SC COBRE in Oxidants, Redox Balance, and Stress Signaling
Eduardo Maldonado, DVM, PhD
Awarded RO1: VDAC Opening Small Molecules to Revert Warburg Metabolism and Induce Oxidative Stress
Abstract: Enhanced glycolysis and suppression of mitochondrial metabolism characterize the Warburg phenomenon in cancer cells. Metabolites enter and exit mitochondria through one channel in the outer membrane: the volt-age dependent anion channel (VDAC). The central hypothesis of this proposal is that high free tubulin levels in cancer cells blocks VDAC and suppresses oxidative phosphorylation in Warburg metabolism and that reversal of tubulin inhibition of VDAC has an anti-Warburg effect that enhances oxidative phosphorylation, promotes oxidative stress and decreases glycolysis. We further hypothesize that small molecules antagonists of the inhibitory effect of VDAC on tubulin hyperpolarize mitochondria and in-crease generation of reactive oxygen species (ROS), leading to mitochondrial dysfunction and cell death. Accordingly in Specific Aim 1, we will characterize the effects of erastin and other VDAC-tubulin antag-onists on cellular bioenergetics (ATP, ADP, AMP, Pi, NADH redox state, AMP kinase, and rates of respiration and glycolysis) in HepG2, Huh7 and FOCUS human hepatocarcinoma (HCC) cells. We will also assess in a Huh7 mouse xenograft model the effect of erastin/VDAC-tubulin antagonists on mitochondrial membrane po-tential (ΔΨ) and the glycolytic phenotype. Lead compounds identified in a high throughput screening will be confirmed by electrophysiology as VDAC-tubulin antagonists, evaluated for effects on cellular bioenergetics and used to create a pharmacophore. In Specific Aim 2, we will assess the effects of protein kinase A (PKA)-dependent VDAC phosphorylation on the bioenergetic of HCC cells. We will use agonists and inhibitors of PKA as well as PKA overexpression and siRNA silencing in the presence and absence of erastin/VDAC-tubulin an-tagonists and after VDAC isoform double knockdown. Additionally, proteomic analysis will determine specific sites of VDAC isoform phosphorylation. In Specific Aim 3, we will determine the cytotoxic mechanisms of VDAC-tubulin antagonists. We expect that erastin and lead compounds will increase ΔΨ and ROS formation, leading to the mitochondrial permeability transition, bioenergetic failure, and cell death. We will also determine if cell death occurs by apoptosis, necrosis or necroptosis and if mitochondrial function can be preserved by an-tioxidants. Overall, the project will generate fundamental new knowledge on mechanisms causing sup-pression of mitochondrial metabolism in HCC and will identify new agents that block VDAC-tubulin in-teraction to revert the pro-proliferative Warburg metabolic phenotype and selectively promote cytotox-ic oxidative stress.
Marcelo Vargas, PhD
Awarded RO1: NAD metabolism and mitochondrial dysfunction in ALS models (R01NS089640)
Abstract: The long-term goal of the proposal is to develop new therapeutic strategies using mechanistic insights drawn from understanding astrocyte-motor neuron interaction in amyotrophic lateral sclerosis (ALS). In particular, the primary objective of this proposal is to establish whether increased nicotinamide adenine dinucleotide (NAD) availability ameliorates motor neuron degeneration in ALS models. ALS or Lou Gehrig's disease accounts for about 1 in 500 to 1 in 1,000 adult deaths in the United States and is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Motor neuron death leads to muscle weakness and paralysis causing death in one to five years from the time of symptoms onset. Most ALS cases are sporadic (SALS) and exposure to yet unidentified environmental toxicants might be responsible for SALS. About 5-10% of the cases are inherited (familial ALS, FALS) but FALS and SALS are phenotypically indistinguishable, and a significant share of our understanding come from the study of rodent models over-expressing ALS-linked mutant human superoxide dismutase 1 (hSOD1). Primary astrocytes isolated from mutant hSOD1 over-expressing mice induce motor neuron death in co-culture, and it has been demonstrated that astrocytes differentiated from spinal cord autopsy-derived neuronal progenitor cells from FALS and SALS patients are also toxic for motor neurons in co-culture. Sirtuins are a family of enzymes capable of catalyzing NAD-dependent deacylation and mono(ADPribosyl)ation reactions. Remarkably, NAD-dependent sirtuin-mediated deacetylation has been shown to modulate all major mitochondrial processes. Since mitochondrial dysfunction has been linked to ALS and the toxicity of ALS-astrocytes, we seek to better define the role of NAD-dependent signaling in motor neuron degeneration and determine if the modulation of NAD levels may be a potential therapeutic strategy for ALS. Our ongoing experiments demonstrate that increasing NAD content in ALS-astrocytes reverts its toxicity towards co-cultured motor neurons, while NAD synthesis and NAD-dependent signaling may be compromised in mutant hSOD1 mice.
Thus, the specific aims of the proposal are: Aim 1-To determine the role of NAD content in the toxicity of astrocytes expressing ALS-linked mutant hSOD1s toward co-cultured motor neurons. Aim 2-To evaluate the effect of transgenic models with altered NAD synthesis and degradation on the onset and progression of the disease in ALS mouse models. Aim 3-To evaluate the effect of treatment with a key NAD precursor on the onset and progression of the disease in ALS mouse models.
The outcome of the proposal will contribute to the current understanding of NAD metabolism and mitochondrial dysfunction in neurodegeneration. More important, since we have shown that therapeutic targets identified in our astrocyte-motor neuron co-culture system have a beneficial effect when translated into animal models of ALS, the proposal is likely to provide in vivo proof of the value of modulating NAD metabolism as a therapeutic target in ALS.
COBRE Project: Mitochondrial antioxidant status and mitochondrial protein lysine acetylation in the central nervous system
Abstract: Numerous lines of evidence suggest that mitochondria have a central role in aging and age-related neurodegenerative diseases. However, a major obstacle for the development of new therapies is our inadequate knowledge of basic mitochondrial biology of neurons and glial cells and its contribution to neurodegenerative conditions. Although reversible Nε-lysine acetylation as a mean of regulating protein function is well characterized for other cellular compartments, lysine acetylation of mitochondrial proteins has only recently been described. Sirtuins are a highly conserved family of proteins capable of catalyzing NAD-dependent deacylation and mono(ADPribosyl)ation reactions. More than 20% of the mitochondrial proteins are subject to reversible acetylation in the ε-amino group of lysine residues and this process modulates their function and activity. SIRT3-mediated deacetylation has been shown to modulate all major mitochondrial processes, including the tricarboxylic acid cycle, fatty acid metabolism, oxidative phosphorylation, and antioxidant response. In addition, SIRT3 has been recently shown to control the levels of mitochondrial reactive oxygen species (ROS) by multiple mechanisms. Therefore, availability of NAD can have a profound impact on mitochondrial function. This proposal intends to investigate how changes in mitochondrial antioxidant status affect protein lysine acetylation in the mitochondria of astrocytes and neurons. To investigate the correlation between mitochondrial antioxidant status and mitochondrial protein lysine acetylation in the central nervous system we are using knockout mice for the glutamate-cysteine ligase modifier subunit (GCLM) and mice that overexpress a catalase targeted to the mitochondria (MCAT). These models allow us to compare changes in the acetylated lysine subproteome in response to decreased antioxidant defenses [GCLM(-/-)] or increased mitochondrial peroxide detoxification (MCAT). In addition, we are using new and established methods to manipulate NAD levels in amyotrophic lateral sclerosis models to better define the role of NAD-dependent signaling in motor neuron degeneration and determine if modulation of NAD levels may be a potential therapeutic strategy for amyotrophic lateral sclerosis.
James Chou, PhD
Awarded RO1: Novel lysine deacetylase 6 Hsp domain inhibitors against AML (1R01CA163452-01A1)
Acute myelogenous leukemia (AML) is one of the most common and aggressive forms of acute leukemia affecting 30,000 people per year. Greater than 5 year survival rate still remains around 10-30%, and this depends greatly on the patient’s ability to tolerate the combination of cytotoxic chemotherapies, which suppresses much needed haemopoiesis. The limits of current treatment modalities indicate a need for innovative therapies directed against relevant biological targets in AML to improve the clinical outcome. AML is a heterogeneous disease; recent studies have identified a set of activating kinase mutations in FLT-3, c-Kit, and Ras and constitutively active transcription factors such as STAT5 and chimera MLL relevant to disease outcome. One of the major challenges in treating AML is developing therapies that are capable of affecting multiple biological pathways promoting AML proliferation and survival. Histone deacetylase isozyme 6 (HDAC6) is over-expressed in AML patients and is hypothesized to play a key role in maintaining oncogenic signaling through the regulation of heat-shock protein functions that are critical for AML pathogenesis and survival. HDAC6 is also required for malignant cell transformation both in transformed cells and in vivo. Moreover, HDAC6 knock-out mice have been shown to resist mutagen induced tumors and develop normally, which further underscore the potential of HDAC6 as a better tolerated and less toxic therapeutic target for AML. We have identified novel HDAC6-Hsp domain inhibitors, which preferentially inhibit the HDAC6-Hsp deacetylation domain at low µM concentration and induce Hsp90 acetylation, unlike the canonical hydroxamate HDAC6 inhibitors, tubastatin A and Tubacin. Our overarching hypothesis is that HDAC6-Hsp domain activity promotes AML proliferation and survival by maintaining proper function of multiple heat-shock proteins. Selective HDAC6-Hsp domain inhibition deactivates multiple Hsp activities, attenuates aberrant AML oncogenic signaling, and promotes AML apoptosis. This proposal intends to use multifaceted and innovative approaches to develop novel HDAC6-Hsp domain inhibitors and to investigate the role that HDAC6 plays in the regulation of Hsp activities and apoptosis initiation in AML. In Aim 1, we will refine our HDAC6-Hsp inhibitors through a phylogenetic library synthesis in order to characterize the structure activity relationship of the HDAC6-Hsp domain. In Aim 2, we will investigate the HDAC6-Hsp pathway axis, study HDAC6 dependent Hsp70 regulation, and the roles of Hsp70 acetylation play in apoptosome formation. Finally, we will examine our HDAC6-Hsp deacetylation domain inhibitors in vivo using a physiologically relevant disseminated AML xenograft model. We will determine the efficacy of our lead HDAC6 inhibitor candidates against AML, performing pharmacokinetic and dynamic studies on lead inhibitor candidates, and validate the inhibitor’s ability to influence biomarkers in vivo.
Public Health Relevance Statement:
Acute myelogenous leukemia (AML) is one of the most common and aggressive forms of acute leukemia affecting 30,000 people annually. With an expected increase in American life expectancy, the numbers of AML cases are also expected to increase, as the majority of AML patients are over the age of 60. Little has changed in the standard AML therapies for the last 30 years, and treatments with low cytotoxicity and high therapeutic windows are highly desirable. By studying molecular mechanisms of HDAC6-Hsp domain inhibition and induction of selective AML apoptosis, the development of novel HDAC-Hsp domain inhibitors against AML can be a significant advancement from the current treatment disadvantages.
2011 - 2013
COBRE Project: Novel AML therapy targeting HDAC6 and Hsp90 Chaperone Complex
Abstract: Acute myelogenous leukemia (AML) is one of the most common and aggressive forms of acute leukemia affecting 30,000 people per year. Survival greater than 5 years still remains around 10-30%, and this depends greatly on the patient’s ability to tolerate the combination of cytotoxic chemotherapies, which suppress much needed haemopoiesis. The limits of current treatment modalities indicate a need for innovative therapies directed against relevant biological targets in AML to improve the clinical outcome in AML. Heat-shock protein 90 (Hsp90) and histone deacetylase isozyme 6 (HDAC6) are required for the maintenance of AML oncogenic signaling and stability of oncogenic chimera transcription factors. Hsp90 and HDAC6 are constitutively over-expressed in AML patient blood mononucleocytes and bone marrows. HDAC6 is also required for malignant cell transformation both in transformed cells and in vivo. HDAC6/Hsp90 complex/chaperone activity requires active HDAC6, and inhibition of HDAC6 destabilizes Hsp90 complex and abolish its chaperone function. We have identified a novel class of selective HDAC6/Hsp90 inhibitors, which preferentially inhibit HDAC6 at low µM concentrations and rapidly induce inactive acetylated Hsp90 thereby resulting in Hsp90 client protein depletion. Our overarching hypothesis is that a functional HDAC6/Hsp90 complex is an essential part of AML pathogenesis. The proposal intends to use multifaceted and innovative approaches to develop novel HDAC6/Hsp90 complex inhibitors and to investigate the roles of HDAC6/Hsp90 complex in AML.
Scott Eblen, Ph.D.
Jennifer Isaacs, Ph.D.
Carola Neumann, M.D.