- Open Access
Capillary nano-immunoassay for Akt 1/2/3 and 4EBP1 phosphorylation in acute myeloid leukemia
© Sabnis et al.; licensee BioMed Central Ltd. 2014
- Received: 16 January 2014
- Accepted: 14 May 2014
- Published: 12 June 2014
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.
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).
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.
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.
- Capillary electrophoresis
Acute myeloid leukemia (AML) affects 16,000 -18,000 people annually in the United States and approximately 75% will succumb to the illness . 6% of all patients affected are under the age of 20 years . In spite of the advances made in the treatment of acute myeloid leukemia with chemotherapy as well as hematopoietic stem cell transplantation, overall cure rates remain at 60-65% with relapse being a major cause of mortality . Of those relapsed patients, only a third are salvageable with current treatment regimens [3, 4]. Discovery of both cytogenetic and molecular abnormalities in AML has resulted in the development of the current prognostic sub-groups in AML  and the molecular abnormalities play an important role in leukemogenesis, especially in patients with normal cytogenetics .
Downstream of these molecular aberrations in leukemic cells, highly complex and inter-linked networks of signaling pathways control cell survival growth, proliferation, self renewal and differentiation. Up-regulation of the PI3K-Akt-mTOR (PI3K-Akt-mammalian target of rapamycin) pathway occurs via mutations in surface receptors like FLT3, c-Kit or by mutations in the genes encoding pathway constituents like PI3K, PTEN or Akt [7, 8] . Akt is a serine/threonine protein kinase that exists in three conserved isoforms: Akt 1, 2 and 3. Of the three iso-forms present, Akt 1 and 2 are expressed to a higher extent in hematopoietic stem cells . Akt is phosphorylated at Thr 308 by up-stream phosphoinositide-dependent protein kinase 1 (PDK-1) and at Ser 473 by mTOR complex 2 (mTORC2). Akt plays an important role in key cellular processes such as protein translation, cell proliferation, cell cycle, and apoptosis through its multiple downstream targets however activating mutations in Akt have not been described in AML  . Akt can be constitutively phosphorylated in AML which results in depletion of normal hematopoietic stem cells .
Activation of the mTOR pathway is seen in up to 80% of AML patients and is associated with a shortened overall survival. mTOR kinase is also a serine/threonine kinase that complexes with other proteins . mTORC2 mainly functions to phosphorylate and activate Akt whereas mTORC1 plays a central role in the translational machinery of normal and leukemic cells via its downstream targets - p70S6 Kinase and eukaryotic initiation factor (eIF) 4E binding protein-1 (4EBP1) [12, 13]. p70S6 Kinase phosphorylates the 40S ribosomal subunit protein S6 and thereby allows translation of proteins involved in cell growth and hypertrophy. 4EBP1 phosphorylation results in release of the inhibition of eIF4E and enables the formation of eIF4F complex. This complex is necessary for the cap-dependent translation of highly structured mRNAs which encode genes such as c-Myc, Mcl-1 and VEGF that are involved in cell survival . In certain subtypes of AML (FAB M4/M5) eIF4E itself has been shown to function as an oncogene via transcriptional up-regulation by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) . Both p70S6 Kinase and 4EBP1 are downstream targets of mTOR however, inhibition of 4EBP1 phosphorylation is key for ensuring efficacy of mTOR antagonists . Thus inhibiting downstream mTOR targets has played a prominent role in anti-leukemic therapy for several years and continues to be an active area of research .
Molecular differences in Akt-mTOR pathway with AML patients may provide key information to better define the pathogenesis of disease, especially in patients with normal cytogenetics. Traditionally, techniques such as Western Blot and intra-cellular flow cytometry have been used for this purpose but these have several limitations - they require large number of cells, require technical expertise and quantitative results are difficult to obtain. The NanoPro 1000 system (ProteinSimple) enables a rapid and quantitative analysis of specific proteins from small quantities of sample (dependent on cell size and percentage of protein). The NanoPro provides precise and quantitative data of the multi-site phosphorylation states of a specific protein of interest. This degree of phospho-protein specificity and rapid turn-around time are unique. The system is built on an automated, capillary based immunoassay platform. Proteins are separated by isoelectric focusing separation (PI) to resolve the various modification states of proteins, immobilized, and probed with specific antibodies. Signal intensity is detected by a horseradish peroxidase-conjugated chemiluminescence system and the data is shown as an electropherogram. It can examine numerous proteins within few cells using the same antibodies used in traditional Western blots. This feature of the NanoPro 1000 makes it a unique tool to look at signaling within rare leukemic cell populations as it requires very small amounts (as low as 80 ng) of protein per capillary. It is also capable of simultaneously detecting multi-phosphorylation states of the same protein which is impossible to do by Western Blot or intracellular flow cytometry. We used the NanoPro 1000 platform on AML cell lines to standardize the assays for 4EBP1 and Akt 1/2/3.
MV4-11, MOLM-14, OCI-AML3 and HL-60 cell lines were used for this analysis. Cell lines were grown in RPMI (MOLM-14), alpha-MEM (OCI-AML3) or IMDM (MV4-11 and HL-60) and supplemented with 10% or 20% fetal bovine serum with 1% penicillin-streptomycin. Cell density was maintained at 0.1 million cells/ml and cultures were split every 48 hours maintaining cell viability. Cells were harvested when confluent at 48 hours for analysis of baseline signal using the NanoPro 1000 assay as outlined below. Cell lines were treated for 24 hours in the presence of specific inhibitors prior to analysis.
Primary patient samples
Primary AML bone marrow samples were obtained from the Children’s Oncology Group (COG) Myeloid Diseases Reference Laboratory on pilot study protocol AAML12B13. The study was approved by Emory Institutional Review Board and the COG Myeloid Diseases Committee. These bone marrow samples were obtained from pediatric AML patients after written informed consent at the time of diagnosis and were de-identified prior to storage. Samples were ficolled to isolate mono-nuclear cells and frozen with 10% DMSO. Information provided to the investigators included age, gender, sample mono-nuclear cell count and FLT3 mutation status. Samples were thawed at 37°C and placed in RPMI media supplemented with 20% fetal bovine serum and 100 ng/ml cytokines (IL-3, G-CSF, GM-CSF, SCF – Gemini Bio Products Cat. #300-151P, #300-123P, #300-124P and #300-185P respectively) for 24 hour drug effects. For baseline analysis, samples were analyzed immediately after thawing.
AZD-8055 was obtained from Chemietek Biochemicals (Cat. No. CT-A8055). It was dissolved in DMSO and stored at -20 C. Both cell lines and primary AML samples were treated with AZD-8055 with concentrations ranging from 25–1000 nM for varying times to demonstrate target inhibition.
MV4-11 cells (5×106 cells) were treated for 1 hour with AZD-8055 at concentrations ranging from 25–1000 nM and with vehicle DMSO. Cell pellets were lysed in 150 μl Bicine Chaps lysis buffer (containing protease and phosphatase inhibitor cocktail made as per Protein Simple specifications). Protein concentrations were determined by Bio-Rad protein assay. Proteins were separated using SDS-polyacrylamide gels, transferred to polyvinylidene diflouride membranes (EMD Millipore) and blocked in 5% non-fat dry milk. Primary antibody incubations were performed overnight at 4°C, followed by incubation in secondary horseradish peroxidase-linked anti-rabbit or anti-mouse secondary antibody at room temperature for 1 hour. Primary antibodies used were total 4EBP1 (Cell Signal Cat #9644 s), phospho-serine 65 4EBP1(Cell Signal Cat #9451 s), phospho-threonine 37/46 4EBP1 (Cell Signal Cat #2855), total Akt 1/2/3 (Santa Cruz Cat #sc-8312), β-2 microglobulin (Abcam Cat #ab75853) and β-actin (Sigma Cat #A5441). These were used at a concentration of 1:1000 except for β-actin (conc. 1:10,000) and secondary antibodies (Anti-mouse Cell Signal Cat #7076S, Anti-rabbit Cell Signal Cat #7074S) were used at a concentration of 1:2000.
All isoelectric separations were performed on the NanoPro 1000 (ProteinSimple, Santa Clara, CA) by mixing 1 part lysate with 3 parts of ProteinSimple’s Generation 2 pH 5–8 (nested) separation gradient which contains a pH 2–4 plug (Cat #040–972). Standard pI Ladder 3 (ProteinSimple Cat #040–646) supplemented with individual pI Standard 5.5 (ProteinSimple Cat #040–028) diluted 60 fold was added to the ampholyte pre-mix. Lysates were then separated for 40 min at 21,000 μW in individual capillaries. After separation the proteins in the lysate were immobilized to the capillary wall by subjecting them to UV exposure for a period of 80 seconds. After two washes of 150 seconds each, primary antibodies were introduced into the capillaries for a period of 2 hours. Antibodies for 4EBP-1 were used at a 1:25 dilution, whereas antibodies for AKT 1/2/3 and β-2 Microglobulin were used at 1:100 dilutions. After another two washes of 150 seconds each, samples were run either with or without amplification reagents. Secondary anti-rabbit-HRP-conjugated antibodies (ProteinSimple Cat #040–656) or secondary anti-rabbit-biotin-conjugated antibodies (ProteinSimple’s amplified rabbit secondary antibody kit - Cat #041–126) were loaded into the capillary for 1 hour. Amplification was performed only for 4EBP1 antibodies using primary patient samples and AML cell lines. After a third set of two washes of 150 seconds each, either streptavidin, conjugated with horse radish peroxidase (ProteinSimple Cat #041–126), or antibody diluent was loaded into the capillary for 2 hours or 10 minutes respectively. After a final two wash cycle of 150 seconds each, a luminol-peroxidase 1:1 mix (ProteinSimple Cat #040–0652 and 040–684) was flowed through the capillaries and chemiluminescence was detected at 30, 60, 120, 240, 480, and 960 seconds. Primary 4EBP1 antibodies used were similar to those used for Western blotting and in addition rabbit polyclonal total Akt1/2/3 (Santa Cruz Cat #sc-8312) was used for the assay. To determine phospho-peaks, sample lysates were pre-treated with 100 U lambda phosphatase or vehicle according to the manufacturer’s instructions (Millipore, Cat #14-405). Lysates were incubated in 1× DTT-containing lambda phosphatase buffer for 1 hour at 37°C before running on the NanoPro.
All data was derived as a result of three independent experiments, unless stated otherwise. Two tailed t-test was used to calculate p-values and values less than 0.05 were considered to be significant.
The NanoPro 1000 platform can be used to measure 4EBP1 phosphorylation within AML cell lines and to demonstrate target inhibition
Total Akt 1/2/3 antibody can be used to measure total and phosphorylated forms using nano-immunoassay in AML cell lines
Nano-immunoassay provides a reliable and sensitive measurement of 4EBP1 and Akt activation in primary patient samples
Acute myeloid leukemia continues to be a therapeutic challenge with low overall survival rates and high incidence of relapse. Development of improved biological correlates that define the disease may allow for risk stratification of patients that can be incorporated into current risk strata based on cytogenetic and molecular abnormalities. Defining the activation status of the 4EBP1 and Akt 1/2/3 proteins can serve as an important indicator of signal transduction in AML and can potentially provide information of prognostic significance. Using the nano-immunoassay platform we were able to standardize assays for both 4EBP1 and Akt 1/2/3 in AML cell lines.
Traditionally, Western blotting and intra-cellular flow cytometry have been used to study individual protein activation. However both of these techniques need a large number of cells, are unable to look at multiple sites in the same protein in the same assay, and in the case of flow cytometry require significant operator expertise. The NanoPro assays have the advantage of being automated and are amenable to clinical translation due to the rapid turn-around time and the routine methods for data acquisition. While not capable of providing the depth of information on single cells that can be achieved from flow cytometry analysis, the nano-immunoassay platform can be combined with flow cytometry sorting to characterize rare AML sub-populations. Enrichment of whole AML bone marrow or peripheral blood for important cell subsets such as CD34+CD38- populations can be achieved by flow cytometry and subsequently these cells can be lysed and analyzed using the NanoPro platform. Multi-color flow cytometry confers the advantage of being able to study different phospho-proteins in gated populations but can also be limited by the number of phospho-proteins studied simultaneously (typically <12) and can be technically challenging. The NanoPro technology supplements flow cytometry with the ability to quantify phosphorylation patterns and examine other post-translational modifications such as acetylation and methylation. Since the separation of the protein is based on iso-electric pH, this platform can use total protein antibodies to determine multi-phosphorylation events as demonstrated here for 4EBP1 and Akt1/2/3 since heavily phosphorylated proteins tend to have lower iso-electric pH.
More recently, Reverse Phase Protein Array (RPPA) analysis has been used to study the effects of protein expression and modification in tumor samples . RPPA is a high-throughput antibody based technique capable of screening a large number of antibodies in a single assay however our nano-immunoassay has certain advantages. Detection of multiple phosphorylation states of a protein using RPPA requires the use of multiple phospho-specific antibodies while in case of the nano-immunoassay, a single total antibody (eg. Akt 1/2/3) can provide accurate information about multiple phosphorylation sites. RPPA also has a much longer turn-around time of several weeks whereas the NanoPro can provide results within 24 hours making it a viable option for real-time analysis of patient samples.
Considering the above mentioned advantages of the NanoPro system, we standardized the assays for 4EBP1 and Akt 1/2/3. Depending on the antibody and the degree of expression, we were able to detect a signal for most antibodies with as low as 80 ng of protein per capillary and in certain cases (total 4EBP1) even as low as 20 ng per capillary. The assays were robust with a short turn-around time of 24 hours providing results that were quantitative and easy to interpret. We further tested these assays in primary bone marrow samples from pediatric AML patients and found the results to be consistent and reproducible. Primary cells are smaller than cell lines but we were able to reliably detect signal in as low as 40–96 ng of protein. Although the amount of protein needed for performing these assays per capillary is low, there are certain limiting factors that can affect the sample preparation that need consideration. Firstly, though cell counts for AML blasts might be high in samples, these cells are still fairly small in size as compared to AML cell lines. The recommended amount of protein per capillary is 80 ng which corresponds roughly to 2500 primary AML cells and ranged from 900–1400 cells from cultured AML cell lines. Secondly, in order to achieve these numbers, we had to lyse at least 1.6 million primary cells in lysis buffer to be able to obtain adequate signal for each antibody. Therefore, future studies that focus on leukemia stem cells or minimal residual disease would require incorporation of methods for concentrating protein as part of the sample preparation prior to running on the NanoPro.
Both 4EBP1 and Akt are proteins that are phosphorylated on multiple sites. Using the nano-immunoassay we were able to distinguish these multiple phosphorylated forms in cell lines and primary AML samples. We tested both AML cell lines and primary AML samples with AZD-8055 mTOR 1/2 inhibitor and found that the drug was effective at inhibiting 4EBP1 phosphorylation thus making this technology useful to determine specific target inhibition. We are also currently working on developing assays that cover the entire PI3K-Akt-mTOR pathway. Assays for PI3K activation or p70S6K activation have been problematic and require additional development. Therefore, it is possible that our assay could miss some p70S6K activation through the ERK signaling pathway. We could therefore combine the analysis of Akt 1/2/3 and 4EBP1 phosphorylation with analysis of pERK in future studies. The assay for pERK is well described by several groups in non-hematologic cancers  but thus far has not worked on AML samples in our laboratory.
Since this was a pilot study utilizing a small number of samples to highlight an emerging new technology, we were unable to make any relevant prognostic conclusions correlating signal strength to overall survival/relapse rates. Our future studies will involve studying larger numbers of patient samples with correlative outcome data as well as comparison of samples at diagnosis and relapse to determine changes in protein activation. The utility of our overall approach to study signal activation is broad and could apply not only to leukemia but also to other cancers where tumor samples might be limiting in number.
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. 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). 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.
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