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: Mohammed Dany, Salih Gencer, Rose Nganga, Raquela J Thomas, Natalia Oleinik, Kyla D Baron, Zdzislaw M Szulc, Peter Ruvolo, Steven Kornblau, Michael Andreeff, Besim Ogretmen
• Journal: Blood (Blood) Vol. 128 Issue 15 Pg. 1944-1958 (10 13 2016) ISSN: 1528-0020 [Electronic] United States
• PMID: 27540013 (Publication Type: Journal Article)
• Copyright: © 2016 by The American Society of Hematology.

Chemical References

• Benzimidazoles
• Ceramides
• MAP1LC3A protein, human
• Microtubule-Associated Proteins
• Mitochondrial Proteins
• Piperidines
• RNA, Small Interfering
• FLT3 protein, human
• fms-Like Tyrosine Kinase 3
• Cyclic AMP-Dependent Protein Kinases
• GTP Phosphohydrolases
• DNM1L protein, human
• Dynamins
• crenolanib

Topics

• Animals
• Benzimidazoles (pharmacology)
• Ceramides (genetics, metabolism)
• Cyclic AMP-Dependent Protein Kinases (genetics, metabolism)
• Drug Resistance, Neoplasm (drug effects, genetics)
• Dynamins
• GTP Phosphohydrolases (genetics, metabolism)
• Humans
• Leukemia, Myeloid, Acute (drug therapy, genetics, metabolism)
• Male
• Mice
• Mice, Inbred NOD
• Microtubule-Associated Proteins (antagonists & inhibitors, genetics, metabolism)
• Mitochondria (genetics, metabolism)
• Mitochondrial Proteins (genetics, metabolism)
• Mitophagy (drug effects, genetics)
• Mutation
• Phosphorylation (drug effects, genetics)
• Piperidines (pharmacology)
• RNA, Small Interfering (pharmacology)
• Signal Transduction (drug effects, genetics)
• fms-Like Tyrosine Kinase 3 (antagonists & inhibitors, genetics, metabolism)