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Author Notes:

Correspondence: Kevin D Bunting, Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Emory University, 1760 Haygood Drive NE, Atlanta, Georgia; Email: kevin.bunting@emory.edu

Authors' Contributions: HSS and HLB performed the experiments.

HSS, HLB, STB, TMC, and KDB analyzed the data.

HSS and KDB drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgments: All pediatric AML samples were obtained from Children’s Oncology Group – Myeloid Disease Laboratory.

The authors would also like to thank Deborah Pritchett (ProteinSimple) for her invaluable technical support in using the NanoPro 1000 platform.

Disclosures: The authors declare that they have no competing interests.

Subjects:

Research Funding:

This work was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR000454 and ACTSI KL2 Award number TR000455 (H.S. Sabnis).

The work was also supported by the Aflac Cancer and Blood Disorders Center (K.D. Bunting), Cure Childhood Cancer Foundation (K.D. Bunting) and Children’s Healthcare of Atlanta Friends Research Fund (S.T. Bunting).

Keywords:

  • Nano-immunoassay
  • Biomarker
  • Leukemia
  • Capillary electrophoresis
  • mTOR

Capillary nano-immunoassay for Akt 1/2/3 and 4EBP1 phosphorylation in acute myeloid leukemia

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Journal Title:

Journal of Translational Medicine

Volume:

Volume 12, Number 166

Publisher:

Type of Work:

Article | Final Publisher PDF

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. Conclusion 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.

Copyright information:

© 2014 Sabnis et al.; licensee BioMed Central Ltd.

This is an Open Access work distributed under the terms of the Creative Commons Universal : Public Domain Dedication License (http://creativecommons.org/publicdomain/zero/1.0/).

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