title: Capillary nano-immunoassay for Akt 1/2/3 and 4EBP1 phosphorylation in acute myeloid leukemia.
abstract: BACKGROUND
  Overall cure rates in acute myeloid leukemia (AML) continue to range between 60-65% with disease relapse being a major cause of mortality. The PI3K-Akt-mTOR kinase pathway plays a vital role in pro-survival signals within leukemic cells and inhibition of this pathway is being investigated to improve patient outcomes. Tracking activation of multiple signaling proteins simultaneously in patient samples can be challenging especially with limiting cell numbers within rare sub-populations.
  
  
  METHODS
  The NanoPro 1000 system (ProteinSimple) is built on an automated, capillary-based immunoassay platform and enables a rapid and quantitative analysis of specific proteins and their phosphorylation states. We have utilized this nano-immunoassay to examine activation of Akt 1/2/3 and downstream mTOR target--eukaryotic initiation factor 4E-Binding Protein 1 (4EBP1).
  
  
  RESULTS
  Assays for Akt 1/2/3 and 4EBP1 were standardized using AML cell lines (MV4-11, MOLM-14, OCI-AML3 and HL-60) prior to testing in patient samples. Target inhibition was studied using mTOR 1/2 inhibitor AZD-8055 and results were corroborated by Western blotting. The assay was able to quantify nanogram amounts of 4EBP1 and Akt 1/2/3 in AML cell lines and primary pediatric AML samples and results were quantifiable, consistent and reproducible.
  
  
  CONCLUSIONS
  Our data provides a strong basis for testing this platform on a larger scale and our long term aim is to utilize this nano-immunoassay prospectively in de-novo AML to be able to identify poor responders who might benefit from early introduction of targeted therapy.
authors.forename: Himalee
authors.initials: H
authors.lastname: Sabnis
authors.forename: Heath L
authors.initials: HL
authors.lastname: Bradley
authors.forename: Silvia T
authors.initials: ST
authors.lastname: Bunting
authors.forename: Todd M
authors.initials: TM
authors.lastname: Cooper
authors.affiliation: Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Emory University, 1760 Haygood Drive NE, Atlanta, Georgia, USA. kevin.bunting@emory.edu.
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Bunting
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D018797:Cell Cycle Proteins; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D015470:Leukemia, Myeloid, Acute / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; D010750:Phosphoproteins; D011494:Protein Kinases