumours taken from a satellite group at day 7 of the study shown in Fig. 5 and at the end of the same study at day 28. • Targeted degradation of androgen receptor (AR) and androgen receptor variants (AR-SV) remains an important therapeutic opportunity for patients with castration resistant prostate cancer. • E1A binding protein (p300) and CREB binding protein (CBP) are two closely related histone acetyl transferase proteins that act as translational co-activators of AR. • We have developed the clinical candidate, CCS1477, which is a potent, selective and orally active small molecule inhibitor of the bromodomain of p300/CBP and we report here its impact on AR, AR-SV and c-Myc expression and function. • We have also extended the evaluation of CCS1477 into other disease settings, including haematological cancers, and those tumours with loss of function mutations in p300/CBP providing susceptibility to synthetic lethality (e.g. bladder cancer). 5. In vivo efficacy in 22Rv1 xenograft: Including continued tumour growth inhibition following drug withdrawal • CCS1477, a p300/CBP inhibitor and clinical candidate, demonstrates complete tumour growth inhibition in selected xenograft models at doses which are well tolerated. • The tumour growth inhibition caused by CCS1477 is sustained following drug withdrawal. • These data support the clinical testing of CCS1477 in patients in three settings (i) CRPC through down-regulating AR, AR-SV and c-Myc; (ii) haematological cancers, in particular AML, through effects on cell cycle arrest and myeloid differentiation, and (iii) in patients with loss of function mutations in p300 or CBP by driving synthetic lethality. Copies of this poster obtained through Quick Response (QR) Code are for personal use only and may not be reproduced without permission from AACR® and the author of this poster. 4. Good oral exposure in mouse: blood levels exceed 22Rv1 proliferation IC50 for several hours a) Mouse pharmacokinetics 0.5mg/kg iv; 3mg/kg po T1/2 (hr) 0.96 Cl obs (ml/min/kg) 14 Vss (L/kg) 1.1 F (%) 73 b) Free drug exposure @ 30mg/kg po b) a) Pharmacokinetic parameters after iv (0.5mg/kg in 5% DMSO/HPBCD) and oral (3mg/kg in 5% DMSO/MC) dosing; b) Plasma levels of CCS1477 (free drug) following an oral dose of 30mg/kg in mouse (blue); IC50 value for CCS1477 for inhibition of proliferation of 22Rv1 cells (black); IC50 value for in cell binding of CCS1477 to p300 using BRET assay (green); IC50 value for in cell binding of CCS1477 to BRD4 using BRET assay (red) 0 250 500 750 1000 1250 1500 1 5 9 13 17 21 25 29 33 37 41 45 49 53 Vehicle QD CCS1477 10mg/kg QD CCS1477 30mg/kg QOD CCS1477 20mg/kg QD Tumour volume (mm 3 ) Drug treatment period No drug treatment Time (days) Proliferation was measured with a cell viability assay (CellTiter Blue) in bladder cancer cells (maintained in 10% FCS) after compound treatment for 72h. Data are from two separate experiments represented by different shading. 8. Differential sensitivity to CCS1477 in bladder cancer cell lines with p300 or CBP mutations compared to wild type Cdt1-RFP geminin-GFP Vehicle (DMSO) 10. CCS1477 inhibits the proliferation of patient derived primary AML cells and promotes myeloid differentiation (a) Inhibition of proliferation of THP-1 cells after 48hrs incubation; (b) Fucci flow analysis of THP-1 cells following incubation with DMSO vehicle or CCS1477 (100nM) for 48 hrs . a) b) (a) Inhibition of proliferation of patient derived primary AML cells after 7 days treatment with 100nM CCS1477; (b) Flow cytometry analysis of differentiation markers (CD86 and CD11b) in patient derived cells (patient BB108) following treatment with DMSO vehicle or CCS1477 (100nM) for 7 days. CD86 PerCP-EF710 CD11b PE CD11b PE CD86 PerCP-EF710 a) b) LNCaP-AR F876L cells stably-express AR F876L and demonstrate enhanced AR target gene expression in response to both DHT and enzalutamide. Cells were grown in steroid-depleted media for 48 hours prior to stimulation with 1nM DHT or 1μM enzalutamide in the presence and absence of 0.5μM CCS1477 for 24hrs. 3. CCS1477 inhibits DHT and enzalutamide agonist activity at AR F876L 11. In vivo efficacy in AML MOLM16 xenograft: Including continued tumour growth inhibition following drug withdrawal MOLM-16 tumour bearing NOD / SCID mice were treated with CCS1477 by oral gavage, once daily (5/10/20mg/kg). Vehicle (5% DMSO:95% methylcellulose [0.5%w/v]) was dosed once daily. 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 % viabilityrelative to 0.1% DMSO [CCS1477] uM VM-CUB 2 – CBP mutant 0.00 20.00 40.00 60.00 80.00 100.00 120.00 % viability relative to 0.1% DMSO [CCS1477] uM RT112 - wildtype qPCR of analysis of AR, c-Myc and AR-target genes after 24h incubation in 22Rv1 cells Cdt1-RFP geminin-GFP D G1 S G2M CCS1477 (100nM) 0.00 20.00 40.00 60.00 80.00 100.00 120.00 % viability relative to 0.1% DMSO [CCS1477] uM 647V – p300 mutant
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Transcript of Abstract # 3991 Novel small molecule inhibitor of p300/CBP ... · Novel small molecule inhibitor of...
Novel small molecule inhibitor of p300/CBP down-regulates androgen receptor (AR) and c-Myc for the treatment of prostate cancer and beyond
Neil Pegg1, Jenny Worthington2, Barbara Young3, Amy Prosser3, Luke Gaughan4, Gary Spencer5, Tim Somervaille5, Julie Burns6, Margaret Knowles6, Nigel Brooks1.1CellCentric Ltd, Cambridge UK; 2Axis Bioservices, Coleraine, UK; 3Sygnature Discovery, Nottingham UK; 4Northern Institute for Cancer Research, Newcastle, UK; 5CRUK Manchester Institute,
Manchester, UK; 6The University of Leeds, UK
Abstract # 3991
Cell Line AR status Model CCS1477Proliferation IC50 mM
LNCaP AR-FL Hormone responsive 0.230
LNCaP-AR AR-FL over-expressed CRPC 0.150
VCaP AR-FL, AR-SV CRPC 0.049
22Rv1 AR-FL, AR-SV CRPC 0.096
DU145 AR negative Hormone independent 1.280
PC3 AR negative Hormone independent 1.490
1. CCS1477: Inhibits prostate cancer cell proliferation
Conclusions
CellCentric
Proliferation was measured with a cell viability assay (CyQuant Direct Cell Proliferation or CellTiter Glo) in prostate cancer cells (maintained in 10% FCS) after compound treatment for 72h.
Proliferation was measured with a cell viability assay (CellTiter Glo) in panel of 277 cancer cell lines after compound treatment for 7 days.
2. CCS1477 degrades AR-FL, AR-SV & c-Myc protein in 22Rv1 cells: Including downstream genes
a)
Representative Western analysis of AR-FL, AR-SV (V7) and c-Myc protein in 22Rv1 cells after 24h treatment with CCS1477
6. Protein biomarkers are reduced in 22Rv1 tumour bearing animals treated with CCS1477 for 7 and 28 days
22Rv1 tumour bearing athymic nude mice were treated with CCS1477 by oral gavage, once daily (10/20mg/kg) or once every other day (30mg/kg). Vehicle (5% DMSO:95% methylcellulose [0.5%w/v]) was dosed once daily.
9. CCS1477 inhibits the proliferation of AML cells mediated by G1 cell cycle arrestIntroduction
7. Breadth of efficacy screening with CCS1477 demonstrates subsets of cell lines which are sensitive: including prostate cancer and beyond
Western analysis of AR-FL, AR-SV, c-Myc in 22Rv1 tumours taken from a satellite group at day 7 of the study shown in Fig. 5 and at the end of the same study at day 28.
• Targeted degradation of androgen receptor (AR) and androgen receptor variants (AR-SV) remains an important therapeutic opportunity for patients with castration resistant prostate cancer.
• E1A binding protein (p300) and CREB binding protein (CBP) are two closely related histone acetyl transferase proteins that act as translational co-activators of AR.
• We have developed the clinical candidate, CCS1477, which is a potent, selective and orally active small molecule inhibitor of the bromodomain of p300/CBP and we report here its impact on AR, AR-SV and c-Mycexpression and function.
• We have also extended the evaluation of CCS1477 into other disease settings, including haematological cancers, and those tumours with loss of function mutations in p300/CBP providing susceptibility to synthetic lethality (e.g. bladder cancer).
5. In vivo efficacy in 22Rv1 xenograft: Including continued tumour growth inhibition following drug withdrawal
• CCS1477, a p300/CBP inhibitor and clinical candidate, demonstrates complete tumour growth inhibition in selected xenograft models at doses which are well tolerated.
• The tumour growth inhibition caused by CCS1477 is sustained following drug withdrawal.
• These data support the clinical testing of CCS1477 in patients in three settings (i) CRPC through down-regulating AR, AR-SV and c-Myc; (ii) haematological cancers, in particular AML, through effects on cell cycle arrest and myeloid differentiation, and (iii) in patients with loss of function mutations in p300 or CBP by driving synthetic lethality.
Copies of this poster obtained through Quick Response (QR) Code are for personal use only and may not be reproduced without permission from AACR® and the author of this poster.
4. Good oral exposure in mouse: blood levels exceed 22Rv1 proliferation IC50 for several hours
a) Mouse pharmacokinetics
0.5mg/kg iv; 3mg/kg po
T1/2 (hr) 0.96
Cl obs (ml/min/kg) 14
Vss (L/kg) 1.1
F (%) 73
b) Free drug exposure @ 30mg/kg po
b)
a) Pharmacokinetic parameters after iv (0.5mg/kg in 5% DMSO/HPBCD) and oral (3mg/kg in 5% DMSO/MC) dosing; b) Plasma levels of CCS1477 (free drug) following an oral dose of 30mg/kg in mouse (blue); IC50 value for CCS1477 for inhibition of proliferation of 22Rv1 cells (black); IC50 value for in cell binding of CCS1477 to p300 using BRET assay (green); IC50 value for in cell binding of CCS1477 to BRD4 using BRET assay (red)
0
250
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1 5 9 13 17 21 25 29 33 37 41 45 49 53
Vehicle QD
CCS1477 10mg/kg QD
CCS1477 30mg/kg QOD
CCS147720mg/kg QD
Tum
ou
r vo
lum
e (m
m3 )
Drug treatment period No drug treatment
Time (days)
Proliferation was measured with a cell viability assay (CellTiter Blue) in bladder cancer cells (maintained in 10% FCS) after compound treatment for 72h. Data are from two separate experiments represented by different shading.
8. Differential sensitivity to CCS1477 in bladder cancer cell lines with p300 or CBP mutations compared to wild type
Cdt1-RFP
gem
inin
-GFP
Vehicle (DMSO)
10. CCS1477 inhibits the proliferation of patient derived primary AML cells and promotes myeloid differentiation
(a) Inhibition of proliferation of THP-1 cells after 48hrs incubation; (b) Fucci flow analysis of THP-1 cells following incubation with DMSO vehicle or CCS1477 (100nM) for 48 hrs .
a) b)
(a) Inhibition of proliferation of patient derived primary AML cells after 7 days treatment with 100nM CCS1477; (b) Flow cytometry analysis of differentiation markers (CD86 and CD11b) in patient derived cells (patient BB108) following treatment with DMSO vehicle or CCS1477 (100nM) for 7 days.
CD86 PerCP-EF710
CD
11
b P
E
CD
11
b P
E
CD86 PerCP-EF710
a) b)
LNCaP-ARF876L cells stably-express ARF876L and demonstrate enhanced AR target gene expression in response to both DHT and enzalutamide. Cells were grown in steroid-depleted media for 48 hours prior to stimulation with 1nM DHT or 1µM enzalutamide in the presence and absence of 0.5µM CCS1477 for 24hrs.
3. CCS1477 inhibits DHT and enzalutamide agonist activity at AR F876L
11. In vivo efficacy in AML MOLM16 xenograft: Including continued tumour growth inhibition following drug withdrawal
MOLM-16 tumour bearing NOD / SCID mice were treated with CCS1477 by oral gavage, once daily (5/10/20mg/kg). Vehicle (5% DMSO:95% methylcellulose [0.5%w/v]) was dosed once daily.
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RT112 - wildtype
qPCR of analysis of AR, c-Myc and AR-target genes after 24h incubation in 22Rv1 cells
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gem
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647V – p300 mutant
