TY - JOUR
T1 - Effect of a smac mimetic (TL32711, birinapant) on the apoptotic program and apoptosis biomarkers examined with validated multiplex immunoassays fit for clinical use
AU - Srivastava, Apurva K.
AU - Jaganathan, Soumya
AU - Stephen, Laurie
AU - Hollingshead, Melinda G.
AU - Layhee, Adam
AU - Damour, Eric
AU - Govindharajulu, Jeevan Prasaad
AU - Donohue, Jennifer
AU - Esposito, Dominic
AU - Mapes, James P.
AU - Kinders, Robert J.
AU - Takebe, Naoko
AU - Tomaszewski, Joseph E.
AU - Kummar, Shivaani
AU - Doroshow, James H.
AU - Parchment, Ralph E.
N1 - Publisher Copyright:
© 2015 AACR.
PY - 2016/2/15
Y1 - 2016/2/15
N2 - Purpose: To support clinical pharmacodynamic evaluation of the Smac mimetic TL32711 (birinapant) and other apoptosistargeting drugs, we describe the development, validation, and application of novel immunoassays for 15 cytosolic and membrane- associated proteins indicative of the induction, onset, and commitment to apoptosis in human tumors. Experimental Design: The multiplex immunoassays were constructed on the Luminex platform with apoptosis biomarkers grouped into three panels. Panel 1 contains Bak, Bax, total caspase-3, total lamin-B (intact and 45 kDa fragment), and Smac; panel 2 contains Bad, Bax-Bcl-2 heterodimer, Bcl-xL, Bim, and Mcl1; and panel 3 contains active (cleaved) caspase-3, Bcl-xL-Bak heterodimer, Mcl1-Bak heterodimer, pS99-Bad, and survivin. Antibody specificity was confirmed by immunoprecipitation and Western blot analysis. Results: Two laboratories analytically validated the multiplex immunoassays for application with core-needle biopsy samples processed to control preanalytical variables; the biologic variability for each biomarker was estimated from xenograft measurements. Studies of TL32711 in xenograft models confirmed a dosedependent increase in activated caspase-3 6 hours after dosing and provided assay fit-for-purpose confirmation. Coincident changes in cytosolic lamin-B and subsequent changes in Bcl-xL provided correlative evidence of caspase-3 activation. The validated assay is suitable for use with clinical specimens; 14 of 15 biomarkers were quantifiable in patient core-needle biopsies. Conclusions: The validated multiplex immunoassays developed for this study provided proof of mechanism data for TL32711 and are suitable for quantifying apoptotic biomarkers in clinical trials.
AB - Purpose: To support clinical pharmacodynamic evaluation of the Smac mimetic TL32711 (birinapant) and other apoptosistargeting drugs, we describe the development, validation, and application of novel immunoassays for 15 cytosolic and membrane- associated proteins indicative of the induction, onset, and commitment to apoptosis in human tumors. Experimental Design: The multiplex immunoassays were constructed on the Luminex platform with apoptosis biomarkers grouped into three panels. Panel 1 contains Bak, Bax, total caspase-3, total lamin-B (intact and 45 kDa fragment), and Smac; panel 2 contains Bad, Bax-Bcl-2 heterodimer, Bcl-xL, Bim, and Mcl1; and panel 3 contains active (cleaved) caspase-3, Bcl-xL-Bak heterodimer, Mcl1-Bak heterodimer, pS99-Bad, and survivin. Antibody specificity was confirmed by immunoprecipitation and Western blot analysis. Results: Two laboratories analytically validated the multiplex immunoassays for application with core-needle biopsy samples processed to control preanalytical variables; the biologic variability for each biomarker was estimated from xenograft measurements. Studies of TL32711 in xenograft models confirmed a dosedependent increase in activated caspase-3 6 hours after dosing and provided assay fit-for-purpose confirmation. Coincident changes in cytosolic lamin-B and subsequent changes in Bcl-xL provided correlative evidence of caspase-3 activation. The validated assay is suitable for use with clinical specimens; 14 of 15 biomarkers were quantifiable in patient core-needle biopsies. Conclusions: The validated multiplex immunoassays developed for this study provided proof of mechanism data for TL32711 and are suitable for quantifying apoptotic biomarkers in clinical trials.
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U2 - 10.1158/1078-0432.CCR-14-3156
DO - 10.1158/1078-0432.CCR-14-3156
M3 - Article
C2 - 26446940
AN - SCOPUS:84964413016
SN - 1078-0432
VL - 22
SP - 1000
EP - 1010
JO - Clinical Cancer Research
JF - Clinical Cancer Research
IS - 4
ER -