Abstract C200: Rapid nano-immunoassay for evaluation of mTOR pathway activation in acute myeloid leukemia.
Authors: Sabnis Himalee , Bradley Heath , Cooper Todd , Bunting Kevin 
Published: August 2014

Overall cure rates in acute myeloid leukemia (AML) continue to range between 60-65% with disease relapse in AML being a major cause of mortality. Risk stratification by cytogenetic and molecular aberrations has changed the prognosis for some patients, but for a majority of the patients (especially with normal cytogenetics) improvement in therapy is needed. Understanding key differences in survival pathways such as the mTOR pathway within leukemic cells could be potentially exploited to develop new drug therapy and design relapse therapy. Tracking pathway activation in patient samples is challenging in AML since samples are difficult to obtain, contain fewer cells, and are heterogeneous in nature. 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 used this novel nano-technology based system to study the mTOR pathway in AML samples (n=8) obtained from the Children's Oncology Group (COG) Myeloid Disease Reference Laboratory. Assays for downstream mTOR target protein-eukaryotic initiation factor 4E binding protein 1 (4EBP1) were standardized using AML cell lines (MV4-11, MOLM-14, OCI-AML3 and HL-60) prior to testing in patient samples. The assay was able to detect as little as 80 ng of protein which corresponded to 1000 cells per assay. MV4-11 cells were treated with the dual mTOR 1/2 inhibitor AZD-8055 for 24 hours and reduced phosphorylation of 4EBP1 was confirmed by Western Blotting and corroborated by the NanoPro assay. This nano-immunoassay was able to reliably quantify amounts of 4EBP1 (total and phosphorylated forms) in primary pediatric AML samples (n=8) thawed following cryopreservation. The relative luminescence units (measure of chemiluminescence and protein signal) were plotted against exposure time and the linearity of the graph provided evidence that the signal is sensitive over a range of three logs. Further, these samples were treated with AZD-8055 for 24 hours followed by analysis of changes in signaling profiles for total and phosphorylated Ser65 and Thr37/46. As expected, complete reduction of phosphorylation was noted in the treated samples (n=4). The assays obtained were quantifiable, consistent, and reproducible and enabled us to delineate activation patterns of the mTOR pathway in primary AML samples and evaluate drug efficacy for targeted agents. Our data provides a strong basis for testing this platform on a larger scale and our long term aim is to utilize this novel nano-immunoassay based platform prospectively in de-novo AML to be able to identify poor responders who might benefit from early introduction of targeted therapy.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C200.

Citation Format: Himalee Sabnis, Heath Bradley, Todd Cooper, Kevin Bunting. Rapid nano-immunoassay for evaluation of mTOR pathway activation in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C200.