Plasma S-Evs Mirnas in Pediatric B Lineage Leukemia: Transforming Factors of Bone Marrow Niche

Abstract

BACKGROUND Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and mature B-cell leukemia are characterized by dismal prognosis in case of relapse. Exosomes are crucial small extracellular vesicles (S-EVs) used by cells for intercellular communication, capable to modulate recipient cells through the release of small biological molecules (DNA, RNA, proteins). Tumor derived exosomes play an important role in leukemogenesis, disease progression, and organ invasion, due to their ability to induce molecular and functional changes within the bone marrow microenvironment (BME). Since little is known behind the mechanism used by leukemic cells to sustain tumor progression through exosomes release, this has become an exciting area to be investigated. OBJECTIVES This study aimed to provide novel insight about S-EVs-mediated small-RNA (sRNA) transfer within tumor niche and clarify how the crosstalk between tumor cells and cells from the microenvironment promote leukemia cells survival and progression. The characterization of leukemic S-EVs cargo led to the identification of new potential biomarkers of disease progression and treatments efficacy. DESIGN/METHODS RNA from plasma S-EVs samples of 17 mature B-cell leukemia, 16 BCP-ALL at diagnosis and 8 healthy donors (HD) were isolated and small-RNA sequencing on Illumina platform was performed; data were analyzed by miR&moRe2 pipeline. MiRNAs levels were quantified by qRT-PCR in an extended cohort including HDs and patients both at diagnosis and during treatment. Mesenchymal stroma cells (MSCs) derived from healthy BM were co-cultured with tumor cell lines by using transwell. MSCs were also directly treated with S-EVs isolated from mature B-cell leukemia and BCP-ALL cell lines. S-EVs exchange was detected by confocal microscopy. Cell viability, migration and adipocyte differentiation assays were then assessed. Finally, pharmacological treatment of tumor cells co-cultured or not with adipocytes was administered. RESULTS Analysis of the profile of sRNAs contained in plasma exosomes according to sRNA-sequencing showed a peculiar miRNA cargo in both BCP-ALL and mature B cell leukemia groups compared to HD, and revealed 15 and 40 sRNAs significantly deregulated, respectively. We prioritized and validated the upregulation of 6 miRNAs (3 in BCP-ALL and 3 in mature B-cell leukemia) in an enlarged cohort patients' S-EVs obtained at diagnosis and in reference cell lines. Of note, the expression of these miRNAs during follow-up significantly decreases, suggesting their tumor origin and corroborating their usefulness as tumor biomarkers. To sustain this hypothesis sRNA-seq data were integrated with gene expression profiles obtained from leukemia blasts, underlining the tumor origin of these miRNAs. The prediction analysis of the miRNAs-target genes highlighted the alteration of specific networks related to cell survival, migration and cell differentiation. Furthermore, we demonstrated the communication via exosome release between leukemic cells and the surrounding cells and how S-EVs play a crucial role in the modulation of BME components. In particular, treatment with tumor derived S-EVs increases MSCs migration ability and prompts their adipogenic differentiation. These results were also confirmed by prioritized miRNAs transfection in MSCs. Co-culture of leukemic cells with adipocytes demonstrated their ability to protect BCP-ALL and mature B-cell leukemia cell lines from chemotherapy: leukemia cells showed a decrease in cell proliferation and a reduction of apoptosis. In line, poor responders BCP-ALL patients showed at diagnosis a positive enrichment in their transcriptome of mechanisms related to adipogenesis and lipids beta-oxidations. CONCLUSIONS Our data demonstrated that leukemic cells and BM niche cells efficiently communicate between each other through S-EVs release and uptake, inducing MSCs rewiring, with enhanced migratory and differentiative properties in vitro. We noted a strong enrichment of common pathways stroked by all the miRNA validated and related to lipid metabolism and sustenance of adipogenesis. Our study demonstrated that the bone marrow modulation is due to leukemic miRNAs shuttled by S-EVs in MSC cells and that the BME rewiring prompt leukemia chemoresistance.