Molecular Mechanisms Associated with Autophagy Inhibition to Overcome Resistance to FLT3 Inhibitors
Authors: Albuquerque De Melo Manuela , Pereira-Martins Diego A. , Macedo Brunno Gilberto Santos , de Miranda Livia Bassani Lins , Scheucher Priscila Santos , Weinhäuser Isabel , Huls Gerwin , Schuringa Jan Jacob , Machado-Neto João Agostinho , Traina Fabiola 
Published: December 2024

Autophagy induction has recently emerged as a mechanism of resistance to FLT3 inhibitors (FLT3i) in patients with FLT3-ITD mutant acute myeloid leukemia (AML). Molecularly, this resistance is facilitated by autophagy-mediated degradation of drug-targeted proteins and via activation of pro-survival pathways in FLT3-mutant cells. Chloroquine (CQ), an FDA-approved antimalarial drug, is a safe alternative to inhibit autophagy and to overcome resistance in myeloid neoplasms. By applying a multiomic approach, we assessed the molecular mechanisms by which CQ increases the efficacy of FLT3i in FLT3-ITD resistant models. Transcriptome analysis of ex vivo AML samples (n=78) treated with FLT3i (midostaurin, PKC and quizartinib, AC220) revealed enriched signatures related to mitochondrial metabolism and autophagy in resistant samples. We validated these findings in a second cohort of patients treated with PKC (n=13), where we observed an increased mitochondrial membrane potential in CD34+ cells from poor responders. Additionally, proteome profiling of CD34+/CD117+ cells revealed high expression of proteins associated with LSC-UP and LMPP programs in poor responders. In FLT3-ITD AML cell line models (MOLM13 and MV4-11), treatment with PKC and AC220 led to an increase in acidic vesicular organelles (AVOs) and reduction in the levels of autophagy-related proteins, such as LC3BI/II and p62. This indicated induction of autophagy by FLT3i. Combining FLT3i (PKC and AC220) with CQ further decreased cell survival compared to FLT3i monotherapy in both cell lines and primary patient samples. In vivo, the combination of PKC+CQ improved overall survival and preserved healthy hematopoiesis in MOLM13-transplanted mice, which was not observed with PKC (6 mg/Kg, q.d. orally) or CQ (20 mg/Kg q.d. intraperitoneally) monotherapies. To determine whether CQ induces cell death through autophagy inhibition or via other off-target effects, we performed genetic knockdown (KD) of the autophagy-related ATG5 and ATG7 genes. ATG5- and ATG7-KD increased sensitivity to both FLT3i, with no additional effects from CQ, indicating that CQ-induced cell death relies on the pharmacological inhibition of autophagy. To investigate the potential of CQ in overcoming FLT3i resistance, we generated MV4-11 cells resistant to AC220 (MV4-11 QR) through selective pressure. MV4-11 QR cells displayed higher basal levels of autophagy compared to normal MV4-11 cells. The combination CQ+AC220 demonstrated a synergistic effect in MV4-11 QR cells (ZIP score 10.8) enhancing cell death more than the monotherapies. This effect was associated with greater inhibition of FLT3, pSTAT5 and pP70S6K compared to the monotherapies. At the molecular level, CQ potentiates the efficacy of AC220 by significantly inhibiting the autophagy mediator ATG7, with this effect being more pronounced in the combination treatment than with CQ alone. To identify new targets associated with the response to CQ+FLT3i, we performed label-free quantification proteomics on MOLM13 cells treated with CQ, PKC, and their combination. We found that 46 proteins were downregulated across all 03 conditions (CQ, PKC and CQ+PKC), mostly related to cell proliferation and survival (BAX, SMARCA4 and SUMO1). RCF4 (an autophagy regulator linked to increased chemosensitivity) and GATD3 proteins were upregulated only in the CQ+PKC group. Additionally, 12 proteins were downregulated in the CQ+PKC group, which are associated with FLT3i resistance in FLT3-ITD models. Finally, we showed thatFLT3i treatment induces autophagy flux, which can limit the inhibitory effectiveness of the drug. Combining FLT3i with CQ enhances its antileukemic efficacy by overcoming this pharmacological resistance. While CQ is generally well-tolerated clinically, patient heterogeneity must be considered. Assessing the autophagic response upon FLT3i in AML could significantly improve personalized treatment strategies.