Abstract 4447: Autophagy inhibition overcomes resistance to FLT3 inhibitors in <i>FLT3-</i>ITD acute myeloid leukemia models

Abstract Autophagy has recently been postulated as a potential mechanism that may contribute to the development of resistance to FLT3 inhibitors (FLT3i). Our aim was to investigate the role of pharmacologic autophagy inhibition in enhancing the efficacy of FLT3i in the FLT3-ITD acute myeloid leukemia (AML) resistant model and its impact on the survival and molecular response to FLT3i. In MOLM13 and MV4-11 (FLT3-ITD mutated cell lines), the administration of FLT3i first (midostaurin) or second (quizartinib) generation significantly elevates autophagic flux by inducing the formation of acidic vesicular organelles visualized by flux cytometry. We also observed a reduction in negative autophagy regulators, evidenced by the inhibition of AKT (p-Ser473), mTOR (p-Ser2448), and its downstream protein p70S6K (p-Thr421/Ser424), as well as a reduction of ULK1 (p-Ser757) by western blot assay. FLT3i treatment resulted in the conversion of LC3B-I to LC3B-II, accompanied by the degradation of LC3B-II and p62, indicating autophagy induction. The combination of FLT3i with autophagy inhibitors chloroquine (CQ), bafilomycin A1 and ROC-325 significantly reduced cell viability and survival in comparison with vehicle and monotherapies in vitro in MOLM13 and MV4-11 cells and ex vivo in primary cells from bone marrow of FLT3-ITD AML patients. To elucidate the role of inhibition of autophagy in FLT3i-resistant cells, we generated MV4-11 cells resistant to quizartinib (MV4-11 QR) trough selective pressure and we noted that MV4-11 QR cells exhibited a higher basal level of autophagy compared to conventional MV4-11 cells, as indicated by the increased formation of acidic vesicular organelles, and confirmed by the reduced expression of LC3B and p62 proteins. In MV4-11 QR cells, we observed that CQ effectively reduced the IC50 of quizartinib and increased FLT3i-induced apoptosis, suggesting its use to mediate resistance to FLT3i in AML. We also observed an increased molecular effect of quizartinib when associated with CQ, manifested by a significant increase in Cleaved-Caspase3 and a reduction of STAT5 (p-Tyr694) and P70S6K in comparison with vehicle and quizartinib monotherapy. The combination of quizartinib with CQ showed a strong synergistic effect in the resistant lineage demonstrated by a ZIP score value = 10.8, while in the non-resistant lineage it demonstrated an additive effect (ZIPscore = 8.4), suggesting that CQ is autophagy-dependent and has its effect potentiated in conditions of high autophagic flux, as observed in MV4-11 QR, being effective, mainly, in conditions of resistance generated by the induction of autophagy. In conclusion, FLT3i induces autophagy flux, limiting drug inhibitory activity, and autophagy inhibition can enhance the antileukemic effect of FLT3i and reverts cellular resistance. Analysis of the autophagic profile in AML can play a crucial role in individualized treatment. Citation Format: Manuela Albuquerque de Melo, Diego A. Pereira-Martins, Brunno G. Macedo, Priscila S. Scheucher, João A. Machado-Neto, Fabíola Traina. Autophagy inhibition overcomes resistance to FLT3 inhibitors in FLT3-ITD acute myeloid leukemia models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4447.