Activin-a Increases B-Cell Acute Lymphoblastic Leukemia (B-ALL) Vesiculation and Modifies the Micro-RNA Cargo: Special Focus on MiR-491-5p

Abstract

We recently demonstrated that ActivinA (ActA) is abundant in the leukemic bone marrow microenvironment where it exerts a pro-leukemic action by increasing the migratory and invasive properties of B-ALL cells. Extracellular vesicles (EV) are a new way of intercellular communication. They can be categorized in small-EV (sEV) and large-EV (lEV) based on size and origin. Similarly to migration, EV release is dependent upon cytoskeleton activation and membrane remodeling. EV play a prominent role in carcinogenesis, depending on the molecules they enclose, including miRNA that can downmodulate the expression of target mRNAs in recipient cells. The aim of the project was to study the impact of ActA on B-ALL vesiculation: quantity and miRNA cargo. Moreover, we investigated whether deregulated miRNAs could contribute to ActA-mediated leukemic progression. To investigate the role of ActA on B-ALL vesiculation, we used 697 and Nalm-6 cells lines. By means of nanoparticle tracking analysis, we demonstrated that after 24h, ActA significantly increased of 2.5 and 1.4 folds the number of sEV (p<0.0001) and lEV (p<0.0026, n=9) produced by 697 cells, compared to not stimulated cells (NS). A significant increase of both EV was also observed after 48h of ActA stimulation. The ability of ActA to promote B-ALL cell vesiculation was also confirmed on Nalm-6. Furthermore, we investigated the miRNA cargo of EV derived from 697 cells stimulated or not with ActA by OpenArray. After 24h, ActA significantly upregulated miR491-5p, let-7i-3p, and downregulated miR-639, miR135b-3p and miR139-5p. On the other hand, after 48h, ActA significantly upregulated miR15b-3p, let-7-e-5p, miR23b-3p and downregulated miR1236-3p, miR-18a-3p. We focused on miR491-5p, the most upregulated in ActA EV (3 folds at 24h), compared to NS (p<0.0001). ActA-mediated increase of miR491-5p was observed also in 697 cells (FC=1.5, p<0.0001, n=14). To investigate miR491-5p modulation also in Nalm-6 cells, they were stimulated or not for 24h and miRNA levels were evaluated in cells and their EV, isolated by ultracentrifugation. We preliminary observed that ActA increased miRNA expression of 479 folds in Nalm-6 EV (p=0.09, n=3), but only slightly in cells. Interestingly, absolute miRNA levels were higher of 4 folds in NS EV compared to NS cells (p=0.05), and showed an increasing trend in stimulated condition (FC=1552, p=0.09, n=3), suggesting that upon ActA stimulation, miR491-5p is upregulated and mostly encapsulated in EV. It has been described that miR491-5p promotes cell survival and proliferation in solid tumors. Indeed, we investigated whether ActA could impact on B-ALL cell viability through miR491-5p modulation. Indeed, 697 cells were stimulated every 3 days with ActA. Cell counts showed no differences between cells stimulated or not in the exponential growth phase. Interestingly, at plateau (day 7), ActA-stimulated cells were more viable (76%, range: 69%-80%) compared to NS cells (56%, range: 50%-66%, p=0.05, n=3). Alongside, we preliminary observed higher miR491-5p in ActA-stimulated cells (FC=3, n=1). It has also been reported that miR491-5p regulates chemoresistance in several cancers. Interestingly, by using Annexin-V/7-AAD assay, we demonstrated that ActA-stimulated 697 cells had a viability advantage of 11.3% (p=0.006, n=3), compared to NS cells, after 72h of ASNase-based chemotherapy, suggesting a chemoprotective role of ActA in B-ALL. Moreover, we observed that the combination of ActA and ASNase treatments induced the overexpression of miR491-5p compared to NS (FC=2.5, p=0.001) and to cells stimulated with ActA alone (FC=2.3, p=0.04) or ASNase alone (FC=1.4, p=0.004, n=8). To investigate the possible role of miR491-5p, we downregulated it into 697 cells by transfecting a miR491-5p inhibitor. Interestingly, under ASNase treatment, ActA still induced a viability advantage in cell transfected with a random inhibitor (negative control, 9.8%, p=0.006), comparable to untransfected cells. On the contrary, in miR491-5p inhibitor transfected cells, the viability advantage mediated by ActA decreased to 5.9% (p=0.03). Therefore, miR491-5p inhibition reduced of 30% the chemoprotective ability of ActA (p=0.04, n=3). In conclusion, these results suggest that ActA not only increases EV production, but also modifies their miRNA cargo and that miR491-5p could be one of the key players involved in ActA-mediated pro-leukemic action.