title: Single-cell pharmacodynamic monitoring of S6 ribosomal protein phosphorylation in AML blasts during a clinical trial combining the mTOR inhibitor sirolimus and intensive chemotherapy.
abstract: OBJECTIVE
  Integration of signal transduction inhibitors into chemotherapy regimens generally has generally not led to anticipated increases in response and survival. However, it remains unclear whether this is because of inadequate or inconsistent inhibition of target or other complex biology. The mTOR signaling pathway is frequently activated in acute myelogenous leukemia (AML) and we previously showed the safety of combining the mTOR inhibitor, sirolimus, with mitoxantrone, etoposide, and cytarabine (MEC) chemotherapy. However, we did not reliably determine the extent of mTOR inhibition on that study. Here, we sought to develop an assay that allowed us to serially quantify the activation state of mTOR kinase during therapy.
  
  
  METHODS
  To provide evidence of mTOR kinase activation and inhibition, we applied a validated whole blood fixation/permeabilization technique for flow cytometry to serially monitor S6 ribosomal protein (S6) phosphorylation in immunophenotypically identified AML blasts.
  
  
  RESULTS
  With this approach, we show activation of mTOR signaling in 8 of 10 subjects' samples (80%) and conclusively show inhibition of mTOR in the majority of subjects' tumor cell during therapy. Of note, S6 phosphorylation in AML blasts is heterogeneous and, in some cases, intrinsically resistant to rapamycin at clinically achieved concentrations.
  
  
  CONCLUSIONS
  The methodology described is rapid and reproducible. We show the feasibility of real-time, direct pharmacodynamic monitoring by flow cytometry during clinical trials combining intensive chemotherapy and signal transduction inhibitors. This approach greatly clarifies pharmacokinetic/pharmacodynamic relationships and has broad application to preclinical and clinical testing of drugs whose direct or downstream effects disrupt PI3K/AKT/mTOR signaling.
authors.affiliation: Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA. alexander.perl@uphs.upenn.edu
authors.forename: Alexander E
authors.initials: AE
authors.lastname: Perl
authors.forename: Margaret T
authors.initials: MT
authors.lastname: Kasner
authors.forename: Doris
authors.initials: D
authors.lastname: Shank
authors.forename: Selina M
authors.initials: SM
authors.lastname: Luger
authors.forename: Martin
authors.initials: M
authors.lastname: Carroll
mesh_terms: D000328:Adult; D000368:Aged; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D003561:Cytarabine / Q000627:therapeutic use; D004334:Drug Administration Schedule; D005047:Etoposide / Q000627:therapeutic use; D005260:Female; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D008942:Mitoxantrone / Q000627:therapeutic use; D010766:Phosphorylation; D010865:Pilot Projects; D038601:Ribosomal Protein S6 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000627:therapeutic use*; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors*
chemical_list: D038601:Ribosomal Protein S6; D003561:Cytarabine; D005047:Etoposide; D008942:Mitoxantrone; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus