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

• Published in: Blood 2016-10, Vol.128 (15), p.1944-1958
• Main Authors: Dany, Mohammed, Gencer, Salih, Nganga, Rose, Thomas, Raquela J., Oleinik, Natalia, Baron, Kyla D., Szulc, Zdzislaw M., Ruvolo, Peter, Kornblau, Steven, Andreeff, Michael, Ogretmen, Besim
• Format: Article
• Language: English
• Subjects: Animals; Benzimidazoles - pharmacology; Ceramides - genetics; Ceramides - metabolism; Cyclic AMP-Dependent Protein Kinases - genetics; Cyclic AMP-Dependent Protein Kinases - metabolism; Drug Resistance, Neoplasm - drug effects; Drug Resistance, Neoplasm - genetics; Dynamins; fms-Like Tyrosine Kinase 3 - antagonists & inhibitors; fms-Like Tyrosine Kinase 3 - genetics; fms-Like Tyrosine Kinase 3 - metabolism; GTP Phosphohydrolases - genetics; GTP Phosphohydrolases - metabolism; Humans; Leukemia, Myeloid, Acute - drug therapy; Leukemia, Myeloid, Acute - genetics; Leukemia, Myeloid, Acute - metabolism; Male; Mice; Mice, Inbred NOD; Microtubule-Associated Proteins - antagonists & inhibitors; Microtubule-Associated Proteins - genetics; Microtubule-Associated Proteins - metabolism; Mitochondria - genetics; Mitochondria - metabolism; Mitochondrial Proteins - genetics; Mitochondrial Proteins - metabolism; Mitophagy - drug effects; Mitophagy - genetics; Mutation; Myeloid Neoplasia; Phosphorylation - drug effects; Phosphorylation - genetics; Piperidines - pharmacology; RNA, Small Interfering - pharmacology; Signal Transduction - drug effects; Signal Transduction - genetics
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2016-04-708750

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. •FLT3-ITD mutations suppress ceramide generation, and FLT3-ITD inhibition mediates ceramide-dependent mitophagy, leading to AML cell death.•Alteration of mitochondrial ceramide prevents mitophagy, resulting in resistance to FLT3-ITD inhibition which is attenuated by LCL-461.