P064 | TARGETING AUTOPHAGY ENHANCES MIDOSTAURIN'S EFFICACY AGAINST FLT3-MUTATED ACUTE MYELOID LEUKEMIA UNDER CONDITIONS MIMICKING THE HYPOXIC BONE MERROW MICROENVIRONMENT

Beckground: FMS-like tyrosine kinase 3 (FLT3) activating mutations are found in about 30% of AML patients and are associated with poor prognosis. Midostaurin (Mido) has improved outcomes for FLT3-mutated (FLT3m) AML, yet relapse driven by resistant leukemic cells remains the most frequent cause of treatment failure. In the last decade, it has been proposed that the bone merrow microenvironment in general and the hypoxic niche specifically, plays a crucial role in drug-response, acting as a sanctuary for leukemic stem cells (LSCs). Aim: This study explores how Mido affects FLT3m AML cells under conditions mimicking the hypoxic bone marrow niche and investigates whether combining it with the autophagy inhibitor chloroquine (Cq) could better target leukemic cells. Methods: FLT3-ITD AML cell lines MV-4-11 and MOLM-13 were treated with Mido and/or Cq under normoxic (20% O2) and hypoxic (1% O2) conditions for 24-48 h. Apoptosis induction, cell proliferation, metabolic activity, gene expression changes, and protein expression were evaluated. Results: In FLT3-mutated AML cell lines, Mido activates autophagy—a potential survival mechanism—by causing non-essential amino acid depletion and suppressing mTORC1 signaling. Combining Mido with the autophagy inhibitor Cq enhanced apoptosis and cell cycle arrest under both normoxic and hypoxic conditions. This combination also significantly reduced lactate levels, although ROS and glutathione levels remained unchanged. Under hypoxia, co-treatment notably decreased phosphorylated FLT3 and autophagy-related proteins (BECLIN, ATG3, LC3I-II). Lastly, gene set enrichment analysis showed that the combination reversed Mido-induced mTORC1 inhibition and activated DNA repair pathways, including ATM and p53 signaling. Conclusion: Combining Mido with autophagy inhibition (Cq) offers a promising strategy to eradicate FLT3m cells in the hypoxic niche, potentially overcoming drug resistance. Supported by the AIRC-Fellowship for Italy, Project Code: 26986.