P25 | RESPONSE TO HEDGEHOG AND PI3K/AKT/MTOR PATHWAY INHIBITORS IN PREB-ALL: ANALYSIS OF THE INTRACELLULAR DISTRIBUTION OF PATHWAY ENZYMES AND OF THE AUTOPHAGY PROCESS

M. Sicurella¹, M. De Chiara², E. Battaglia², F. Franco², M. De Vita², L.M. Neri² | ¹Department of Environmental and Prevention Sciences, University of Ferrara, Ferrara, Italy; ²Department of Translational Medicine, University of Ferrara, Ferrara, Italy

PreB-Acute Lymphoblastic Leukemia (ALL) is characterized by the uncontrolled proliferation of lymphoid precursors and the dysregulation of key intracellular signaling pathways. Among these, the PI3K/AKT/mTOR and Hedgehog (Hh) pathways play critical roles in promoting leukemic cell growth, survival, and resistance to therapy^1,2. While the PI3K/AKT/ mTOR axis has been extensively studied, the contribution of GLI1 a key downstream transcriptional effector of the Hh pathway remains largely unexplored in hematologic malignancies. To identify the role of these signaling pathways in development, maintenance and progression of preBALL lines. To investigate the effect of single or combined inhibition of the PI3K/AKT/mTOR and Hh/GLI1 pathways, in preB-ALL cell lines (NALM-6, KOPN8 and SEM) were treated with the GLI1 inhibitor GANT-61 and/or the AKT inhibitor MK-2206. Cell viability was assessed using the CCK-8 assay. Apoptosis, cell cycle distribution, and autophagy were analyzed by flow cytometry. Protein expression and localization of Gli1 and Akt was evaluated by Western blotting and immunofluorescence, respectively. Dual pathway inhibition significantly decreased cell viability when compared with single treatments in ALL cell lines, underscoring a synergistic cytotoxic effect. The combined treatment induced G0/G1 cell cycle arrest, increased apoptotic cell death and significantly triggered autophagy, as evidenced by elevated LC3B expression. Western blot analyses revealed a relevant downregulation of the total form GLI1 and of phosphorylated AKT (p-AKT), GSK3β (p-GSK3β), p70S6K (pp70S6K). Before treatments immunofluorescence analysis showed a nuclear localization of GLI1 whereas p-Akt was mainly in the cytoplasm. After treatments the fluorescent signals reduced their intensity. Co-inhibition of the PI3K/AKT/mTOR and Hh/GLI1 pathways showed a synergistic cytotoxic effect in preB-ALL models, indicating a potential strategy to overcome therapeutic resistance in leukemia.