Modulation of Bax and mTOR for cancer therapeutics

Article abstract of DOI:10.1158/0008-5472.CAN-16-2356

A rationale exists for pharmacologic manipulation of the serine (S)184 phosphorylation site of the proapoptotic Bcl2 family member Bax as an anticancer strategy. Here, we report the refinement of the Bax agonist SMBA1 to generate CYD-2-11, which has characteristics of a suitable clinical lead compound. CYD-2-11 targeted the structural pocket proximal to S184 in the C-terminal region of Bax, directly activating its proapoptotic activity by inducing a conformational change enabling formation of Bax homooligomers in mitochondrial membranes. In murine models of small-cell and non-small cell lung cancers, including patient-derived xenograft and the genetically engineered mutant KRAS-driven lung cancer models, CYD-2-11 suppressed malignant growth without evident significant toxicity to normal tissues. In lung cancer patients treated with mTOR inhibitor RAD001, we observed enhanced S184 Bax phosphorylation in lung cancer cells and tissues that inactivates the propaoptotic function of Bax, contributing to rapalog resistance. Combined treatment of CYD-2-11 and RAD001 in murine lung cancer models displayed strong synergistic activity and overcame rapalog resistance in vitro and in vivo. Taken together, our findings provide preclinical evidence for a pharmacologic combination of Bax activation and mTOR inhibition as a rational strategy to improve lung cancer treatment.

Full text of DOI:10.1158/0008-5472.CAN-16-2356

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ0RGXODWLRQꢀRIꢀ%D[ꢀDQGꢀP725ꢀIRUꢀFDQFHUꢀWKHUDSHXWLFVꢀ
5XLꢀ/Lꢁꢂꢀꢃꢂꢀ&KXQ\RQJꢀ'LQJꢄꢂꢀꢃꢂꢀꢀ-XQꢀ=KDQJꢀꢅꢂꢀꢃꢂꢀꢆꢂꢀ0DRKXDꢀ;LHꢁꢀꢂꢀ'RQJN\RRꢀ3DUNꢁꢂꢀ<Hꢀ'LQJꢄꢂꢀ*XRꢀ
&KHQꢁꢂꢀ*XRMLQJꢀ=KDQJꢅꢂꢀꢀ0HOLVVDꢀ*LOEHUWꢇ5RVVꢅꢂꢀ:HLꢀ=KRXꢅꢂꢀ$GDPꢀ,ꢈꢀ0DUFXVꢅꢂꢀꢀ6KLꢇ<RQJꢀ6XQꢅꢂꢀꢀ
=KXRꢀ*ꢈꢀ&KHQꢅꢂꢀ*DEULHOꢀ/ꢈꢀ6LFDꢉꢂꢀ6XUHVKꢀ6ꢈꢀ5DPDOLQJDPꢅꢂꢀ$QGUHZꢀ7ꢈꢀ0DJLVꢊꢂꢀ+DLDQꢀ)Xꢅꢂꢀ)DGORꢀ
5ꢈꢀ.KXULꢅꢂꢀ:DOWHUꢀ-ꢈꢀ&XUUDQꢁꢂꢀ7DRIHHNꢀ.ꢈꢀ2ZRQLNRNRꢅꢂꢀ'RQJꢀ0ꢈꢀ6KLQꢅꢋꢂꢀ-LDꢀ=KRXꢄꢋꢂꢀꢀDQGꢀ
;LQJPLQJꢀ'HQJꢁꢋꢀ
ꢀ
'HSDUWPHQWVꢀRIꢀꢁ5DGLDWLRQꢀ2QFRORJ\ꢂꢀꢅ+HPDWRORJ\ꢀDQGꢀ0HGLFDOꢀ2QFRORJ\ꢀDQGꢀꢉ3DWKRORJ\ꢂꢀ
(PRU\ꢀ8QLYHUVLW\ꢀ6FKRROꢀRIꢀ0HGLFLQHꢀDQGꢀ:LQVKLSꢀ&DQFHUꢀ,QVWLWXWHꢀRIꢀ(PRU\ꢀ8QLYHUVLW\ꢂꢀ$WODQWDꢂꢀ
*$ꢀꢉꢌꢉꢅꢅꢍꢀꢄ&KHPLFDOꢀ%LRORJ\ꢀ3URJUDPꢂꢀ'HSDUWPHQWꢀRIꢀ3KDUPDFRORJ\ꢀDQGꢀ7R[LFRORJ\ꢂꢀ
8QLYHUVLW\ꢀRIꢀ7H[DVꢀ0HGLFDOꢀ%UDQFKꢂꢀ*DOYHVWRQꢂꢀ7;ꢀꢆꢆꢊꢊꢊꢍꢀꢊ,QVWLWXWHꢀIRUꢀ6\VWHPVꢀ%LRORJ\ꢂꢀ
6HDWWOHꢂꢀ:$ꢀꢎꢏꢁꢌꢎꢂꢀ86$ꢀ
ꢀ
ꢃ&RꢇILUVWꢀDXWKRUꢀ
ꢀ
ꢆ3UHVHQWꢀ$GGUHVVꢐꢀ'HSDUWPHQWꢀRIꢀ,QWHUQDOꢀ0HGLFLQHꢂꢀ'LYLVLRQꢀRIꢀ+HPDWRORJ\ꢂꢀ2QFRORJ\ꢀDQGꢀ
%ORRGꢀ ꢀ0DUURZꢀ7UDQVSODQWDWLRQꢂꢀ+ROGHQꢀ&RPSUHKHQVLYHꢀ&DQFHUꢀ&HQWHUꢂꢀ8QLYHUVLW\ꢀRIꢀ,RZDꢀ
&DUYHUꢀ&ROOHJHꢀRIꢀ0HGLFLQHꢂꢀꢅꢌꢌꢀ+DZNLQVꢀ'ULYHꢂꢀ,RZDꢀ&LW\ꢂꢀ,$ꢀꢊꢅꢅꢄꢅꢀ
ꢀ
5XQQLQJꢀ7LWOHꢐꢀ%D[ꢀDJRQLVWꢀV\QHUJL]HVꢀZLWKꢀ5$'ꢌꢌꢁꢀDJDLQVWꢀFDQFHUꢀ
ꢀ
'LVFORVXUHꢀRIꢀ3RWHQWLDOꢀ&RQIOLFWVꢀRIꢀ,QWHUHVWꢀ
7KHꢀDXWKRUVꢀGLVFORVHꢀQRꢀSRWHQWLDOꢀFRQIOLFWVꢀRIꢀLQWHUHVWꢀ
ꢀꢀ
ꢁ&RUUHVSRQGLQJꢀ$XWKRUꢐꢀ;LQJPLQJꢀ'HQJꢂꢀ'LYLVLRQꢀRIꢀ&DQFHUꢀ%LRORJ\ꢂꢀ'HSDUWPHQWꢀRIꢀ5DGLDWLRQꢀ
2QFRORJ\ꢂꢀ(PRU\ꢀ8QLYHUVLW\ꢀ6FKRROꢀRIꢀ0HGLFLQHꢂꢀ:LQVKLSꢀ&DQFHUꢀ,QVWLWXWHꢀRIꢀ(PRU\ꢀ8QLYHUVLW\ꢂꢀ
$WODQWDꢂꢀ*$ꢀꢉꢌꢉꢅꢅꢂꢀ86$ꢈꢀ3KRQHꢐꢀꢑꢄꢌꢄꢒꢆꢆꢏꢇꢉꢉꢎꢏꢂꢀ(ꢇPDLOꢐꢀ[GHQJꢄ#HPRU\ꢈHGXꢂꢀ)D[ꢐꢀꢑꢄꢌꢄꢒꢆꢆꢏꢇ
ꢁꢎꢌꢎꢍꢀ-LDꢀ=KRXꢂꢀ&KHPLFDOꢀ%LRORJ\ꢀ3URJUDPꢂꢀ'HSDUWPHQWꢀRIꢀ3KDUPDFRORJ\ꢀDQGꢀ7R[LFRORJ\ꢂꢀ
8QLYHUVLW\ꢀRIꢀ7H[DVꢀ0HGLFDOꢀ%UDQFKꢂꢀ*DOYHVWRQꢂꢀ7;ꢀꢆꢆꢊꢊꢊꢈꢀ3KRQHꢐꢀꢑꢄꢌꢎꢒꢀꢆꢆꢅꢇꢎꢆꢄꢏꢂꢀ(ꢇPDLOꢐꢀ
ML]KRX#XWPEꢈHGXꢂꢀ)D[ꢐꢀꢑꢄꢌꢎꢒꢀꢆꢆꢅꢇꢎꢃꢄꢏꢍꢀꢀ'RQJꢀ0ꢈꢀ6KLQꢂꢀ'HSDUWPHQWꢀRIꢀ+HPDWRORJ\ꢀDQGꢀ
0HGLFDOꢀ2QFRORJ\ꢂꢀ(PRU\ꢀ8QLYHUVLW\ꢀ6FKRROꢀRIꢀ0HGLFLQHꢂꢀ:LQVKLSꢀ&DQFHUꢀ,QVWLWXWHꢀRIꢀ(PRU\ꢀ
8QLYHUVLW\ꢂꢀ$WODQWDꢂꢀ*Dꢀꢉꢌꢉꢅꢅꢂꢀ86$ꢈꢀ3KRQHꢐꢑꢄꢌꢄꢒꢆꢆꢏꢇꢊꢎꢎꢌꢈꢀ(PDLOꢐꢀGPVKLQ#HPRU\ꢈHGXꢂꢀ
)D[ꢐꢄꢌꢄꢇꢆꢆꢏꢇꢊꢅꢅꢌꢈꢀ
ꢀ
7KLVꢀZRUNꢀZDVꢀVXSSRUWHGꢀE\ꢀ1DWLRQDOꢀ,QVWLWXWHVꢀRIꢀ+HDOWKꢀJUDQWVꢀꢅ5ꢌꢁ&$ꢁꢉꢃꢊꢉꢄꢇꢌꢃꢀꢑ;ꢈꢀ'HQJꢒꢂꢀ
5ꢌꢁ&$ꢁꢎꢉꢏꢅꢏꢀꢑ;ꢈꢀ'HQJꢒꢂꢀꢁ5ꢌꢁ&$ꢅꢌꢌꢎꢌꢊꢇꢌꢁ$ꢁꢀꢑ;ꢈꢀ'HQJꢒꢂꢀ5ꢌꢁ&$ꢁꢄꢌꢊꢆꢁꢀꢑ:ꢈꢀ=KRXꢒꢂꢀ3ꢊꢌꢀ
&$ꢌꢎꢆꢌꢌꢆꢀꢑ-ꢈꢀ=KRXꢒꢂꢀ3ꢉꢌꢀ'$ꢌꢅꢏꢏꢅꢁꢀꢑ-ꢈꢀ=KRXꢒꢀDQGꢀ7ꢉꢅ&$ꢁꢃꢌꢌꢄꢌꢀꢑ'ꢈ0ꢈꢀ6KLQꢒꢀDVꢀZHOOꢀDVꢀ5ꢈꢀ$ꢈꢀ
:HOFKꢀ)RXQGDWLRQꢀ&KHPLVWU\ꢀDQGꢀ%LRORJ\ꢀ&ROODERUDWLYHꢀ*UDQWꢀIURPꢀ*XOIꢀ&RDVWꢀ&RQVRUWLDꢀ
ꢑ*&&ꢒꢀIRUꢀ&KHPLFDOꢀ*HQRPLFVꢂꢀ&DQFHUꢀ3UHYHQWLRQꢀDQGꢀ5HVHDUFKꢀ,QVWLWXWHꢀRIꢀ7H[DVꢀꢑ&35,7ꢒꢀ
DZDUGVꢀꢑ-ꢈꢀ=KRXꢒꢂꢀDQGꢀ-RKQꢀ6HDO\ꢀ0HPRULDOꢀ(QGRZPHQWꢀ)XQGꢀꢑ-ꢈꢀ=KRXꢒꢈꢀ5HVHDUFKꢀUHSRUWHGꢀLQꢀ
WKLVꢀSXEOLFDWLRQꢀZDVꢀDOVRꢀVXSSRUWHGꢀLQꢀSDUWꢀE\ꢀE\ꢀWKHꢀ:LQVKLSꢀ5HVHDUFKꢀ3DWKRORJ\ꢀDQGꢀ
,QWHUJUDWHGꢀ&HOOXODUꢀ,PDJLQJꢀVKDUHGꢀUHVRXUFHꢂꢀWKHꢀFRUHVꢀVXSSRUWHGꢀE\ꢀWKHꢀ:LQVKLSꢀ&DQFHUꢀ
,QVWLWXWHꢀRIꢀ(PRU\ꢀ8QLYHUVLW\ꢀꢑ3ꢉꢌ&$-ꢀꢉꢏꢅꢎꢅꢒꢂꢀDQGꢀE\ꢀWKHꢀ:LQVKLSꢀ)DVKLRQꢀDꢀ&XUHꢀ5HVHDUFKꢀ
6FKRODUꢀ$ZDUGꢀꢑ;ꢈꢀ'HQJꢒꢂꢀDꢀSKLODQWKURSLFꢀDZDUGꢀSURYLGHGꢀE\ꢀWKHꢀ:LQVKLSꢀ&DQFHUꢀ,QVWLWXWHꢀRIꢀ
(PRU\ꢀ8QLYHUVLW\ꢈꢀ
ϭꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
$EVWUDFWꢀ
$ꢀUDWLRQDOHꢀH[LVWVꢀIRUꢀSKDUPDFRORJLFꢀPDQLSXODWLRQꢀRIꢀWKHꢀVHULQHꢀꢂ6ꢃꢄꢅꢆꢀSKRVSKRU\ODWLRQꢀ
VLWHꢀ RIꢀ WKHꢀ SURDSRSWRWLFꢀ %FOꢇꢀ IDPLO\ꢀ PHPEHUꢀ %D[ꢀ DVꢀ DQꢀ DQWLFDQFHUꢀ VWUDWHJ\ꢈꢀ +HUHꢀ ZHꢀ
UHSRUWꢀ WKHꢀ UHILQHPHQWꢀ RIꢀ WKHꢀ %D[ꢀ DJRQLVWꢀ 60%$ꢄꢀ WRꢀ JHQHUDWHꢀ &<'ꢉꢇꢉꢄꢄꢊꢀ ZKLFKꢀ KDVꢀ
FKDUDFWHULVWLFVꢀ RIꢀ Dꢀ VXLWDEOHꢀ FOLQLFDOꢀ OHDGꢀ FRPSRXQGꢈꢀ &<'ꢉꢇꢉꢄꢄꢀ WDUJHWHGꢀ WKHꢀ VWUXFWXUDOꢀ
SRFNHWꢀ SUR[LPDOꢀ WRꢀ 6ꢄꢅꢆꢀ LQꢀ WKHꢀ &ꢉWHUPLQDOꢀ UHJLRQꢀ RIꢀ %D[ꢊꢀ GLUHFWO\ꢀ DFWLYDWLQJꢀ LWVꢀ
SURDSRSWRWLFꢀ DFWLYLW\ꢀ E\ꢀ LQGXFLQJꢀ Dꢀ FRQIRUPDWLRQDOꢀ FKDQJHꢀ HQDEOLQJꢀ IRUPDWLRQꢀ RIꢀ %D[ꢀ
KRPRROLJRPHUVꢀLQꢀPLWRFKRQGULDOꢀPHPEUDQHVꢈꢀ,QꢀPXULQHꢀPRGHOVꢀRIꢀVPDOOꢀFHOOꢀDQGꢀQRQꢉ
VPDOOꢀ FHOOꢀ OXQJꢀ FDQFHUVꢊꢀ LQFOXGLQJꢀ SDWLHQWꢉGHULYHGꢀ [HQRJUDIWꢀ DQGꢀ WKHꢀ JHQHWLFDOO\ꢀ
HQJLQHHUHGꢀPXWDQWꢀ.5$6ꢉGULYHQꢀOXQJꢀFDQFHUꢀPRGHOVꢊꢀ&<'ꢉꢇꢉꢄꢄꢀVXSSUHVVHGꢀPDOLJQDQWꢀ
JURZWKꢀ ZLWKRXWꢀ HYLGHQWꢀ VLJQLILFDQWꢀ WR[LFLW\ꢀ WRꢀ QRUPDOꢀ WLVVXHVꢈꢀ ,Qꢀ OXQJꢀ FDQFHUꢀ SDWLHQWVꢀ
WUHDWHGꢀZLWKꢀP725ꢀLQKLELWRUꢀ5$'ꢋꢋꢄꢊꢀZHꢀREVHUYHGꢀHQKDQFHGꢀ6ꢄꢅꢆꢀ%D[ꢀSKRVSKRU\ODWLRQꢀ
LQꢀ OXQJꢀ FDQFHUꢀ FHOOVꢀ DQGꢀ WLVVXHVꢀ ZKLFKꢀ LQDFWLYDWHVꢀ WKHꢀ SURSDRSWRWLFꢀ IXQFWLRQꢀ RIꢀ %D[ꢊꢀ
FRQWULEXWLQJꢀ WRꢀ UDSDORJꢀ UHVLVWDQFHꢈꢀ &RPELQHGꢀ WUHDWPHQWꢀ RIꢀ &<'ꢉꢇꢉꢄꢄꢀ DQGꢀ 5$'ꢋꢋꢄꢀ LQꢀ
PXULQHꢀOXQJꢀFDQFHUꢀPRGHOVꢀGLVSOD\HGꢀVWURQJꢀV\QHUJLVWLFꢀDFWLYLW\ꢀDQGꢀRYHUFDPHꢀUDSDORJꢀ
UHVLVWDQFHꢀLQꢀYLWURꢀDQGꢀLQꢀYLYRꢈꢀ7DNHQꢀWRJHWKHUꢊꢀRXUꢀILQGLQJVꢀSURYLGHꢀSUHFOLQLFDOꢀHYLGHQFHꢀ
IRUꢀDꢀSKDUPDFRORJLFꢀFRPELQDWLRQꢀRIꢀ%D[ꢀDFWLYDWLRQꢀDQGꢀP725ꢀLQKLELWLRQꢀDVꢀDꢀUDWLRQDOꢀ
VWUDWHJ\ꢀWRꢀLPSURYHꢀOXQJꢀFDQFHUꢀWUHDWPHQWꢈꢀꢀ
ꢀ
ꢀ
Ϯꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
ꢀ,QWURGXFWLRQꢀꢀ
/XQJꢀ FDQFHUꢀ KDVꢀ SRRUꢀ SURJQRVLVꢀ LQꢀ SDUWꢀ EHFDXVHꢀ RIꢀ LQQDWHꢀ DQGꢀ DFTXLUHGꢀ UHVLVWDQFHꢀ WRꢀ
FRQYHQWLRQDOꢀ WKHUDSLHVꢈꢀ ,Qꢀ RUGHUꢀ WRꢀ LPSURYHꢀ WKHꢀ VXUYLYDOꢀ RIꢀ OXQJꢀ FDQFHUꢀ SDWLHQWVꢂꢀ EDVLFꢀ
PROHFXODUꢀPHFKDQLVPVꢀUHVSRQVLEOHꢀIRUꢀUHVLVWDQFHꢀWRꢀWKHUDS\ꢀPXVWꢀEHꢀFDUHIXOO\ꢀHOXFLGDWHGꢀDQGꢀ
VXFKꢀ NQRZOHGJHꢀ H[SORLWHGꢀ IRUꢀ WKHꢀ GHYHORSPHQWꢀ RIꢀ PRUHꢀ HIIHFWLYHꢀ WKHUDSHXWLFꢀ DJHQWVꢈꢀ %D[ꢀ
IXQFWLRQVꢀ DVꢀ DQꢀ HVVHQWLDOꢀ JDWHZD\ꢀ WRꢀ DSRSWRWLFꢀ FHOOꢀ GHDWKꢀ ꢑꢁꢒꢀ DQGꢀ WKHUHIRUHꢀ UHSUHVHQWVꢀ DQꢀ
DWWUDFWLYHꢀWDUJHWꢀIRUꢀDQWLFDQFHUꢀWKHUDS\ꢈꢀ:HꢀDQGꢀRWKHUVꢀUHFHQWO\ꢀGLVFRYHUHGꢀWKDWꢀ$.7ꢇPHGLDWHGꢀ
SKRVSKRU\ODWLRQꢀ RIꢀ %D[ꢀ DWꢀ VHULQHꢀ ꢑ6ꢒꢁꢏꢄꢀ UHVXOWVꢀ LQꢀ WKHꢀ IDLOXUHꢀ RIꢀ %D[ꢀ WRꢀ WDUJHWꢀ RUꢀ LQVHUWꢀ LQWRꢀ
PLWRFKRQGULDOꢀ PHPEUDQHVꢂꢀ OHDGLQJꢀ WRꢀ ORVVꢀ RIꢀ LWVꢀ FHOOꢀ NLOOLQJꢀ DFWLYLW\ꢀ ꢑꢅꢇꢄꢒꢈꢀ &RQYHUVHO\ꢂꢀ
GHSKRVSKRU\ODWLRQꢀRIꢀ%D[ꢀDWꢀ6ꢁꢏꢄꢀFDXVHVꢀDꢀFRQIRUPDWLRQDOꢀFKDQJHꢀWKDWꢀSURPRWHVꢀWKHꢀLQVHUWLRQꢀ
RIꢀ%D[ꢀLQWRꢀPLWRFKRQGULDOꢀPHPEUDQHVꢀDQGꢀIRUPDWLRQꢀRIꢀ%D[ꢀROLJRPHUVꢀOHDGLQJꢀWRꢀF\WRFKURPHꢀFꢀ
UHOHDVHꢀ DQGꢀ DSRSWRVLVꢀ ꢑꢊꢒꢈꢀ 7KXVꢂꢀ WKHꢀ 6ꢁꢏꢄꢀ SKRVSKRU\ODWLRQꢀ VLWHꢀ LVꢀ Dꢀ FULWLFDOꢀ VZLWFKꢀ WRꢀ
IXQFWLRQDOO\ꢀFRQWUROꢀWKHꢀSURDSRSWRWLFꢀDFWLYLW\ꢀRIꢀ%D[ꢀꢑꢉꢂꢀꢊꢒꢈꢀ1RWDEO\ꢂꢀWKHꢀVWUXFWXUDOꢀSRFNHWꢀDURXQGꢀ
WKHꢀ6ꢁꢏꢄꢀUHVLGXHꢀLQꢀWKHꢀ%D[ꢀSURWHLQꢀUHSUHVHQWVꢀDQꢀLGHDOꢀWDUJHWꢀIRUꢀVPDOOꢀPROHFXOHꢀGRFNLQJꢀꢑꢃꢂꢀꢆꢒꢈꢀ
7KLVꢀELQGLQJꢀSRFNHWꢀLVꢀORFDWHGꢀLQꢀWKHꢀK\GURSKRELFꢀ&ꢇWHUPLQDOꢀWDLOꢀRIꢀ%D[ꢂꢀZKLFKꢀUHJXODWHVꢀQRWꢀ
RQO\ꢀWKHꢀVXEFHOOXODUꢀORFDWLRQꢀRIꢀ%D[ꢀEXWꢀDOVRꢀLWVꢀDELOLW\ꢀWRꢀLQVHUWꢀLQWRꢀPLWRFKRQGULDOꢀPHPEUDQHVꢀꢑꢉꢂꢀ
ꢊꢂꢀꢏꢒꢈꢀꢀ
ꢀꢀꢀꢀ$OWKRXJKꢀP725ꢀLVꢀDꢀSURPLVLQJꢀWKHUDSHXWLFꢀWDUJHWꢀLQꢀOXQJꢀFDQFHUꢀꢑꢎꢇꢁꢁꢒꢂꢀP725ꢀLQKLELWLRQꢀE\ꢀ
UDSDORJVꢀ KDVꢀ EHHQꢀ UHSRUWHGꢀ WRꢀ EORFNꢀ DQꢀ 6ꢃ.ꢁꢇ,56ꢁꢀ QHJDWLYHꢀ IHHGEDFNꢀ ORRSꢀ OHDGLQJꢀ WRꢀ WKHꢀ
DFWLYDWLRQꢀRIꢀ3,ꢉ.ꢓ$.7ꢀSUROLIHUDWLYHꢀDQGꢀSURVXUYLYDOꢀVLJQDOVꢂꢀDQGꢀWKXVꢀFRXQWHULQJꢀWKHꢀDQWLFDQFHUꢀ
HIILFDF\ꢀ RIꢀ UDSDORJVꢀ ꢑꢁꢅꢂꢀ ꢁꢉꢒꢈꢀ $.7ꢀ LVꢀ Dꢀ NQRZQꢀ XSVWUHDPꢀ %D[ꢀ NLQDVHꢀ WKDWꢀ FDQꢀ GLUHFWO\ꢀ
SKRVSKRU\ODWHꢀ%D[ꢀDWꢀWKHꢀ6ꢁꢏꢄꢀVLWHꢀOHDGLQJꢀWRꢀ%D[ꢀLQDFWLYDWLRQꢀꢑꢄꢒꢈꢀ+HUHꢀZHꢀGLVFRYHUHGꢀWKDWꢀWKHꢀ
UDSDORJꢇLQGXFHGꢀ DFWLYDWLRQꢀ RIꢀ $.7ꢀ HQKDQFHVꢀ %D[ꢀ SKRVSKRU\ODWLRQꢀ DQGꢀ LQDFWLYDWHVꢀ WKHꢀ
ϯꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
SURDSRSWRWLFꢀ IXQFWLRQꢀ RIꢀ %D[ꢈꢀ 8VLQJꢀ DQꢀ in silicoꢀ VFUHHQLQJꢀ DSSURDFKꢂꢀ ZHꢀ KDYHꢀ UHFHQWO\ꢀꢀ
LGHQWLILHGꢀ WKUHHꢀ VPDOOꢀ PROHFXOHꢀ %D[ꢀ DJRQLVWVꢀ ꢑ60%$ꢁꢇꢉꢒꢀ WKDWꢀ VSHFLILFDOO\ꢀ ELQGꢀ WKHꢀ VWUXFWXUDOꢀ
SRFNHWꢀ DURXQGꢀ WKHꢀ 6ꢁꢏꢄꢀ UHVLGXHꢀ RIꢀ %D[ꢀ SURWHLQꢀ OHDGLQJꢀ WRꢀ DFWLYDWLRQꢀ RIꢀ WKHꢀ SURDSRSWRWLFꢀ
IXQFWLRQꢀRIꢀ%D[ꢀꢑꢆꢒꢈꢀ%DVHGꢀRQꢀFKHPLFDOꢀVWUXFWXUHꢀDQGꢀGUXJꢇOLNHQHVVꢂꢀꢀZHꢀFKRVHꢀ60%$ꢁꢀDVꢀWKHꢀ
OHDGꢀ FRPSRXQGꢀ DQGꢀ JHQHUDWHGꢀ Dꢀ PRUHꢀ HIIHFWLYHꢀ DQDORJꢀ &<'ꢇꢅꢇꢁꢁꢂꢀ ZKLFKꢀ QRWꢀ RQO\ꢀ UHYHUVHVꢀ
UDSDORJꢇUHVLVWDQFHꢀEXWꢀDOVRꢀV\QHUJL]HVꢀZLWKꢀWKHꢀP725ꢀLQKLELWRUꢀ5$'ꢌꢌꢁꢀDJDLQVWꢀOXQJꢀFDQFHUꢈꢀ
ꢀ
0DWHULDOVꢀDQGꢀ0HWKRGVꢀ
0DWHULDOV
60%$ꢁꢀ DQGꢀ &<'ꢇꢅꢇꢁꢁꢀ FRPSRXQGVꢀ ZHUHꢀ FKHPLFDOO\ꢀ V\QWKHVL]HGꢀ E\ꢀ 'Uꢈꢀ =KRX¶Vꢀ ODERUDWRU\ꢂꢀ
8QLYHUVLW\ꢀ RIꢀ 7H[DVꢀ 0HGLFDOꢀ %UDQFKꢈꢀ 5$'ꢌꢌꢁꢀ DQGꢀ %(=ꢅꢉꢊꢀ ZHUHꢀ SXUFKDVHGꢀ IURPꢀ 6HOOHFNꢀ
&KHPLFDOVꢀꢑ+RXVWRQꢂꢀ7;ꢒꢈꢀ$QWLꢇ%D[ꢀꢑFDWDORJꢀꢔꢀVFꢇꢄꢎꢉꢒꢀDQGꢀȕꢇDFWLQꢀZHUHꢀSXUFKDVHGꢀIURPꢀ6DQWDꢀ
&UX]ꢀ%LRWHFKQRORJ\ꢀꢑ6DQWDꢀ&UX]ꢂꢀ&$ꢒꢈꢀ%D[ꢀꢃ$ꢆꢀDQGꢀ&\WꢀFꢀDQWLERGLHVꢀZHUHꢀREWDLQHGꢀIURPꢀ%'ꢀ
3KDU0LQJHQꢀ ꢑ6DQꢀ 'LHJRꢂꢀ &$ꢒꢈꢀ 3KRVSKRꢇVSHFLILFꢀ 6ꢁꢏꢄꢀ %D[ꢀ ꢑS%D[ꢒꢀ DQWLERG\ꢀ ZDVꢀ SXUFKDVHGꢀ
IURPꢀ$EFDPꢀꢑ&DPEULGJHꢂꢀ0$ꢒꢈꢀ$QWLERGLHVꢀDJDLQVWꢀDFWLYHꢀFDVSDVHꢀꢉꢂꢀ$NWꢂꢀP725ꢂꢀS$.7ꢀꢑ6ꢄꢆꢉꢒꢀ
DQGꢀ SꢇSꢆꢌ6ꢃ.ꢀ ZHUHꢀ SXUFKDVHGꢀ IURPꢀ &HOOꢀ 6LJQDOLQJꢀ 7HFKQRORJ\ꢀ ꢑ%HYHUO\ꢂꢀ 0$ꢒꢈꢀ )OXRUHVFHQWꢀ
%DNꢀ SHSWLGHꢀ ꢑ)$0ꢇ*49*54/$,,*'',15ꢒꢀ DQGꢀ %FOꢇ;/ꢀ SURWHLQꢀ ZHUHꢀ SXUFKDVHGꢀ IURPꢀ
1HR%LR6FL70ꢀ ꢑ&DPEULGJHꢂꢀ 0$ꢒꢈꢀ 3XULILHGꢀ UHFRPELQDQWꢀ IXOOꢇOHQJWKꢀ KXPDQꢀ %FOꢅꢀ SURWHLQꢀ ZDVꢀ
REWDLQHGꢀ IURPꢀ 3URWHLQXꢀ /DEꢀ ꢑ6DQꢀ 'LHJRꢂꢀ &$ꢒꢈꢀ 3XULILHGꢀ IXOOꢇOHQJWKꢀ KXPDQꢀ 0FOꢇꢁꢀ SURWHLQꢀ ZDVꢀ
SXUFKDVHGꢀ IURPꢀ *HQ:D\ꢀ %LRWHFKꢂꢀ ,QFꢈꢀ ꢑ6DQꢀ 'LHJRꢂꢀ &$ꢒꢈꢀ 3XULILHGꢀ IXOOꢇOHQJWKꢀ KXPDQꢀ %IOꢇꢁꢓ$ꢁꢀ
SURWHLQꢀ ZDVꢀ REWDLQHGꢀ IURPꢀ 5 'ꢀ V\VWHPVꢀ ꢑ0LQQHDSROLVꢂꢀ 01ꢒꢈꢀ 3XULILHGꢀ IXOOꢇOHQJWKꢀ KXPDQꢀ %D[ꢀ
SURWHLQꢀ ZDVꢀ SXUFKDVHGꢀ IURPꢀ 1RYXVꢀ ꢑ/LWWOHWRQꢂꢀ &2ꢒꢈꢀ %LVꢑPDOHLPLGRꢒꢀ KH[DQHꢀ ꢑ%0+ꢒꢀ ZDVꢀ
SXUFKDVHGꢀIURPꢀ7KHUPRꢀ6FLHQWLILFꢈꢀ4'ꢃꢌꢊꢀJRDWꢀDQWLꢇUDEELWꢀ,J*ꢀFRQMXJDWHꢀꢑUHGꢒꢂꢀ4'ꢆꢌꢊꢀJRDWꢀ
ϰꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
DQWLꢇPRXVHꢀ,J*ꢀFRQMXJDWHꢀꢑJUHHQꢒꢀDQGꢀ3UR/RQJ£ꢀ*ROGꢀDQWLIDGHꢀUHDJHQWꢀZLWKꢀꢄꢕꢂꢀꢃꢇGLDPLGLQRꢇꢅꢇ
SKHQ\OLQGROHꢀꢑ'$3,ꢒꢀZHUHꢀREWDLQHGꢀIURPꢀ,QYLWURJHQꢀ/LIHꢀ7HFKQRORJLHVꢀ,QFꢀꢑ&DUOVEDGꢂꢀ&$ꢒꢈꢀ&UHꢇ
DGHQRYLUXVꢀ ꢑ$Gꢊ&09&UHꢍꢀ &DWꢀ ꢔꢐꢀ 99&ꢇ8ꢀ RIꢀ ,RZDꢇꢊꢒꢀ ZDVꢀ REWDLQHGꢀ IURPꢀ 9LUDOꢀ 9HFWRUꢀ &RUHꢀ
)DFLOLW\ꢀRIꢀWKHꢀ8QLYHUVLW\ꢀRIꢀ,RZDꢈꢀ$OOꢀUHDJHQWVꢀXVHGꢀZHUHꢀREWDLQHGꢀIURPꢀFRPPHUFLDOꢀVRXUFHVꢀ
XQOHVVꢀRWKHUZLVHꢀVWDWHGꢈꢀ
&HOOꢀOLQHVꢀDQGꢀFHOOꢀFXOWXUH
16&/&ꢂꢀ6&/&ꢀDQGꢀQRUPDOꢀKXPDQꢀEURQFKLDOꢀHSLWKHOLDOꢀꢑ%($6ꢇꢅ%ꢒꢀFHOOꢀOLQHVꢀZHUHꢀREWDLQHGꢀIURPꢀ
WKHꢀ $PHULFDQꢀ 7\SHꢀ &XOWXUHꢀ &ROOHFWLRQꢀ ꢑ0DQDVVDVꢂꢀ 9$ꢒꢈꢀ +XPDQꢀ 16&/&ꢀ FHOOꢀ OLQHVꢀ ꢑ+ꢁꢅꢎꢎꢂꢀ
+ꢅꢎꢅꢂꢀ+ꢁꢊꢆꢂꢀ&DOXꢇꢁꢂꢀ+ꢄꢃꢌꢀDQGꢀ+ꢁꢎꢆꢊꢒꢀZHUHꢀPDLQWDLQHGꢀLQꢀ530,ꢀꢁꢃꢄꢌꢀZLWKꢀꢁꢌꢖꢀIHWDOꢀERYLQHꢀ
VHUXPꢀꢑ)%6ꢒꢈꢀ$ꢊꢄꢎꢀFHOOVꢀZHUHꢀFXOWXUHGꢀLQꢀ)ꢇꢁꢅ.ꢀPHGLXPꢀZLWKꢀꢁꢌꢖꢀ)%6ꢈꢀ%($6ꢇꢅ%ꢀFHOOVꢀZHUHꢀ
FXOWXUHGꢀLQꢀ'0(0/)ꢇꢁꢅꢀPHGLXPꢀZLWKꢀꢁꢌꢖꢀ)%6ꢈꢀ6&/&ꢀFHOOꢀOLQHVꢀ'06ꢊꢉꢂꢀ'06ꢁꢊꢉꢂꢀ'06ꢁꢁꢄꢂꢀ
+ꢁꢅꢏꢂꢀ +ꢁꢄꢃꢀ DQGꢀ +ꢃꢎꢀ ZHUHꢀ FXOWXUHGꢀ LQꢀ :H\PRXWKꢕVꢀ PHGLXPꢀ ZLWKꢀ ꢊꢖꢀ )%6ꢀ DQGꢀ ꢊꢖꢀ ERYLQHꢀ
VHUXPꢀ ꢑ%6ꢒꢀ DVꢀ GHVFULEHGꢀ ꢑꢁꢄꢒꢈꢀ 7KHꢀ UDSDP\FLQꢀ UHVLVWDQWꢀ $ꢊꢄꢎꢀ FHOOꢀ OLQHꢀ ꢑ$ꢊꢄꢎꢇ55ꢒꢀ ZDVꢀ
HVWDEOLVKHGꢀDVꢀGHVFULEHGꢀꢑꢁꢌꢂꢀꢁꢊꢒꢂꢀDQGꢀLVꢀDOVRꢀUHVLVWDQWꢀWRꢀUDSDORJꢀ5$'ꢌꢌꢁꢈꢀ7KHVHꢀFHOOꢀOLQHVꢀ
ZHUHꢀHPSOR\HGꢀIRUꢀWKHꢀGHVFULEHGꢀH[SHULPHQWVꢀZLWKRXWꢀIXUWKHUꢀDXWKHQWLFDWLRQꢀE\ꢀDXWKRUVꢈꢀꢀ
+XPDQꢀ FOLQLFDOꢀ WULDOꢀ SDWLHQWꢀ VDPSOHVꢀ DQGꢀ HVWDEOLVKPHQWꢀ RIꢀ SDWLHQWꢉGHULYHGꢀ [HQRJUDIWVꢀ
ꢂ3';ꢃꢀ
,QIRUPHGꢀ FRQVHQWꢀ ZDVꢀ REWDLQHGꢀ IURPꢀ DOOꢀ KXPDQꢀ VXEMHFWVꢂꢀ DQGꢀ XVHꢀ RIꢀ KXPDQꢀ VDPSOHVꢀ IRUꢀ
LPPXQRKLVWRFKHPLVWU\ꢀ DQGꢀ 3';ꢀ ZDVꢀ DSSURYHGꢀ E\ꢀ WKHꢀ ,QVWLWXWLRQDOꢀ 5HYLHZꢀ %RDUGꢀ ꢑ,5%ꢒꢀ RIꢀ
(PRU\ꢀ 8QLYHUVLW\ꢈꢀ 3DLUHGꢀ VDPSOHVꢀ RIꢀ 16&/&ꢀ WLVVXHVꢀ ZHUHꢀ FROOHFWHGꢀ DVꢀ SDUWꢀ RIꢀ Dꢀ FRPSOHWHGꢀ
SKDVHꢀ,ꢀFOLQLFDOꢀWULDOꢀHYDOXDWLQJꢀWKHꢀSKDUPDFRG\QDPLFꢀHIIHFWVꢀRIꢀ5$'ꢌꢌꢁꢀLQꢀDGXOWꢀSDWLHQWVꢀZLWKꢀ
UHVHFWDEOHꢀ16&/&ꢈꢀ(QUROOHGꢀHOLJLEOHꢀSDWLHQWVꢀUHFHLYHGꢀ5$'ꢌꢌꢁꢀꢑꢁꢌꢀPJꢒꢀRUDOO\ꢀRQFHꢀGDLO\ꢀIRUꢀꢅꢏꢀ
GD\Vꢀꢑꢁꢃꢒꢈꢀ3UHWUHDWPHQWꢀELRSV\ꢀVDPSOHVꢀDQGꢀSRVWꢀWUHDWPHQWꢀVXUJLFDOꢀUHVHFWLRQꢀVSHFLPHQVꢀZHUHꢀ
ϱꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
DQDO\]HGꢀIRUꢀS%D[ꢀꢑ6ꢁꢏꢄꢒꢀH[SUHVVLRQꢈꢀ3';Vꢀꢑ7.2ꢇꢌꢌꢅꢀDQGꢀ7.2ꢇꢌꢌꢊꢒꢀZHUHꢀREWDLQHGꢀDVꢀSDUWꢀRIꢀ
DQꢀ ,5%ꢇDSSURYHGꢀ SKDVHꢀ ,,ꢀ FRꢇFOLQLFDOꢀ WULDOꢀ RIꢀ DUVHQLFꢀ WULR[LGHꢀ LQꢀ SDWLHQWVꢀ ZLWKꢀ UHODSVHGꢀ 6&/&ꢀ
ZKRꢀ KDGꢀ IDLOHGꢀ VWDQGDUGꢀ SODWLQXPꢇEDVHGꢀ FKHPRWKHUDS\ꢈꢀ 7XPRUꢀ VDPSOHVꢀ ZHUHꢀ REWDLQHGꢀ E\ꢀ
LPDJHꢇJXLGHGꢀ ELRSV\ꢀ DQGꢀ ZHUHꢀ GLUHFWO\ꢀ LPSODQWHGꢀ LQWRꢀ 1Xꢓ1Xꢀ QXGHꢀ PLFHꢀ ZLWKRXWꢀ LQWHUYHQLQJꢀ
SURSDJDWLRQꢀLQꢀSODVWLFꢀFXOWXUHꢀSODWHVꢈꢀ'LUHFWꢀVHULDOꢀSURSDJDWLRQꢀRIꢀJURZLQJꢀWXPRUꢀRFFXUUHGꢀIRUꢀXSꢀ
WRꢀꢊꢀJHQHUDWLRQVꢈꢀ7KHꢀDQLPDOꢀSURSDJDWLRQꢀSURWRFROꢀZDVꢀDSSURYHGꢀE\ꢀWKHꢀ(PRU\ꢀ,$&8&ꢀDQGꢀWKHꢀ
(PRU\ꢀ$QLPDOꢀ(WKLFVꢀ&RPPLWWHHꢈꢀ
*HQHWLFDOO\ꢀ HQJLQHHUHGꢀ PRXVHꢀ PRGHOꢀ ꢂ*(00ꢃꢊꢀ WUHDWPHQWꢀ DQGꢀ WXPRUꢀ EXUGHQꢀ
TXDQWLILFDWLRQ
0RXVHꢀ VWUDLQVꢀ KDUERULQJꢀ Dꢀ FRQGLWLRQDOꢀ DFWLYDWLQJꢀ PXWDWLRQꢀ ꢑ*ꢁꢅ'ꢒꢀ DWꢀWKHꢀ HQGRJHQRXVꢀ .5$6ꢀ
ORFXVꢀ DQGꢀ FRQGLWLRQDOꢀ /.%ꢁꢀ NQRFNRXWꢀ ZHUHꢀ SUHYLRXVO\ꢀ GHVFULEHGꢀ ꢑꢁꢆꢒꢈꢀ /R[ꢇVWRSꢇOR[ꢀ ꢑ/6/)ꢇ
.5$6*ꢁꢅ'ꢀ/.%ꢁIOꢓIOꢀꢑi.eꢈꢀ./ꢒꢀPLFHꢀZHUHꢀJHQHUDWHGꢀE\ꢀLQWHUFURVVLQJꢀWKHꢀ%ꢃꢈꢁꢅꢎ6ꢄꢇ.UDVWPꢉ7\Mꢓ-ꢀ
ꢑ.ꢇ5DV*ꢁꢅ'ꢒꢀ DQGꢀ )9%ꢍꢀ ꢁꢅꢎ6ꢃꢇ6WNꢁꢁWPꢁ5GSꢓ1FLꢀ ꢑ/.%ꢁꢒꢀ VWUDLQVꢀ IRUꢀ WZRꢀ JHQHUDWLRQVꢀ DQGꢀ
JHQRW\SHGꢀWRꢀFRQILUPꢀKRPR]\JRVLW\ꢀIRUꢀWKHꢀ/.%ꢁꢀDOOHOHꢀDVꢀGHVFULEHGꢀꢑꢁꢆꢇꢅꢌꢒꢈꢀ$OOꢀVWXGLHVꢀZHUHꢀ
SHUIRUPHGꢀRQꢀSURWRFROVꢀDSSURYHGꢀE\ꢀWKHꢀ(PRU\ꢀ8QLYHUVLW\ꢀ,$&8&ꢈꢀ6L[ꢀZHHNVꢀDIWHUꢀLQWUDQDVDOꢀ
DGPLQLVWUDWLRQꢀRIꢀꢊ[ꢁꢌꢃꢀSIXꢀ$GH&UHꢂꢀPLFHꢀZHUHꢀWUHDWHGꢀZLWKꢀ&<'ꢇꢅꢇꢁꢁꢀLꢈSꢀIRUꢀꢏꢀZHHNVꢈꢀ0LFHꢀ
ZHUHꢀVDFULILFHGꢀE\ꢀLQKDOHGꢀ&2ꢅꢀDWꢀWKHꢀHQGꢀRIꢀWUHDWPHQWꢈꢀ$IWHUꢀOXQJꢀSHUIXVLRQꢀZLWKꢀ3%6ꢂꢀWKHꢀOHIWꢀ
OXQJꢀOREHVꢀZHUHꢀKDUYHVWHGꢀIURPꢀPLFHꢀLQꢀFRQWUROꢀDQGꢀ&<'ꢇꢅꢇꢁꢁꢀWUHDWHGꢀJURXSVꢀDQGꢀLPPHGLDWHO\ꢀ
IL[HGꢀLQꢀꢁꢌꢖꢀQHXWUDOꢀEXIIHUHGꢀIRUPDOLQꢀꢑ)LVKHUꢀ6FLHQWLILFꢂꢀ.DODPD]RRꢂꢀ0,ꢒꢂꢀKRUL]RQWDOO\ꢀFXWꢀLQWRꢀ
WKUHHꢀHTXDOꢀSDUWVꢀDQGꢀHPEHGGHGꢀLQꢀSDUDIILQꢀEORFNVꢈꢀ7KUHHꢀSDUWVꢀRIꢀOXQJꢀWLVVXHVꢀUHSUHVHQWLQJꢀ
GLIIHUHQWꢀUHJLRQVꢀRIꢀWKHꢀOXQJꢀZHUHꢀYHUWLFDOO\ꢀSXWꢀLQWRꢀSDUDIILQꢀEORFNVꢀIRUꢀKHPDWR[LQꢇHRVLQꢀꢑ+ (ꢒꢀ
VWDLQLQJꢈꢀ/XQJꢀWLVVXHꢀVDPSOHVꢀZHUHꢀVHFWLRQHGꢀDWꢀꢉꢀ—PꢀWKUHHꢀWLPHVꢀIRUꢀSODFHPHQWꢀRIꢀVOLGHVꢀDQGꢀ
VWDLQHGꢀ ZLWKꢀ + (ꢈꢀ + (ꢀ VWDLQHGꢀ VDPSOHVꢀ ZHUHꢀ VFDQQHGꢀ XVLQJꢀ 1DQRꢀ =RRPHUꢀ ꢅꢈꢌꢇ+7ꢀ
ϲꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
ꢑ+DPDPDWVXꢂꢀ-DSDQꢒꢀDQGꢀLPDJHVꢀZHUHꢀDQDO\]HGꢀXVLQJꢀ,PDJH6FRSHꢀYLHZLQJꢀVRIWZDUHꢀꢑ/HLFDꢀ
%LRV\VWHPVꢂꢀ%XIIDORꢀ*URYHꢂꢀ,/ꢒꢈꢀ7XPRUꢀQXPEHUVꢀZHUHꢀFRXQWHGꢀXQGHUꢀDꢀPLFURVFRSHꢀDQGꢀWXPRUꢀ
DUHDꢀZDVꢀTXDQWLILHGꢀXVLQJꢀ2SHQODEꢀPRGXODUꢀLPDJLQJꢀVRIWZDUHꢀꢑ3HUNLQ(OPHUꢂꢀ:DOWKDPꢂꢀ0$ꢒꢈ ꢀ
6WDWLVWLFDOꢀDQDO\VLV
7KHꢀ VWDWLVWLFDOꢀ VLJQLILFDQFHꢀ RIꢀ GLIIHUHQFHVꢀ EHWZHHQꢀ WZRꢀ JURXSVꢀ ZDVꢀ DQDO\]HGꢀ ZLWKꢀ WZRꢇVLGHGꢀ
XQSDLUHGꢀ VWXGHQW¶Vꢀ WꢇWHVWꢈꢀ 5HVXOWVꢀ ZHUHꢀ FRQVLGHUHGꢀ WRꢀ EHꢀ VWDWLVWLFDOO\ꢀ VLJQLILFDQWꢀ DWꢀ Sꢀ ꢗꢀ ꢌꢈꢌꢊꢈꢀ
6WDWLVWLFDOꢀDQDO\VLVꢀZDVꢀSHUIRUPHGꢀZLWKꢀ*UDSKSDGꢀ,Q6WDWꢀꢉꢀVRIWZDUHꢀꢑ6DQꢀ'LHJRꢂꢀ&$ꢒꢀꢑꢅꢁꢒꢈꢀ
ꢀ
5HVXOWVꢀꢀ
*HQHUDWLRQꢀRIꢀQHZꢀ60%$ꢀDQDORJꢀ&<'ꢉꢇꢉꢄꢄ
:Hꢀ KDYHꢀ UHFHQWO\ꢀ LGHQWLILHGꢀ WKUHHꢀ VPDOOꢀ PROHFXOHꢀ %D[ꢀ DJRQLVWꢀ FRPSRXQGVꢀ ꢑ60%$ꢁꢂꢀ 60%$ꢅꢀ
DQGꢀ60%$ꢉꢒꢀꢑꢆꢒꢈꢀ%DVHGꢀRQꢀFRPSDULVRQꢀDQDO\VHVꢀRIꢀ6ꢁꢏꢄꢀGRFNLQJꢀDQGꢀWKHꢀFKHPLFDOꢀVWUXFWXUHVꢀ
DVꢀ ZHOOꢀ DVꢀ GUXJꢇOLNHQHVVꢂꢀ ZHꢀ VHOHFWHGꢀ 60%$ꢁꢀ DVꢀ WKHꢀ EHVWꢀ OHDGꢀ FRPSRXQGꢀ RIꢀ WKHꢀ WKUHHꢀ
PROHFXOHVꢀIRUꢀIXUWKHUꢀ6$5ꢀVWXGLHVꢈꢀ2XUꢀPROHFXODUꢀGRFNLQJꢀDQDO\VLVꢀUHYHDOHGꢀWKDWꢀLQWURGXFWLRQꢀRIꢀ
DQꢀ OꢇDON\ODPLQRꢀ VLGHꢀ FKDLQꢀ WHWKHUHGꢀ WRꢀ WKHꢀ K\GUR[\Oꢀ JURXSꢀ RQꢀ WKHꢀ SKHQ\Oꢀ ULQJꢀ RIꢀ OHDGꢀ
FRPSRXQGꢀ60%$ꢁꢀꢑ)LJXUHꢀꢄ$ꢀDQGꢀ%ꢒꢀFRXOGꢀHQKDQFHꢀWKHꢀSKDUPDFRSKRUHꢀJURXSꢀWRꢀDFFHVVꢀDꢀ
GHHSHUꢀELQGLQJꢀSRFNHWꢀQHDUꢀ6ꢁꢏꢄꢀDQGꢀLPSURYHꢀ%D[ꢀELQGLQJꢈꢀ7KHꢀUHVXOWLQJꢀDQDORJꢀ&<'ꢇꢅꢇꢁꢁꢀ
GRFNVꢀZHOOꢀLQWRꢀWKHꢀELQGLQJꢀSRFNHWꢀRIꢀ%D[ꢀꢑ)LJXUHꢀ ꢄ&ꢃꢂꢀDQGꢀWKHꢀOꢇDON\ODPLQRꢀVLGHꢀFKDLQꢀFDQꢀ
IRUPꢀ K\GURJHQꢇERQGVꢀ ZLWKꢀ WKHꢀ DPLQRꢀ DFLGꢀ UHVLGXHVꢀ DURXQGꢀ WKHꢀ 6ꢁꢏꢄꢀ VLWHꢀ ꢑ)LJXUHꢀ ꢄ&ꢒꢈꢀ
0RUHRYHUꢂꢀWKHꢀWHUPLQDOꢀDPLQRꢀJURXSꢀRIꢀ&<'ꢇꢅꢇꢁꢁꢀFDQꢀIRUPꢀDQꢀ+&OꢀVDOWꢀWRꢀLPSURYHꢀDTXHRXVꢀ
VROXELOLW\ꢀ DQGꢀ OHDGꢀ WRꢀ Dꢀ IDYRUDEOHꢀ /RJ3ꢀ YDOXHꢀ RIꢀ WKHꢀ PROHFXOHꢀ IRUꢀ EHWWHUꢀ FHOOꢀ SHUPHDELOLW\ꢈꢀ
60%$ꢁꢀZDVꢀV\QWKHVL]HGꢀE\ꢀFRXSOLQJꢀRIꢀWKHꢀFRPPHUFLDOO\ꢀDYDLODEOHꢀPDWHULDOꢀꢅꢇQLWURꢇꢎHꢇIOXRUHQHꢀ
ZLWKꢀVDOLF\ODOGHK\GHꢀLQꢀWKHꢀSUHVHQFHꢀRIꢀ.)ꢇ$Oꢅ2ꢉꢀDVꢀDꢀVROLGꢀEDVHꢀLQꢀPHWKDQROꢀZLWKꢀꢆꢅꢖꢀ\LHOGꢈꢀ
ϳꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
$QDORJXHꢀ&<'ꢇꢅꢇꢁꢁꢀZDVꢀV\QWKHVL]HGꢀXVLQJꢀWKHꢀ0LWVXQREXꢀUHDFWLRQꢀIURPꢀSKHQROꢀ60%$ꢁꢀZLWKꢀ
%RFꢇSURWHFWHGꢀDPLQRHWKDQROꢂꢀIROORZHGꢀE\ꢀVXEVHTXHQWꢀ%RFꢇGHSURWHFWLRQꢀLQꢀDꢀWZRꢇVWHSꢀSURFHVVꢂꢀ
ZLWKꢀDꢀ\LHOGꢀRIꢀꢆꢉꢖꢈꢀ
ꢀ
&<'ꢉꢇꢉꢄꢄꢀVSHFLILFDOO\ꢀELQGVꢀWRꢀ%D[ꢀSURWHLQꢀDQGꢀLQGXFHVꢀDSRSWRVLVꢀRIꢀKXPDQꢀOXQJꢀFDQFHUꢀ
FHOOV
&RPSXWDWLRQDOꢀPRGHOLQJꢀDQDO\VLVꢀUHYHDOVꢀWKDWꢀ&<'ꢇꢅꢇꢁꢁꢀGRFNVꢀDWꢀWKHꢀ6ꢁꢏꢄꢀELQGLQJꢀSRFNHWꢀRIꢀ
%D[ꢀ ꢑ)LJXUHꢀ ꢄ&ꢒꢈꢀ %D[ꢀ DQGꢀ %DNꢀ SURWHLQVꢀ FDQꢀ KHWHURGLPHUL]Hꢀ YLDꢀ WKHLUꢀ %+ꢉꢀ GRPDLQVꢀ WRꢀ IRUPꢀ
%D[ꢓ%DNꢀKHWHURGLPHUVꢀꢑꢅꢅꢒꢈꢀ$ꢀIOXRUHVFHQWO\ꢇODEHOHGꢀ%DNꢀ%+ꢉꢀGRPDLQꢀSHSWLGHꢀELQGVꢀWRꢀWKHꢀ%D[ꢀ
SURWHLQꢀZLWKꢀKLJKꢇDIILQLW\ꢀꢑꢆꢒꢈꢀ7KLVꢀ%DNꢀ%+ꢉꢀGRPDLQꢀSHSWLGHꢀLVꢀORFDWHGꢀLQꢀ%DNꢀ%+ꢉꢀGRPDLQꢀDVꢀ
VKRZQꢀ LQꢀ )LJXUHꢀ 6ꢄꢈꢀ :Hꢀ XVHGꢀ WKLVꢀ SHSWLGHꢀ WRꢀ TXDQWLI\ꢀ &<'ꢇꢅꢇꢁꢁꢓ%D[ꢀ ELQGLQJꢀ DIILQLW\ꢀ LQꢀ Dꢀ
FRPSHWLWLRQꢀIOXRUHVFHQFHꢀSRODUL]DWLRQꢀDVVD\ꢀDVꢀGHVFULEHGꢀꢑꢅꢉꢇꢅꢊꢒꢈꢀ5HVXOWVꢀLQGLFDWHꢀWKDWꢀ&<'ꢇꢅꢇ
ꢁꢁꢀGLUHFWO\ꢀELQGVꢀWRꢀ%D[ꢀSURWHLQꢀZLWKꢀKLJKꢀDIILQLW\ꢀꢑKLꢀꢐꢀꢉꢄꢈꢁꢀ“ꢀꢏꢈꢊꢄꢀQ0ꢒꢀꢀꢑ)LJXUHꢀꢄ'ꢒꢈꢀ&<'ꢇꢅꢇꢁꢁꢀ
IDLOHGꢀWRꢀELQGꢀRWKHUꢀ%FOꢅꢀIDPLO\ꢀPHPEHUVꢀꢑi.eꢈꢀ%DNꢂꢀ%LGꢂꢀ%FOꢅꢂꢀ%FOꢇ;/ꢂꢀ%FOꢇZꢂꢀ0FOꢇꢁꢀDQGꢀ$ꢁꢒꢀin
vitroꢀꢑ)LJXUHꢀꢄ'ꢒꢂꢀLQGLFDWLQJꢀDꢀELQGLQJꢀVHOHFWLYLW\ꢀIRUꢀ%D[ꢀSURWHLQꢈꢀ7KHꢀDSRSWRWLFꢀHIIHFWꢀRIꢀ&<'ꢇ
ꢅꢇꢁꢁꢀ ZDVꢀ PHDVXUHGꢀ LQꢀ ERWKꢀ 6&/&ꢀ DQGꢀ 16&/&ꢀ FHOOꢀ OLQHVꢀ WKDWꢀ H[SUHVVꢀ YDULRXVꢀ OHYHOVꢀ RIꢀ
HQGRJHQRXVꢀ S%D[ꢓ%D[ꢀ E\ꢀ DQDO\VLVꢀ RIꢀ $QQH[LQꢇ9ꢓ3,ꢀ ELQGLQJꢀ E\ꢀ IOXRUHVFHQFHꢇDFWLYDWHGꢀ FHOOꢀ
VRUWHUꢀꢑ)$&6ꢒꢀDVꢀZHꢀSUHYLRXVO\ꢀGHVFULEHGꢀꢑꢅꢃꢂꢀꢅꢆꢒꢈꢀ,WꢀDSSHDUVꢀWKDWꢀKXPDQꢀOXQJꢀFDQFHUꢀFHOOVꢀ
H[SUHVVLQJꢀKLJKHUꢀOHYHOVꢀRIꢀWRWDOꢀ%D[ꢀDOVRꢀFRQWDLQꢀUHODWLYHO\ꢀKLJKHUꢀOHYHOVꢀRIꢀS%D[ꢀꢑ)LJXUHꢀꢄ(ꢒꢈꢀ
&<'ꢇꢅꢇꢁꢁꢀZDVꢀPRUHꢀSRWHQWꢀWKDQꢀ60%$ꢁꢀLQꢀLQGXFLQJꢀDSRSWRVLVꢀLQꢀERWKꢀ16&/&ꢀDQGꢀ6&/&ꢀFHOOꢀ
OLQHVꢈꢀ16&/&ꢀFHOOꢀOLQHVꢀꢑi.e. +ꢅꢎꢅꢂꢀ+ꢁꢊꢆꢂꢀ+ꢁꢎꢆꢊꢂꢀ$ꢊꢄꢎꢒꢀDQGꢀ6&/&ꢀFHOOꢀOLQHVꢀꢑi.eꢈꢀ'06ꢊꢉꢀDQGꢀ
'06ꢁꢊꢉꢒꢀWKDWꢀH[SUHVVꢀUHODWLYHO\ꢀKLJKꢀOHYHOVꢀRIꢀS%D[ꢓ%D[ꢀZHUHꢀPRUHꢀVHQVLWLYHꢀWRꢀLQGXFWLRQꢀRIꢀ
DSRSWRVLVꢀE\ꢀ&<'ꢇꢅꢇꢁꢁꢈꢀ,QꢀFRQWUDVWꢂꢀOXQJꢀFDQFHUꢀFHOOꢀOLQHVꢀH[SUHVVLQJꢀUHODWLYHO\ꢀORZꢀOHYHOVꢀRIꢀ
ϴꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
S%D[ꢓ%D[ꢀꢑi.eꢈꢀ16&/&ꢀFHOOꢀOLQHꢐꢀ+ꢄꢃꢌꢍꢀ6&/&ꢀFHOOꢀOLQHVꢐꢀ'06ꢁꢁꢄꢀDQGꢀ+ꢁꢅꢏꢒꢀZHUHꢀUHODWLYHO\ꢀOHVVꢀ
VHQVLWLYHꢀWRꢀ&<'ꢇꢅꢇꢁꢁꢀꢑ)LJXUHꢀꢄ(ꢀDQGꢀ)ꢒꢈꢀ7RꢀFRQILUPꢀWKHꢀDQWLꢇSKRVSKRꢀ%D[ꢀꢑ6ꢁꢏꢄꢒꢀDQWLERG\ꢀLVꢀ
WRWDOO\ꢀVHOHFWLYHꢀWRꢀWKHꢀSKRVSKRU\ODWHGꢀIRUPꢀRIꢀ%D[ꢂꢀZHꢀXVHGꢀSURWHLQꢀSKRVSKDWDVHꢀꢑλ33DVHꢒꢀWRꢀ
WUHDWꢀ LPPXQRSUHFLSLWDWHGꢀ %D[ꢀ IURPꢀ $ꢊꢄꢎꢀ FHOOVꢈꢀ S%D[ꢀ ZDVꢀ DQDO\]HGꢀ E\ꢀ DQWLꢇSKRVSKRꢇVSHFLILFꢀ
%D[ꢀDQWLERG\ꢈꢀ5HVXOWVꢀUHYHDOꢀWKDWꢀλ33DVHꢀUHPRYHGꢀSKRVSKDWHꢀIURPꢀ6ꢁꢏꢄꢀSKRVSKRU\ODWHGꢀ%D[ꢀ
EXWꢀKDVꢀQRꢀHIIHFWꢀRQꢀWRWDOꢀ%D[ꢀOHYHOꢀꢑ)LJXUHꢀ6ꢇꢒꢂꢀVXJJHVWLQJꢀWKDWꢀWKHꢀDQWLꢇSKRVSKRꢀ%D[ꢀꢑ6ꢁꢏꢄꢒꢀ
DQWLERG\ꢀZHꢀXVHGꢀLVꢀWRWDOO\ꢀVHOHFWLYHꢀWRꢀWKHꢀS%D[ꢈꢀꢀ%DVHGꢀRQꢀRXUꢀILQGLQJVꢂꢀZHꢀSURSRVHꢀWKDWꢀWKHꢀ
F\WRWR[LFLW\ꢀRIꢀ&<'ꢇꢅꢇꢁꢁꢀPD\ꢀEHꢀGHSHQGHQWꢀRQꢀWKHꢀH[SUHVVLRQꢀOHYHOVꢀRIꢀS%D[ꢀDQGꢓRUꢀWRWDOꢀ%D[ꢀ
LQꢀ OXQJꢀ FDQFHUꢀ FHOOꢀ OLQHVꢈꢀ ,PSRUWDQWO\ꢂꢀ &<'ꢇꢅꢇꢁꢁꢀ VKRZHGꢀ OHVVꢀ F\WRWR[LFLW\ꢀ LQꢀ WKHꢀ QRUPDOꢀ OXQJꢀ
HSLWKHOLDOꢀFHOOꢀOLQHꢀꢑ%($6ꢇꢅ%ꢒꢂꢀZKLFKꢀH[SUHVVHVꢀDꢀORZꢀOHYHOꢀRIꢀHQGRJHQRXVꢀS%D[ꢀꢑ)LJXUHꢀ ꢄ(ꢀ
DQGꢀ)ꢒꢂꢀLQGLFDWLQJꢀDꢀUHODWLYHꢀVHOHFWLYLW\ꢀDJDLQVWꢀFDQFHUꢀFHOOVꢀFRPSDUHGꢀWRꢀQRUPDOꢀFHOOVꢈꢀ7KHVHꢀ
ILQGLQJVꢀVXJJHVWꢀWKDWꢀ&<'ꢇꢅꢇꢁꢁꢀPD\ꢀKDYHꢀVWURQJꢀSRWHQWLDOꢀWRꢀEHꢀGHYHORSHGꢀDVꢀDꢀQHZꢀFODVVꢀRIꢀ
DQWLꢇFDQFHUꢀDJHQWVꢈꢀꢀꢀ
&<'ꢉꢇꢉꢄꢄꢀGLUHFWO\ꢀDFWLYDWHVꢀWKHꢀSURDSRSWRWLFꢀIXQFWLRQꢀRIꢀ%D[ꢀYLDꢀROLJRPHUL]DWLRQꢀOHDGLQJꢀ
WRꢀF\WRFKURPHꢀFꢀꢂ&\WꢀFꢃꢀUHOHDVHꢀIURPꢀPLWRFKRQGULD
2XUꢀUHVXOWVꢀ LQGLFDWHꢀ WKDWꢀ WUHDWPHQWꢀ RIꢀ KXPDQꢀ OXQJꢀ FDQFHUꢀ $ꢊꢄꢎꢀ FHOOVꢀ ZLWKꢀ &<'ꢇꢅꢇꢁꢁꢀ GLGꢀ QRWꢀ
DIIHFWꢀ %D[ꢀ SURWHLQꢀ OHYHOꢀ ꢑ)LJXUHꢀ ꢇ$ꢒꢈꢀ ,Wꢀ LVꢀ NQRZQꢀ WKDWꢀ %D[ꢀ ROLJRPHUL]DWLRQꢀ FDQꢀ DFWLYDWHꢀ WKHꢀ
SURDSRSWRWLFꢀ IXQFWLRQꢀ RIꢀ %D[ꢀ EHFDXVHꢀ %D[ꢀ ROLJRPHUVꢀ FDQꢀ IRUPꢀ Dꢀ SRUHꢀ ZLWKꢀ Dꢀ VL]Hꢀ WKDWꢀ LVꢀ
FDSDEOHꢀ RIꢀ WUDQVSRUWLQJꢀ &\Wꢀ Fꢀ ꢑꢅꢏꢒꢈꢀ 7Rꢀ DVVHVVꢀ ZKHWKHUꢀ &<'ꢇꢅꢇꢁꢁꢀ UHJXODWHVꢀ WKHꢀ DELOLW\ꢀ RIꢀ
PHPEUDQHꢇLQVHUWHGꢀ %D[ꢀ WRꢀ IRUPꢀ ROLJRPHUVꢀ LQꢀ WKHꢀ PLWRFKRQGULDOꢀ PHPEUDQHꢂꢀ Dꢀ FURVVꢇOLQNLQJꢀ
VWXG\ꢀZLWKꢀ%LVꢀꢑPDOHLPLGRꢒꢀKH[DQHꢀꢑ%0+ꢒꢀZDVꢀSHUIRUPHGꢀWRꢀPHDVXUHꢀ%D[ꢀROLJRPHUL]DWLRQꢀDVꢀ
ZHꢀSUHYLRXVO\ꢀDVꢀGHVFULEHGꢀꢑꢆꢂꢀꢅꢅꢂꢀꢅꢎꢒꢈꢀ,QWULJXLQJO\ꢂꢀGLUHFWꢀWUHDWPHQWꢀRIꢀPLWRFKRQGULDꢀLVRODWHGꢀ
IURPꢀ $ꢊꢄꢎꢀ FHOOVꢀ ZLWKꢀ &<'ꢇꢅꢇꢁꢁꢀ ꢑꢊꢀ μ0ꢒꢀ IDFLOLWDWHGꢀ WKHꢀ IRUPDWLRQꢀ RIꢀ %D[ꢀ GLPHUVꢀ DQGꢀ WULPHUVꢀ
ϵꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
ꢑ)LJXUHꢀ ꢇ%ꢂꢀ OHIWꢀ SDQHOꢒꢈꢀ 60%$ꢁꢀ ZDVꢀ XVHGꢀ DVꢀ Dꢀ SRVLWLYHꢀ FRQWUROꢀ ꢑꢆꢒꢈꢀ 7KHꢀ PROHFXODUꢀ VL]HVꢀ RIꢀ
WKHVHꢀ DGGXFWVꢀ ZHUHꢀ HVWLPDWHGꢀ WRꢀ EHꢀ PXOWLSOHVꢀ RIꢀ ׽ꢅꢁꢀ N'Dꢂꢀ VXJJHVWLQJꢀ WKHꢀ IRUPDWLRQꢀRIꢀ %D[ꢀ
KRPRꢇROLJRPHUVꢀ LQꢀ LVRODWHGꢀ PLWRFKRQGULDꢈꢀ 7Rꢀ IXUWKHUꢀ WHVWꢀ ZKHWKHUꢀ &<'ꢇꢅꢇꢁꢁꢀ DFWLYDWHVꢀ %D[ꢀ
WKURXJKꢀ WKHꢀ IRUPDWLRQꢀ RIꢀ ROLJRPHUVꢀ LQꢀ $ꢊꢄꢎꢀ FHOOVꢂꢀ FURVVꢀ OLQNLQJꢀ VWXGLHVꢀ ZLWKꢀ %+0ꢀ ZHUHꢀ
HPSOR\HGꢀIROORZLQJꢀWUHDWPHQWꢀRIꢀ$ꢊꢄꢎꢀFHOOVꢀZLWKꢀ&<'ꢇꢅꢇꢁꢁꢀDVꢀGHVFULEHGꢀꢑꢆꢂꢀꢅꢅꢂꢀꢅꢎꢒꢈꢀ5HVXOWVꢀ
LQGLFDWHꢀWKDWꢀWUHDWPHQWꢀRIꢀ$ꢊꢄꢎꢀFHOOVꢀZLWKꢀ&<'ꢇꢅꢇꢁꢁꢀRUꢀ60%$ꢁꢀUHVXOWHGꢀLQꢀWKHꢀIRUPDWLRQꢀRIꢀ%D[ꢀ
GLPHUVꢀDQGꢀWULPHUVꢀLQꢀ$ꢊꢄꢎꢀFHOOVꢀꢑ)LJXUHꢀꢇ%ꢂꢀULJKWꢀSDQHOꢒꢈꢀ7KHꢀSDWWHUQꢀRIꢀ%D[ꢀROLJRPHUVꢀZDVꢀ
VLPLODUꢀ WRꢀ WKDWꢀ LQꢀ WKHꢀ WUHDWHGꢀ PLWRFKRQGULDꢀ DERYHꢈꢀ 6LQFHꢀ &<'ꢇꢅꢇꢁꢁꢀ IDLOHGꢀ WRꢀ LQGXFHꢀ %$.ꢀ
ROLJRPHUL]DWLRQꢀLQꢀLVRODWHGꢀPLWRFKRQGULDꢀꢑ)LJXUHꢀ6ꢌꢒꢂꢀWKLVꢀLQGLFFDWHVꢀWKDWꢀVXFKꢀHIIHFWꢀRIꢀ&<'ꢇꢅꢇ
ꢁꢁꢀLVꢀVSHFLILFꢀIRUꢀ%D[ꢈꢀ7KHVHꢀILQGLQJVꢀSURYLGHꢀHYLGHQFHꢀWKDWꢀ&<'ꢇꢅꢇꢁꢁꢀFDQꢀGLUHFWO\ꢀDFWLYDWHꢀ%D[ꢀ
EXWꢀQRWꢀ%$.ꢀWKURXJKꢀLWVꢀROLJRPHUL]DWLRQꢀLQꢀLVRODWHGꢀPLWRFKRQGULDOꢀPHPEUDQHVꢈꢀꢀ
6XFHOOXODUꢀ IUDFWLRQDWLRQꢀ H[SHULPHQWVꢀ IXUWKHUꢀ GHPRQVWUDWHGꢀ WKDWꢀ WUHDWPHQWꢀ RIꢀ $ꢊꢄꢎꢀ FHOOVꢀ ZLWKꢀ
&<'ꢇꢅꢇꢁꢁꢀ OHGꢀ WRꢀ &\Wꢀ Fꢀ UHOHDVHꢀ IURPꢀ PLWRFKRQGULDꢀ LQWRꢀ F\WRVROꢀ LQꢀ DVVRFLDWLRQꢀ ZLWKꢀ %D[ꢀ
DFFXPXODWLRQꢀLQꢀPLWRFKRQGULDꢀꢑ)LJXUHꢀꢇ&ꢒꢈꢀꢀ
&<'ꢉꢇꢉꢄꢄꢀ VXSSUHVVHVꢀ OXQJꢀ FDQFHUꢀ JURZWKꢀ YLDꢀ LQGXFWLRQꢀ RIꢀ DSRSWRVLVꢀ LQꢀ 16&/&ꢀ DQGꢀ
6&/&ꢀ[HQRJUDIWVꢀDVꢀZHOOꢀDVꢀ3';ꢀPRGHOV
7RꢀWHVWꢀWKHꢀin vivoꢀHIILFDF\ꢀRIꢀ&<'ꢇꢅꢇꢁꢁꢂꢀZHꢀXVHGꢀQXGHꢀPLFHꢀDQGꢀWKHꢀKXPDQꢀOXQJꢀFDQFHUꢀ$ꢊꢄꢎꢀ
FHOOꢀ OLQHꢀ WRꢀ SURGXFHꢀ VXEFXWDQHRXVꢀ ꢑVꢈFꢈꢒꢀ OXQJꢀ WXPRUꢀ [HQRJUDIWVꢀ DVꢀ GHVFULEHGꢀ ꢑꢉꢌꢂꢀ ꢉꢁꢒꢈꢀ 0LFHꢀ
ZHUHꢀWUHDWHGꢀZLWKꢀLQFUHDVLQJꢀGRVHVꢀRIꢀ&<'ꢇꢅꢇꢁꢁꢀLQWUDSHULWRQHDOO\ꢀꢑLꢈSꢈꢒꢀIRUꢀꢁꢌꢀGD\Vꢈꢀ&<'ꢇꢅꢇꢁꢁꢀ
LQGXFHGꢀ Dꢀ GRVHꢇGHSHQGHQWꢀ UHSUHVVLRQꢀ RIꢀ OXQJꢀ FDQFHUꢀ JURZWKꢀ in vivoꢀ ꢑ)LJXUHꢀ ꢇ'ꢒꢈꢀ 7Rꢀ WHVWꢀ
ZKHWKHUꢀ &<'ꢇꢅꢇꢁꢁꢀ VXSSUHVVLRQꢀ RIꢀ OXQJꢀ FDQFHUꢀ RFFXUVꢀ WKURXJKꢀ DFWLYDWLRQꢀ RIꢀ %D[ꢀ OHDGLQJꢀ WRꢀ
DSRSWRVLVꢀ LQꢀ OXQJꢀ WXPRUꢀ WLVVXHVꢂꢀ DFWLYHꢀ FDVSDVHꢀ ꢉꢀ DQGꢀ %D[ꢀ ROLJRPHUL]DWLRQꢀ LQꢀ WXPRUꢀ WLVVXHVꢀ
ZHUHꢀPHDVXUHGꢀE\ꢀ,+&ꢀRUꢀFURVVꢇOLQNLQJꢂꢀUHVSHFWLYHO\ꢈꢀ7UHDWPHQWꢀRIꢀOXQJꢀFDQFHUꢀ[HQRJUDIWꢀPLFHꢀ
ϭϬꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
ZLWKꢀ&<'ꢇꢅꢇꢁꢁꢀUHVXOWHGꢀLQꢀ%D[ꢀROLJRPHUL]DWLRQꢀDQGꢀFDVSDVHꢀꢉꢀDFWLYDWLRQꢀꢑ)LJXUHꢀꢇ(ꢊꢀ)ꢀDQGꢀ*ꢒꢈꢀ
1RꢀZHLJKWꢀORVVꢂꢀQRꢀVLJQLILFDQWꢀLQFUHDVHꢀLQꢀ$/7ꢂꢀ$67ꢀDQGꢀ%81ꢀRUꢀGHFUHDVHꢀLQꢀ:%&ꢂꢀ5%&ꢂꢀ+Eꢀ
DQGꢀ3/7ꢀZHUHꢀREVHUYHGꢀIROORZLQJꢀ&<'ꢇꢅꢇꢁꢁꢀWUHDWPHQWꢀꢑ)LJXUHꢀ6ꢆꢒꢈꢀ+LVWRSDWKRORJLFꢀHYDOXDWLRQꢀ
RIꢀKDUYHVWHGꢀQRUPDOꢀWLVVXHVꢀꢑEUDLQꢂꢀKHDUWꢂꢀOXQJꢂꢀOLYHUꢂꢀVSOHHQꢂꢀNLGQH\ꢀDQGꢀLQWHVWLQHꢒꢀUHYHDOHGꢀQRꢀ
HYLGHQFHꢀRIꢀQRUPDOꢀWLVVXHꢀWR[LFLW\ꢀꢑ)LJXUHꢀ6ꢆꢒꢈꢀꢀ
7RꢀFRPSDUHꢀWKHꢀin vivoꢀHIILFDF\ꢀRIꢀ&<'ꢇꢅꢇꢁꢁꢀZLWKꢀWKDWꢀRIꢀ60%$ꢁꢂꢀ 16&/&ꢀꢑ$ꢊꢄꢎꢒꢀDQGꢀ6&/&ꢀ
ꢑ'06ꢊꢉꢒꢀ[HQRJUDIWVꢀZHUHꢀWUHDWHGꢀZLWKꢀꢄꢌPJꢓNJꢀRIꢀ&<'ꢇꢅꢇꢁꢁꢀRUꢀ60%$ꢁꢀLꢈSꢈꢀIRUꢀꢁꢄꢀGD\Vꢈꢀ&<'ꢇ
ꢅꢇꢁꢁꢀ H[KLELWHGꢀ PRUHꢀ SRWHQWꢀ DQWLꢇWXPRUꢀ DFWLYLW\ꢀ WKDQꢀ 60%$ꢁꢀ LQꢀ ERWKꢀ 16&/&ꢀ DQGꢀ 6&/&ꢀ
[HQRJUDIWVꢀꢑ)LJXUHꢀꢌꢒꢈꢀꢀ
7Rꢀ HYDOXDWHꢀ WKHꢀ DQWLꢇWXPRUꢀ DFWLYLW\ꢀ RIꢀ &<'ꢇꢅꢇꢁꢁꢀ LQꢀ SDWLHQWꢇGHULYHGꢀ [HQRJUDIWVꢀ ꢑ3';ꢒꢂꢀ ZHꢀ
HPSOR\HGꢀWZRꢀ3';Vꢀꢑ7.2ꢇꢌꢌꢅꢀDQGꢀ7.2ꢇꢌꢌꢊꢒꢀGHULYHGꢀIURPꢀWXPRUꢀWLVVXHꢀELRSVLHVꢀIURPꢀSDWLHQWVꢀ
ZLWKꢀ UHODSVHGꢀ 6&/&ꢈꢀ *LYHQꢀ WKDWꢀ WKHVHꢀ WZRꢀ 3';Vꢀ ZHUHꢀ GHULYHGꢀ IURPꢀ SDWLHQWVꢀ ZLWKRXWꢀ
LQWHUYHQLQJꢀin vitroꢀFXOWXUHꢂꢀWKH\ꢀDUHꢀH[SHFWHGꢀWRꢀFORVHO\ꢀUHFDSLWXODWHꢀWKHꢀ6&/&ꢀWXPRUꢀVHWWLQJꢈꢀ
0LFHꢀ ZLWKꢀ 7.2ꢇꢌꢌꢅꢀ DQGꢀ 7.2ꢇꢌꢌꢊꢀ 3';Vꢀ ZHUHꢀ WUHDWHGꢀ ZLWKꢀ &<'ꢇꢅꢇꢁꢁꢀ ꢑꢄꢌPJꢓNJꢓGꢒꢀ LꢈSꢈꢀ IRUꢀ ꢅꢀ
ZHHNVꢈꢀ&<'ꢇꢅꢇꢁꢁꢀSRWHQWO\ꢀVXSSUHVVHGꢀWKHꢀJURZWKꢀRIꢀERWKꢀ6&/&ꢀ7.2ꢇꢌꢌꢅꢀDQGꢀ7.2ꢇꢌꢌꢊꢀ3';Vꢀ
ꢑ)LJXUHꢀꢆ$ꢒꢈꢀ7RꢀH[DPLQHꢀZKHWKHUꢀ&<'ꢇꢅꢇꢁꢁꢀFDQꢀFRQYHUWꢀ%D[ꢀIURPꢀLWVꢀLQDFWLYHꢀꢑS6ꢁꢏꢄꢀ%D[ꢒꢀWRꢀ
DFWLYHꢀ ꢑꢃ$ꢆꢀ %D[ꢒꢀ IRUPꢀ LQꢀ 3';ꢀ WXPRUꢀ WLVVXHVꢂꢀ TXDQWXPꢀ GRWꢇEDVHGꢀ LPPXQRKLVWRÀXRUHVFHQFHꢀ
ꢑ4'ꢇ,+)ꢒꢀ ZDVꢀ SHUIRUPHGꢀ DVꢀ ZHꢀ SUHYLRXVO\ꢀ GHVFULEHGꢀ ꢑꢆꢂꢀ ꢉꢅꢒꢈꢀ S%D[ꢀ DQGꢀ ꢃ$ꢆꢀ %D[ꢀ ZHUHꢀ
DQDO\]HGꢀVLPXOWDQHRXVO\ꢀE\ꢀ4'ꢇ,+)ꢀRQꢀWKHꢀVDPHꢀWLVVXHꢀVOLGHꢀXVLQJꢀSULPDU\ꢀDQWLERGLHVꢀIURPꢀ
GLIIHUHQWꢀ VRXUFHVꢀ ꢑUDEELWꢀ DQWLꢇS%D[ꢀ DQGꢀ PRXVHꢀ DQWLꢇꢃ$ꢆꢒꢀ DQGꢀ 4'ꢇFRQMXJDWHGꢀ DQWLꢇUDEELWꢀ
[4'ꢃꢌꢊꢀ ꢑUHGꢒ]ꢀ DQGꢀ DQWLꢇPRXVHꢀ [4'ꢆꢌꢊꢀ ꢑJUHHQꢒ]ꢀ VHFRQGDU\ꢀ DQWLERGLHVꢀ ZLWKꢀ WZRꢀ GLIIHUHQWꢀ
HPLVVLRQꢀZDYHOHQJWKVꢈꢀ6HSDUDWHGꢀDQGꢀFRPELQHGꢀ4'ꢀLPDJHVꢀZHUHꢀREWDLQHGꢀDIWHUꢀGHWHUPLQLQJꢀ
WKHꢀ 4'ꢀ VSHFWUDOꢀ OLEUDU\ꢀ DQGꢀ XQPL[LQJꢀ WKHꢀ FXEHGꢀ LPDJHꢈꢀ 7UHDWPHQWꢀ RIꢀ 6&/&ꢀ 3';ꢀ PLFHꢀ ZLWKꢀ
ϭϭꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
&<'ꢇꢅꢇꢁꢁꢀ ꢑꢄꢌPJꢓNJꢓGꢒꢀ UHVXOWHGꢀ LQꢀ GRZQUHJXODWLRQꢀ RIꢀ SKRVSKRU\ODWHGꢀ %D[ꢀ ꢑS%D[ꢒꢀ DQGꢀ
XSUHJXODWLRQꢀRIꢀFRQIRUPDWLRQDOO\ꢀFKDQJHGꢀ%D[ꢀꢑꢃ$ꢆꢀ%D[ꢒꢀLQꢀWXPRUꢀWLVVXHVꢀꢑ)LJXUHꢀ ꢆ%ꢒꢈꢀ7RWDOꢀ
%D[ꢀOHYHOVꢀKDGꢀQRꢀVLJQLILFDQWꢀFKDQJHꢀꢑ)LJXUHꢀ 6ꢍꢒꢈꢀ7KHVHꢀILQGLQJVꢀVXJJHVWꢀWKDWꢀ&<'ꢇꢅꢇꢁꢁꢀLVꢀ
DEOHꢀWRꢀFRQYHUWꢀ%D[ꢀIURPꢀWKHꢀLQDFWLYHꢀꢑS%D[ꢒꢀWRꢀDFWLYDWHGꢀꢑꢃ$ꢆꢀ%D[ꢒꢀIRUPꢂꢀHYHQWXDOO\ꢀOHDGLQJꢀWRꢀ
DSRSWRVLVꢀ LQꢀ WXPRUꢀ WLVVXHVꢈꢀ )XUWKHUPRUHꢂꢀ %D[ꢀ ROLJRPHUL]DWLRQꢀ DQGꢀ DSRSWRVLVꢀ ꢑi.eꢈꢀ DFWLYHꢀ
FDVSDVHꢀꢉꢒꢀZHUHꢀDOVRꢀREVHUYHGꢀLQꢀWXPRUꢀWLVVXHVꢀꢑ)LJXUHꢀ ꢆ&'ꢒꢈꢀ1RꢀVLJQLILFDQWꢀQRUPDOꢀWLVVXHꢀ
WR[LFLW\ꢀZDVꢀREVHUYHGꢀꢑ)LJXUHꢀ6ꢎꢒꢈꢀ7KHVHꢀILQGLQJVꢀLQGLFDWHꢀWKDWꢀ&<'ꢇꢅꢇꢁꢁꢀPD\ꢀSRWHQWLDOO\ꢀEHꢀ
HIIHFWLYHꢀLQꢀSDWLHQWVꢀZLWKꢀ6&/&ꢂꢀIRUꢀZKLFKꢀWKHUHꢀDUHꢀFXUUHQWO\ꢀOLPLWHGꢀWUHDWPHQWꢀRSWLRQVꢈꢀꢀ
ꢀ
&<'ꢉꢇꢉꢄꢄꢀ GLVSOD\Vꢀ SRWHQWꢀ DQWLꢉWXPRUꢀ DFWLYLW\ꢀ DJDLQVWꢀ .5$6ꢉGULYHQꢀ OXQJꢀ FDQFHUꢀ LQꢀ
JHQHWLFDOO\ꢀHQJLQHHUHGꢀPRXVHꢀPRGHOVꢀꢂ*(00ꢃ
.5$6ꢀLVꢀWKHꢀPRVWꢀFRPPRQO\ꢀPXWDWHGꢀRQFRJHQHꢂꢀ\HWꢀQRꢀHIIHFWLYHꢀWDUJHWHGꢀWKHUDSLHVꢀH[LVWꢀIRUꢀ
.5$6ꢇPXWDQWꢀFDQFHUVꢀꢑꢉꢉꢒꢈꢀ+HUHꢀZHꢀIRXQGꢀWKDWꢀH[SUHVVLRQꢀRIꢀH[RJHQRXVꢀFRQVWLWXWLYHO\ꢀDFWLYHꢀ
.5$6ꢀꢑ*ꢁꢅ'ꢒꢀPXWDQWꢀLQꢀ+ꢁꢎꢄꢄꢀFHOOVꢀZLWKꢀZLOGꢇW\SHꢀ.5$6ꢀEDFNJURXQGꢀVLJQLILFDQWO\ꢀHQKDQFHGꢀ
%D[ꢀ SKRVSKRU\ODWLRQꢀ DWꢀ 6ꢁꢏꢄꢀ LQꢀ DVVRFLDWLRQꢀ ZLWKꢀ DFWLYDWLRQꢀ RIꢀ XSVWUHDPꢀ %D[ꢀ NLQDVHꢀ $.7ꢀ
ꢑ)LJXUHꢀ 6ꢏ$ꢒꢈꢀ 6LQFHꢀ &<'ꢇꢅꢇꢁꢁꢀ FDQꢀ FRQYHUWꢀ %D[ꢀ IURPꢀ LWVꢀ LQDFWLYHꢀ IRUPꢀ ꢑi.eꢈꢀ S%D[ꢒꢀ WRꢀ WKHꢀ
DFWLYDWHGꢀIRUPꢀꢑi.eꢈꢀꢃ$ꢆꢀ%D[ꢒꢀWRꢀLQGXFHꢀDSRSWRVLVꢀꢑ)LJXUHꢀ ꢆ%ꢒꢂꢀWKLVꢀLQGLFDWHVꢀWKDWꢀ&<'ꢇꢅꢇꢁꢁꢀ
PD\ꢀEHꢀHIIHFWLYHꢀIRUꢀWKHꢀWUHDWPHQWꢀRIꢀ.5$6ꢀPXWDQWꢇGULYHQꢀFDQFHUꢈꢀ7RꢀWHVWꢀWKHꢀSRWHQF\ꢀRIꢀ&<'ꢇ
ꢅꢇꢁꢁꢀLQꢀ.5$6ꢀPXWDQWꢇGULYHQꢀOXQJꢀFDQFHUꢂꢀOR[ꢇVWRSꢇOR[ꢀꢑ/6/)ꢇ.5$6*ꢁꢅ'ꢀ/.%ꢁIOꢓIOꢀꢑi.eꢈꢀ./ꢒꢀPLFHꢀ
ZHUHꢀJHQHUDWHGꢀDQGꢀEUHGꢀRXWꢀDVꢀSUHYLRXVO\ꢀGHVFULEHGꢀꢑꢁꢆꢇꢅꢌꢒꢈꢀ7KHVHꢀPLFHꢀFRQWDLQꢀDꢀ.5$6*ꢁꢅ'
ꢀ
/6/ꢀ NQRFNꢇLQꢀ DOOHOHꢀ DQGꢀ Dꢀ IOR[HGꢀ DOOHOHꢀ RIꢀ /.%ꢁꢀ ꢑ/.%ꢁIOꢓIOꢒꢀ ꢑꢁꢆꢂꢀ ꢁꢏꢒꢈꢀ 3ULPDU\ꢀ OXQJꢀ
DGHQRFDUFLQRPDꢀZDVꢀGHWHFWDEOHꢀDVꢀHDUO\ꢀDVꢀꢃꢀZHHNVꢀDIWHUꢀLQWUDQDVDOꢀDGPLQLVWUDWLRQꢀRIꢀꢊ[ꢁꢌꢃꢀ
SIXꢀDGHQRYLUXVꢀH[SUHVVLQJꢀ&UHꢀUHFRPELQDVHꢀꢑ$GH&UHꢒꢀLQꢀ.5$6*ꢁꢅ'ꢀ/.%ꢁIOꢓIOꢀꢑ./ꢒꢀPLFHꢀꢑ)LJXUHꢀ
ϭϮꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
6ꢏ%&ꢒꢈꢀ,WꢀKDVꢀEHHQꢀSUHYLRXVO\ꢀUHSRUWHGꢀWKDWꢀS$.7ꢀLVꢀXSUHJXODWHGꢀLQꢀOXQJꢀWXPRUꢀWLVVXHVꢀIURPꢀ./ꢀ
PLFHꢀꢑꢉꢄꢒꢈꢀ6LQFHꢀ$.7ꢀLVꢀDꢀSK\VLRORJLFDOꢀ%D[ꢀNLQDVHꢀꢑꢉꢂꢀꢄꢒꢂꢀDFWLYDWHGꢀ$.7ꢀPD\ꢀSKRVSKRU\ODWHꢀ
%D[ꢀLQꢀ./ꢀPLFHꢈꢀꢀ$VꢀH[SHFWHGꢂꢀLQFUHDVHGꢀ%D[ꢀSKRVSKRU\ODWLRQꢀꢑ6ꢁꢏꢄꢒꢀZDVꢀREVHUYHGꢀLQꢀWXPRUꢀ
WLVVXHVꢀ IURPꢀ ./ꢀ PLFHꢀ DVꢀ FRPSDUHGꢀ WRꢀ DGMDFHQWꢀ QRUPDOꢀ OXQJꢀ WLVVXHVꢀ ꢑ)LJXUHꢀ 6ꢏ'ꢒꢂꢀ WKHUHE\ꢀ
SURYLGLQJꢀDꢀJRRGꢀUDWLRQDOHꢀWRꢀHPSOR\ꢀ%D[ꢀDJRQLVWꢀꢑ&<'ꢇꢅꢇꢁꢁꢒꢀIRUꢀWKHꢀWUHDWPHQWꢀRIꢀ.5$6ꢇGULYHQꢀ
OXQJꢀFDQFHUꢈꢀ,QꢀRUGHUꢀWRꢀIXUWKHUꢀDVVHVVꢀWKHꢀSRWHQWLDOꢀRIꢀ&<'ꢇꢅꢇꢁꢁꢀDVꢀWKHUDS\ꢀIRUꢀ.5$6ꢇGULYHQꢀ
OXQJꢀFDQFHUꢂꢀ&<'ꢇꢅꢇꢁꢁꢀꢑꢄꢌPJꢓNJꢓGꢒꢀRUꢀYHKLFOHꢀZDVꢀDGPLQLVWHUHGꢀLꢈSꢈꢀVWDUWLQJꢀDWꢀꢃꢀZHHNVꢀSRVWꢀ
$GH&UHꢀ GHOLYHU\ꢀ DVꢀ SUHYLRXVO\ꢀ VXJJHVWHGꢀ ꢑꢁꢏꢒꢈꢀ $IWHUꢀ WUHDWPHQWꢀ IRUꢀ ꢏꢀ ZHHNVꢂꢀ PLFHꢀ ZHUHꢀ
HXWKDQL]HGꢀZLWKꢀ&2ꢅꢀDVSK\[LDWLRQꢈꢀ/XQJVꢀZLWKꢀWXPRUꢀDQGꢀQRUPDOꢀOXQJꢀWLVVXHVꢀZHUHꢀFROOHFWHGꢀ
IRUꢀIXUWKHUꢀDQDO\VLVꢈꢀ7RꢀTXDQWLI\ꢀWXPRUꢀEXUGHQꢀDQGꢀWXPRUꢀPXOWLSOLFLW\ꢀLQꢀPLFHꢂꢀ+ (ꢇVWDLQHGꢀOXQJVꢀ
ZHUHꢀLPDJHGꢀZLWKꢀPRUSKRPHWULFꢀVRIWZDUHꢀWRꢀTXDQWLI\ꢀWKHꢀVXUIDFHꢀDUHDꢀFRPSRVHGꢀRIꢀWXPRUꢀDVꢀ
RSSRVHGꢀWRꢀQRUPDOꢀWLVVXHꢀRIꢀUHSUHVHQWDWLYHꢀFURVVꢇVHFWLRQVꢀRIꢀHDFKꢀOXQJꢀOREHꢀIRUꢀHDFKꢀPRXVHꢀDVꢀ
VKRZQꢀLQꢀ)LJXUHꢀ6ꢅꢀDQGꢀSUHYLRXVO\ꢀGHVFULEHGꢀꢀꢑꢁꢏꢒꢈꢀ5HVXOWVꢀLQGLFDWHꢀWKDWꢀWUHDWPHQWꢀRIꢀ./ꢀPLFHꢀ
ZLWKꢀ&<'ꢇꢅꢇꢁꢁꢀIRUꢀꢏꢀZHHNVꢀUHVXOWHGꢀLQꢀVLJQLILFDQWꢀUHGXFWLRQꢀRIꢀWXPRUꢀEXUGHQꢀDQGꢀPXOWLSOLFLW\ꢀLQꢀ
WKHꢀ OXQJꢀ YLDꢀ DSRSWRVLVꢀ ꢑ)LJXUHꢀ ꢍ$ꢊꢀ %ꢀ DQGꢀ &ꢒꢈꢀ ,PSRUWDQWO\ꢂꢀ WUHDWPHQWꢀ ZLWKꢀ &<'ꢇꢅꢇꢁꢁꢀ
VLJQLILFDQWO\ꢀSURORQJHGꢀVXUYLYDOꢀRIꢀ./ꢀPLFHꢀZKHQꢀFRPSDUHGꢀZLWKꢀWKHꢀFRQWUROꢀJURXSꢀꢑ)LJXUHꢀꢍ'ꢒꢈꢀ
7KHUHꢀZHUHꢀꢊꢀGHDWKVꢀRXWꢀRIꢀꢏꢀPLFHꢀLQꢀWKHꢀFRQWUROꢀJURXSꢀYHUVXVꢀꢅꢀGHDWKVꢀRXWꢀRIꢀꢆꢀPLFHꢀLQꢀWKHꢀ
&<'ꢇꢅꢇꢁꢁꢀ WUHDWPHQWꢀ JURXSꢀ ꢑSꢀ <ꢀ ꢌꢈꢌꢁꢒꢂꢀ FDOFXODWHGꢀ XSꢀ WRꢀ ꢏꢀ ZHHNVꢀ EHIRUHꢀ HXWKDQL]DWLRQꢈꢀ 1Rꢀ
QRUPDOꢀWLVVXHꢀWR[LFLW\ꢀZDVꢀREVHUYHGꢀGXULQJꢀWUHDWPHQWꢀꢑ)LJXUHꢀ6ꢐꢒꢈꢀꢀ
P725ꢀ LQKLELWLRQꢀ E\ꢀ 5$'ꢋꢋꢄꢀ HQKDQFHVꢀ %D[ꢀ SKRVSKRU\ODWLRQꢀ LQꢀ OXQJꢀ FDQFHUꢀ FHOOVꢀ DQGꢀ
WXPRUꢀWLVVXHVꢀIURPꢀ16&/&ꢀSDWLHQWVꢀ
5DSDP\FLQꢀDQGꢀLWVꢀGHULYDWLYHꢀ5$'ꢌꢌꢁꢀꢑi.eꢈꢀHYHUROLPXVꢒꢀDUHꢀSRWHQWꢀDOORVWHULFꢀLQKLELWRUVꢀRIꢀP725ꢀ
ꢑꢉꢊꢒꢈꢀ5$'ꢌꢌꢁꢀLVꢀZHOOꢀWROHUDWHGꢀEXWꢀVKRZVꢀOLPLWHGꢀDQWLWXPRUꢀDFWLYLW\ꢀDVꢀDꢀVLQJOHꢀDJHQWꢀLQꢀSDWLHQWVꢀ
ϭϯꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
ZLWKꢀOXQJꢀFDQFHUꢀꢑꢉꢃꢇꢉꢏꢒꢈꢀ3UHYLRXVꢀUHSRUWVꢀLQGLFDWHꢀWKDWꢀLQKLELWLRQꢀRIꢀP725ꢀE\ꢀUDSDP\FLQꢀRUꢀ
5$'ꢌꢌꢁꢀ UHVXOWVꢀ LQꢀ DFWLYDWLRQꢀ RIꢀ $.7ꢀ ꢑꢁꢅꢂꢀ ꢁꢉꢒꢂꢀ ZKLFKꢀ LVꢀ DQꢀ XSVWUHDPꢀ %D[ꢀ NLQDVHꢀ ꢑꢉꢂꢀ ꢄꢒꢈꢀ 7Rꢀ
IXUWKHUꢀ WHVWꢀ ZKHWKHUꢀ P725ꢀ LQKLELWLRQꢀ DFWXDOO\ꢀ SURPRWHVꢀ %D[ꢀ SKRVSKRU\ODWLRQꢀ LQꢀ KXPDQꢀ OXQJꢀ
FDQFHUꢀ FHOOVꢂꢀ $ꢊꢄꢎꢀ FHOOVꢀ ZHUHꢀ WUHDWHGꢀ ZLWKꢀ LQFUHDVLQJꢀ FRQFHQWUDWLRQVꢀ RIꢀ 5$'ꢌꢌꢁꢀ IRUꢀ ꢅꢄKUꢈꢀ
:HVWHUQꢀ EORWꢀ DQGꢀ 4'ꢇ,+)ꢀ VWXGLHVꢀ LQGLFDWHꢀ WKDWꢀ LQKLELWLRQꢀ RIꢀ P725ꢀ E\ꢀ 5$'ꢌꢌꢁꢀ UHVXOWHGꢀ LQꢀ
DFWLYDWLRQꢀRIꢀ$.7ꢀDQGꢀXSUHJXODWLRQꢀRIꢀ6ꢁꢏꢄꢀ%D[ꢀSKRVSKRU\ODWLRQꢀꢑ)LJXUHꢀꢎ$ꢀDQGꢀ%ꢒꢈꢀ7RꢀWHVWꢀ
ZKHWKHUꢀ Dꢀ VLPLODUꢀ HIIHFWꢀ RIꢀ 5$'ꢌꢌꢁꢀ RQꢀ %D[ꢀ SKRVSKRU\ODWLRQꢀ RFFXUVꢀ LQꢀ SDWLHQWꢀ WXPRUVꢂꢀ ZHꢀ
DQDO\]HGꢀS%D[ꢀE\ꢀ,+&ꢀLQꢀEDVHOLQHꢀDQGꢀSRVWꢇWUHDWPHQWꢀWLVVXHꢀVDPSOHVꢀREWDLQHGꢀIURPꢀꢁꢌꢀ16&/&ꢀ
SDWLHQWVꢀ WUHDWHGꢀ ZLWKꢀ 5$'ꢌꢌꢁꢀ ꢑꢁꢌꢀ PJꢓGD\ꢒꢀ IRUꢀ ꢅꢏꢀ GD\Vꢀ DVꢀ SDUWꢀ RIꢀ Dꢀ QHRDGMXYDQWꢀ SKDVHꢀ ,Eꢀ
FOLQLFDOꢀVWXG\ꢀRIꢀHYHUROLPXVꢀLQꢀSDWLHQWVꢀZLWKꢀUHVHFWDEOHꢀ16&/&ꢀꢑꢁꢃꢒꢈꢀ7KHUHꢀZDVꢀLQFUHDVHGꢀ%D[ꢀ
SKRVSKRU\ODWLRQꢀ LQꢀ SRVWꢇWUHDWPHQWꢀ WXPRUꢀ WLVVXHVꢀ FRPSDUHGꢀ WRꢀ EDVHOLQHꢀ VDPSOHVꢀ ꢑ)LJXUHꢀ ꢎ&ꢀ
DQGꢀ 'ꢒꢈꢀ 7KHVHꢀ ILQGLQJVꢀ VXJJHVWꢀ WKDWꢀ P725ꢀ LQKLELWLRQꢇLQGXFHGꢀ %D[ꢀ SKRVSKRU\ODWLRQꢀ DQGꢀ
LQDFWLYDWLRQꢀ RIꢀ WKHꢀ SURDSRSWRWLFꢀ IXQFWLRQꢀ RIꢀ %D[ꢀ PD\ꢀ QHJDWLYHO\ꢀ DIIHFWꢀ WKHꢀ VHQVLWLYLW\ꢀ RIꢀ OXQJꢀ
FDQFHUꢀWRꢀP725ꢀLQKLELWRUꢈꢀ7KLVꢀVXJJHVWVꢀWKDWꢂꢀDVꢀDꢀ%D[ꢀDJRQLVWꢂꢀ&<'ꢇꢅꢇꢁꢁꢀPD\ꢀKDYHꢀVWURQJꢀ
SRWHQWLDOꢀWRꢀHQKDQFHꢀWKHꢀSRWHQF\ꢀRIꢀP725ꢀLQKLELWRUꢀDJDLQVWꢀOXQJꢀFDQFHUꢈꢀꢀ
ꢀꢀꢀꢀꢀ%(=ꢅꢉꢊꢀLVꢀDꢀGXDOꢀ3,ꢉ.ꢓP725ꢀLQKLELWRUꢀꢑꢉꢎꢒꢈꢀ:HꢀDOVRꢀWHVWHGꢀWKHꢀHIIHFWꢀRIꢀ%(=ꢅꢉꢊꢀRQꢀ$.7ꢀ
DFWLYLW\ꢀDQGꢀ%D[ꢀSKRVSKRU\ODWLRQꢈꢀꢀ$VꢀH[SHFWHGꢂꢀ%(=ꢅꢉꢊꢀQRWꢀRQO\ꢀLQKLELWHGꢀP725ꢀDFWLYLW\ꢀꢑi.eꢈꢀ
UHGXFHGꢀSꢇSꢆꢌ6ꢃ.ꢒꢀEXWꢀDOVRꢀVXSSUHVVHGꢀ$.7ꢀDFWLYLW\ꢀLQꢀ$ꢊꢄꢎꢀFHOOVꢈꢀ,QWULJXLQJO\ꢂꢀWUHDWPHQWꢀRIꢀ
$ꢊꢄꢎꢀ FHOOVꢀ ZLWKꢀ %(=ꢅꢉꢊꢀ GLGꢀ QRWꢀ HQKDQFHꢀ %D[ꢀ SKRVSKRU\ODWLRQꢂꢀ LQYHUVHO\ꢂꢀ UHGXFHGꢀ %D[ꢀ
SKRVSKRU\ODWLRQꢀ ꢑ)LJXUHꢀ 6ꢄꢋꢒꢈꢀ 7KLVꢀ PD\ꢀ RFFXUꢀ WKURXJKꢀ LQKLELWLRQꢀ RIꢀ $.7ꢀ ꢑi.eꢈꢀ %D[ꢀ NLQDVHꢒꢀ
DFWLYLW\ꢂꢀIXUWKHUꢀGHPRQVWUDWHGꢀWKDWꢀ$.7ꢀLVꢀFULWLFDOꢀIRUꢀ%D[ꢀSKRVSKRU\ODWLRQꢈꢀ
&<'ꢉꢇꢉꢄꢄꢀ DQGꢀ 5$'ꢋꢋꢄꢀ V\QHUJLVWLFDOO\ꢀ UHSUHVVꢀ OXQJꢀ FDQFHUꢀ JURZWKꢀ DQGꢀ RYHUFRPHꢀ
UDSDORJꢀUHVLVWDQFHꢀin vitroꢀDQGꢀin vivo
ϭϰꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
7RꢀGHWHUPLQHꢀZKHWKHUꢀ%D[ꢀSKRVSKRU\ODWLRQꢀFRQWULEXWHVꢀWRꢀDFTXLUHGꢀUHVLVWDQFHꢀWRꢀ5$'ꢌꢌꢁꢂꢀZHꢀ
HVWDEOLVKHGꢀDꢀ5$'ꢌꢌꢁꢇUHVLVWDQWꢀVWUDLQꢀIURPꢀWKHꢀSDUHQWDOꢀ$ꢊꢄꢎꢀFHOOꢀOLQHꢀꢑi.eꢈꢀ$ꢊꢄꢎꢇ55ꢒꢀDVꢀZHꢀ
SUHYLRXVO\ꢀ GHVFULEHGꢀ ꢑꢁꢊꢒꢈꢀ ,QFUHDVHGꢀ OHYHOVꢀ RIꢀ S%D[ꢀ ZHUHꢀ REVHUYHGꢀ LQꢀ $ꢊꢄꢎꢇ55ꢀ FHOOVꢀ DVꢀ
FRPSDUHGꢀ WRꢀ SDUHQWDOꢀ $ꢊꢄꢎꢀ FHOOVꢀ ꢑ)LJXUHꢀ 6ꢄꢄ$ꢀ DQGꢀ %ꢒꢈꢀ $Vꢀ H[SHFWHGꢂꢀ SDUHQWDOꢀ $ꢊꢄꢎꢀ FHOOVꢀ
UHPDLQHGꢀ VHQVLWLYHꢀ ZKLOHꢀ $ꢊꢄꢎꢇ55ꢀ FHOOVꢀ ZHUHꢀ UHVLVWDQWꢀ WRꢀ 5$'ꢌꢌꢁꢀ ꢑ)LJXUHꢀ 6ꢄꢄ&ꢀ DQGꢀ 'ꢒꢈꢀ
7KHVHꢀ UHVXOWVꢀ SURYLGHꢀ VWURQJꢀ HYLGHQFHꢀ WKDWꢀ 5$'ꢌꢌꢁꢇHQKDQFHGꢀ %D[ꢀ SKRVSKRU\ODWLRQꢀ
FRQWULEXWHVꢀ WRꢀ DFTXLUHGꢀ 5$'ꢌꢌꢁꢀ UHVLVWDQFHꢈꢀ ,QWULJXLQJO\ꢂꢀ $ꢊꢄꢎꢇ55ꢀ FHOOVꢀ ZHUHꢀ UHVLVWDQWꢀ WRꢀ
5$'ꢌꢌꢁꢀEXWꢀUHPDLQHGꢀVHQVLWLYHꢀWRꢀ&<'ꢇꢅꢇꢁꢁꢀꢑ)LJXUHꢀ6ꢄꢄ&ꢀDQGꢀ'ꢒꢂꢀVXJJHVWLQJꢀWKDWꢀ&<'ꢇꢅꢇꢁꢁꢀ
FDQꢀUHYHUVHꢀDFTXLUHGꢀ5$'ꢌꢌꢁꢀUHVLVWDQFHꢀWKURXJKꢀFRQYHUVLRQꢀRIꢀ%D[ꢀIURPꢀWKHꢀLQDFWLYHꢀWRꢀDFWLYHꢀ
IRUPꢈꢀ 7Rꢀ WHVWꢀ ZKHWKHUꢀ &<'ꢇꢅꢇꢁꢁꢀ RYHUFRPHVꢀ 5$'ꢌꢌꢁꢀ UHVLVWDQFHꢀ in vivoꢂꢀ 16&/&ꢀ [HQRJUDIWVꢀ
GHULYHGꢀ IURPꢀ $ꢊꢄꢎꢀ DQGꢀ $ꢊꢄꢎꢇ55ꢀ FHOOꢀ OLQHVꢀ ZHUHꢀ WUHDWHGꢀ ZLWKꢀ FRQWLQXRXVꢀ GDLO\ꢀ GRVLQJꢀ RIꢀ
5$'ꢌꢌꢁꢀ ꢑꢁꢀ PJꢓNJꢒꢀ DQGꢀ &<'ꢇꢅꢇꢁꢁꢀ ꢑꢄꢌꢀ PJꢓNJꢒꢀ DORQHꢀ RUꢀ LQꢀ FRPELQDWLRQꢀ IRUꢀ ꢅꢀ ZHHNVꢈꢀ :Hꢀ
REVHUYHGꢀWKDWꢀOXQJꢀFDQFHUꢀ[HQRJUDIWVꢀGHULYHGꢀIURPꢀ$ꢊꢄꢎꢀSDUHQWDOꢀFHOOVꢀZHUHꢀVHQVLWLYHꢀZKHUHDVꢀ
[HQRJUDIWVꢀGHULYHGꢀIURPꢀ$ꢊꢄꢎꢇ55ꢀFHOOVꢀZHUHꢀUHVLVWDQWꢀWRꢀ5$'ꢌꢌꢁꢀꢑ)LJXUHꢀ ꢏ$ꢊꢀ OHIWꢀ YVꢈꢀ ULJKWꢀ
SDQHOꢒꢈꢀ &RQVLVWHQWꢀ ZLWKꢀ WKHꢀ in vitroꢀ REVHUYDWLRQVꢂꢀ &<'ꢇꢅꢇꢁꢁꢀ DQGꢀ 5$'ꢌꢌꢁꢀ V\QHUJLVWLFDOO\ꢀ
UHSUHVVHGꢀ ERWKꢀ $ꢊꢄꢎꢀ DQGꢀ $ꢊꢄꢎꢇ55ꢀ [HQRJUDIWVꢀ ꢑ)LJXUHꢀ ꢏ$ꢒꢂꢀ LQGLFDWLQJꢀ WKDWꢀ &<'ꢇꢅꢇꢁꢁꢀ FDQꢀ
RYHUFRPHꢀ DFTXLUHGꢀ 5$'ꢌꢌꢁꢀ UHVLVWDQFHꢀ in vivoꢈꢀ 4'ꢇ,+)ꢀ VWXGLHVꢀ UHYHDOHGꢀ WKDWꢀ &<'ꢇꢅꢇꢁꢁꢀ
EORFNHGꢀ5$'ꢌꢌꢁꢇVWLPXODWHGꢀ%D[ꢀSKRVSKRU\ODWLRQꢀDQGꢀFRYHUWHGꢀ%D[ꢀIURPꢀWKHꢀLQDFWLYHꢀꢑS%D[ꢒꢀWRꢀ
DFWLYHꢀIRUPꢀꢑꢃ$ꢆꢀ%D[ꢒꢀLQꢀWXPRUꢀWLVVXHVꢀꢑ)LJXUHꢀ ꢏ%ꢒꢂꢀZKLFKꢀSURPRWHGꢀFDVSDVHꢀDFWLYDWLRQꢀDQGꢀ
DSRSWRVLVꢀꢑ)LJXUHꢀꢏ&ꢀDQGꢀ'ꢒꢈꢀ7RWDOꢀ%D[ꢀOHYHOVꢀKDGꢀQRꢀVLJQLILFDQWꢀFKDQJHꢀꢑ)LJXUHꢀ6ꢄꢇꢒꢈ7KHUHꢀ
ZHUHꢀ QRꢀ VLJQLILFDQWꢀ QRUPDOꢀ WLVVXHꢀ WR[LFLWLHVꢀ ꢑ)LJXUHꢀ 6ꢄꢌꢒꢈꢀ )XUWKHUPRUHꢂꢀ &<'ꢇꢅꢇꢁꢁꢀ LQꢀ
FRPELQDWLRQꢀ ZLWKꢀ 5$'ꢌꢌꢁꢀ H[KLELWHGꢀ VLJQLILFDQWO\ꢀ PRUHꢀ SRWHQWꢀ DQWLWXPRUꢀ DFWLYLW\ꢀ WKDQꢀ HLWKHUꢀ
&<'ꢇꢅꢇꢁꢁꢀRUꢀ5$'ꢌꢌꢁꢀDORQHꢀLQꢀDꢀ6&/&ꢀ3';ꢀPRGHOꢀꢑ)LJXUHꢀ6ꢄꢆꢒꢈꢀꢀ
ϭϱꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
'LVFXVVLRQꢀ
,QGXFWLRQꢀ RIꢀ DSRSWRVLVꢀ LVꢀ Dꢀ FULWLFDOꢀ PHFKDQLVPꢀ XQGHUO\LQJꢀ WKHꢀ GXUDEOHꢀ HIILFDF\ꢀ RIꢀ FDQFHUꢀ
WUHDWPHQWꢀ DJHQWVꢈꢀ %D[ꢀ LVꢀ Dꢀ PDMRUꢀ SURDSRSWRWLFꢀ SURWHLQꢀ ZKRVHꢀ DFWLYDWLRQꢀ LVꢀ UHTXLUHGꢀ IRUꢀ FHOOꢀ
GHDWKꢀ ꢑꢁꢒꢈꢀ 8QGHUVWDQGLQJꢀ WKHꢀ FHOOXODUꢀ PHFKDQLVPVꢀ UHTXLUHGꢀ IRUꢀ %D[ꢀ DFWLYDWLRQꢀ LVꢀ RIꢀ JUHDWꢀ
VLJQLILFDQFHꢀEHFDXVHꢀRIꢀWKHꢀSRWHQWLDOꢀWRꢀH[SORLWꢀWKLVꢀNQRZOHGJHꢀWRꢀGHYHORSꢀQHZꢀDSSURDFKHVꢀDQGꢀ
DJHQWVꢀ IRUꢀ FDQFHUꢀ WKHUDS\ꢈꢀ :Hꢀ KDYHꢀ UHFHQWO\ꢀ LGHQWLILHGꢀ WKUHHꢀ VPDOOꢀ PROHFXOHꢀ %D[ꢀ DJRQLVWVꢂꢀ
LQFOXGLQJꢀ60%$ꢁꢂꢀ60%$ꢅꢀDQGꢀ60%$ꢉꢂꢀWKDWꢀWDUJHWꢀWKHꢀVWUXFWXUDOꢀSRFNHWꢀDURXQGꢀ6ꢁꢏꢄꢀLQꢀWKHꢀ&ꢇ
WHUPLQDOꢀWDLOꢀRIꢀ%D[ꢀDQGꢀDFWLYDWHꢀWKHꢀSURSDSRSWRWLFꢀIXQFWLRQꢀRIꢀ%D[ꢀꢑꢆꢒꢈꢀ%DVHGꢀRQꢀLWVꢀFKHPLFDOꢀ
VWUXFWXUHꢀDQGꢀGUXJꢇOLNHQHVVꢂꢀZHꢀFKRVHꢀ60%$ꢁꢀDVꢀWKHꢀOHDGꢀDQGꢀJHQHUDWHGꢀDQꢀ60%$ꢁꢀDQDORJꢀ
&<'ꢇꢅꢇꢁꢁꢀWKDWꢀKDVꢀLPSURYHGꢀSRWHQF\ꢀDJDLQVWꢀOXQJꢀFDQFHUꢈꢀ&<'ꢇꢅꢇꢁꢁꢀVHOHFWLYHO\ꢀELQGVꢀWRꢀ%D[ꢀ
LQꢀ WKHꢀ SRFNHWꢀ DURXQGꢀ WKHꢀ 6ꢁꢏꢄꢀ SKRVSKRU\ODWLRQꢀ VLWHꢂꢀ ZLWKꢀ QRꢀ ELQGLQJꢀ WRꢀ RWKHUꢀ %FOꢅꢀ IDPLO\ꢀ
PHPEHUVꢂꢀDQGꢀLQGXFHVꢀDSRSWRVLVꢀRIꢀYDULRXVꢀOXQJꢀFDQFHUꢀFHOOꢀOLQHVꢀPRUHꢀSRWHQWO\ꢀWKDQꢀ60%$ꢁꢀ
ꢑ)LJXUHꢀ ꢄꢒꢈꢀ 0HFKDQLVWLFDOO\ꢂꢀ &<'ꢇꢅꢇꢁꢁꢀ GLUHFWO\ꢀ LQGXFHVꢀ %D[ꢀ ROLJRPHUL]DWLRQꢀ LQꢀ LVRODWHGꢀ
PLWRFKRQGULDꢀDQGꢀOXQJꢀFDQFHUꢀ$ꢊꢄꢎꢀFHOOVꢀꢑ)LJXUHꢀꢇ%ꢒꢀDVꢀZHOOꢀDVꢀLQꢀWXPRUꢀWLVVXHVꢀIURPꢀ$ꢊꢄꢎꢀ
[HQRJUDIWVꢀRUꢀ3';Vꢀꢑ)LJXUHVꢀꢇꢉꢆꢒꢂꢀLQGLFDWLQJꢀDꢀGLUHFWꢀPHFKDQLVPꢀE\ꢀZKLFKꢀ&<'ꢇꢅꢇꢁꢁꢀDFWLYDWHVꢀ
%D[ꢀ SURWHLQꢈꢀ ,Wꢀ LVꢀ NQRZQꢀ WKDWꢀ ROLJRPHUL]DWLRQꢀ RIꢀ %D[ꢀ PROHFXOHVꢀ LQꢀ PLWRFKRQGULDꢀ LVꢀ Dꢀ PDMRUꢀ
PHFKDQLVPꢀWKURXJKꢀZKLFKꢀ%D[ꢀLQGXFHVꢀF\WRFKURPHꢀFꢀꢑ&\WꢀFꢒꢀUHOHDVHꢀꢑꢅꢏꢒꢈꢀ7KHVHꢀILQGLQJVꢀKHOSꢀ
H[SODLQꢀ KRZꢀ &<'ꢇꢅꢇꢁꢁꢀ WULJJHUVꢀ &\Wꢀ Fꢀ UHOHDVHꢀ LQꢀ OXQJꢀ FDQFHUꢀ $ꢊꢄꢎꢀ FHOOVꢀ ꢑ)LJXUHꢀ ꢇ&ꢒꢈꢀ
,QWULJXLQJO\ꢂꢀ &<'ꢇꢅꢇꢁꢁꢀ VXSSUHVVHVꢀ $ꢊꢄꢎꢀ [HQRJUDIWꢀ WXPRUꢀ JURZWKꢀ YLDꢀ %D[ꢀ DFWLYDWLRQꢀ DQGꢀ
LQGXFWLRQꢀRIꢀDSRSWRVLVꢀLQꢀWXPRUꢀWLVVXHVꢀLQꢀDꢀGRVHꢇGHSHQGHQWꢀPDQQHUꢀꢑ)LJXUHꢀꢇ'ꢉ*ꢒꢈꢀ&<'ꢇꢅꢇꢁꢁꢀ
GLVSOD\Vꢀ PRUHꢀ SRWHQWꢀ DQWLWXPRUꢀ DFWLYLW\ꢀ WKDQꢀ 60%$ꢁꢀ DJDLQVWꢀ ERWKꢀ 16&/&ꢀ DQGꢀ 6&/&ꢀ
[HQRJUDIWVꢀ ꢑ)LJXUHꢀ ꢌꢒꢈꢀ 4'ꢇEDVHGꢀ LPDJHVꢀ VKRZHGꢀ WKDWꢀ &<'ꢇꢅꢇꢁꢁꢀ FDQꢀ UHGXFHꢀ OHYHOVꢀ RIꢀ WKHꢀ
LQDFWLYHꢀ IRUPꢀ RIꢀ %D[ꢀ ꢑS6ꢁꢏꢄꢀ %D[ꢒꢀ DQGꢀ HQKDQFHꢀ WKHꢀ DFWLYDWHGꢀ IRUPꢀ RIꢀ %D[ꢀ ꢑi.eꢈꢂꢀ ꢃ$ꢆꢀ
ϭϲꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
FRQIRUPDWLRQDOO\ꢀFKDQJHGꢀ%D[ꢒꢀLQꢀWXPRUꢀWLVVXHVꢀIURPꢀ3';VꢀDQGꢀ$ꢊꢄꢎꢀ[HQRJUDIWVꢀꢑ)LJXUHVꢀꢆꢀ
DQGꢀ ꢏꢒꢈꢀ %DVHGꢀ RQꢀ WKHVHꢀ ILQGLQJVꢂꢀ ZHꢀ SURSRVHꢀ WKDWꢀ &<'ꢇꢅꢇꢁꢁꢇLQGXFHGꢀ VXSSUHVVLRQꢀ RIꢀ OXQJꢀ
FDQFHUꢀ PD\ꢀ UHVXOWꢀ IURPꢀ VHOHFWLYHꢀ DFWLYDWLRQꢀ RIꢀ WKHꢀ SURDSRSWRWLFꢀ IXQFWLRQꢀ RIꢀ %D[ꢀ OHDGLQJꢀ WRꢀ
LQFUHDVHGꢀDSRSWRVLVꢀLQꢀWXPRUVꢈꢀꢀ
3';ꢀ PRGHOVꢀ KDYHꢀ EHHQꢀ FRQVLGHUHGꢀ DVꢀ PRUHꢀ DGYDQFHGꢀ SUHFOLQLFDOꢀ FDQFHUꢀ PRGHOVꢂꢀ ZKLFKꢀꢀ
PRVWO\ꢀ UHWDLQꢀ WKHꢀ SULQFLSDOꢀ KLVWRORJLFꢀ DQGꢀ JHQHWLFꢀ FKDUDFWHULVWLFVꢀ RIꢀ WKHLUꢀ GRQRUꢀ WXPRUVꢀ DQGꢀ
UHPDLQꢀVWDEOHꢀDFURVVꢀSDVVDJHVꢈꢀ&RPELQHGꢀFOLQLFDOꢀDQGꢀ3';ꢀVWXGLHVꢀKDYHꢀVKRZQꢀDꢀKLJKꢀGHJUHHꢀ
RIꢀFRUUHODWLRQꢀEHWZHHQꢀFOLQLFDOꢀUHVSRQVHꢀWRꢀF\WRWR[LFꢀDJHQWVꢀLQꢀDGXOWꢀSDWLHQWVꢀZLWKꢀOXQJꢀFDQFHUꢀ
DQGꢀUHVSRQVHꢀWRꢀWKHꢀVDPHꢀDJHQWVꢀLQꢀ3';ꢀPRGHOVꢀJHQHUDWHGꢀIURPꢀWKHVHꢀSDWLHQWVꢀꢑꢄꢌꢒꢈꢀ3';ꢀ
PRGHOVꢀ KDYHꢀ EHHQꢀ VKRZQꢀ WRꢀ EHꢀ SUHGLFWLYHꢀ RIꢀ FOLQLFDOꢀ RXWFRPHVꢀ DQGꢀ DUHꢀ EHLQJꢀ XVHGꢀ IRUꢀ
SUHFOLQLFDOꢀDQGꢀFOLQLFDOꢀGUXJꢀHYDOXDWLRQꢀꢑꢄꢁꢂꢀꢄꢅꢒꢈꢀ6LQFHꢀꢀ&<'ꢇꢅꢇꢁꢁꢀHIIHFWLYHO\ꢀVXSSUHVVHGꢀWKHꢀ
JURZWKꢀRIꢀWZRꢀ3';Vꢀꢑ7.2ꢇꢌꢌꢅꢀDQGꢀ7.2ꢇꢌꢌꢊꢒꢀUDLVHGꢀIURPꢀWZRꢀSDWLHQWVꢀZLWKꢀUHIUDFWRU\ꢀ6&/&ꢀ
ꢑ)LJXUHꢀ ꢆꢒꢂꢀ WKHUHꢀ LVꢀ JRRGꢀ SRWHQWLDOꢀ IRUꢀ &<'ꢇꢅꢇꢁꢁꢀ WRꢀ SURYLGHꢀ FOLQLFDOꢀ SRWHQF\ꢀ LQꢀ SDWLHQWVꢀ ZLWKꢀ
FKHPRUHIUDFWRU\ꢀ6&/&ꢀLQꢀWKHꢀIXWXUHꢈꢀꢀ
,QꢀDGGLWLRQꢀWRꢀ3';ꢀPRGHOVꢂꢀJHQHWLFDOO\ꢀHQJLQHHUHGꢀPRXVHꢀPRGHOVꢀꢑ*(00ꢒꢀDUHꢀDOVRꢀFRQVLGHUHGꢀ
WRꢀEHꢀDGYDQFHGꢀSUHFOLQLFDOꢀFDQFHUꢀPRGHOVꢈꢀ,WꢀLVꢀNQRZQꢀWKDWꢀJHQHWLFDOO\ꢀHQJLQHHUHGꢀPLFHꢀꢑ*(0ꢒꢀ
DUHꢀ WKHꢀ PRVWꢀ VRSKLVWLFDWHGꢀ DQLPDOꢀ PRGHOVꢀ RIꢀ KXPDQꢀ FDQFHUꢂꢀ ZKLFKꢀ FORVHO\ꢀ UHFDSLWXODWHꢀ WKHꢀ
SDWKRSK\VLRORJLFDOꢀ SURFHVVꢀ RIꢀ KXPDQꢀ PDOLJQDQFLHVꢀ LQꢀ JHQHWLFDOO\ꢀ SUHFLVHO\ꢀ GHILQHGꢀ V\VWHPVꢀ
ꢑꢄꢉꢒꢈꢀ 7KHUHIRUHꢂꢀ WKHꢀ SRWHQF\ꢀ RIꢀ &<'ꢇꢅꢇꢁꢁꢀ ZDVꢀ DOVRꢀ HYDOXDWHGꢀ LQꢀ JHQHWLFDOO\ꢀ HQJLQHHUHGꢀ ./ꢀ
PLFHꢈꢀ6LQFHꢀS6ꢁꢏꢄꢀ%D[ꢀꢑLQDFWLYHꢀIRUPꢒꢀLVꢀVLJQLILFDQWO\ꢀXSUHJXODWHGꢀꢀLQꢀWXPRUꢀWLVVXHVꢀIURPꢀ./ꢀ
PLFHꢀ ꢑ)LJXUHꢀ 6ꢏꢒꢂꢀ &<'ꢇꢅꢇꢁꢁꢂꢀ DVꢀ Dꢀ %D[ꢀ DJRQLVWꢂꢀ VKRXOGꢀ EHꢀ DQꢀ LGHDOꢀ DJHQWꢀ IRUꢀ WUHDWPHQWꢀ RIꢀ
.5$6ꢇGULYHQꢀ OXQJꢀ FDQFHUꢈꢀ ,QWULJXLQJO\ꢂꢀ &<'ꢇꢅꢇꢁꢁꢀ VLJQLILFDQWO\ꢀ UHGXFHVꢀ WXPRUꢀ EXUGHQꢀ LQꢀ WKHꢀ
OXQJVꢀ RIꢀ ./ꢀ PLFHꢀ OHDGLQJꢀ WRꢀ SURORQJHGꢀ VXUYLYDOꢀ ZKHQꢀ FRPSDUHGꢀ ZLWKꢀ WKHꢀ XQWUHDWHGꢀ FRQWUROꢀ
ϭϳꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
JURXSꢀꢑ)LJXUHꢀꢍꢒꢂꢀVXJJHVWLQJꢀWKDWꢀ&<'ꢇꢅꢇꢁꢁꢀKDVꢀWKHꢀSRWHQWLDOꢀWRꢀLPSURYHꢀWKHꢀSURJQRVLVꢀRIꢀOXQJꢀ
FDQFHUꢀSDWLHQWVꢀZLWKꢀ.5$6ꢀPXWDWLRQVꢈꢀꢀꢀ
P725ꢀLVꢀDꢀSURPLVLQJꢀWKHUDSHXWLFꢀWDUJHWꢀLQꢀOXQJꢀFDQFHUꢀꢑꢎꢇꢁꢁꢒꢈꢀ+RZHYHUꢂꢀP725ꢀLQKLELWLRQꢀE\ꢀ
UDSDORJVꢀKDVꢀEHHQꢀUHSRUWHGꢀWRꢀDFWLYDWHꢀ3,ꢉ.ꢓ$.7ꢀSUROLIHUDWLYHꢀDQGꢀSURVXUYLYDOꢀVLJQDOVꢀWKHUHE\ꢀ
DWWHQXDWLQJꢀDQWLFDQFHUꢀHIILFDF\ꢀꢑꢁꢅꢂꢀꢁꢉꢂꢀꢄꢄꢒꢈꢀ+HUHꢂꢀZHꢀGLVFRYHUHGꢀWKDWꢀLQKLELWLRQꢀRIꢀP725ꢀE\ꢀ
5$'ꢌꢌꢁꢀQRWꢀRQO\ꢀDFWLYDWHVꢀ$.7ꢀEXWꢀDOVRꢀOHDGVꢀWRꢀ6ꢁꢏꢄꢀ%D[ꢀSKRVSKRU\ODWLRQꢀDQGꢀLQDFWLYDWLRQꢀLQꢀ
DꢀOXQJꢀFDQFHUꢀFHOOꢀOLQHꢀDQGꢀLQꢀWXPRUꢀWLVVXHVꢀIURPꢀ16&/&ꢀSDWLHQWVꢀWUHDWHGꢀZLWKꢀ5$'ꢌꢌꢁꢀꢑ)LJXUHꢀ
ꢎꢒꢈꢀ,WꢀLVꢀSRVVLEOHꢀWKDWꢀ%D[ꢀSKRVSKRU\ODWLRQꢀLQGXFHGꢀE\ꢀP725ꢀLQKLELWRUꢀWKHUDS\ꢀPD\ꢀQHJDWLYHO\ꢀ
DIIHFWꢀWKHꢀHIILFDF\ꢀRIꢀP725ꢀLQKLELWRUꢀLQꢀFDQFHUꢀWKHUDS\ꢈꢀ7KLVꢀPD\ꢀH[SODLQꢀWKHꢀOLPLWHGꢀHIILFDF\ꢀRIꢀ
VLQJOHꢇDJHQWꢀ5$'ꢌꢌꢁꢀLQꢀOXQJꢀFDQFHUꢀSDWLHQWVꢀꢑꢉꢃꢂꢀꢉꢆꢒꢈꢀ)XUWKHUPRUHꢂꢀWKHꢀSKRVSKRU\ODWHGꢀIRUPꢀ
RIꢀ%D[ꢀꢑS%D[ꢒꢀZDVꢀIRXQGꢀWRꢀDFFXPXODWHꢀLQꢀ5$'ꢌꢌꢁꢀUHVLVWDQWꢀFHOOVꢀꢑ$ꢊꢄꢎꢇ55ꢒꢀꢑ)LJXUHꢀ6ꢄꢄ$%ꢒꢂꢀ
LQGLFDWLQJꢀ WKDWꢀ S%D[ꢀ PD\ꢀ FRQWULEXWHꢀ WRꢀ UDSDORJꢀ UHVLVWDQFHꢈꢀ 2XUꢀ ILQGLQJVꢀ SURYLGHꢀ Dꢀ VWURQJꢀ
UDWLRQDOHꢀIRUꢀWKHꢀFRPELQDWLRQꢀRIꢀP725ꢀLQKLELWRUꢀꢑ5$'ꢌꢌꢁꢒꢀZLWKꢀ%D[ꢀDJRQLVWꢀꢑ&<'ꢇꢅꢇꢁꢁꢒꢂꢀZKLFKꢀ
PD\ꢀ KDYHꢀ VXSHULRUꢀ WKHUDSHXWLFꢀ EHQHILWVꢀ DQGꢀ PD\ꢀ UHYHUVHꢀ UDSDORJꢀ UHVLVWDQFHꢈꢀ $Vꢀ H[SHFWHGꢂꢀ
&<'ꢇꢅꢇꢁꢁꢀLQꢀFRPELQDWLRQꢀZLWKꢀ5$'ꢌꢌꢁꢀQRWꢀRQO\ꢀH[KLELWVꢀVWURQJꢀV\QHUJLVWLFꢀDFWLYLW\ꢀDJDLQVWꢀOXQJꢀ
FDQFHUꢀ EXWꢀ DOVRꢀ RYHUFRPHVꢀ UDSDORJꢀ UHVLVWDQFHꢀ in vitroꢀ ꢑ)LJXUHꢀ 6ꢄꢄ&ꢀ DQGꢀ 'ꢒꢀ DQGꢀ in vivoꢀ
ꢑ)LJXUHꢀꢏꢒꢈꢀꢀ
,Qꢀ VXPPDU\ꢂꢀ ZHꢀ KDYHꢀ JHQHUDWHGꢀ Dꢀ QHZꢀ %D[ꢀ DJRQLVWꢀ &<'ꢇꢅꢇꢁꢁꢀ WKDWꢀ VHOHFWLYHO\ꢀ WDUJHWVꢀ WKHꢀ
VWUXFWXUDOꢀELQGLQJꢀSRFNHWꢀDURXQGꢀWKHꢀ6ꢁꢏꢄꢀSKRVSKRU\ODWLRQꢀVLWHꢀLQꢀ%D[ꢈꢀ7KHꢀELQGLQJꢀRIꢀ&<'ꢇꢅꢇ
ꢁꢁꢀZLWKꢀ%D[ꢀUHVXOWVꢀLQꢀDꢀꢃ$ꢆꢀFRQIRUPDWLRQDOꢀFKDQJHꢀDQGꢀROLJRPHUL]DWLRQꢀRIꢀ%D[ꢀSURWHLQꢀOHDGLQJꢀ
WRꢀ DFWLYDWLRQꢀ RIꢀ LWVꢀ SURDSRSWRWLFꢀ IXQFWLRQꢈꢀ 7KHꢀ %D[ꢀ DJRQLVWꢀ &<'ꢇꢅꢇꢁꢁꢀ H[KLELWVꢀ SRWHQWꢀ HIILFDF\ꢀ
DJDLQVWꢀKXPDQꢀOXQJꢀFDQFHUꢀin vivoꢀZLWKRXWꢀQRUPDOꢀWLVVXHꢀWR[LFLW\ꢈꢀP725ꢀLQKLELWLRQꢀE\ꢀ5$'ꢌꢌꢁꢀ
OHDGVꢀWRꢀ6ꢁꢏꢄꢀ%D[ꢀSKRVSKRU\ODWLRQꢀFRQWULEXWLQJꢀWRꢀUDSDORJꢀUHVLVWDQFHꢈꢀ&RꢇWDUJHWLQJꢀP725ꢀDQGꢀ
ϭϴꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
%D[ꢀ YLDꢀ Dꢀ PHFKDQLVPꢇGULYHQꢀ FRPELQDWLRQꢀ RIꢀ P725ꢀ LQKLELWRUꢀ DQGꢀ %D[ꢀ DJRQLVWꢀ UHSUHVHQWVꢀ Dꢀ
PRUHꢀHIIHFWLYHꢀWKHUDSHXWLFꢀDSSURDFKꢀIRUꢀRYHUFRPLQJꢀWUHDWPHQWꢀUHVLVWDQFHꢀDQGꢀLPSURYLQJꢀOXQJꢀ
FDQFHUꢀRXWFRPHꢈꢀꢀꢀ
ꢀ
$&.12:/('*0(176ꢀꢀ
:HꢀWKDQNꢀ$QWKHDꢀ+DPPRQGꢀIRUꢀHGLWLQJꢀRIꢀWKHꢀPDQXVFULSWꢈꢀꢀ
ꢀ
5HIHUHQFHVꢀ
ꢁꢈꢀ
:HLꢀ0&ꢂꢀ=RQJꢀ:;ꢂꢀ&KHQJꢀ(+ꢂꢀ/LQGVWHQꢀ7ꢂꢀ3DQRXWVDNRSRXORXꢀ9ꢂꢀ5RVVꢀ$-ꢂꢀHWꢀDOꢈꢀ
3URDSRSWRWLFꢀ%$;ꢀDQGꢀ%$.ꢐꢀDꢀUHTXLVLWHꢀJDWHZD\ꢀWRꢀPLWRFKRQGULDOꢀG\VIXQFWLRQꢀDQGꢀGHDWKꢈꢀ
6FLHQFHꢈꢀꢅꢌꢌꢁꢍꢅꢎꢅꢐꢆꢅꢆꢇꢉꢌꢈꢀ
ꢅꢈꢀ
GLVWLQFWO\ꢀUHJXODWHVꢀWKHꢀSURDSRSWRWLFꢀIXQFWLRQꢀRIꢀ%D[ꢈꢀ3/R6ꢀ2QHꢈꢀꢅꢌꢁꢌꢍꢊꢐHꢁꢉꢉꢎꢉꢈꢀ
ꢉꢈꢀ ;LQꢀ0ꢂꢀ'HQJꢀ;ꢈꢀ1LFRWLQHꢀLQDFWLYDWLRQꢀRIꢀWKHꢀSURDSRSWRWLFꢀIXQFWLRQꢀRIꢀ%D[ꢀWKURXJKꢀ
SKRVSKRU\ODWLRQꢈꢀ-ꢀ%LROꢀ&KHPꢈꢀꢅꢌꢌꢊꢍꢅꢏꢌꢐꢁꢌꢆꢏꢁꢇꢎꢈꢀ
ꢄꢈꢀ *DUGDLꢀ6-ꢂꢀ+LOGHPDQꢀ'$ꢂꢀ)UDQNHOꢀ6.ꢂꢀ:KLWORFNꢀ%%ꢂꢀ)UDVFKꢀ6&ꢂꢀ%RUUHJDDUGꢀ1ꢂꢀHWꢀDOꢈꢀ
:DQJꢀ4ꢂꢀ6XQꢀ6<ꢂꢀ.KXULꢀ)ꢂꢀ&XUUDQꢀ:-ꢂꢀ'HQJꢀ;ꢈꢀ0RQRꢇꢀRUꢀGRXEOHꢇVLWHꢀSKRVSKRU\ODWLRQꢀ
3KRVSKRU\ODWLRQꢀRIꢀ%D[ꢀ6HUꢁꢏꢄꢀE\ꢀ$NWꢀUHJXODWHVꢀLWVꢀDFWLYLW\ꢀDQGꢀDSRSWRVLVꢀLQꢀQHXWURSKLOVꢈꢀ-ꢀ%LROꢀ
&KHPꢈꢀꢅꢌꢌꢄꢍꢅꢆꢎꢐꢅꢁꢌꢏꢊꢇꢎꢊꢈꢀ
ꢊꢈꢀ
WKURXJKꢀGHSKRVSKRU\ODWLRQꢈꢀ-ꢀ%LROꢀ&KHPꢈꢀꢅꢌꢌꢃꢍꢅꢏꢁꢐꢁꢏꢏꢊꢎꢇꢃꢆꢈꢀ
ꢃꢈꢀ 6X]XNLꢀ0ꢂꢀ<RXOHꢀ5-ꢂꢀ7MDQGUDꢀ1ꢈꢀ6WUXFWXUHꢀRIꢀ%D[ꢐꢀFRUHJXODWLRQꢀRIꢀGLPHUꢀIRUPDWLRQꢀDQGꢀ
LQWUDFHOOXODUꢀORFDOL]DWLRQꢈꢀ&HOOꢈꢀꢅꢌꢌꢌꢍꢁꢌꢉꢐꢃꢄꢊꢇꢊꢄꢈꢀ
ꢆꢈꢀ ;LQꢀ0ꢂꢀ/Lꢀ5ꢂꢀ;LHꢀ0ꢂꢀ3DUNꢀ'ꢂꢀ2ZRQLNRNRꢀ7.ꢂꢀ6LFDꢀ*/ꢂꢀHWꢀDOꢈꢀ6PDOOꢇPROHFXOHꢀ%D[ꢀDJRQLVWVꢀ
IRUꢀFDQFHUꢀWKHUDS\ꢈꢀ1DWXUHꢀFRPPXQLFDWLRQVꢈꢀꢅꢌꢁꢄꢍꢊꢐꢄꢎꢉꢊꢈꢀ
ꢏꢈꢀ 1HFKXVKWDQꢀ$ꢂꢀ6PLWKꢀ&/ꢂꢀ+VXꢀ<7ꢂꢀ<RXOHꢀ5-ꢈꢀ&RQIRUPDWLRQꢀRIꢀWKHꢀ%D[ꢀ&ꢇWHUPLQXVꢀ
UHJXODWHVꢀVXEFHOOXODUꢀORFDWLRQꢀDQGꢀFHOOꢀGHDWKꢈꢀ(0%2ꢀ-ꢈꢀꢁꢎꢎꢎꢍꢁꢏꢐꢅꢉꢉꢌꢇꢄꢁꢈꢀ
ꢎꢈꢀ -RKQVRQꢀ%(ꢂꢀ-DFNPDQꢀ'ꢂꢀ-DQQHꢀ3$ꢈꢀ5DWLRQDOHꢀIRUꢀDꢀSKDVHꢀ,ꢀWULDOꢀRIꢀHUORWLQLEꢀDQGꢀWKHꢀ
;LQꢀ0ꢂꢀ'HQJꢀ;ꢈꢀ3URWHLQꢀSKRVSKDWDVHꢀꢅ$ꢀHQKDQFHVꢀWKHꢀSURDSRSWRWLFꢀIXQFWLRQꢀRIꢀ%D[ꢀ
PDPPDOLDQꢀWDUJHWꢀRIꢀUDSDP\FLQꢀLQKLELWRUꢀHYHUROLPXVꢀꢑ5$'ꢌꢌꢁꢒꢀIRUꢀSDWLHQWVꢀZLWKꢀUHODSVHGꢀQRQꢀ
VPDOOꢀFHOOꢀOXQJꢀFDQFHUꢈꢀ&OLQꢀ&DQFHUꢀ5HVꢈꢀꢅꢌꢌꢆꢍꢁꢉꢐVꢄꢃꢅꢏꢇꢉꢁꢈꢀ
ꢁꢌꢈꢀ :DQJꢀ;ꢂꢀ<XHꢀ3ꢂꢀ.LPꢀ<$ꢂꢀ)Xꢀ+ꢂꢀ.KXULꢀ)5ꢂꢀ6XQꢀ6<ꢈꢀ(QKDQFLQJꢀPDPPDOLDQꢀWDUJHWꢀRIꢀ
UDSDP\FLQꢀꢑP725ꢒꢇWDUJHWHGꢀFDQFHUꢀWKHUDS\ꢀE\ꢀSUHYHQWLQJꢀP725ꢓUDSWRUꢀLQKLELWLRQꢇLQLWLDWHGꢂꢀ
P725ꢓULFWRUꢇLQGHSHQGHQWꢀ$NWꢀDFWLYDWLRQꢈꢀ&DQFHUꢀ5HVꢈꢀꢅꢌꢌꢏꢍꢃꢏꢐꢆꢄꢌꢎꢇꢁꢏꢈꢀ
ꢁꢁꢈꢀ :DQJSDLFKLWUꢀ0ꢂꢀ:Xꢀ&ꢂꢀ<RXꢀ0ꢂꢀ.XRꢀ07ꢂꢀ)HXQꢀ/ꢂꢀ/DPSLGLVꢀ7ꢂꢀHWꢀDOꢈꢀ,QKLELWLRQꢀRIꢀP725ꢀ
UHVWRUHVꢀFLVSODWLQꢀVHQVLWLYLW\ꢀWKURXJKꢀGRZQꢇUHJXODWLRQꢀRIꢀJURZWKꢀDQGꢀDQWLꢇDSRSWRWLFꢀSURWHLQVꢈꢀ(XUꢀ
-ꢀ3KDUPDFROꢈꢀꢅꢌꢌꢏꢍꢊꢎꢁꢐꢁꢅꢄꢇꢆꢈꢀ
ϭϵꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
ꢁꢅꢈꢀ 2ꢕ5HLOO\ꢀ.(ꢂꢀ5RMRꢀ)ꢂꢀ6KHꢀ4%ꢂꢀ6ROLWꢀ'ꢂꢀ0LOOVꢀ*%ꢂꢀ6PLWKꢀ'ꢂꢀHWꢀDOꢈꢀP725ꢀLQKLELWLRQꢀLQGXFHVꢀ
XSVWUHDPꢀUHFHSWRUꢀW\URVLQHꢀNLQDVHꢀVLJQDOLQJꢀDQGꢀDFWLYDWHVꢀ$NWꢈꢀ&DQFHUꢀ5HVꢈꢀꢅꢌꢌꢃꢍꢃꢃꢐꢁꢊꢌꢌꢇꢏꢈꢀ
ꢁꢉꢈꢀ 6XQꢀ6<ꢂꢀ5RVHQEHUJꢀ/0ꢂꢀ:DQJꢀ;ꢂꢀ=KRXꢀ=ꢂꢀ<XHꢀ3ꢂꢀ)Xꢀ+ꢂꢀHWꢀDOꢈꢀ$FWLYDWLRQꢀRIꢀ$NWꢀDQGꢀ
H,)ꢄ(ꢀVXUYLYDOꢀSDWKZD\VꢀE\ꢀUDSDP\FLQꢇPHGLDWHGꢀPDPPDOLDQꢀWDUJHWꢀRIꢀUDSDP\FLQꢀLQKLELWLRQꢈꢀ
&DQFHUꢀ5HVꢈꢀꢅꢌꢌꢊꢍꢃꢊꢐꢆꢌꢊꢅꢇꢏꢈꢀ
ꢁꢄꢈꢀ 3DUNꢀ'ꢂꢀ0DJLVꢀ$7ꢂꢀ/Lꢀ5ꢂꢀ2ZRQLNRNRꢀ7.ꢂꢀ6LFDꢀ*/ꢂꢀ6XQꢀ6<ꢂꢀHWꢀDOꢈꢀ1RYHOꢀVPDOOꢇPROHFXOHꢀ
LQKLELWRUVꢀRIꢀ%FOꢇ;/ꢀWRꢀWUHDWꢀOXQJꢀFDQFHUꢈꢀ&DQFHUꢀ5HVꢈꢀꢅꢌꢁꢉꢍꢆꢉꢐꢊꢄꢏꢊꢇꢎꢃꢈꢀ
ꢁꢊꢈꢀ /LXꢀ<ꢂꢀ6XQꢀ6<ꢂꢀ2ZRQLNRNRꢀ7.ꢂꢀ6LFDꢀ*/ꢂꢀ&XUUDQꢀ:-ꢂꢀ.KXULꢀ)5ꢂꢀHWꢀDOꢈꢀ5DSDP\FLQꢀLQGXFHVꢀ
%DGꢀSKRVSKRU\ODWLRQꢀLQꢀDVVRFLDWLRQꢀZLWKꢀLWVꢀUHVLVWDQFHꢀWRꢀKXPDQꢀOXQJꢀFDQFHUꢀFHOOVꢈꢀ0ROꢀ&DQFHUꢀ
7KHUꢈꢀꢅꢌꢁꢅꢍꢁꢁꢐꢄꢊꢇꢊꢃꢈꢀ
ꢁꢃꢈꢀ 2ZRQLNRNRꢀ7.ꢂꢀ5DPDOLQJDPꢀ66ꢂꢀ0LOOHUꢀ'/ꢂꢀ)RUFHꢀ6'ꢂꢀ6LFDꢀ*/ꢂꢀ0HQGHOꢀ-ꢂꢀHWꢀDOꢈꢀ$ꢀ
7UDQVODWLRQDOꢂꢀ3KDUPDFRG\QDPLFꢂꢀDQGꢀ3KDUPDFRNLQHWLFꢀ3KDVHꢀ,%ꢀ&OLQLFDOꢀ6WXG\ꢀRIꢀ(YHUROLPXVꢀ
LQꢀ5HVHFWDEOHꢀ1RQꢇ6PDOOꢀ&HOOꢀ/XQJꢀ&DQFHUꢈꢀ&OLQꢀ&DQFHUꢀ5HVꢈꢀꢅꢌꢁꢊꢍꢅꢁꢐꢁꢏꢊꢎꢇꢃꢏꢈꢀ
ꢁꢆꢈꢀ -Lꢀ+ꢂꢀ5DPVH\ꢀ05ꢂꢀ+D\HVꢀ'1ꢂꢀ)DQꢀ&ꢂꢀ0F1DPDUDꢀ.ꢂꢀ.R]ORZVNLꢀ3ꢂꢀHWꢀDOꢈꢀ/.%ꢁꢀPRGXODWHVꢀ
OXQJꢀFDQFHUꢀGLIIHUHQWLDWLRQꢀDQGꢀPHWDVWDVLVꢈꢀ1DWXUHꢈꢀꢅꢌꢌꢆꢍꢄꢄꢏꢐꢏꢌꢆꢇꢁꢌꢈꢀ
ꢁꢏꢈꢀ 6KDFNHOIRUGꢀ'%ꢂꢀ$EWꢀ(ꢂꢀ*HUNHQꢀ/ꢂꢀ9DVTXH]ꢀ'6ꢂꢀ6HNLꢀ$ꢂꢀ/HEODQFꢀ0ꢂꢀHWꢀDOꢈꢀ/.%ꢁꢀ
LQDFWLYDWLRQꢀGLFWDWHVꢀWKHUDSHXWLFꢀUHVSRQVHꢀRIꢀQRQꢇVPDOOꢀFHOOꢀOXQJꢀFDQFHUꢀWRꢀWKHꢀPHWDEROLVPꢀGUXJꢀ
SKHQIRUPLQꢈꢀ&DQFHUꢀ&HOOꢈꢀꢅꢌꢁꢉꢍꢅꢉꢐꢁꢄꢉꢇꢊꢏꢈꢀ
ꢁꢎꢈꢀ -DFNVRQꢀ(/ꢂꢀ:LOOLVꢀ1ꢂꢀ0HUFHUꢀ.ꢂꢀ%URQVRQꢀ57ꢂꢀ&URZOH\ꢀ'ꢂꢀ0RQWR\Dꢀ5ꢂꢀHWꢀDOꢈꢀ$QDO\VLVꢀRIꢀ
OXQJꢀWXPRUꢀLQLWLDWLRQꢀDQGꢀSURJUHVVLRQꢀXVLQJꢀFRQGLWLRQDOꢀH[SUHVVLRQꢀRIꢀRQFRJHQLFꢀ.ꢇUDVꢈꢀ*HQHVꢀ
'HYꢈꢀꢅꢌꢌꢁꢍꢁꢊꢐꢉꢅꢄꢉꢇꢏꢈꢀ
ꢅꢌꢈꢀ 7XYHVRQꢀ'$ꢂꢀ6KDZꢀ$7ꢂꢀ:LOOLVꢀ1$ꢂꢀ6LOYHUꢀ'3ꢂꢀ-DFNVRQꢀ(/ꢂꢀ&KDQJꢀ6ꢂꢀHWꢀDOꢈꢀ(QGRJHQRXVꢀ
RQFRJHQLFꢀ.ꢇUDVꢑ*ꢁꢅ'ꢒꢀVWLPXODWHVꢀSUROLIHUDWLRQꢀDQGꢀZLGHVSUHDGꢀQHRSODVWLFꢀDQGꢀGHYHORSPHQWDOꢀ
GHIHFWVꢈꢀ&DQFHUꢀ&HOOꢈꢀꢅꢌꢌꢄꢍꢊꢐꢉꢆꢊꢇꢏꢆꢈꢀ
ꢅꢁꢈꢀ :DQJꢀ;ꢂꢀ+DZNꢀ1ꢂꢀ<XHꢀ3ꢂꢀ.DXKꢀ-ꢂꢀ5DPDOLQJDPꢀ66ꢂꢀ)Xꢀ+ꢂꢀHWꢀDOꢈꢀ2YHUFRPLQJꢀP725ꢀ
LQKLELWLRQꢇLQGXFHGꢀSDUDGR[LFDOꢀDFWLYDWLRQꢀRIꢀVXUYLYDOꢀVLJQDOLQJꢀSDWKZD\VꢀHQKDQFHVꢀP725ꢀ
LQKLELWRUVꢕꢀDQWLFDQFHUꢀHIILFDF\ꢈꢀ&DQFHUꢀ%LROꢀ7KHUꢈꢀꢅꢌꢌꢏꢍꢆꢐꢁꢎꢊꢅꢇꢏꢈꢀ
ꢅꢅꢈꢀ 'HZVRQꢀ*ꢂꢀ0Dꢀ6ꢂꢀ)UHGHULFNꢀ3ꢂꢀ+RFNLQJVꢀ&ꢂꢀ7DQꢀ,ꢂꢀ.UDWLQDꢀ7ꢂꢀHWꢀDOꢈꢀ%D[ꢀGLPHUL]HVꢀYLDꢀDꢀ
V\PPHWULFꢀ%+ꢉꢐJURRYHꢀLQWHUIDFHꢀGXULQJꢀDSRSWRVLVꢈꢀ&HOOꢀ'HDWKꢀ'LIIHUꢈꢀꢅꢌꢁꢅꢍꢁꢎꢐꢃꢃꢁꢇꢆꢌꢈꢀ
ꢅꢉꢈꢀ :DQJꢀ-/ꢂꢀ/LXꢀ'ꢂꢀ=KDQJꢀ=-ꢂꢀ6KDQꢀ6ꢂꢀ+DQꢀ;ꢂꢀ6ULQLYDVXODꢀ60ꢂꢀHWꢀDOꢈꢀ6WUXFWXUHꢇEDVHGꢀ
GLVFRYHU\ꢀRIꢀDQꢀRUJDQLFꢀFRPSRXQGꢀWKDWꢀELQGVꢀ%FOꢇꢅꢀSURWHLQꢀDQGꢀLQGXFHVꢀDSRSWRVLVꢀRIꢀWXPRUꢀ
FHOOVꢈꢀ3URFꢀ1DWOꢀ$FDGꢀ6FLꢀ8ꢀ6ꢀ$ꢈꢀꢅꢌꢌꢌꢍꢎꢆꢐꢆꢁꢅꢄꢇꢎꢈꢀ
ꢅꢄꢈꢀ =KDQJꢀ+ꢂꢀ1LPPHUꢀ3ꢂꢀ5RVHQEHUJꢀ6+ꢂꢀ1Jꢀ6&ꢂꢀ-RVHSKꢀ0ꢈꢀ'HYHORSPHQWꢀRIꢀDꢀKLJKꢇ
WKURXJKSXWꢀIOXRUHVFHQFHꢀSRODUL]DWLRQꢀDVVD\ꢀIRUꢀ%FOꢇ[ꢑ/ꢒꢈꢀ$QDOꢀ%LRFKHPꢈꢀꢅꢌꢌꢅꢍꢉꢌꢆꢐꢆꢌꢇꢊꢈꢀ
ꢅꢊꢈꢀ %UXQFNRꢀ0ꢂꢀ2RVWꢀ7.ꢂꢀ%HOOLꢀ%$ꢂꢀ'LQJꢀ+ꢂꢀ-RVHSKꢀ0.ꢂꢀ.XQ]HUꢀ$ꢂꢀHWꢀDOꢈꢀ6WXGLHVꢀOHDGLQJꢀWRꢀ
SRWHQWꢂꢀGXDOꢀLQKLELWRUVꢀRIꢀ%FOꢇꢅꢀDQGꢀ%FOꢇ[/ꢈꢀ-ꢀ0HGꢀ&KHPꢈꢀꢅꢌꢌꢆꢍꢊꢌꢐꢃꢄꢁꢇꢃꢅꢈꢀ
ꢅꢃꢈꢀ 'HQJꢀ;ꢂꢀ;LDRꢀ/ꢂꢀ/DQJꢀ:ꢂꢀ*DRꢀ)ꢂꢀ5XYRORꢀ3ꢂꢀ0D\ꢀ:6ꢂꢀ-Uꢈꢀ1RYHOꢀUROHꢀIRUꢀ-1.ꢀDVꢀDꢀVWUHVVꢇ
DFWLYDWHGꢀ%FOꢅꢀNLQDVHꢈꢀ-ꢀ%LROꢀ&KHPꢈꢀꢅꢌꢌꢁꢍꢅꢆꢃꢐꢅꢉꢃꢏꢁꢇꢏꢈꢀ
ꢅꢆꢈꢀ 'HQJꢀ;ꢂꢀ*DRꢀ)ꢂꢀ)ODJJꢀ7ꢂꢀ$QGHUVRQꢀ-ꢂꢀ0D\ꢀ:6ꢈꢀ%FOꢅꢕVꢀIOH[LEOHꢀORRSꢀGRPDLQꢀUHJXODWHVꢀ
SꢊꢉꢀELQGLQJꢀDQGꢀVXUYLYDOꢈꢀ0ROꢀ&HOOꢀ%LROꢈꢀꢅꢌꢌꢃꢍꢅꢃꢐꢄꢄꢅꢁꢇꢉꢄꢈꢀ
ꢅꢏꢈꢀ 6DLWRꢀ0ꢂꢀ.RUVPH\HUꢀ6-ꢂꢀ6FKOHVLQJHUꢀ3+ꢈꢀ%$;ꢇGHSHQGHQWꢀWUDQVSRUWꢀRIꢀF\WRFKURPHꢀFꢀ
UHFRQVWLWXWHGꢀLQꢀSXUHꢀOLSRVRPHVꢈꢀ1DWꢀ&HOOꢀ%LROꢈꢀꢅꢌꢌꢌꢍꢅꢐꢊꢊꢉꢇꢊꢈꢀ
ꢅꢎꢈꢀ 'HZVRQꢀ*ꢂꢀ.UDWLQDꢀ7ꢂꢀ6LPꢀ+:ꢂꢀ3XWKDODNDWKꢀ+ꢂꢀ$GDPVꢀ-0ꢂꢀ&ROPDQꢀ30ꢂꢀHWꢀDOꢈꢀ7RꢀWULJJHUꢀ
DSRSWRVLVꢂꢀ%DNꢀH[SRVHVꢀLWVꢀ%+ꢉꢀGRPDLQꢀDQGꢀKRPRGLPHUL]HVꢀYLDꢀ%+ꢉꢐJURRYHꢀLQWHUDFWLRQVꢈꢀ0ROꢀ
&HOOꢈꢀꢅꢌꢌꢏꢍꢉꢌꢐꢉꢃꢎꢇꢏꢌꢈꢀ
ϮϬꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
ꢉꢌꢈꢀ 2OWHUVGRUIꢀ7ꢂꢀ(OPRUHꢀ6:ꢂꢀ6KRHPDNHUꢀ$5ꢂꢀ$UPVWURQJꢀ5&ꢂꢀ$XJHULꢀ'-ꢂꢀ%HOOLꢀ%$ꢂꢀHWꢀDOꢈꢀ$Qꢀ
LQKLELWRUꢀRIꢀ%FOꢇꢅꢀIDPLO\ꢀSURWHLQVꢀLQGXFHVꢀUHJUHVVLRQꢀRIꢀVROLGꢀWXPRXUVꢈꢀ1DWXUHꢈꢀꢅꢌꢌꢊꢍꢄꢉꢊꢐꢃꢆꢆꢇꢏꢁꢈꢀ
ꢉꢁꢈꢀ 3XULꢀ1ꢂꢀ.KUDPWVRYꢀ$ꢂꢀ$KPHGꢀ6ꢂꢀ1DOODVXUDꢀ9ꢂꢀ+HW]HOꢀ-7ꢂꢀ-DJDGHHVZDUDQꢀ5ꢂꢀHWꢀDOꢈꢀ$ꢀ
VHOHFWLYHꢀVPDOOꢀPROHFXOHꢀLQKLELWRUꢀRIꢀFꢇ0HWꢂꢀ3+$ꢃꢃꢊꢆꢊꢅꢂꢀLQKLELWVꢀWXPRULJHQLFLW\ꢀDQGꢀ
DQJLRJHQHVLVꢀLQꢀPRXVHꢀOXQJꢀFDQFHUꢀ[HQRJUDIWVꢈꢀ&DQFHUꢀ5HVꢈꢀꢅꢌꢌꢆꢍꢃꢆꢐꢉꢊꢅꢎꢇꢉꢄꢈꢀ
ꢉꢅꢈꢀ +DQꢀ%ꢂꢀ3DUNꢀ'ꢂꢀ/Lꢀ5ꢂꢀ;LHꢀ0ꢂꢀ2ZRQLNRNRꢀ7.ꢂꢀ=KDQJꢀ*ꢂꢀHWꢀDOꢈꢀ6PDOOꢇ0ROHFXOHꢀ%FOꢅꢀ%+ꢄꢀ
$QWDJRQLVWꢀIRUꢀ/XQJꢀ&DQFHUꢀ7KHUDS\ꢈꢀ&DQFHUꢀ&HOOꢈꢀꢅꢌꢁꢊꢍꢅꢆꢐꢏꢊꢅꢇꢃꢉꢈꢀ
ꢉꢉꢈꢀ &RUFRUDQꢀ5%ꢂꢀ&KHQJꢀ.$ꢂꢀ+DWDꢀ$1ꢂꢀ)DEHUꢀ$&ꢂꢀ(ELꢀ+ꢂꢀ&RIIHHꢀ(0ꢂꢀHWꢀDOꢈꢀ6\QWKHWLFꢀOHWKDOꢀ
LQWHUDFWLRQꢀRIꢀFRPELQHGꢀ%&/ꢇ;/ꢀDQGꢀ0(.ꢀLQKLELWLRQꢀSURPRWHVꢀWXPRUꢀUHJUHVVLRQVꢀLQꢀ.5$6ꢀ
PXWDQWꢀFDQFHUꢀPRGHOVꢈꢀ&DQFHUꢀFHOOꢈꢀꢅꢌꢁꢉꢍꢅꢉꢐꢁꢅꢁꢇꢏꢈꢀ
ꢉꢄꢈꢀ &KHQꢀ=ꢂꢀ&KHQJꢀ.ꢂꢀ:DOWRQꢀ=ꢂꢀ:DQJꢀ<ꢂꢀ(ELꢀ+ꢂꢀ6KLPDPXUDꢀ7ꢂꢀHWꢀDOꢈꢀ$ꢀPXULQHꢀOXQJꢀFDQFHUꢀ
FRꢇFOLQLFDOꢀWULDOꢀLGHQWLILHVꢀJHQHWLFꢀPRGLILHUVꢀRIꢀWKHUDSHXWLFꢀUHVSRQVHꢈꢀ1DWXUHꢈꢀꢅꢌꢁꢅꢍꢄꢏꢉꢐꢃꢁꢉꢇꢆꢈꢀ
ꢉꢊꢈꢀ /HJULHUꢀ0(ꢂꢀ<DQJꢀ&3ꢂꢀ<DQꢀ+*ꢂꢀ/RSH]ꢇ%DUFRQVꢀ/ꢂꢀ.HOOHUꢀ60ꢂꢀ3HUH]ꢇ6ROHUꢀ5ꢂꢀHWꢀDOꢈꢀ
7DUJHWLQJꢀSURWHLQꢀWUDQVODWLRQꢀLQꢀKXPDQꢀQRQꢀVPDOOꢀFHOOꢀOXQJꢀFDQFHUꢀYLDꢀFRPELQHGꢀ0(.ꢀDQGꢀ
PDPPDOLDQꢀWDUJHWꢀRIꢀUDSDP\FLQꢀVXSSUHVVLRQꢈꢀ&DQFHUꢀ5HVꢈꢀꢅꢌꢌꢆꢍꢃꢆꢐꢁꢁꢉꢌꢌꢇꢏꢈꢀ
ꢉꢃꢈꢀ 7DUKLQLꢀ$ꢂꢀ.RWVDNLVꢀ$ꢂꢀ*RRGLQJꢀ:ꢂꢀ6KXDLꢀ<ꢂꢀ3HWURꢀ'ꢂꢀ)ULHGODQGꢀ'ꢂꢀHWꢀDOꢈꢀ3KDVHꢀ,,ꢀVWXG\ꢀ
RIꢀHYHUROLPXVꢀꢑ5$'ꢌꢌꢁꢒꢀLQꢀSUHYLRXVO\ꢀWUHDWHGꢀVPDOOꢀFHOOꢀOXQJꢀFDQFHUꢈꢀ&OLQꢀ&DQFHUꢀ5HVꢈꢀ
ꢅꢌꢁꢌꢍꢁꢃꢐꢊꢎꢌꢌꢇꢆꢈꢀ
ꢉꢆꢈꢀ 5DPDOLQJDPꢀ66ꢂꢀ2ZRQLNRNRꢀ7.ꢂꢀ%HKHUDꢀ0ꢂꢀ6XEUDPDQLDQꢀ-ꢂꢀ6DEDꢀ1)ꢂꢀ.RQRꢀ6$ꢂꢀHWꢀDOꢈꢀ
3KDVHꢀ,,ꢀVWXG\ꢀRIꢀGRFHWD[HOꢀLQꢀFRPELQDWLRQꢀZLWKꢀHYHUROLPXVꢀIRUꢀVHFRQGꢇꢀRUꢀWKLUGꢇOLQHꢀWKHUDS\ꢀRIꢀ
DGYDQFHGꢀQRQꢇVPDOOꢇFHOOꢀOXQJꢀFDQFHUꢈꢀ-ꢀ7KRUDFꢀ2QFROꢈꢀꢅꢌꢁꢉꢍꢏꢐꢉꢃꢎꢇꢆꢅꢈꢀ
ꢉꢏꢈꢀ %HVVHꢀ%ꢂꢀ/HLJKOꢀ1ꢂꢀ%HQQRXQDꢀ-ꢂꢀ3DSDGLPLWUDNRSRXORXꢀ9$ꢂꢀ%ODLVꢀ1ꢂꢀ7UD\QRUꢀ$0ꢂꢀHWꢀDOꢈꢀ
3KDVHꢀ,,ꢀVWXG\ꢀRIꢀHYHUROLPXVꢇHUORWLQLEꢀLQꢀSUHYLRXVO\ꢀWUHDWHGꢀSDWLHQWVꢀZLWKꢀDGYDQFHGꢀQRQꢇVPDOOꢇ
FHOOꢀOXQJꢀFDQFHUꢈꢀ$QQꢀ2QFROꢈꢀꢅꢌꢁꢄꢍꢅꢊꢐꢄꢌꢎꢇꢁꢊꢈꢀ
ꢉꢎꢈꢀ +HUUHUDꢀ9$ꢂꢀ=HLQGOꢇ(EHUKDUWꢀ(ꢂꢀ-XQJꢀ$ꢂꢀ+XEHUꢀ50ꢂꢀ%HUJQHUꢀ$ꢈꢀ7KHꢀGXDOꢀ3,ꢉ.ꢓP725ꢀ
LQKLELWRUꢀ%(=ꢅꢉꢊꢀLVꢀHIIHFWLYHꢀLQꢀOXQJꢀFDQFHUꢀFHOOꢀOLQHVꢈꢀ$QWLFDQFHUꢀUHVHDUFKꢈꢀꢅꢌꢁꢁꢍꢉꢁꢐꢏꢄꢎꢇꢊꢄꢈꢀ
ꢄꢌꢈꢀ )LHELJꢀ++ꢂꢀ1HXPDQQꢀ+$ꢂꢀ+HQVVꢀ+ꢂꢀ.RFKꢀ+ꢂꢀ.DLVHUꢀ'ꢂꢀ$UQROGꢀ+ꢈꢀ'HYHORSPHQWꢀRIꢀWKUHHꢀ
KXPDQꢀVPDOOꢀFHOOꢀOXQJꢀFDQFHUꢀPRGHOVꢀLQꢀQXGHꢀPLFHꢈꢀ5HFHQWꢀ5HVXOWVꢀ&DQFHUꢀ5HVꢈꢀꢁꢎꢏꢊꢍꢎꢆꢐꢆꢆꢇ
ꢏꢃꢈꢀ
ꢄꢁꢈꢀ +LGDOJRꢀ0ꢂꢀ$PDQWꢀ)ꢂꢀ%LDQNLQꢀ$9ꢂꢀ%XGLQVNDꢀ(ꢂꢀ%\UQHꢀ$7ꢂꢀ&DOGDVꢀ&ꢂꢀHWꢀDOꢈꢀ3DWLHQWꢇ
GHULYHGꢀ[HQRJUDIWꢀPRGHOVꢐꢀDQꢀHPHUJLQJꢀSODWIRUPꢀIRUꢀWUDQVODWLRQDOꢀFDQFHUꢀUHVHDUFKꢈꢀ&DQFHUꢀ
'LVFRYꢈꢀꢅꢌꢁꢄꢍꢄꢐꢎꢎꢏꢇꢁꢌꢁꢉꢈꢀ
ꢄꢅꢈꢀ 0RQWHURꢀ-ꢂꢀ6DURVLHNꢀ.$ꢂꢀ'H$QJHORꢀ-'ꢂꢀ0DHUWHQVꢀ2ꢂꢀ5\DQꢀ-ꢂꢀ(UFDQꢀ'ꢂꢀHWꢀDOꢈꢀ'UXJꢇ
LQGXFHGꢀGHDWKꢀVLJQDOLQJꢀVWUDWHJ\ꢀUDSLGO\ꢀSUHGLFWVꢀFDQFHUꢀUHVSRQVHꢀWRꢀFKHPRWKHUDS\ꢈꢀ&HOOꢈꢀ
ꢅꢌꢁꢊꢍꢁꢃꢌꢐꢎꢆꢆꢇꢏꢎꢈꢀ
ꢄꢉꢈꢀ )UHVHꢀ..ꢂꢀ7XYHVRQꢀ'$ꢈꢀ0D[LPL]LQJꢀPRXVHꢀFDQFHUꢀPRGHOVꢈꢀ1DWꢀ5HYꢀ&DQFHUꢈꢀ
ꢅꢌꢌꢆꢍꢆꢐꢃꢄꢊꢇꢊꢏꢈꢀ
ꢄꢄꢈꢀ 6FKXXUELHUVꢀ2&ꢂꢀ.DDQGHUVꢀ-+ꢂꢀYDQꢀGHUꢀ+HLMGHQꢀ+)ꢂꢀ'HNKXLM]HQꢀ53ꢂꢀ2\HQꢀ:-ꢂꢀ%XVVLQNꢀ
-ꢈꢀ7KHꢀ3,ꢉꢇ.ꢓ$.7ꢇSDWKZD\ꢀDQGꢀUDGLDWLRQꢀUHVLVWDQFHꢀPHFKDQLVPVꢀLQꢀQRQꢇVPDOOꢀFHOOꢀOXQJꢀFDQFHUꢈꢀ
-ꢀ7KRUDFꢀ2QFROꢈꢀꢅꢌꢌꢎꢍꢄꢐꢆꢃꢁꢇꢆꢈꢀ
Ϯϭꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
)LJXUHꢀ/HJHQGVꢀ
)LJXUHꢀꢄꢈꢀ&<'ꢉꢇꢉꢄꢄꢀFRPSRXQGꢀVHOHFWLYHO\ꢀELQGVꢀWRꢀ%D[ꢀSURWHLQꢀDQGꢀLQGXFHVꢀDSRSWRVLVꢀLQꢀ
YDULRXVꢀ16&/&ꢀDQGꢀ6&/&ꢀFHOOꢀOLQHVꢈꢀ$ꢂꢀ'HVLJQꢀRIꢀ&<'ꢇꢅꢇꢁꢁꢀEDVHGꢀRQꢀ60%$ꢁꢈꢀ%ꢂꢀ6\QWKHVLVꢀ
RIꢀ &<'ꢇꢅꢇꢁꢁꢈꢀ 5HDFWLRQVꢀ DQGꢀ FRQGLWLRQVꢐꢀ Dꢒꢀ VDOLF\ODOGHK\GHꢂꢀ .)ꢇ$Oꢅ2ꢉꢂꢀ 0H2+ꢂꢀ ꢆꢅꢖꢀ \LHOGꢍꢀ Eꢒꢀ
tertꢇEXW\OꢀꢑꢅꢇK\GUR[\HWK\OꢒFDUEDPDWHꢂꢀ',$'ꢂꢀ33Kꢉꢂꢀ7+)ꢂꢀUWꢂꢀꢁꢅꢀKꢍꢀFꢒꢀ7)$ꢂꢀ&+ꢅ&OꢅꢂꢀUWꢂꢀꢆꢉꢖꢀ\LHOGꢀ
ꢑꢅꢀVWHSVꢒꢈꢀ&ꢂꢀ0ROHFXODUꢀGRFNLQJꢀRIꢀ&<'ꢇꢅꢇꢁꢁꢀLQWRꢀWKHꢀ6ꢁꢏꢄꢀELQGLQJꢀSRFNHWꢀLQꢀWKHꢀ&ꢇWHUPLQXVꢀRIꢀ
%D[ꢀSURWHLQꢈꢀ'ꢂꢀ%LQGLQJꢀDIILQLWLHVꢀRIꢀ&<'ꢇꢅꢇꢁꢁꢀZLWKꢀ%D[ꢀSURWHLQꢀDQGꢀRWKHUꢀ%FOꢅꢀIDPLO\ꢀPHPEHUVꢀ
ZHUHꢀDQDO\]HGꢀXVLQJꢀDꢀFRPSHWLWLRQꢀIOXRUHVFHQFHꢀSRODUL]DWLRQꢀDVVD\ꢈꢀ(UURUꢀEDUVꢀUHSUHVHQWꢀ“6ꢈ'ꢈꢀ
(ꢂꢀ /HYHOVꢀ RIꢀ S%D[ꢀ DQGꢀ WRWDOꢀ %D[ꢀ ZHUHꢀ DQDO\]HGꢀ E\ꢀ :HVWHUQꢀ EORWꢀ LQꢀ %($6ꢇꢅ%ꢂꢀ 16&/&ꢀ DQGꢀ
6&/&ꢀFHOOꢀOLQHVꢈꢀ)ꢂꢀ$ꢀSDQHOꢀRIꢀ16&/&ꢀDQGꢀ6&/&ꢀFHOOꢀOLQHVꢀZHUHꢀWUHDWHGꢀZLWKꢀ60%$ꢁꢀꢑꢊꢀμ0ꢒꢀRUꢀ
&<'ꢇꢅꢇꢁꢁꢀꢑꢊꢀ—0ꢒꢀIRUꢀꢄꢏꢀKUꢈꢀ$SRSWRWLFꢀFHOOꢀGHDWKꢀZDVꢀGHWHUPLQHGꢀE\ꢀDQDO\]LQJꢀ$QQH[LQꢇ9ꢓ3,ꢀ
ELQGLQJꢀE\ꢀIOXRUHVFHQFHꢇDFWLYDWHGꢀFHOOꢀVRUWHUꢀꢑ)$&6ꢒꢈꢀ(UURUꢀEDUVꢀUHSUHVHQWꢀ“ꢀVWDQGDUGꢀGHYLDWLRQꢀ
ꢑ6'ꢒꢀRIꢀWKUHHꢀVHSDUDWHꢀGHWHUPLQDWLRQVꢈꢀ
)LJXUHꢀ ꢇꢈꢀ &<'ꢉꢇꢉꢄꢄꢀ GLUHFWO\ꢀ LQGXFHVꢀ %D[ꢀ ROLJRPHUL]DWLRQꢊꢀ IDFLOLWDWHVꢀ &\Wꢀ Fꢀ UHOHDVHꢀ DQGꢀ
VXSSUHVVHVꢀOXQJꢀFDQFHUꢀLQꢀGRVHꢉGHSHQGHQWꢀPDQQHUꢀLQꢀYLYRꢈꢀ$ꢂꢀ%D[ꢀDQGꢀ%$.ꢀH[SUHVVLRQꢀ
ZDVꢀ DQDO\]HGꢀ E\ꢀ :HVWHUQꢀ EORWꢀ IROORZLQJꢀ WUHDWPHQWꢀ RIꢀ $ꢊꢄꢎꢀ FHOOVꢀ ZLWKꢀ &<'ꢇꢅꢇꢁꢁꢀ IRUꢀ ꢅꢄKꢈꢀ %ꢂꢀ
0LWRFKRQGULDꢀZHUHꢀLVRODWHGꢀIURPꢀ$ꢊꢄꢎꢀFHOOVꢀDQGꢀWKHQꢀWUHDWHGꢀZLWKꢀ&<'ꢇꢅꢇꢁꢁꢀRUꢀ60%$ꢁꢀꢑꢊꢀ—0ꢒꢀ
LQꢀ FURVVꢇOLQNLQJꢀ EXIIHUꢀ IRUꢀ ꢉꢌꢀ PLQꢀ DWꢀ ꢉꢌꢀ R&ꢂꢀ IROORZHGꢀ E\ꢀ FURVVꢇOLQNLQJꢀ XVLQJꢀ %0+ꢈꢀ %D[ꢀ ZDVꢀ
DQDO\]HGꢀE\ꢀ:HVWHUQꢀEORWꢀꢑ%ꢂꢀOHIWꢀSDQHOꢒꢈꢀ$ꢊꢄꢎꢀFHOOVꢀZHUHꢀWUHDWHGꢀZLWKꢀ&<'ꢇꢅꢇꢁꢁꢀRUꢀ60%$ꢁꢀꢑꢊꢀ
—0ꢒꢀ IRUꢀ ꢅꢄKꢂꢀ IROORZHGꢀ E\ꢀ LVRODWLRQꢀ RIꢀ PLWRFKRQGULDꢀ DQGꢀ FURVVꢇOLQNLQJꢀ XVLQJꢀ %0+ꢈꢀ %D[ꢀ ZDVꢀ
DQDO\]HGꢀ E\ꢀ :HVWHUQꢀ EORWꢀ ꢑ%ꢂꢀ ULJKWꢀ SDQHOꢒꢈꢀ &ꢂꢀ $ꢊꢄꢎꢀ FHOOVꢀ ZHUHꢀ WUHDWHGꢀ ZLWKꢀ LQFUHDVLQJꢀ
FRQFHQWUDWLRQVꢀRIꢀ&<'ꢇꢅꢇꢁꢁꢀIRUꢀꢅꢄKꢈꢀ0LWRFKRQGULDOꢀDQGꢀF\WRVROLFꢀIUDFWLRQVꢀZHUHꢀLVRODWHGꢈꢀ/HYHOVꢀ
RIꢀ&\WꢀcꢀDQGꢀ%D[ꢀLQꢀWKHVHꢀWZRꢀIUDFWLRQVꢀZHUHꢀDQDO\]HGꢀE\ꢀ:HVWHUQꢀEORWꢈꢀ3URKLELWLQꢀZHUHꢀXVHGꢀDVꢀ
ϮϮꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
SRVLWLYHꢀFRQWUROꢀWRꢀYHULI\ꢀPLWRFKRQGULDOꢀIUDFWLRQꢀZLWKRXWꢀFRQWDPLQDWLRQꢈꢀ'ꢂꢀ1Xꢓ1XꢀPLFHꢀZLWKꢀ$ꢊꢄꢎꢀ
OXQJꢀFDQFHUꢀ[HQRJUDIWVꢀZHUHꢀWUHDWHGꢀZLWKꢀLQFUHDVLQJꢀGRVHVꢀRIꢀ&<'ꢇꢅꢇꢁꢁꢀꢑꢅaꢄꢌꢀPJꢓNJꢓGꢒꢀLꢈSꢈꢀIRUꢀ
ꢁꢌꢀGD\Vꢈꢀ(DFKꢀJURXSꢀLQFOXGHGꢀꢏꢀPLFHꢈꢀꢀ7XPRUꢀYROXPHꢀZDVꢀPHDVXUHGꢀRQFHꢀHYHU\ꢀꢅꢀGD\Vꢈꢀ7KHꢀ
HUURUꢀEDUVꢀLQGLFDWHꢀ ꢀ6'ꢀꢑQ ꢀꢏꢒꢈꢀ(ꢀDQGꢀ)ꢂꢀ$FWLYHꢀFDVSDVHꢀꢉꢀZDVꢀDQDO\]HGꢀLQꢀWXPRUꢀWLVVXHVꢀDWꢀWKHꢀ
HQGꢀ RIꢀ H[SHULPHQWVꢀ E\ꢀ ,+&ꢀ VWDLQLQJꢈꢀ 7KHꢀ HUURUꢀ EDUVꢀ LQGLFDWHꢀ ꢀ 6'ꢀ RIꢀ WKUHHꢀ VHSDUDWHꢀ
GHWHUPLQDWLRQVꢈꢀ *ꢂꢀ 0LWRFKRQGULDꢀ ZHUHꢀ LVRODWHGꢀ IURPꢀ WXPRUꢀ WLVVXHVꢂꢀ DQGꢀ IROORZHGꢀ E\ꢀ FURVVꢇ
OLQNLQJꢀXVLQJꢀ%0+ꢈꢀ%D[ꢀZDVꢀDQDO\]HGꢀE\ꢀ:HVWHUQꢀEORWꢈꢀꢀ
)LJXUHꢀ ꢌꢈꢀ (IILFDF\ꢀ RIꢀ 60%$ꢄꢀ DQGꢀ &<'ꢉꢇꢉꢄꢄꢀ ZDVꢀ FRPSDUHGꢀ LQꢀ 16&/&ꢀ DQGꢀ 6&/&ꢀ
[HQRJUDIWVꢈꢀ$ꢂꢀ1Xꢓ1XꢀPLFHꢀZLWKꢀ16&/&ꢀꢑ$ꢊꢄꢎꢒꢀRUꢀ6&/&ꢀꢑ'06ꢊꢉꢒꢀꢀ[HQRJUDIWVꢀZHUHꢀWUHDWHGꢀ
ZLWKꢀꢄꢌꢀPJꢓNJꢀRIꢀ60%$ꢁꢀRUꢀ&<'ꢇꢅꢇꢁꢁꢀLꢈSꢈꢀIRUꢀꢁꢄꢀGD\Vꢈꢀ(DFKꢀJURXSꢀLQFOXGHGꢀꢏꢀPLFHꢈꢀ7XPRUꢀ
YROXPHꢀ ZDVꢀ PHDVXUHGꢀ RQFHꢀ HYHU\ꢀ ꢅꢀ GD\Vꢈꢀ 7KHꢀ HUURUꢀ EDUVꢀ LQGLFDWHꢀ ꢀ 6'ꢀ ꢑQ ꢏꢒꢈꢀ %ꢂꢀ $FWLYHꢀ
FDVSDVHꢀꢉꢀZDVꢀDQDO\]HGꢀLQꢀWXPRUꢀWLVVXHVꢀDWꢀWKHꢀHQGꢀRIꢀH[SHULPHQWVꢀE\ꢀ,+&ꢀVWDLQLQJꢈꢀ7KHꢀHUURUꢀ
EDUVꢀLQGLFDWHꢀ ꢀ6'ꢀRIꢀWKUHHꢀVHSDUDWHꢀGHWHUPLQDWLRQVꢈꢀ&ꢂꢀ0LWRFKRQGULDꢀZHUHꢀLVRODWHGꢀIURPꢀWXPRUꢀ
WLVVXHVꢀDIWHUꢀWUHDWPHQWꢀRIꢀPLFHꢀZLWKꢀ60%$ꢁꢀRUꢀ&<'ꢇꢅꢇꢁꢁꢂꢀDQGꢀIROORZHGꢀE\ꢀFURVVꢇOLQNLQJꢀXVLQJꢀ
%0+ꢈꢀ%D[ꢀZDVꢀDQDO\]HGꢀE\ꢀ:HVWHUQꢀEORWꢈꢀ
)LJXUHꢀꢆꢈꢀꢀ&<'ꢉꢇꢉꢄꢄꢀUHSUHVVHVꢀWXPRUꢀJURZWKꢀLQꢀ6&/&ꢀSDWLHQWꢇGHULYHGꢀ[HQRJUDIWVꢈꢀ$ꢂꢀ0LFHꢀ
FDUU\LQJꢀ [HQRJUDIWVꢀ GHULYHGꢀ IURPꢀ WZRꢀ SDWLHQWVꢀ ZLWKꢀ UHIUDFWRU\ꢀ 6&/&ꢀ ꢑ7.2ꢇꢌꢌꢅꢀ RUꢀ 7.2ꢇꢌꢌꢊꢒꢀ
ZHUHꢀ WUHDWHGꢀ ZLWKꢀ &<'ꢇꢅꢇꢁꢁꢀ ꢑꢄꢌꢀ PJꢓNJꢓGꢒꢀ LꢈSꢈꢀ IRUꢀ ꢅꢀ ZHHNVꢈꢀ 7XPRUꢀ YROXPHVꢀ ZHUHꢀ DQDO\]HGꢈꢀ
(UURUꢀEDUVꢀUHSUHVHQWꢀ ꢀ6ꢈ'ꢀꢑQ ꢃꢒꢈꢀ%ꢂꢀꢀS%D[ꢀDQGꢀꢃ$ꢆꢀ%D[ꢀZHUHꢀDQDO\]HGꢀLQꢀWXPRUꢀWLVVXHVꢀDWꢀWKHꢀ
HQGꢀRIꢀH[SHULPHQWVꢀE\ꢀ4'ꢇ,+)ꢀDVꢀGHVFULEHGꢀLQꢀ³6XSSOHPHQWDOꢀ0HWKRGV´ꢈꢀ&ꢂꢀ0LWRFKRQGULDꢀZHUHꢀ
LVRODWHGꢀ IURPꢀ WXPRUꢀ WLVVXHVꢀ DIWHUꢀ WUHDWPHQWꢀ RIꢀ PLFHꢀ ZLWKꢀ &<'ꢇꢅꢇꢁꢁꢂꢀ DQGꢀ IROORZHGꢀ E\ꢀ FURVVꢇ
OLQNLQJꢀXVLQJꢀ%0+ꢈꢀ%D[ꢀZDVꢀDQDO\]HGꢀE\ꢀ:HVWHUQꢀEORWꢈꢀ'ꢂꢀ$FWLYHꢀFDVSDVHꢀꢉꢀZDVꢀDQDO\]HGꢀE\ꢀ
Ϯϯꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
,+&ꢀDQGꢀTXDQWLILHGꢀLQꢀWXPRUꢀWLVVXHVꢀDWꢀWKHꢀHQGꢀRIꢀH[SHULPHQWVꢈꢀ7KHꢀHUURUꢀEDUVꢀLQGLFDWHꢀ ꢀ6'ꢀRIꢀ
WKUHHꢀVHSDUDWHꢀGHWHUPLQDWLRQVꢈꢀꢀ
)LJXUHꢀꢍꢈꢀ&<'ꢉꢇꢉꢄꢄꢀVXSSUHVVHVꢀOXQJꢀFDQFHUꢀJURZWKꢀDQGꢀSURORQJVꢀVXUYLYDOꢀLQꢀJHQHWLFDOO\ꢀ
HQJLQHHUHGꢀPRXVHꢀPRGHOVꢀꢂ*(00ꢃꢈꢀ$ꢂꢀ$IWHUꢀDGPLQLVWUDWLRQꢀRIꢀDGHQRYLUXVꢀ&UHꢀUHFRPELQDVHꢀ
LQꢀ.5$6*ꢁꢅ'ꢀ/.%ꢁIOꢓIOꢀꢑ./ꢒꢀPLFHꢀIRUꢀꢃꢀZHHNVꢂꢀ./ꢀPLFHꢀZHUHꢀWUHDWHGꢀZLWKꢀ&<'ꢇꢅꢇꢁꢁꢀIRUꢀꢏꢀZHHNVꢀ
ꢑFRQWUROꢀ JURXSꢂꢀ ꢏꢀ PLFHꢂꢀ WUHDWPHQWꢀ JURXSꢂꢀ ꢆꢀ PLFHꢒꢈꢀ 7XPRUꢀ QXPEHUVꢀ ZHUHꢀ FRXQWHGꢀ XQGHUꢀ WKHꢀ
PLFURVFRSHꢀDQGꢀWXPRUꢀDUHDꢀZDVꢀTXDQWLILHGꢀXVLQJꢀ2SHQODEꢀPRGXODUꢀLPDJLQJꢀVRIWZDUHꢈꢀ%ꢂꢀ7KUHHꢀ
UHSUHVHQWDWLYHꢀ+ (ꢀLPDJHVꢀIURPꢀFRQWUROꢀRUꢀWUHDWPHQWꢀJURXSꢀZHUHꢀVKRZQꢈꢀ&ꢂꢀ$FWLYHꢀFDVSDVHꢀꢉꢀ
ZDVꢀDQDO\]HGꢀE\ꢀ,+&ꢀDWꢀWKHꢀHQGꢀRIꢀH[SHULPHQWVꢈꢀ'ꢂꢀ6XUYLYDOꢀRIꢀPLFHꢀZDVꢀFDOFXODWHGꢀXSꢀWRꢀꢏꢀ
ZHHNVꢀEHIRUHꢀHXWKDQL]DWLRQꢀLQꢀWKHꢀFRQWUROꢀJURXSꢀYHUVXVꢀ&<'ꢇꢅꢇꢁꢁꢀWUHDWPHQWꢀJURXSꢈꢀꢀ
)LJXUHꢀꢎꢈꢀ7UHDWPHQWꢀRIꢀOXQJꢀFDQFHUꢀFHOOVꢀRUꢀ16&/&ꢀSDWLHQWVꢀZLWKꢀ5$'ꢋꢋꢄꢀXSUHJXODWHVꢀ
%D[ꢀSKRVSKRU\ODWLRQꢈꢀ$ꢀDQGꢀ%ꢂꢀ$ꢊꢄꢎꢀFHOOVꢀZHUHꢀWUHDWHGꢀZLWKꢀ5$'ꢌꢌꢁꢀIRUꢀꢅꢄKꢈꢀS%D[ꢂꢀS$.7ꢀ
DQGꢀSꢇSꢆꢌ6ꢃ.ꢀZHUHꢀDQDO\]HGꢀE\ꢀ:HVWHUQꢀEORWꢀRUꢀ4'ꢇ,+)ꢈꢀ&ꢀDQGꢀ'ꢂꢀꢁꢌꢀSDWLHQWVꢀZLWKꢀ16&/&ꢀ
ZHUHꢀWUHDWHGꢀZLWKꢀ5$'ꢌꢌꢁꢀꢑꢁꢌꢀPJꢓGD\ꢒꢀIRUꢀꢅꢏꢀGD\VꢈꢀS%D[ꢀLQꢀWXPRUꢀWLVVXHVꢀZDVꢀDQDO\]HGꢀE\ꢀ
,+&ꢀDQGꢀTXDQWLILHGꢀE\ꢀDQDO\]LQJꢀLPPXQRVFRUHꢈꢀ(UURUꢀEDUVꢀUHSUHVHQWꢀ ꢀ6'ꢀRIꢀWKUHHꢀVHSDUDWHꢀ
GHWHUPLQDWLRQVꢈꢀ
)LJXUHꢀꢏꢈꢀ&RPELQDWLRQꢀRIꢀ&<'ꢉꢇꢉꢄꢄꢀDQGꢀ5$'ꢋꢋꢄꢀV\QHUJLVWLFDOO\ꢀUHSUHVVHVꢀOXQJꢀFDQFHUꢀ
DQGꢀRYHUFRPHVꢀUDSDORJꢀUHVLVWDQFHꢀin vivoꢈꢀ1Xꢓ1XꢀPLFHꢀZLWKꢀ$ꢊꢄꢎꢀRUꢀ$ꢊꢄꢎꢇ55ꢀOXQJꢀFDQFHUꢀ
[HQRJUDIWVꢀ ZHUHꢀ WUHDWHGꢀ ZLWKꢀ 5$'ꢌꢌꢁꢂꢀ &<'ꢇꢅꢇꢁꢁꢀ RUꢀ WKHꢀ FRPELQDWLRQꢀ LꢈSꢈꢀ IRUꢀ ꢁꢄꢀ GD\Vꢈꢀ (DFKꢀ
JURXSꢀLQFOXGHGꢀꢃꢀPLFHꢈꢀꢑ$ꢒ7XPRUꢀYROXPHVꢀZHUHꢀDQDO\]HGꢈꢀ(UURUꢀEDUVꢀUHSUHVHQWꢀ ꢀ6ꢈ'ꢀꢑQ ꢃꢒꢈꢀ
ꢑ%ꢉ&ꢒꢀS%D[ꢂꢀꢃ$ꢆꢀ%D[ꢂꢀS$.7ꢂꢀSꢇSꢆꢌ6ꢃ.ꢀDQGꢀDFWLYHꢀFDVSDVHꢀꢉꢀLQꢀWXPRUꢀWLVVXHVꢀꢀZHUHꢀDQDO\]HGꢀ
E\ꢀ4'ꢇ,+)ꢀRUꢀ,+&ꢈꢀ(UURUꢀEDUVꢀUHSUHVHQWꢀ ꢀ6'ꢀRIꢀWKUHHꢀVHSDUDWHꢀGHWHUPLQDWLRQVꢈꢀ
ꢀ
Ϯϰꢀ
ꢀ
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.

Author Manuscript Published OnlineFirst on April 5, 2017; DOI: 10.1158/0008-5472.CAN-16-2356
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Downloaded from cancerres.aacrjournals.org on April 7, 2017. © 2017 American Association for Cancer Research.