Mutations in FLT3 receptor tyrosine kinase are common targets in Acute Myeloid Leukemia (AML); however, FLT3 targeted therapy shows limited success due to development of resistance. Ceramide, a bioactive sphingolipid, is synthesized de novo by Ceramide Synthases (CerS) and mediates cancer cell death in response to various chemotherapeutic agents. This study investigates the biological role of ceramide lipid in the response of AML to FLT3 targeted therapy and aims at finding mechanism-based alternative therapeutic strategies to overcome resistance to FLT3 inhibitors. We found that AML cell lines and patient samples expressing FLT3 have suppressed CerS1 expression and lower levels of its product C18-ceramide compared with FLT3 negative AML cells. Silenced FLT3 expression or its pharmacological inhibition increased CerS1 and C18-ceramide levels while FLT3 overexpression suppressed them. The increase in C18-ceramide after FLT3 inhibition is required for cell death as silencing CerS1 expression or inhibiting its enzymatic activity protected from FLT3 inhibitors-induced cell death in vitro and in vivo. Mechanistically, FLT3 inhibition resulted in CerS1 translocation from cytosol to mitochondria resulting in generation and mitochondrial accumulation of C18-ceramide. The mitochondrial C18-ceramide then binds directly to LC3B-II to recruit autophagosomal membranes to mitochondria for the execution of lethal mitophagy and degradation of mitochondria. We also show that this process is regulated upstream by early Drp1 activation and p-Drp1 S637 de-phosphorylation, whereby silencing Drp1 expression or preventing its S637 dephosphorylation blocked the translocation of CerS1 to mitochondria, prevented ceramide mitochondrial accumulation, halted the events of lethal mitophagy, and protected from FLT3 inhibitors-induced cell death. Due to the importance of ceramide accumulation in mitochondria for AML cells to respond to FLT3 inhibition, we proposed a synthetic lipid compound, LCL-461, composed of C18-ceramide conjugated to a pyridinium ring in the sphingosine backbone. Mass spectrometry proved that LCL-461 accumulates selectively in mitochondrial fractions of AML cells due to the positively charged conjugated pyridinium ring. LCL-461 was effective in inducing cell death in several AML cell lines of different FLT3 mutation statuses and resistance profiles as well as in patient samples and in vivo xenografts, with minimal cytotoxicity effects on normal human bone marrow cells. LCL-461 induced cell death via the same LC3B dependent lethal mitophagy mechanism detected following FLT3 inhibition. This highlights the potential of LCL-461 as an agent that can bypass FLT3 signaling by accumulating in mitochondria to induce lethal mitophagy and AML cell death regardless of whether patients are sensitive or resistant to FLT3 targeted therapy.
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