A Phase 1 study of intravenous infusions of tigecycline in patients with acute myeloid leukemia.

Acute myeloid leukemia (AML) cells meet the higher energy, metabolic, and signaling demands of the cell by increasing mitochondrial biogenesis and mitochondrial protein translation. Blocking mitochondrial protein synthesis through genetic and chemical approaches kills human AML cells at all stages of development in vitro and in vivo. Tigecycline is an antimicrobial that we found inhibits mitochondrial protein synthesis in AML cells. Therefore, we conducted a phase 1 dose-escalation study of tigecycline administered intravenously daily 5 of 7 days for 2 weeks to patients with AML. A total of 27 adult patients with relapsed and refractory AML were enrolled in this study with 42 cycles being administered over seven dose levels (50-350 mg/day). Two patients experienced DLTs related to tigecycline at the 350 mg/day level resulting in a maximal tolerated dose of tigecycline of 300 mg as a once daily infusion. Pharmacokinetic experiments showed that tigecycline had a markedly shorter half-life in these patients than reported for noncancer patients. No significant pharmacodynamic changes or clinical responses were observed. Thus, we have defined the safety of once daily tigecycline in patients with refractory AML. Future studies should focus on schedules of the drug that permit more sustained target inhibition

The mitochondrial peptidase, neurolysin, regulates respiratory chain supercomplex formation and is necessary for AML viability

Neurolysin (NLN) is a zinc metallopeptidase whose mitochondrial function is unclear. We found that NLN was overexpressed in almost half of patients with acute myeloid leukemia (AML), and inhibition of NLN was selectively cytotoxic to AML cells and stem cells while sparing normal hematopoietic cells. Mechanistically, NLN interacted with the mitochondrial respiratory chain. Genetic and chemical inhibition of NLN impaired oxidative metabolism and disrupted the formation of respiratory chain supercomplexes (RCS). Furthermore, NLN interacted with the known RCS regulator, LETM1, and inhibition of NLN disrupted LETM1 complex formation. RCS were increased in patients with AML and positively correlated with NLN expression. These findings demonstrate that inhibiting RCS formation selectively targets AML cells and stem cells and highlights the therapeutic potential of pharmacologically targeting NLN in AML

The metabolic enzyme hexokinase 2 localizes to the nucleus in AML and normal haematopoietic stem and progenitor cells to maintain stemness

Thomas, Egan et al. report that hexokinase 2 localizes to the nucleus of leukaemic and normal haematopoietic cells to maintain stemness by interacting with nuclear proteins and modulating chromatin accessibility independently of its kinase activity. Mitochondrial metabolites regulate leukaemic and normal stem cells by affecting epigenetic marks. How mitochondrial enzymes localize to the nucleus to control stem cell function is less understood. We discovered that the mitochondrial metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus in leukaemic and normal haematopoietic stem cells. Overexpression of nuclear HK2 increases leukaemic stem cell properties and decreases differentiation, whereas selective nuclear HK2 knockdown promotes differentiation and decreases stem cell function. Nuclear HK2 localization is phosphorylation-dependent, requires active import and export, and regulates differentiation independently of its enzymatic activity. HK2 interacts with nuclear proteins regulating chromatin openness, increasing chromatin accessibilities at leukaemic stem cell-positive signature and DNA-repair sites. Nuclear HK2 overexpression decreases double-strand breaks and confers chemoresistance, which may contribute to the mechanism by which leukaemic stem cells resist DNA-damaging agents. Thus, we describe a non-canonical mechanism by which mitochondrial enzymes influence stem cell function independently of their metabolic function

Apoptosis prevention of non-Hodgkin's lymphoma cells in culture by huam plasma

grantor: University of TorontoNon Hodgkin's Lymphoma (NHL) is a disease characterized by the clonal expansion of malignant cells of B or T origin. A culture system was developed in our laboratory that allowed the establishment of 20 NHL cell lines from direct samples from patients with NHL. Their further characterization in terms of growth regulation and genetic changes provided the opportunity to examine pathogenic mechanisms. I was able to demonstrate that human platelet poor plasma (PPP) is required by NHL cells for proliferation and survival. Its withdrawal induces programmed cell death in all tested cell lines except one. Proliferative and survival signals provided by PPP could not be replaced by two fully defined media: one composed of bovine serum albumin, human transferrin and bovine insulin and a second, a commercially available preparation (Stem Pro 34\sp\circler ) containing proprietary reagents. The addition of cytokines to both serum replacement media did neither improve proliferation nor survival.M.Sc

Activity of tigecycline (1 mg/mL) when freshly prepared in saline.

<p>*Tigecycline solution was preincubated at room temperature for 4 days, which was then used to determine the minimum inhibitory concentration (MIC) in <i>Escherichia coli</i> (MG1655) and mean half-maximal inhibitory concentration (IC₅₀) in TEX cells using the CellTiter viability assay following incubations with bacteria (24 hours) or cells (72 hours), respectively.</p>†<p>Tigecycline solution was preincubated at room temperature for 5 days, and TEX cells were then incubated with 5<b> µ</b>M tigecycline for 48 hours. The IC₅₀ for cell viability was then assessed by the Alamar blue assay, and respiratory complex IV activity was determined in cell lysates. Activity was normalized to citrate synthase content as a proxy for mitochondrial mass.</p><p>All solutions were adjusted to pH 7 and all incubations were performed in the dark. Numbers indicate mean ± standard deviation of at least 3 independent experiments.</p><p>Tig, tigecycline; Pyr, pyruvate; CD, 2-hydroxypropyl-β-cyclodextrin; AA, ascorbic acid.</p

Stability and activity of tigecycline reconstituted in saline with or without additives.

<p>*Tigecycline solution was preincubated at room temperature for 4 days, which was then used to determine the minimum inhibitory concentration (MIC) in <i>Escherichia coli</i> (MG1655) and mean half-maximal inhibitory concentration (IC₅₀) in TEX cells using the CellTiter viability assay following incubations with bacteria (24 hours) or cells (72 hours), respectively.</p>†<p>Tigecycline solution was preincubated at room temperature for 5 days, and TEX cells were then incubated with 5<b> µ</b>M tigecycline for 48 hours. The IC₅₀ for cell viability was then assessed by the Alamar blue assay, and respiratory complex IV activity was determined in cell lysates. Activity was normalized to citrate synthase content as a proxy for mitochondrial mass.</p><p>All solutions were adjusted to pH 7 and all incubations were performed in the dark. Numbers indicate mean ± standard deviation of at least 3 independent experiments, where applicable.</p><p>Tig, tigecycline; Pyr, pyruvate; CD, 2-hydroxypropyl-β-cyclodextrin; AA, ascorbic acid.</p