Targeting FLT3-ITD signaling mediates ceramide-dependent mitophagy and attenuates drug resistance in AML

Abstract

Signaling pathways regulated by mutant Fms-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD), which mediate resistance to acute myeloid leukemia (AML) cell death, are poorly understood. Here, we reveal that pro-cell death lipid ceramide generation is suppressed by FLT3-ITD signaling. Molecular or pharmacologic inhibition of FLT3-ITD reactivated ceramide synthesis, selectively inducing mitophagy and AML cell death. Mechanistically, FLT3-ITD targeting induced ceramide accumulation on the outer mitochondrial membrane, which then directly bound autophagy-inducing light chain 3 (LC3), involving its I35 and F52 residues, to recruit autophagosomes for execution of lethal mitophagy. Short hairpin RNA (shRNA)-mediated knockdown of LC3 prevented AML cell death in response to FLT3-ITD inhibition by crenolanib, which was restored by wild-type (WT)-LC3, but not mutants of LC3 with altered ceramide binding (I35A-LC3 or F52A-LC3). Mitochondrial ceramide accumulation and lethal mitophagy induction in response to FLT3-ITD targeting was mediated by dynamin-related protein 1 (Drp1) activation via inhibition of protein kinase A-regulated S637 phosphorylation, resulting in mitochondrial fission. Inhibition of Drp1 prevented ceramide-dependent lethal mitophagy, and reconstitution of WT-Drp1 or phospho-null S637A-Drp1 but not its inactive phospho-mimic mutant (S637D-Drp1), restored mitochondrial fission and mitophagy in response to crenolanib in FLT3-ITD AML cells expressing stable shRNA against endogenous Drp1. Moreover, activating FLT3-ITD signaling in crenolanib-resistant AML cells suppressed ceramide-dependent mitophagy and prevented cell death. FLT3-ITD AML drug resistance is attenuated by LCL-461, a mitochondria-targeted ceramide analog drug, in vivo, which also induced lethal mitophagy in human AML blasts with clinically relevant FLT3 mutations. Thus, these data reveal a novel mechanism which regulates AML cell death by ceramide-dependent mitophagy in response to FLT3-ITD targeting.

Dual Role of Mitophagy in Cancer Drug Resistance

“…FLT3-ITD inhibitors, such as sorafenib, quizartinib (AC220), and crenolanib, showed efficacy for therapy in preclinical models of AML, but not in clinical trials because of the development of drug resistance (30). Interestingly, LCL-461, a mitochondria-targeted ceramide analog drug, is efficacious in attenuating crenolanib resistance by inducing lethal mitophagy in FLT3-ITD + AML in vitro and in vivo (27). This lethal pathway of mitophagy induction by ceramide and ceramide analog drug is likely be a potential target in FLT3-ITD+ AML, and further studies will be needed to determine if ceramide will be effective in other cancer types.…”

“…Studies show that CerS1/C18-ceramide selectively mediates lethal mitophagy, which is B-cell lymphoma 2-associated X (BAX)/BCL2 antagonist/killer 1 (BAK1)-and caspase-independent (24,27). Ceramide induces the formation of lipidated LC3, which then binds ceramide on the mitochondrial membrane upon dynaminrelated protein 1 (DRP1)-mediated mitochondrial fission, targeting autophagolysosomes to mitochondria and leading to lethal mitophagy in cancer cells (24,27). Excessive mitochondrial clearance in the absence of mitochondrial biogenesis or metabolic plasticity likely induce cell metabolic disorders and death in cancer cells (8).…”

Fighting fat in AML

“…1 Inhibition of FLT3-ITD signaling through altering ceramide-involved lipid metabolism provides a new strategy for treating FLT3-ITD-positive AML and overcoming drug resistance by pharmacologically blocking FLT-3-ITD downstream signaling to enhance ceramide-mediated mitophagy.…”

Balance between senescence and apoptosis is regulated by telomere damage–induced association between p16 and caspase-3

“…SA-ß-galactosidase activity was also measured for paw tissues of mice from indicated genotypes using the same kit. All animal experiment protocols were approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina (27,(54)(55)(56).…”

“…After post-fixation in 2% (v/v) osmium tetroxide, specimens were embedded in Epon 812, and sections were cut orthogonally to the cell monolayer with a diamond knife. Thin sections were visualized in a JEOL 1010 TEM (55,56).…”