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

FLT3-ITD mutations are reported in approximately one third of patients diagnosed with Acute Myeloid Leukemia (AML). Patients with these mutations are also reported to have poor outcomes as well. While there are more FLT3-ITD inhibitor drugs being developed and used to treat these patients, the emergence of drug resistance is still an issue in relapsed AML patients. In order to improve patient outcomes, new small molecule inhibitors are being developed to try to overcome the problem of resistance. In addition, researchers are investigating novel mechanisms for a more targeted approach to FLT3-ITD therapy in AML.

One of these novel approaches is the use of mitophagy. Lethal mitophagy is a form of selective autophagy where damaged mitochondria are targeted and eliminated. It has reported that this form of autophagy could have therapeutic benefits for cancer treatment. In addition, data have demonstrated that some anti-AML therapies have produced their effects through an autophagic process.

Ceramide is a lipid effector which has been reported to induce mitochondrial autophagy and tumor suppression. M. Dany from the Medical University of South Carolina has reported that ceramide is involved in lethal mitophagy. However, there are currently no data to show the efficacy of using mitophagy to target FLT3 signaling in AML and the mechanisms by which this could happen are not known.

 This novel therapeutic approach could potentially benefit adult patients with relapsed AML. Also, there could be broader clinical implications for anti-cancer therapy. Dany et al. have conducted the first investigation on the mechanisms by which FLT3-ITD regulates ceramide metabolism and cell death via the modulation of C18-ceramide-dependent mitophagy in AML human samples and mouse models. They showed that AML cells pretreated with inhibitors of autophagy were resistant to FLT3-ITD inhibitors. They confirmed that FLT3-ITD inhibitors induced formation of autophagosomes, supporting mitophagy induction. They also investigated whether the C18-ceramide analogue drug LCL-461 could induce lethal mitophagy in AML cells resistant to crenolanib (a FLT3-ITD inhibitor) and AML blasts from FLT3-ITD+ patients. The key results demonstrated that LCL-461 induced mitophagy in vitro and in vivo (xenografts) in AML blasts from FLT3-ITD+ AML patients, but not in nonleukemic human hematopoietic bone marrow cells.

The results of this study have further elucidated mechanisms by which targeting mitochondria can result in improved therapeutic approaches for AML. The authors reported that pharmacologically targeting of FLT3-ITD signalling led to ceramide mediated mitophagy dependent cell death in AML cell lines. In conclusion, a novel target for FLT3 therapy in AML has been discovered. Also of importance is the discovery that LCL-461 had an effect of reducing resistance to crenolanib through its mitophagic effects.

Their study was published in Blood in October 2016.

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.