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

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.