Molecular Landscaping of Exosomal Protein and miRNA Cargo in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by the accumulation and expansion of immature myeloid cells in the bone marrow and peripheral blood. Although genomic and epigenomic screenings have increased our understanding of leukemogenesis, there are multiple factors that cumulatively result in relapse and low therapeutic response. It is known that both soluble factors and direct cell-cell interactions are critical to leukemia cell survival. However, we have a limited understanding of this bidirectional interaction and the components that facilitate or drive leukemia cell survival. Recently, it has been shown that cells communicate continuously among themselves and with their neighboring cells through the release of small nanovesicles called exosomes. Exosomes have an average diameter of ~100 nanometers and carry molecular signals such as miRNAs, long non-coding RNA (lncRNAs), mRNAs, proteins, and peptides. Based on their cargo and surface phenotype, exosomes have great potential of being used as early diagnostic markers or therapeutic targets for different malignancies, especially cancer.

Here, we conducted a comprehensive analysis of the protein and miRNA profiles within the exosomes from AML samples. The exosomes were isolated using the size exclusion chromatography method from plasma samples collected from 20 patients with AML and healthy donors. Isolated exosomes were characterized by nanoparticle tracking analysis and transmission electron microscopy. We performed proteomics and genomics analyses to identify specific biomarkers. We identified the expression levels of exosomal proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) based on label-free quantification, and miRNA targets were identified by small RNA library preparation and sequencing. Multiple bioinformatics packages were used to analyze proteomic and miRNA data to identify significant proteins and miRNA profiles. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to predict the biological functions and potential mechanisms of differentially expressed proteins and miRNAs in AML exosomes. Additionally, we explored the protein-miRNA interaction network to evaluate the functional role of these networks in AML. We also identified important, mutated proteins from our data to predict liquid biopsy-based neoantigens for AML.

Isolated exosomes were in the size range of 60-80 nm with a concentration of approximately 10¹¹ particles/mL. The result disclosed that the exosomal proteins of the AML samples had a significantly different expression profile compared to healthy individuals. We demonstrated that 3194 proteins were expressed in exosomes with the major abundance of transforming growth factor (TGF-β), complement system, integrin subunit (ITG), and heat shock proteins (Hsps) that differed between exosomes derived from patients and healthy individuals. These proteins showed significantly higher sensitivity, specificity, and association with disease progression, recurrence, and chemoresistance in AML. Interestingly, higher expression of these proteins showed a good correlation with poor survival in AML patients. Besides, we found that these proteins were highly enriched in intercellular pathways. Molecular function analysis revealed that the proteins related to protein binding, transcription regulator activity, and DNA-binding transcription factor activity were highly enriched (P < 0.05). Most importantly, we found significantly mutated proteins such as mucin 1 (MUC1), isocitrate dehydrogenase 1 (IDH1), apolipoprotein B (APOB), and atypical cadherin 4 (FAT4). Our data provide a proof of concept for future studies that evaluate liquid biopsy-based neoantigens for AML. Our findings propose the potential of these exosomal proteins as diagnostic and prognostic biomarkers for AML patients. Hopefully, our data will provide valuable information on relapse and treatment failure in AML and its correlation with exosomal biomarkers. We are concluding the miRNA sequencing and analysis for isolated exosomes, and we will perform correlative analysis to present in the meeting. Additional in-depth studies are going on in our lab to explain the notable biological functions of the exosomal contents in the microenvironmental crosstalk of AML cancer.

No relevant conflicts of interest to declare.