Modulation of Lipid Metabolism Through Inhibition of ... · Modulation of Lipid Metabolism Through...
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Modulation of Lipid Metabolism Through Inhibition of Acetyl-CoA Carboxylase with ND-646 Leads to Potent Inhibition of Breast Cancer Cell Growth in vitro and in vivo Jennifer L. Rocnik 1 , Wenyan Miao 1 , Geraldine Harriman 1 , Jeremy Greenwood 2 , Sathesh Bhat 2 , H. James Harwood 1 , Rosana Kapeller 1 , William F. Westlin 1 1 Nimbus Therapeutics, Cambridge, MA, 2 Schrödinger, New York, NY Abstract #1048 CPT1 Fatty Acids ACC2 Mitochondrion ACC1 Acetyl-CoA + HCO 3 - Malonyl-CoA - C o A - C o A A C C I n h i b i t i o n L e a d s t o : Fatty acid synthesis Tissue triglycerides Body fat and weight Fatty acid oxidation Insulin sensitivity occupies binding site and prevents conformational change Conformational change of the A- domain tail upon phosphorylation A- domain tail ND-646 binds to the same site as Soraphen A L K B 1 A C C A M P K Without Drug: Phosphorylation induces conformational change With Drug: Drug binds to pSer 222 binding pocket, preventing conformational change ND-646 ACC is rapidly dephosphorylated AMPK phosphorylation site (Ser 117/222 ) p-ACC ACC β-actin Vehicle 25 mg/kg BID 50 mg/kg BID p-ACC ACC β-actin Vehicle 25 mg/kg BID 50 mg/kg BID 0.0 0.33 0.6 0.9 1.2 Tumor Normal Breast Tissue p-ACC/ACC Ratio Vehicle 25 mg/kg BID 50 mg/kg BID Vehicle 25 mg/kg BID 50 mg/kg BID 0 1000 2000 3000 4000 ND-646 ND-646 (μM) 0.001 0.01 0.1 1 10 0.00001 0.0001 CPM Inhibition of Fatty Acid Synthesis in HepG2 cells Target potency • ACC1 IC 50 = 3.5 nM • ACC2 IC 50 = 4.1 nM • HepG2 FASyn EC 50 = 8 nM ADMET • Intrinsic Clearance (human, mouse, rat, dog, cyno) = Moderate • Aqueous Solubility >300 mM • Cyp Inhibition >10 mM • Multispecies Protein Binding = 94-98% • Eurofins Panel >1000 fold selective Mouse PK (50 mg/kg, PO) • AUC last = 9602 hr*ng/ml • T 1/2 = 1.5 hr • C MAX = 11080 ng/ml 14 C-acetate Incorporation Assay Tumors Normal Breast Tissue 7 Day Cyquant Proliferation Assay pACC/total ACC Ratio % Caspase-3 Positive Cells 0 1 2 3 4 5 Vehicle x 10 mag 25 mg/kg BID x 10 mag 50 mg/kg BID x 10 mag Vehicle x 10 mag 25 mg/kg BID x 10 mag 50 mg/kg BID x 10 mag Vehicle 25 mg/kg BID 50 mg/kg BID 0 20 40 60 80 100 120 MDA-MB-468 MCF7 BT474 SUM149PT % DMSO Control ND-646 (μM) 0.001 0.01 0.1 1 10 MDA-MB-436 HCC1937 MDA-MB-231 SUM1315MO2 MDA- MB-468 MCF7 BT474 SUM 149PT MDA- MB-436 HCC 1937 MDA- MB-231 SUM131 5MO2 IC 50 (absolute) 38 nM 104 nM 120 nM 44 nM >10 μM >10 μM >10 μM >10 μM 0 200 400 600 0 5 10 15 20 25 30 Days Post Treatment % Tumor Volume 80 90 100 110 % Body Weight Change 0 5 10 15 20 25 30 Days Post Treatment Vehicle --- Doxorubicin, 4mg/kg weekly, IP 68 ND-646, 25 mg/kg BID PO 44 ND-646, 50 mg/kg BID PO 29 % TC Vehicle Doxorubicin, 4mg/kg weekly, IP ND-646, 25 mg/kg BID PO ND-646, 50 mg/kg BID PO 1. Acetyl-CoA Carboxylase (ACC) Regulates Fatty Acid Synthesis and Oxidation 3. ND-646 Compound Profile ABSTRACT CONCLUSIONS 5. ND-646 Inhibits Proliferation of Breast Cancer Cell Lines in vitro with Differing Sensitivities 7. ND-646 Treatment Results in Target Engagement in MDA-MB-468 Tumors but not in Normal Breast Tissue 6. ND-646 Inhibits Tumor Growth of MDA-MB-468 Orthotopic Breast Cancer Xenograft Model 8. ND-646 Treatment Causes a Change in MDA-MB-468 Tumor Architecture and an Increase in Apoptosis 2. ND-646 Mechanism of Action 4. Breast Cancer Cell Line Characteristics • AMPK phosphorylates the A domain tail leading to inactivation of ACC • ND-646 occupies the pSer 117/222 binding site on the BC domain of ACC preventing the phosphopeptide from binding and allowing rapid dephosphorylation • Allows for the use of p-ACC as a target engagement marker in cells and tissues • % tumor volume normalized to pretreatment tumor volume • Western blot analysis of tumors (human) and normal breast tissue (mouse) harvested 12hrs post last dose of ND-646 784 Memorial Dr, Suite 100, Cambridge, MA 02139, USA • www.nimbustx.com • Tel: 857.999.2009 • [email protected] • ND-646 is a potent allosteric inhibitor of ACC1/2 with good drug-like properties • Treatment of human breast cancer cell lines with ND-646 resulted in inhibition of proliferation of selected cell lines in vitro • ND-646 was well tolerated and significantly inhibited growth of MDA-MB-468 orthotopic tumors • ND-646 demonstrated prolonged target engagement in tumor tissue and induction of apoptosis in MDA-MB-468 tumors • Profiling of ND-646 is ongoing in broad panel of breast cancer cell lines to evaluate sensitivity across diverse genetic backgrounds and identify predictors of response Metabolic attenuation is a promising approach to cancer therapy and rate-limiting steps in key biosynthetic pathways are particularly attractive targets. Many cancer types are dependent on fatty acid synthesis as a primary source of energy and for providing lipids for expansion of cell and nuclear membranes in rapidly proliferating cells. The rate- limiting enzyme in fatty acid synthesis, acetyl-CoA carboxylase (ACC), has been shown to be highly expressed in human breast cancer. ACC is thought to be critical for the growth and survival of cancer cells, especially within a tumor microenvironment where ex- ogenous fatty acids might be limited. Effective therapeutic options for triple negative breast cancer are limited and identification of robust targeted agents without overt toxicity for this indication are especially needed. Dual inhibition of the ACC isozymes, ACC1 and ACC2, results in concomitant inhibition of fatty acid synthesis and stimula- tion of fatty acid oxidation. We have identified ND-646, a potent, selective, allosteric inhibitor of ACC with broad tissue distribution that binds to the ACC biotin carboxylase domain and potently inhibits the dimerization and enzymatic activity of both ACC1 (IC 50 = 3.5nM) and ACC2 (IC 50 = 4.1nM). Profiling the potency of ND-646 in vitro in a panel of breast cancer cell lines including triple negative and BRCA1 mutant cell lines demon- strated potent inhibition of cell proliferation with IC50s<100nM. The anti-proliferative effects were more pronounced when cells were cultured in media containing delipidat- ed serum. Daily oral dosing of ND-646 at 25 mg/kg BID,50 mg/kg QD, and 50 mg/kg BID in mice bearing orthotopic triple negative MDA-MB-468 breast cancer xenografts led to tumor growth inhibition of 60-70% that correlated with compound exposure and target engagement in the tumor. Analysis of ND-646 treated tumors demonstrated dis- ruption of tumor tissue architecture and induction of apoptosis and necrosis suggest- ing a direct effect on cell survival. These results provide further evidence that de novo lipogenesis is an important mediator of breast cancer cell growth and survival, and that selective inhibition of ACC is a viable therapeutic strategy for treatment of breast can- H&E Staining Caspase-3 IHC MDA-MB-468 • Triple negative • Ductal carcinoma • Basal origin • p53 and PTEN mutant MCF7 • Luminal • ER + BT474 • Luminal • Her2 amplified SUM149PT • Triple negative • BRCA1 mutant • p53 mutant MDA-MB-436 • Triple negative • BRCA1 mutant • p53 mutant HCC1937 • Triple negative • BRCA1 mutant • p53 mutant MDA-MB-231 • Triple negative • BRCA1 mutant • p53 mutant • Kras mutant SUM1315MO2 • Triple negative • BRCA1 mutant • Myc amplified
Transcript of Modulation of Lipid Metabolism Through Inhibition of ... · Modulation of Lipid Metabolism Through...
Modulation of Lipid Metabolism Through Inhibition of Acetyl-CoA Carboxylase with ND-646 Leads to Potent Inhibition of Breast Cancer Cell Growth in vitro and in vivo
Jennifer L. Rocnik1, Wenyan Miao1, Geraldine Harriman1, Jeremy Greenwood2, Sathesh Bhat2, H. James Harwood1, Rosana Kapeller1, William F. Westlin1
1Nimbus Therapeutics, Cambridge, MA, 2Schrödinger, New York, NYAbstract#1048
CPT1
Fatty Acids
ACC2
Mitochondrion
ACC1
Acetyl-CoA
+ HCO3-
Malonyl-CoA
-CoA -CoA
ACC Inhibition Leads to:
Fatty acid synthesis
Tissue triglycerides
Body fat and weight
Fatty acid oxidation
Insulin sensitivity
occupies binding site and prevents conformational change
Conformational change of the A-domain tail upon phosphorylation
A-domain tail
ND-646 binds to the same site as Soraphen A
LKB1
ACC
AMPK
Without Drug: Phosphorylation induces conformational change
With Drug: Drug binds to pSer222
binding pocket, preventing conformational change
ND-646
ACC is rapidly dephosphorylated AMPK phosphorylation site (Ser117/222)
p-ACC
ACC
β-actin
Vehicle 25 mg/kg BID 50 mg/kg BID
p-ACC
ACC
β-actin
Vehicle 25 mg/kg BID 50 mg/kg BID
0.0
0.33
0.6
0.9
1.2
Tumor Normal Breast Tissue
p-A
CC
/AC
C R
atio
Vehicl
e
25 m
g/kg B
ID
50 m
g/kg B
ID
Vehicl
e
25 m
g/kg B
ID
50 m
g/kg B
ID
0
1000
2000
3000
4000
ND-646
ND-646 (μM)0.001 0.01 0.1 1 100.00001 0.0001
CPM
Inhibition of Fatty Acid Synthesis in HepG2 cellsTarget potency
• ACC1 IC50 = 3.5 nM• ACC2 IC50 = 4.1 nM• HepG2 FASyn EC50 = 8 nM
ADMET• Intrinsic Clearance
(human, mouse, rat, dog, cyno) = Moderate• Aqueous Solubility >300 mM• Cyp Inhibition >10 mM• Multispecies Protein Binding = 94-98%• Eurofins Panel >1000 fold selective
Mouse PK (50 mg/kg, PO)• AUClast = 9602 hr*ng/ml• T1/2 = 1.5 hr• CMAX = 11080 ng/ml
14C-acetate Incorporation Assay
Tumors
Normal Breast Tissue
7 Day Cyquant Proliferation Assay pACC/total ACC Ratio
% C
aspa
se-3
Po
sitiv
e C
ells
0
1
2
3
4
5
Vehiclex 10 mag
25 mg/kg BIDx 10 mag
50 mg/kg BIDx 10 mag
Vehiclex 10 mag
25 mg/kg BIDx 10 mag
50 mg/kg BIDx 10 mag
Vehicle 25 mg/kg BID 50 mg/kg BID
0
20
40
60
80
100
120 MDA-MB-468
MCF7
BT474
SUM149PT
% D
MSO
Con
trol
ND-646 (μM)
0.001 0.01 0.1 1 10
MDA-MB-436
HCC1937
MDA-MB-231
SUM1315MO2
MDA-MB-468 MCF7 BT474 SUM
149PT MDA-MB-436
HCC 1937
MDA-MB-231
SUM1315MO2
IC50 (absolute) 38 nM 104 nM 120 nM 44 nM >10 µM
>10 µM
>10 µM
>10 µM
0
200
400
600
0 5 10 15 20 25 30
Days Post Treatment
% T
umor
Vol
ume
80
90
100
110
% B
ody
Wei
ght C
hang
e
0 5 10 15 20 25 30
Days Post Treatment
Vehicle ---
Doxorubicin, 4mg/kg weekly, IP 68
ND-646, 25 mg/kg BID PO 44
ND-646, 50 mg/kg BID PO 29
% TC
Vehicle
Doxorubicin, 4mg/kg weekly, IP
ND-646, 25 mg/kg BID PO
ND-646, 50 mg/kg BID PO
1. Acetyl-CoA Carboxylase (ACC) Regulates Fatty Acid Synthesis and Oxidation
3. ND-646 Compound Profile
ABSTRACT
CONCLUSIONS
5. ND-646 Inhibits Proliferation of Breast Cancer Cell Lines in vitro with Differing Sensitivities
7. ND-646 Treatment Results in Target Engagement in MDA-MB-468 Tumors but not in Normal Breast Tissue
6. ND-646 Inhibits Tumor Growth of MDA-MB-468 Orthotopic Breast Cancer Xenograft Model
8. ND-646 Treatment Causes a Change in MDA-MB-468 Tumor Architecture and an Increase in Apoptosis
2. ND-646 Mechanism of Action
4. Breast Cancer Cell Line Characteristics
• AMPK phosphorylates the A domain tail leading to inactivation of ACC• ND-646 occupies the pSer117/222 binding site on the BC domain of ACC preventing the phosphopeptide from
binding and allowing rapid dephosphorylation• Allows for the use of p-ACC as a target engagement marker in cells and tissues
• % tumor volume normalized to pretreatment tumor volume
• Western blot analysis of tumors (human) and normal breast tissue (mouse) harvested 12hrs post last dose of ND-646
784 Memorial Dr, Suite 100, Cambridge, MA 02139, USA • www.nimbustx.com • Tel: 857.999.2009 • [email protected]
• ND-646 is a potent allosteric inhibitor of ACC1/2 with good drug-like properties
• Treatment of human breast cancer cell lines with ND-646 resulted in inhibition of proliferation of selected cell lines in vitro
• ND-646 was well tolerated and significantly inhibited growth of MDA-MB-468 orthotopic tumors
• ND-646 demonstrated prolonged target engagement in tumor tissue and induction of apoptosis in MDA-MB-468 tumors
• Profiling of ND-646 is ongoing in broad panel of breast cancer cell lines to evaluate sensitivity across diverse genetic backgrounds and identify predictors of response
Metabolic attenuation is a promising approach to cancer therapy and rate-limiting steps in key biosynthetic pathways are particularly attractive targets. Many cancer types are dependent on fatty acid synthesis as a primary source of energy and for providing lipids for expansion of cell and nuclear membranes in rapidly proliferating cells. The rate- limiting enzyme in fatty acid synthesis, acetyl-CoA carboxylase (ACC), has been shown to be highly expressed in human breast cancer. ACC is thought to be critical for the growth and survival of cancer cells, especially within a tumor microenvironment where ex- ogenous fatty acids might be limited. Effective therapeutic options for triple negative breast cancer are limited and identification of robust targeted agents without overt toxicity for this indication are especially needed. Dual inhibition of the ACC isozymes, ACC1 and ACC2, results in concomitant inhibition of fatty acid synthesis and stimula-tion of fatty acid oxidation. We have identified ND-646, a potent, selective, allosteric inhibitor of ACC with broad tissue distribution that binds to the ACC biotin carboxylase domain and potently inhibits the dimerization and enzymatic activity of both ACC1 (IC50 = 3.5nM) and ACC2 (IC50 = 4.1nM). Profiling the potency of ND-646 in vitro in a panel of breast cancer cell lines including triple negative and BRCA1 mutant cell lines demon-strated potent inhibition of cell proliferation with IC50s<100nM. The anti-proliferative effects were more pronounced when cells were cultured in media containing delipidat-ed serum. Daily oral dosing of ND-646 at 25 mg/kg BID,50 mg/kg QD, and 50 mg/kg BID in mice bearing orthotopic triple negative MDA-MB-468 breast cancer xenografts led to tumor growth inhibition of 60-70% that correlated with compound exposure and target engagement in the tumor. Analysis of ND-646 treated tumors demonstrated dis-ruption of tumor tissue architecture and induction of apoptosis and necrosis suggest-ing a direct effect on cell survival. These results provide further evidence that de novo lipogenesis is an important mediator of breast cancer cell growth and survival, and that selective inhibition of ACC is a viable therapeutic strategy for treatment of breast can-
H&
E St
aini
ng
Caspase-3 IHC
MDA-MB-468• Triple negative• Ductal carcinoma• Basal origin• p53 and PTEN
mutant
MCF7• Luminal• ER+
BT474• Luminal• Her2 amplified
SUM149PT• Triple negative• BRCA1 mutant• p53 mutant
MDA-MB-436• Triple negative• BRCA1 mutant• p53 mutant
HCC1937• Triple negative• BRCA1 mutant• p53 mutant
MDA-MB-231• Triple negative• BRCA1 mutant• p53 mutant• Kras mutant
SUM1315MO2• Triple negative• BRCA1 mutant• Myc amplified
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