Pb1702: activina modulates the microrna cargo of extracellular vesicles released by b-cell precursor acute lymphoblastic leukemia (bcp-all) cells

Topic: 1. Acute lymphoblastic leukemia - Biology & Translational Research Background: ActivinA (ActA) is an important regulator of carcinogenesis and it is involved in chemoresistance in several solid tumors. We recently demonstrated that ActA is able to exert a pro-leukemic action by increasing the migratory and invasive properties of leukemic cells. Similarly to cell migration, Extracellular Vesicles (EV) release is dependent upon cytoskeleton activation and membrane remodelling. EV play a prominent role in cancer pathogenesis and therapy resistance, depending on the functional molecules they enclose. Among them, microRNA (miRNA) can modulate the expression of target mRNAs in recipient cells Aims: We aimed to study the effect of ActA on BCP-ALL cell vesiculation and its impact on microRNA cargo Methods: EV were isolated by ultracentrifugation from the 697 BCP-ALL cell line and characterized bynanoparticles tracking analysis. EV-miRNA cargo was screened by OpenArray technology and miR-491-5p levels were investigated by qRT-PCR. 697 cell viability was evaluated by Annexin-V/7-AAD assay. Specific miR-491-5p inhibitor/mimic were transfected in 697 cells, by means of lipofectamine, to downregulate/upregulate miR-491-5p expression. miR-491-5p biological function and targets were predicted by miR-System and miR-Walk databases Results: ActA significantly increased, after 24h of stimulation, the quantity of both small-EV and large-EV released by 697 cells, compared to unstimulated cells (NS) (Fold Change (FC)=2, p<0.0001 and 1.3,p=0.003, respectively,n=16). Surprisingly, we discovered that ActA strongly impacted on EV miRNA cargo. In particular, we focused on miR-491-5p that was upregulated in EV of 3 folds (p<0.0001) and of 1.5 folds at the intracellular level after 24h(p=0.0004). Interestingly, literature data suggest that miR-491-5p could play a key role in modulating chemoresistance in several cancers. To investigate the potential involvement of the ActA/ miR-491-5p axis in chemoprotection in the context of BCP-ALL, we performed experiments in which 697 cells were stimulated or not with ActA and then treated with Asparaginase (ActA+ASNase versus NS+ASNase), a drug commonly used as treatment of paediatric BCP-ALL. Surprisingly, ActA significantly increased the viability of ASNase-treated 697 cells of 11,3% compared to NS (p=0.006). Moreover, the combination of ActA and ASNase induced the most significant increase of miR-491-5p in the cytoplasm of 697 cells (FC=2.5, p=0.0078 compared to control, n=8). To confirm the involvement of miR-491-5p in chemoresistance we modulated its expression by transfecting a specific inhibitor, obtaining a reduction of 40% of ActA-mediated anti-apoptotic action (p=0.0423,n=3). Through miR-System database, we predicted the miR-491-5p involvement in TP53-mediated apoptosis. At this purpose, gene expression profile data obtained on 697 cells demonstrated that ActA stimulation reduced of about 30% the levels of tp53aip1, a pro-apoptotic molecule with a key role in TP53 pathway. Interestingly, by using miR-Walk database, tp53aip1 was predicted as a direct target gene of miR‑491-5p. In accordance, the inhibition of miR491-5p by transfection of its specific inhibitor increased of 5,8 folds the expression levels of tp53aip1 mRNA (p=0.0313,n=6), while the transfection in 697 cells of a miR-491-5p synthetic mimic molecule decreased tp53aip1 mRNA of 20% (p=0,0283,n=4) Summary/Conclusion: Overall, we demonstrated that ActA is able to increase the production of miR-491-5p enriched EV. Furthermore, we identified ActA/ miR-491-5p axis as a pathway in the modulation of ASNase chemoresistance in 697 BCP-ALL cells. Future studies are needed to understand whether the delivery of miR-491-5p by EV could transfer chemoresistance to other BCP-ALL cells Keywords: Chemoresistance, B cell acute lymphoblastic leukemia, Microvesicles