title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology and Hollings Cancer Center, Medical University of South Carolina, Charleston, SC; and.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Dany
authors.affiliation: Department of Biochemistry and Molecular Biology and Hollings Cancer Center, Medical University of South Carolina, Charleston, SC; and.
authors.forename: Salih
authors.initials: S
authors.lastname: Gencer
authors.affiliation: Department of Biochemistry and Molecular Biology and Hollings Cancer Center, Medical University of South Carolina, Charleston, SC; and.
authors.forename: Rose
authors.initials: R
authors.lastname: Nganga
authors.affiliation: Department of Biochemistry and Molecular Biology and Hollings Cancer Center, Medical University of South Carolina, Charleston, SC; and.
authors.forename: Raquela J
authors.initials: RJ
authors.lastname: Thomas
authors.affiliation: Department of Biochemistry and Molecular Biology and Hollings Cancer Center, Medical University of South Carolina, Charleston, SC; and.
authors.forename: Natalia
authors.initials: N
authors.lastname: Oleinik
authors.affiliation: Department of Biochemistry and Molecular Biology and Hollings Cancer Center, Medical University of South Carolina, Charleston, SC; and.
authors.forename: Kyla D
authors.initials: KD
authors.lastname: Baron
authors.affiliation: Department of Biochemistry and Molecular Biology and.
authors.forename: Zdzislaw M
authors.initials: ZM
authors.lastname: Szulc
authors.affiliation: Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX.
authors.forename: Peter
authors.initials: P
authors.lastname: Ruvolo
authors.affiliation: Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX.
authors.forename: Steven
authors.initials: S
authors.lastname: Kornblau
authors.affiliation: Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX.
authors.forename: Michael
authors.initials: M
authors.lastname: Andreeff
authors.affiliation: Department of Biochemistry and Molecular Biology and Hollings Cancer Center, Medical University of South Carolina, Charleston, SC; and.
authors.forename: Besim
authors.initials: B
authors.lastname: Ogretmen
mesh_terms: D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology*; D002518:Ceramides / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D034281:Dynamins; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D063306:Mitophagy / Q000187:drug effects / Q000235:genetics; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D010880:Piperidines / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D051941:fms-Like Tyrosine Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D001562:Benzimidazoles; D002518:Ceramides; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D024101:Mitochondrial Proteins; D010880:Piperidines; D034741:RNA, Small Interfering; C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3; D017868:Cyclic AMP-Dependent Protein Kinases; D020558:GTP Phosphohydrolases; C110768:DNM1L protein, human; D034281:Dynamins; C577197:crenolanib