In a previous study on elesclomol's mechanism of action 
, the drug was shown to accomplish its cancer killing activity via the induction of untenable levels of intracellular ROS followed by apoptosis. The cellular mechanism and target by which this occurred, however, remained unknown. In those studies, mitochondrial involvement was specifically ruled out on the basis of using isolated mitochondria. However these experiments were performed in the absence of copper rendering them uninformative. In this study, with the importance of copper newly realized 
, we have used an in vivo
yeast system and additional in vitro
human cell studies to identify the mitochondrion as the source of elesclomol-induced ROS and strongly implicate the process of the electron transport as the “target” of the drug.
As we have shown for other cancer drugs 
, the approach of using the yeast deletion collection yielded an accurate indication of elesclomol's mechanism of action in mammalian cancer cells. This is predicated on the similarities of the cellular responses in these different eukaryotic cells, which we found to be the case. Our results showed that both yeast and human cells require copper for elesclomol activity, induce ROS to high levels when sufficient drug is present, and succumb to cell death upon relatively short elesclomol treatment. We also reveal the importance of an active ETC in both systems.
Our data indicate the lack of a unique protein “target” of elesclomol. While the analysis of the heterozygous deletions identified a single sensitive strain, nus1
, its sensitivity was modest compared to other drug-target strain combinations we have analyzed in the past 
, and it is therefore unlikely that the interaction of elesclomol and NUS1p, if any, is responsible for the primary cytotoxic activity of elesclomol in the cell.
In contrast, the analysis of the homozygous deletion set identified a robust and biologically coherent set of activities associated with mitochondrial activities. Both manual and computational (GSEA) analyses identified overlapping classes of genes involved in various elements of electron transport, mitochondrial translation (including mitochondrial ribosome subunits, translation factors, tRNAs, and mRNA splicing enzymes), mitochondrial copper availability and homeostasis, and genes involved in stress responses, particularly oxidative stress. Importantly, equally sensitizing mutations were found distributed throughout the ETC or its associated processes. Sensitive strains containing mutations affecting individual subunits of the various ETC complexes were identified, as were components required for the modification or assembly of the complexes. Given that each of the complexes contain numerous subunits, it is likely that most of these mutations would not completely abrogate ETC function, but more likely, only partially interfere with its activity. A similar argument can be made for the sensitizing mutations affecting mitochondrial translation, which produces a minority of the proteins required for ETC function. Individually, these mutations would likely only have a small effect on the overall translation capability in the organelle, again perturbing but not eliminating ETC activity. Thus, it appears that modulating the electron flow at any of numerous points along the ETC can lead to enhanced elesclomol impact on the cell and therefore, we conclude that it is the disruption of the process of electron flow down the ETC, rather than disruption of a particular protein or activity, that is of relevance.
This effect on cell viability via ETC disruption appears to operate similarly in human cells. In the data presented in , we used a second drug in combination with elesclomol rather that a potentially sensitizing gene disruption and obtained analogous results. Co-treatment of melanoma cells with elesclomol-Cu and either of two ETC inhibitors enhanced cytotoxicity. Again, the modulation of the process, rather than the inhibition of a particular protein, seems paramount for increased elesclomol activity.
The primary cytotoxic effect of elesclomol appears to be confined to the mitochondria and not to involve a cytoplasmic component. For example, none of the genes normally involved in the response to cytoplasmic oxidative stress were identified in the screen. This includes the YAP1 gene, which encodes the transcription factor that is the primary responder to oxidative stress in the cytoplasm and drives the up-regulation of a battery of stress response genes. Deletion of another prominent cytoplasmic stress response protein, superoxide dismutase 1 (SOD1p), actually provided slight resistance to the elesclomol treatment. Deletion of the copper chaperone protein CCS1p, required for SOD1p activity, also provided mild resistance, thereby confirming the result. In striking contrast, deletion of SOD2, the mitochondrial superoxide dismutase, was highly sensitizing.
Elesclomol is thought to kill cells by inducing ROS to levels from which the cell cannot recover. Given that the principal site of action for elesclomol is likely the respiring mitochondrion, we undertook experiments utilizing human cells lacking their mitochondrial genomic DNA to examine ROS production and cytotoxicity of the drug in cells that cannot undergo oxidative phosphorylation. The human mitochondrial genome encodes 13 proteins, all of which are subunits of the various ETC complexes. The absence of all 13 subunits, as is the case in the melanoma cells used here (HBL-ρ0), ablates ETC function. These cells, when treated with elesclomol-Cu, failed to induce ROS or apoptosis. The parental cell line, with its ETC functions intact, responded with potent ROS induction and cell death. Thus, elesclomol-mediated ROS production and apoptosis are tightly linked, confirming previous analyses 
. With the current studies, we can now propose the origin of this cascade is in the mitochondrion.
The mitochondrion is the major site for ROS production in normal cells as well. Complexes I and III are prone to electron leakage, leading to the production of highly toxic superoxide or hydroxyl radicals in the vicinity of the ETC 
. Under most conditions these free radicals are kept in check by the anti-oxidant systems in the organelle. However, this basal level of electron leakage can be amplified by inhibitors of electron chain complexes, such as rotenone (complex I), antimycin A (complex III) or cyanide (complex IV), leading to decreased viability 
. Similarly, the impact of elesclomol-Cu appears to overwhelm the oxidative stress response systems, allowing cytotoxic levels of ROS to accumulate. When the ETC inhibitors and elesclomol are used together during treatment, a degree of synergy is observed indicating that the combination of these drugs augments their effects in the cell as compared to monotherapy.
How might elesclomol-Cu instigate this lethal increase of ROS via its interaction with the ETC? A major clue comes from elesclomol's requirement for copper for its activity. Copper binds to elesclomol in the Cu(II) state. In the cell, elesclomol-Cu can undergo a redox reaction with copper being reduced to the Cu(I) state. By itself, this reaction could produce free radicals by a Fenton reaction. The redox potential for this reaction is −330 mV 
and this potential appears critical for elesclomol activity. Analysis of analogs with similar structures but with different potentials has shown that only those compounds with potentials similar to elesclomol-Cu are cytotoxic 
. Very interestingly, this potential is well aligned with the potential drops along the ETC 
Considering all of these features, there appears to be at least three major avenues by which elesclomol-Cu could lead to heightened levels of ROS. The drug could generate ROS on its own via its copper-based redox chemistry (perhaps using electrons or redox potential “stolen” from the ETC). Alternatively, the drug could interfere with the electron flow along the ETC, leading to elevated levels of electron leakage and free radical formation normally seen in cells, but here at levels that overwhelm the cell's defense systems. Finally, elesclomol-Cu could specifically interfere with copper-requiring events associated with ETC function. Some of the complexes are comprised of subunit proteins that require Cu for their activity and their assembly depends on specific copper chaperone proteins. Elesclomol could compete for or interfere with these processes, thereby impacting electron flow down the chain. These mechanisms are not mutually exclusive and, in fact, more than one may come into play sequentially: the initial impact of elesclomol-Cu could alter the subsequent dynamics of the ETC allowing additional mechanisms to take place that ultimately result in apoptosis. Whichever mechanism is used, we expect that the match of the redox potentials within the ETC to that of elesclomol-Cu is an important driving force.
The ability of elesclomol treatment to quickly lead to cell death, and not just cell arrest, is an important feature of the drug. Drugs that cause cidality are relatively uncommon in yeast, with fewer than 10% of 10,000 drugs that inhibit growth inducing cidality 
. Both yeast and human cells exposed to elesclomol-Cu for a few hours or less ( and ) are destined to die. The ability to kill a cell exposed briefly to the drug is a valuable property for an anticancer agent.
Finally, the improved mechanistic understanding of elesclomol's activity provided by this report has important implications for its therapeutic application in oncology. Specifically, lactate dehydrogenase (LDH) has been identified as a potential biomarker predictive of response in the clinical evaluation of elesclomol. In a Phase 3 trial of elesclomol in combination with paclitaxel, the primary endpoint of progression free survival was achieved in metastatic melanoma patients exhibiting low and normal LDH levels in their bloodstream, with a significant improvement in median progression free survival time. Conversely there was no benefit in the elevated LDH population 
. High serum levels of LDH are thought to reflect a tumor burden with increased reliance on glycolysis for its metabolic needs 
. Conversely, patients with lower LDH levels should have tumor burdens that are more reliant on oxidative phosphorylation, a situation we have shown here to be more sensitive to elesclomol treatment. Thus, the insights established here by our studies on yeast and human cells provide critical understanding into the clinical activity of the drug. It also offers a compelling rationale for a biomarker-based prioritization of patients likely to respond to elesclomol treatment.