REVEALING EXPRESSION, POST-TRANSLATIONAL MODIFICATIONS AND PROTEOLYSIS IN CHILDHOOD ACUTE LEUKEMIA USING A NOVEL FLOW CYTOMETRY-BASED METHOD OF AFFINITY PROTEOMICS

ABSSUB-4029

Background: Acute leukemia (AL) is the most common childhood malignancy. It is driven by a number of aberrations detectable at the DNA and mRNA level, but the functional consequences of these alterations at the cellular level are not fully understood. Proteins are the entities that form connection between gene expression and cellular response. Therefore, more effective and sensitive approaches to detect changes in proteome are needed. In the present study we develop and validate new affinity proteomics based tool for analysis of clinical samples.

Aims: Using Size-exclusion Chromatography - Microsphere-based Affinity Proteomics (SEC-MAP) we are able to resolve expression and activation (e.g. phosphorylation) of proteins in AL cells.

Methods: SEC-MAP array is a set of 1728 populations of fluorescently-labeled latex microbeads each carrying an antibody against a human protein. We isolate the cellular proteins from membranes, nuclei and cytoplasm using detergents, label them with biotin and separate them using gel chromatography into 24 fractions. These fractions are incubated with SEC-MAP microbeads and the antibody-protein binding is detected using fluorescently-labeled streptavidin by flow cytometry.

Results: We have compared the data collected by SEC-MAP array in leukemic cell lines (n=11) and healthy peripheral blood B-cells, T-cells and monocytes with classical flow cytometry-based immunophenotyping. Thirty-four markers for leukemia classification correlated qualitatively within both methods. Next, we evaluated leukemia classification markers in bone marrow or peripheral blood from fifty-seven patients with leukemia at diagnosis. We were able to correctly classify all patients’ samples to myelo-, B- and T-cell origin using SEC-MAP technology.

We have further examined the expression of 499 proteins in 69 diagnostic samples of AL. The analysis was performed using in-house automatic software created in R-project. For the normalization of protein expression we have used loess normalization commonly used in mRNA profiling studies. Due to ability of SEC-MAP to separate proteins according to their size we have not only quantified the expression of proteins but also evaluated proteins' size that could serve as a sign of proteolysis. We have detected signs of proteolysis in twelve samples (based on e.g. cleaved PARP1, BLNK and BAD). These samples were therefore excluded from the final analyses. Moreover we have evaluated the sensitivity to proteolysis of four commonly used house-keeping proteins (β-actin, β2-microglobulin, AKT1 and ABL1). ABL1 and AKT1 were cleaved while β-actin and β2-microglobulin were not detected in their cleaved forms in the proteolytically degraded diagnostic samples. Therefore we propose to use ABL1 and AKT1 as house-keeping proteins whenever proteolysis has to be excluded. So far we have identified 44 proteins (e.g. SH2D1A, TCF7, GLUD1, TCF3, CD72) differentially expressed in different subtypes of AL and validated their expression with different methods (e.g. flow cytometry, western blot, real-time quantitative PCR).

Summary/Conclusion: In summary, SEC-MAP is a high-content method of functional proteomics that combines the capacity of DNA microarray and high-throughput evaluation by flow cytometry. It can detect changes in expression, post-translational modification and subcellular localization of hundreds of proteins in relatively small-size sample (only 10 million cells needed).

Acknowledgements
This work was supported by GAUK 596912, IGA NT13462, P302/12/G101, UNCE 204012, 00064203, IGA NT12397.

Keywords: Acute leukemia, Antibody, Childhood, Proteomics

http://www.ehaweb.org/