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Inhibition of mitochondrial translation as a therapeutic strategy for acute myeloid leukemia Marko Škrtić Doctor of Philosophy Institute of Medical Science University of Toronto 2012 Abstract Intro: Acute myeloid leukemia (AML) therapies have remained unchanged for 20 years, and thus new therapies are needed. Objective: To identify FDA-approved agents with anti-leukemia stem cell activity, we performed a screen and identified the antimicrobial tigecycline (TIG). Methods: Primary AML mononuclear cells were isolated by Ficoll centrifugation from peripheral blood. Flow cytometry dye; JC-1, Carboxy-H2DCFDA, Mitotracker GreenFM. Leukemia stem cell activity was assayed by human AML engraftment in NOD/SCID mice. Results: TIG induced cell death in primary AML patient samples (LD50, 3-6μM n=14), preferentially over normal hematopoietic cells. Likewise, in colony assays, TIG (5μM) reduced the clonogenic growth of AML samples (n=7) by 93%, demonstrating an effect on leukemia progenitor cells, but not normal hematopoietic cells (34% reduction, n=5). A yeast genome-wide screen identified mitochondrial translation inhibition as the mechanism of tigecycline-mediated cell death in eukaryotic cells. TIG decreased the expression of mitochondrial peptides, enzyme activity and membrane potential preferentially in AML cells over normal hematopoietic cells. ShRNA knockdown of TuFM mitochondrial translation factor in leukemia cells reproduced TIG anti-leukemia target effects previously described. We discovered that primary AML CD34+/CD38- stem cells have greater mitochondrial mass (3-fold, n=5) than normal CD34+ cells (n=4). Higher baseline mitochondrial mass in primary AML samples was predictive for tigecycline sensitivity in vitro (r=-0.71, p<0.05). We assessed the effect of TIG on primary AML stem cells defined by their ability to initiate leukemic engraftment in vivo. NOD/SCID mice treated with TIG had decreased human AML engraftment (n=3 AML patients) compared to control. Conclusions: We identified mitochondrial translation inhibition as a novel therapeutic strategy for AML. Currently, a Phase I clinical trial of tigecycline in hematological malignancies is underway.

Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in leukemia cells through the inhibition of mitochondrial protein synthesis. To better understand the mechanisms of sensitivity and resistance to mitochondrial translation inhibition, we treated TEX leukemia cells with increasing concentrations of tigecycline over 4 months, and selected a population of cells resistant to tigecycline (RTEX+TIG). Compared to their wild type counterparts, the resistant RTEX+TIG cells had an altered metabolic profile with diminished oxidative phosphorylation and a greater reliance on glycolysis. Upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism and oxidative phosphorylation to wild type levels. At the molecular level, these cells had increased levels of HIF1a. Strikingly, tigecycline-resistant cells had decreased expression of CD34 and CD117, clonogenic growth potential and engraftment capabilities in vivo. Thus, chronic inhibition of mitochondrial translation leads to the establishment of rho-zero-like metabolic phenotype, and is associated with differentiation of leukemia cells.

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Sirtuins are a family of nicotinamide adenine dinucleotide - dependent enzymes, which have gained recent interest due to their ability to directly or indirectly regulate cell metabolism, oxidative response mechanisms and cellular senescence. A mitochondrial sirtuin SIRT3, although still relatively under-investigated, regulates mitochondrial processes through deacetylation of metabolic enzymes and components of electron transport chain. We hypothesized that SIRT3 is a mitochondrial cytoprotective factor that exerts its function by decreasing reactive oxygen species levels, and protecting cells from oxidative stress. HEK-293 cells over-expressing SIRT3 exhibit reduced mitochondrial membrane potential and reactive oxygen species levels under basal conditions. In addition, cells over-expressing SIRT3 are less sensitive to hydrogen peroxide and glucose deprivation/glucose reperfusion induced-cell death. Since SIRT3 expression in the brain has not yet been investigated, its expression pattern in the rodent brain was characterized. Our results showed that SIRT3 mRNA and protein levels are robustly expressed in different regions of the adult rodent brain and their expression increases with age. Furthermore, SIRT3 is expressed predominantly in astrocytes in cultures derived from rat primary E18 cortical cells. These results suggest that SIRT3 possesses cytoprotective potential, and that its actions in the brain regulate astrocyte physiology.

Sirtuins are a family of nicotinamide adenine dinucleotide - dependent enzymes, which have gained recent interest due to their ability to directly or indirectly regulate cell metabolism, oxidative response mechanisms and cellular senescence. A mitochondrial sirtuin SIRT3, although still relatively under-investigated, regulates mitochondrial processes through deacetylation of metabolic enzymes and components of electron transport chain. We hypothesized that SIRT3 is a mitochondrial cytoprotective factor that exerts its function by decreasing reactive oxygen species levels, and protecting cells from oxidative stress. HEK-293 cells over-expressing SIRT3 exhibit reduced mitochondrial membrane potential and reactive oxygen species levels under basal conditions. In addition, cells over-expressing SIRT3 are less sensitive to hydrogen peroxide and glucose deprivation/glucose reperfusion induced-cell death. Since SIRT3 expression in the brain has not yet been investigated, its expression pattern in the rodent brain was characterized. Our results showed that SIRT3 mRNA and protein levels are robustly expressed in different regions of the adult rodent brain and their expression increases with age. Furthermore, SIRT3 is expressed predominantly in astrocytes in cultures derived from rat primary E18 cortical cells. These results suggest that SIRT3 possesses cytoprotective potential, and that its actions in the brain regulate astrocyte physiology.

Ischemic heart disease is a major cause of death worldwide. Identifying the mechanisms mediating cardiac resistance to injury (‘cardioprotection’) can contribute to therapies for cardiac injury. These studies investigated the role of the membrane channel forming protein connexin43 (Cx43), a downstream effector of PKCε-mediated cardioprotection. In isolated cardiomyocytes FGF-2/PKCε –induced cytoprotection is mediated by Cx43 phosphorylation at PKCε-target site, serine(S)262. Hypothesis (1). PKCε-mediated cardioprotection increases Cx43 phosphorylation at PKCε-target sites and prevents ischemia and/or reperfusion-induced Cx43 remodeling. Rat hearts were subjected to protective treatments (ischemic preconditioning, FGF-2, diazoxide), followed by 30 min global ischemia and/or 60 min reperfusion. All treatments elicited above-physiological levels of phospho(p)S262-Cx43 and pS368-Cx43 (P*Cx43), which were sustained during global ischemia, and reperfusion, and accompanied by attenuation of ischemia-induced Cx43 dephosphorylation, and prevention of Cx43 lateralization. Cx43 is present in cardiac subsarcolemmal (SSM) but not interfibrillar (IFM) mitochondria. Hypothesis (2). FGF-2 exerts protective effects on both mitochondrial populations, but is associated with mitochondrial (mito) P*Cx43 state in SSM, and mediated by mitoCx43 function. FGF-2 treatment increased calcium tolerance in SSM and IFM by 2.9- and 1.7-fold, respectively, compared to controls. In the presence of Gap27, a Cx43 hemi-channel blocker, the salutary effect of FGF-2 were lost in SSM but not IFM, indicating a functional role for Cx43. FGF-2 increased levels of PKCε, pPKCε and Tom-20 translocase in SSM and IFM. In SSM, FGF-2 increased pS262-and pS368-Cx43 by 30-fold and 8-fold, compared to controls. Stimulation of untreated SSM with a PKC activator (phorbol 12-myristate 13-acetate; PMA) also increased pS262-and-S368-Cx43 and calcium tolerance, which was prevented by εV1-2, a PKCε-inhibiting peptide. The effect of FGF-2 on isolated cardiac mitochondria is unknown. Hypothesis (3). FGF-2 exerts a direct protective effects on SSM. Direct stimulation of SSM with FGF-2 increased pS262-and-S368-mitoCx43, and calcium resistance to mPTP, and was dependent on mitoPKCε. FGF receptor inhibitors, SU5402 and FGFR neutralizing antibodies, blocked the effect of FGF-2, suggesting a FGFR1-like protein is responsible for the direct protective effects of FGF-2. This new intracellular mechanism of cytoprotection implies that endogenous intracellular levels of FGF-2 may determine constitutive levels of cardiac mitochondrial resistance to mPTP.