Researchers have identified a drug that resensitizes cancer cells to proteasome inhibitors

Proteasome modulates DA release. The present figure roughly summarizes the roles of UPS in DA release during physiological conditions (A) and following METH (B). In baseline conditions (A), the UPS restrains DA synthesis by degrading TH enzyme. Once filled with DA, SVs dock and prime to the active zone where SNARE complex assembly together with Munc13-1 and RIM-1 foster DA release via Ca2+ driven-exocytosis. The UPS restrains DA release by ubiquitinating and targeting SNARE (VAMP, Syntaxin-1, SNAP-25) as well as Munc13-1 and RIM-1 for endosomal internalization and degradation. This occurs either via direct degradation by the UPS or by the ATG-lysosomal machinery after fusion with endosomes. Such a mechanism is seminal to limit the rapid endosomal recycling of these proteins back to the plasma membrane, which would otherwise favor subsequent rounds of DA release. In these conditions, post-synaptic DRs (DRD1-like and DRD2-like) are physiologically stimulated. At the post-synaptic side, the UPS is functionally operating to degrade these same DRs. Administration of METH disassembles UPS and inhibits its activity (B). This contributes to raising the level of newly synthesized DA. In turn, increased levels of intracellular DA fuel UPS dysfunction. Thus, the UPS cannot provide degradation of SNAREs, Munc13-1 and RIM1, which recycle back to the plasma membrane to prevent de-priming of SVs and boost DA release. This is further amplified by the increased Ca2+ influx, which occurs when UPS is inhibited. Again, under the effects of METH and UPS inhibition, the direction of the DAT is reversed and its degradation is occluded. In turn, increased levels of Syntaxin-1, which binds to the DAT at the plasma membrane, contribute to strengthening DA release via both exocytosis and efflux. These effects translate into peaks of extracellular DA concentration, which over-stimulate postsynaptic DRs. At this level, abnormal stimulation of DRD1 impairs UPS, which leads to occlude degradation of DRs.

The cells of most patients’ cancers are resistant to a class of drugs, called proteasome inhibitors, that should kill them.

When studied in the lab, these drugs are highly effective, yet hundreds of clinical trials testing proteasome inhibitors have failed.

Now scientists may have solved the mystery of these cells’ surprising hardiness.

The key: Resistant cancer cells have shifted how and where they generate their energy.

Using this new insight, researchers have identified a drug that resensitizes cancer cells to proteasome inhibitors and pinpointed a gene that is crucial for that susceptibility.

As cancer cells develop, they accrue multiple genetic alterations that allow the cells to quickly reproduce, spread and survive in distant parts of the body, and recruit surrounding cells and tissues to support the growing tumor.

To perform these functions, cancer cells must produce high volumes of the proteins that support these processes.

The increased protein production and numerous mutated proteins of cancer cells make them particularly dependent on the proteasome, which is the cell’s protein degradation machine.

These huge protein complexes act as recycling machines, gobbling up unwanted proteins and dicing them into their amino acid building blocks, which can be reused for the production of other proteins.

Previously, researchers exploited cancer cells’ increased dependency on their proteasomes to develop anti-cancer therapies that inhibit the proteasomes’ function.

The importance of telomerase, the enzyme that maintains telomere length, has been reported in many malignancies in general and in multiple myeloma (MM) in particular. Proteasome inhibitors are clinically used to combat effectively MM.

Since the mechanism of action of proteasome inhibitors has not been fully described we sought to clarify its potential effect on telomerase activity (TA) in MM cells.

Previously we showed that the first generation proteasome inhibitor bortezomib (Brt) inhibits TA in MM cells by both transcriptional and post-translational mechanisms and has a potential clinical significance.

In the current study we focused around the anti- telomerase activity of the new generation of proteasome inhibitors, epoxomicin (EP) and MG-132 in order to clarify whether telomerase inhibition represents a class effect.

We have exposed MM cell lines, ARP-1, CAG, RPMI 8226 and U266 to EP or MG and the following parameters were assessed: viability; TA, hTERT expression, the binding of hTERT (human telomerase reverse transcriptase) transcription factors and post-translational modifications.

Epoxomicin and MG-132 differentially downregulated the proliferation and TA in all MM cell lines. The downregulation of TA and the expression of hTERT were faster in CAG than in ARP-1 cells. Epoxomicin was more potent than MG-132 and therefore further mechanistic studies were performed using this compound.

The inhibition of TA was mainly transcriptionally regulated. The binding of three positive regulator transcription factors: SP1, c-Myc and NF-κB to the hTERT promoter was decreased by EP in CAG cells as well as their total cellular expression.

In ARP-1 cells the SP1 and c-MYC binding and protein levels were similarly affected by EP while NF-κB was not affected. Interestingly, the transcription factor WT-1 (Wilms’ tumor-1) exhibited an increased binding to the hTERT promoter while its total cellular amount remained unchanged.

Our results combined with our previous study of bortezomib define telomerase as a general target for proteasome inhibitors.

The inhibitory effect of TA is exerted by several regulatory levels, transcriptional and post translational. SP1, C-Myc and NF-κB were involved in mediating these effects. A novel finding of this study is the role of WT-1 in the regulation of telomerase which appears as a negative regulator of hTERT expression.

The results of this study may contribute to future development of telomerase inhibition as a therapeutic modality in MM.


Several distinct proteasome inhibitors have been developed, and when used in the lab, these proteasome inhibitor drugs are indeed highly effective at eradicating tumor cells.

However, when administered to animal models or patients with cancer, such as multiple myeloma, proteasome inhibitors have limited efficacy and even initially vulnerable cancer cells quickly develop resistance to them.

How do cancer cells so adroitly sidestep drugs that should kill them?

Exploring the gene expression of thousands of tumors and hundreds of cancer cell lines, Peter Tsvetkov, a former postdoc in the lab of late Whitehead Institute Member Susan Lindquist, has determined that the answer may lie with how and where the cells produce their energy.

According to his analysis of the active genes and metabolism products generated in proteasome inhibitor-resistant cancer cells and tumors, Tsvetkov, who is currently a postdoc in the lab of Broad Institute Founding Core Member, director of the Cancer Program, and oncologist at the Dana-Farber Cancer Institute Todd Golub, concluded that such cells have shifted how they produce energy—away from breaking down the sugar glucose toward a dependency on processes within the mitochondria, the “powerhouse” part in the cell.

In fact, when Tsvetkov pushed cancer cells’ metabolism to depend on the mitochondria, that change alone was sufficient to make cancer cells immune to proteasome inhibitors. Tsvetkov’s findings are described online this week in the journal Nature Chemical Biology.

In order to understand how a metabolic shift could link to proteasome inhibitor resistance, Tsvetkov screened proteasome inhibitor-resistant breast cancer cells with thousands of small molecules to identify the ones that hamper the cells’ growth or even kill the cells.

One stood out in the screen: elesclomol, a small molecule that that researchers had previously evaluated as an anti-cancer agent in phase 3 clinical trials without knowing with what the drug interacts in cancer cells.

To identify how elesclomol preferentially targets the proteasome inhibitor resistant cells, Tsvetkov did genome-wide CRISPR-Cas9 screens to find out which genes elesclomol requires to incapacitate resistant cancer cells.

Only the gene FDX1, which encodes an enzyme in the mitochondria, came to the fore.

In collaboration with John Markley from the Department of Biochemistry at the University of Wisconsin-Madison, Tsvetkov used biochemical and biophysical systems to demonstrate that elesclomol directly binds to the mitochondrial enzyme FDX1 and impedes its natural function.

In the presence of copper, elesclomol can also be altered by the FDX1 enzyme, which increases the drug’s anti-cancer toxicity.

These findings led the researchers to determine that as cancer cells become overly reliant on their mitochondrial metabolism, they ramp up the FDX1 protein’s activity.

Also, when the FDX1 protein interacts with copper-bound elesclomol, the protein enhances the drug’s copper-dependent toxicity. Thus, copper appears to play an essential role – when Tsvetkov removed copper, elesclomol was no longer effective.

Having established that a metabolic shift and resistance to proteasome inhibitors are linked, Tsvetkov is now interested in understanding how a change in metabolism allows cancer cells to adapt to other anti-cancer therapies and how copper-binding molecules such as elesclomol can be developed as effective anticancer agents.

More information: Peter Tsvetkov et al. Mitochondrial metabolism promotes adaptation to proteotoxic stress, Nature Chemical Biology (2019). DOI: 10.1038/s41589-019-0291-9

Journal information: Nature Chemical Biology
Provided by Whitehead Institute for Biomedical Research


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