19 research outputs found
Mitochondrial Electron Transport Is the Cellular Target of the Oncology Drug Elesclomol
Elesclomol is a first-in-class investigational drug currently undergoing clinical evaluation as a novel cancer therapeutic. The potent antitumor activity of the compound results from the elevation of reactive oxygen species (ROS) and oxidative stress to levels incompatible with cellular survival. However, the molecular target(s) and mechanism by which elesclomol generates ROS and subsequent cell death were previously undefined. The cellular cytotoxicity of elesclomol in the yeast S. cerevisiae appears to occur by a mechanism similar, if not identical, to that in cancer cells. Accordingly, here we used a powerful and validated technology only available in yeast that provides critical insights into the mechanism of action, targets and processes that are disrupted by drug treatment. Using this approach we show that elesclomol does not work through a specific cellular protein target. Instead, it targets a biologically coherent set of processes occurring in the mitochondrion. Specifically, the results indicate that elesclomol, driven by its redox chemistry, interacts with the electron transport chain (ETC) to generate high levels of ROS within the organelle and consequently cell death. Additional experiments in melanoma cells involving drug treatments or cells lacking ETC function confirm that the drug works similarly in human cancer cells. This deeper understanding of elesclomol's mode of action has important implications for the therapeutic application of the drug, including providing a rationale for biomarker-based stratification of patients likely to respond in the clinical setting
A comprehensive platform for highly multiplexed mammalian functional genetic screens
<p>Abstract</p> <p>Background</p> <p>Genome-wide screening in human and mouse cells using RNA interference and open reading frame over-expression libraries is rapidly becoming a viable experimental approach for many research labs. There are a variety of gene expression modulation libraries commercially available, however, detailed and validated protocols as well as the reagents necessary for deconvolving genome-scale gene screens using these libraries are lacking. As a solution, we designed a comprehensive platform for highly multiplexed functional genetic screens in human, mouse and yeast cells using popular, commercially available gene modulation libraries. The Gene Modulation Array Platform (GMAP) is a single microarray-based detection solution for deconvolution of loss and gain-of-function pooled screens.</p> <p>Results</p> <p>Experiments with specially constructed lentiviral-based plasmid pools containing ~78,000 shRNAs demonstrated that the GMAP is capable of deconvolving genome-wide shRNA "dropout" screens. Further experiments with a larger, ~90,000 shRNA pool demonstrate that equivalent results are obtained from plasmid pools and from genomic DNA derived from lentivirus infected cells. Parallel testing of large shRNA pools using GMAP and next-generation sequencing methods revealed that the two methods provide valid and complementary approaches to deconvolution of genome-wide shRNA screens. Additional experiments demonstrated that GMAP is equivalent to similar microarray-based products when used for deconvolution of open reading frame over-expression screens.</p> <p>Conclusion</p> <p>Herein, we demonstrate four major applications for the GMAP resource, including deconvolution of pooled RNAi screens in cells with at least 90,000 distinct shRNAs. We also provide detailed methodologies for pooled shRNA screen readout using GMAP and compare next-generation sequencing to GMAP (i.e. microarray) based deconvolution methods.</p
Development and Application of High-throughput Chemical Genomic Screens for Functional Studies of Cancer Therapeutics
Chemotherapeutic agents act by targeting rapidly dividing cancer cells. The full extent of their cellular mechanisms, which is essential to balance efficacy and toxicity, is often unclear. In addition, the use of many anticancer drugs is limited by dose-limiting toxicities as well as the development of drug resistance. The work presented in this thesis aims to address the basic biology that underlies these issues through the development and application of chemical genomic tools to probe mechanisms of current and novel anticancer compounds. Chemical genomic screens in the yeast Saccharomyces cerevisiae have been used to successfully identify targets and pathways related to a compound’s mode of action. I applied these screens to examine the mode of action of potential anticancer drugs: a class of platinum-acridine compounds and the apoptosis-inducing compound elesclomol. By analogy to the yeast screens, I developed an RNAi-mediated chemical genomic screen in human cells which has the potential to reveal novel targets and drug mechanisms. This screen was applied to further understand doxorubicin’s mode of action. In parallel with the loss-of-function assays, our lab developed a human ORF overexpression screen in human cells. I applied this gain-of-function screen to identify those genes that, when overexpressed, are toxic to cells. Characterization of such genes that cause toxicity can provide insight into human diseases where gene amplification is prevalent.Ph
A Novel Small Molecule Methyltransferase Is Important for Virulence in <i>Candida albicans</i>
<i>Candida albicans</i> is an opportunistic pathogen
capable of causing life-threatening infections in immunocompromised
individuals. Despite its significant health impact, our understanding
of <i>C. albicans</i> pathogenicity is limited, particularly
at the molecular level. One of the largely understudied enzyme families
in <i>C. albicans</i> are small molecule AdoMet-dependent
methyltransferases (smMTases), which are important for maintenance
of cellular homeostasis by clearing toxic chemicals, generating novel
cellular intermediates, and regulating intra- and interspecies interactions.
In this study, we demonstrated that <i>C</i>. <i>albicans</i> Crg1 (CaCrg1) is a <i>bona fide</i> smMTase that interacts
with the toxin <i>in vitro</i> and <i>in vivo.</i> We report that CaCrg1 is important for virulence-related processes
such as adhesion, hyphal elongation, and membrane trafficking. Biochemical
and genetic analyses showed that CaCrg1 plays a role in the complex
sphingolipid pathway: it binds to exogenous short-chain ceramides <i>in vitro</i> and interacts genetically with genes of glucosylceramide
pathway, and the deletion of <i>CaCRG1</i> leads to significant
changes in the abundance of phytoceramides. Finally we found that
this novel lipid-related smMTase is required for virulence in the
waxmoth <i>Galleria mellonella</i>, a model of infection
Comparative Chemogenomics To Examine the Mechanism of Action of DNA-Targeted Platinum-Acridine Anticancer Agents
Platinum-based drugs have been used to successfully treat
diverse
cancers for several decades. Cisplatin, the original compound of this
class, cross-links DNA, resulting in cell cycle arrest and cell death <i>via</i> apoptosis. Cisplatin is effective against several tumor
types, yet it exhibits toxic side effects and tumors often develop
resistance. To mitigate these liabilities while maintaining potency,
we generated a library of non-classical platinum-acridine hybrid agents
and assessed their mechanisms of action using a validated genome-wide
screening approach in <i>Saccharomyces cerevisiae</i> and
in the distantly related yeast <i>Schizosaccharomyces pombe.</i> Chemogenomic profiles from both <i>S. cerevisiae</i> and <i>S. pombe</i> demonstrate that several of the platinum-acridines
damage DNA differently than cisplatin based on their requirement for
distinct modules of DNA repair
Comparative Chemogenomics To Examine the Mechanism of Action of DNA-Targeted Platinum-Acridine Anticancer Agents
Platinum-based drugs have been used to successfully treat
diverse
cancers for several decades. Cisplatin, the original compound of this
class, cross-links DNA, resulting in cell cycle arrest and cell death <i>via</i> apoptosis. Cisplatin is effective against several tumor
types, yet it exhibits toxic side effects and tumors often develop
resistance. To mitigate these liabilities while maintaining potency,
we generated a library of non-classical platinum-acridine hybrid agents
and assessed their mechanisms of action using a validated genome-wide
screening approach in <i>Saccharomyces cerevisiae</i> and
in the distantly related yeast <i>Schizosaccharomyces pombe.</i> Chemogenomic profiles from both <i>S. cerevisiae</i> and <i>S. pombe</i> demonstrate that several of the platinum-acridines
damage DNA differently than cisplatin based on their requirement for
distinct modules of DNA repair
Comparative Chemogenomics To Examine the Mechanism of Action of DNA-Targeted Platinum-Acridine Anticancer Agents
Platinum-based drugs have been used to successfully treat
diverse
cancers for several decades. Cisplatin, the original compound of this
class, cross-links DNA, resulting in cell cycle arrest and cell death <i>via</i> apoptosis. Cisplatin is effective against several tumor
types, yet it exhibits toxic side effects and tumors often develop
resistance. To mitigate these liabilities while maintaining potency,
we generated a library of non-classical platinum-acridine hybrid agents
and assessed their mechanisms of action using a validated genome-wide
screening approach in <i>Saccharomyces cerevisiae</i> and
in the distantly related yeast <i>Schizosaccharomyces pombe.</i> Chemogenomic profiles from both <i>S. cerevisiae</i> and <i>S. pombe</i> demonstrate that several of the platinum-acridines
damage DNA differently than cisplatin based on their requirement for
distinct modules of DNA repair
Comparative Chemogenomics To Examine the Mechanism of Action of DNA-Targeted Platinum-Acridine Anticancer Agents
Platinum-based drugs have been used to successfully treat
diverse
cancers for several decades. Cisplatin, the original compound of this
class, cross-links DNA, resulting in cell cycle arrest and cell death <i>via</i> apoptosis. Cisplatin is effective against several tumor
types, yet it exhibits toxic side effects and tumors often develop
resistance. To mitigate these liabilities while maintaining potency,
we generated a library of non-classical platinum-acridine hybrid agents
and assessed their mechanisms of action using a validated genome-wide
screening approach in <i>Saccharomyces cerevisiae</i> and
in the distantly related yeast <i>Schizosaccharomyces pombe.</i> Chemogenomic profiles from both <i>S. cerevisiae</i> and <i>S. pombe</i> demonstrate that several of the platinum-acridines
damage DNA differently than cisplatin based on their requirement for
distinct modules of DNA repair
Comparative Chemogenomics To Examine the Mechanism of Action of DNA-Targeted Platinum-Acridine Anticancer Agents
Platinum-based drugs have been used to successfully treat
diverse
cancers for several decades. Cisplatin, the original compound of this
class, cross-links DNA, resulting in cell cycle arrest and cell death <i>via</i> apoptosis. Cisplatin is effective against several tumor
types, yet it exhibits toxic side effects and tumors often develop
resistance. To mitigate these liabilities while maintaining potency,
we generated a library of non-classical platinum-acridine hybrid agents
and assessed their mechanisms of action using a validated genome-wide
screening approach in <i>Saccharomyces cerevisiae</i> and
in the distantly related yeast <i>Schizosaccharomyces pombe.</i> Chemogenomic profiles from both <i>S. cerevisiae</i> and <i>S. pombe</i> demonstrate that several of the platinum-acridines
damage DNA differently than cisplatin based on their requirement for
distinct modules of DNA repair
Elesclomol-induced ROS generation and cytoxicity in yeast is dependent on the presence of copper.
<p>(A) Chemical structure of the elesclomol-Cu complex. (B) The parental BY4743 yeast strain was grown in the presence of the indicated concentrations of elesclomol, preformed elesclomol-Cu, and/or copper for 21.5 h at 30°C. Absorbance at 600 nm was used to determine cell density. (C) Flow cytometric analysis measuring ROS in <i>S. cerevisiae</i>. ROS induction, as measured by Dihydrorhodamine 123 fluorescence, was only observed with preformed elesclomol-Cu complex at a concentration (500 nM) above the MIC but not below (100 nM), nor with free elesclomol at either concentration (<i>upper panel</i>). The addition of supplementary copper (via CuCl<sub>2</sub>) to elesclomol was sufficient to induce ROS, again only at the higher concentration (<i>lower panel</i>). (D) Elesclomol is cidal to yeast cells within an hour of treatment. Logarithmically growing cells were incubated with the indicated doses of elesclomol for 1, 2 or 4 h and then plated onto media without elesclomol. 5 µM and 22.5 µM elesclomol rendered cells unviable within 1 h, and lower doses (1.25 µM) killed cells within 4 h.</p