Date of Award

2016

Embargo Period

8-1-2024

Document Type

Dissertation - MUSC Only

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Besim Ogretmen

Second Advisor

Dennis Watson

Third Advisor

Harry Drabkin

Fourth Advisor

James Norris

Fifth Advisor

Amanda C. LaRue

Sixth Advisor

Elizabeth Yeh

Abstract

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. The metabolic product of ceramide, sphingosine 1 phosphate (S1P) generated by sphingosine Kinases (SK1/2), is a tumor promoting lipid. This dissertation investigates the biological role of ceramide and S1P lipids in the response and resistance 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. FLT3 regulated CerS1 expression epigenetically via HDAC1 dependent Sp1 recruitment. The increase in C18-ceramide after inhibition of FLT3 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, inhibition of FLT3 resulted in CerS1 translocation from cytosol to mitochondria to generate C18-ceramide. The mitochondrial C18-ceramide then binds directly to LC3B-II to recruit autophagosomal membranes to mitochondria for the execution of lethal mitophagy. We also show that this process is regulated upstream by early Drp1 activation and PKA mediated p-Drp1 S637 de-phosphorylation, whereby silencing Drp1 expression or preventing its S637 de-phosphorylation 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 inhibition of FLT3, 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 in vivo xenografts and patient samples, with minimal cytotoxicity effects on normal human bone marrow cells. LCL-461 induced cell death via the same LC3B dependent lethal mitophagy mechanism detected upon inhibition of FLT3. 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. In addition, in a model consisting of AML cells resistant to FLT3 inhibitor treatment, we found that resistant cells exhibit overexpression of nuclear SK2 and higher levels of nuclear S1P. SK2-S1P axis was required for the resistance phenotype whereby silencing SK2 sensitized the cells to FLT3 inhibitor treatment. Inhibition of SK2 activity using ABC294640 re-sensitized the cells to FLT3 targeted therapy. This highlights the potential of ABC294640 as a pharmacological agent that can re-sensitize resistant AML to FLT3 targeted therapy.

Rights

All rights reserved. Copyright is held by the author.

Download

Share

COinS