Effect of a smac mimetic (TL32711, birinapant) on the apoptotic program and apoptosis biomarkers examined with validated multiplex immunoassays fit for clinical use

Apurva K. Srivastava, Soumya Jaganathan, Laurie Stephen, Melinda G. Hollingshead, Adam Layhee, Eric Damour, Jeevan Prasaad Govindharajulu, Jennifer Donohue, Dominic Esposito, James P. Mapes, Robert J. Kinders, Naoko Takebe, Joseph E. Tomaszewski, Shivaani Kummar, James H. Doroshow, Ralph E. Parchment

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1000-1010
Number of pages11
JournalClinical Cancer Research
Volume22
Issue number4
DOIs
StatePublished - Feb 15 2016
Externally publishedYes

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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