title: Effect of a Smac Mimetic (TL32711, Birinapant) on the Apoptotic Program and Apoptosis Biomarkers Examined with Validated Multiplex Immunoassays Fit for Clinical Use.
abstract: OBJECTIVE
  To support clinical pharmacodynamic evaluation of the Smac mimetic TL32711 (birinapant) and other apoptosis-targeting 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.
  
  
  METHODS
  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 dose-dependent 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.
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland. srivastavaa4@mail.nih.gov.
authors.forename: Apurva K
authors.initials: AK
authors.lastname: Srivastava
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Soumya
authors.initials: S
authors.lastname: Jaganathan
authors.affiliation: Myriad RBM, Austin, Texas.
authors.forename: Laurie
authors.initials: L
authors.lastname: Stephen
authors.affiliation: Biological Testing Branch, Developmental Therapeutics Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Melinda G
authors.initials: MG
authors.lastname: Hollingshead
authors.affiliation: Myriad RBM, Austin, Texas.
authors.forename: Adam
authors.initials: A
authors.lastname: Layhee
authors.affiliation: Myriad RBM, Austin, Texas.
authors.forename: Eric
authors.initials: E
authors.lastname: Damour
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Jeevan Prasaad
authors.initials: JP
authors.lastname: Govindharajulu
authors.affiliation: Myriad RBM, Austin, Texas.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Donohue
authors.affiliation: Protein Expression Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Dominic
authors.initials: D
authors.lastname: Esposito
authors.affiliation: Myriad RBM, Austin, Texas.
authors.forename: James P
authors.initials: JP
authors.lastname: Mapes
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Kinders
authors.affiliation: Investigational Drug Branch, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.
authors.forename: Naoko
authors.initials: N
authors.lastname: Takebe
authors.affiliation: Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Tomaszewski
authors.affiliation: Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.
authors.forename: Shivaani
authors.initials: S
authors.lastname: Kummar
authors.affiliation: Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.
authors.forename: James H
authors.initials: JH
authors.lastname: Doroshow
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Ralph E
authors.initials: RE
authors.lastname: Parchment
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000378:metabolism*; D045744:Cell Line, Tumor; D004151:Dipeptides / Q000494:pharmacology*; D005260:Female; D006801:Humans; D007118:Immunoassay; D007211:Indoles / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry; D008819:Mice, Nude; D024101:Mitochondrial Proteins / Q000737:chemistry; D018716:Molecular Mimicry; D047368:Tumor Burden / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D014408:Biomarkers, Tumor; C412403:DIABLO protein, human; D004151:Dipeptides; D007211:Indoles; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; C582429:birinapant