Monday, July 21, 2008

The curious case of the proteasome inhibitor Argyrin A

A burning set of questions in my old shop when I was there, and I have every reason to think is still aflame, is why does Velcade work in some tumors but not others and how could you predict which tumors it will work in. Does the sensitivity of myelomas & certain lymphomas generally (and a seemingly random scatter of solid tumor examples) to proteasome inhibition follow a pattern? And is this pattern a reflection of the inner workings of these cells or more how the drug is distributed throughout the body?

An even broader burning question is whether any other proteasome inhibitor would behave differently at either level. Would a more potent inhibitor of the proteasome have a different spectrum of tumors which it hit?

Now, while Velcade (bortezomib, fka PS) is the only proteasome inhibitor on the market, it will probably not always be that. Indeed, since Velcade has proven the therapeutic utility of proteasome inhibition, other companies and academics have been exploring proteasome inhibitors. The most advanced that I am aware of is a natural product being developed by Nereus Pharmaceuticals, which I will freely confess to not really following.

The featured (and therefore free!) article in July's Cancer Cell describes a new proteasome inhibitor, another natural product. Argyrin A was identified in a screen for compounds which stabilize p27Kip1, an important negative regulator of the cell cycle. Kip1 is one of the a host of proteins reported to be an important protein stabilized by proteasome inhibition (one of duties back on Landsdowne Street was to catalog the literature on such candidates). While there are probably many ways to stabilize p27Kip1, what they reported on is this novel proteasome inhibitor.

By straightforward proteasome assays Argyrin A shows a very similar profile to Velcade. That is, the proteasome has multiple protease activities which can be chemically distinguished, and the pattern of inhibition by the two compounds is very similar. However, by a number of approaches they make the case that there are significant biological differences in the response to Velcade & Argyrin A.

Now there is a whole lot of data in this paper & I won't go into detail on most of it. But I will point out something a bit curious -- very curious. They performed transcriptional profiling (using Affymetrix chips) on samples treated with Velcade, Argyrin A, and siRNA vs an ensemble of proteasome subunits, each at different timepoints. In their analysis they saw lots of genes perturbed by Velcade but a very small set perturbed by Argyrin A and the siRNA. Specifically, they claim 10,500(!) "genes" (probably probesets) for Velcade vs 500 for Argyrin A. That's a huge fraction of the array moving!

Now, I'll confess things are a bit murky. Back at MLNM I would have had the right tools at my disposal & could quickly verify things; now I have to rely on my visual cortex & decaying memory. But when I browse through their lists of genes for Argyrin A in the supplementary data, I don't see a bunch of genes which are a distinct part of the proteasome inhibition signature. At MLNM, huge numbers of proteasome inhibition experiments were done & profiled on arrays, using a number of structurally unrelated proteasome inhibitors in many different cell lines. Not only does a consistent signal emerge, but when an independent group published a signature for proteasome inhibition in Drosophila there was a lot of overlap in their signature & our signature once you mapped the orthologs.

What's the explanation? Well, it could be that I'm not recognizing what is there due to poor memory, though I'm pretty sure. One thing that is worrisome is that the Argyrin A group's data is based on a single profile per drug x timepoint; there are no biological replicates. That's not uncommon due to the expense and challenge of microarray studies, but good experiments are easy. Nor was there any follow-up by another technology (e.g. RT-PCR) to show the effects across biological replicates or other cell lines. Given that these are in tissue culture cells, which can behave screwy if you stare at them the wrong way, that's very unfortunate. Even small differences in the culturing of the cells -- such as edge effects on plates or humidity differences, can lead to huge artifacts.

Another possible explanation is that the Bortezomib cells were watched too late; the first Velcade timepoint is at 14 hours. After 14 hours, the cells are decidedly unhealthy and heading for death. The right times to sample were always a point of contention, but one suggestion that there is an issue is the lack of correlation between the different timepoints for Velcade vs the strong correlation for the other treatments (Figure 7). That works (in my head at least) in reverse too -- it's downright odd that their other treatments are so auto-correlated between 14 and 48 hours with Argyrin A -- if cells are not yet dead at 14 hours but committed to die, one would expect there to be some sort of movement away from the original profile.

One other curiosity. They do report looking for the Unfolded Protein Response (UPR) and report seeing it in the Velcade treated cells but not Argyrin A treated ones. The UPR is the cell's response to misfolded proteins -- and since disposal of misfolded proteins is a role of the proteasome, it has never surprised anyone that the UPR is induced by proteasome inhibitors. Can you really have a proteasome inhibitor that doesn't induce the UPR? If this is truly the case, it is very striking and deserves its own study.

Is the paper wrong? Obviously I can't say, but I really wonder about it. I also wonder if the referees brought up the same questions. Hopefully we'll see some more papers in the future which explore this compound in a wider range of cell lines and with more biological replicates

Nickeleit et al
Argyrin a reveals a critical role for the tumor suppressor protein p27(kip1) in mediating antitumor activities in response to proteasome inhibition.
Cancer Cell. 2008 Jul 8;14(1):23-35.


Anonymous said...

"A burning set of questions ... is why does Velcade work in some tumors but not others and how could you predict which tumors it will work in"
Another burning question is why Velcade works at all. I have discussed this question with several people in the field (including Joe Bolen) and heard a wide variety of ideas, with prominent features of NFkB inhibition and cell cycle arrest.
Unfortunately, what you can see in mRNA profiling experiments are only ripples of the original effect. Nevertheless, we have also done our share of microarrays on proteasome-inhibited cells. I agree with you that there is something odd with this paper.

Anonymous said...

depends on cell type, lol
I'm reading this cancer cell paper now and I wanna try this in our cells

Anonymous said...


I am a novice at this proteasome stuff but I had just read article in Nature drug discovery(Sept 2008vol. 3 page 730-proteasome inhibitor unleases three pronged attack) about argyrin A and wondered how it related to Valcade(since Vlacade is a proteasome inhibitor). I googled argyrin a and saw your OMICS OMICS article and decided to read it. Great stuff. I look forward to further comments from you and Kay.Also, I recently read that MLNM had another potent proteasome inhibitor in the works-can you tell me more about it? I realize that Takeda purchased MLNM.



Anonymous said...

Have any of you considered the possibility that perhaps Velcade doesn't quite work via the mechanism you think it does? Setting aside a discussion of the quality and design of the microarray expts which will never be resolved unless we all go do some more experiments, just maybe Velcade is doing something more/something else besides simply inhibiting the 20S proteasome. Maybe what is odd in this paper is Velcade?? One of the biggest mistakes cellular pharmacologists make is in assuming that how a drug works in a patient is a cold hard fact...I didn't dig into the supp info but from the correlation analysis, i'd say the siRNA against the subunits and the argyrin look good, the outlier is velcade..

Keith Robison said...

Anonymous raises a very valid point -- perhaps it is Velcade acting strangely. Maybe we're seeing the effect of some other target being hit, or boron toxicity, or something else.

I can't rule it out. But my memory is that proteasome inhibitors of completely different structural classes (and non-boron containing) produce a signature consistent with Velcade. The Drosophila work was done with MG132, which is a peptide analog but not a boronic acid.

It is safe to say there is a muddle.

Could be an interesting class exercise -- there do appear to be a handful of experiments with MG132 in the public expression databases -- can you find a consistent signature for MG132 treatment & how does it coincide with signatures for other proteasome inhibitors such as argyrin A or inhibitors of similar pathways?

RDM said...

I wonder, with proteasome being so intricately involved in many signaling processes, regulation of apoptosis, transcription, translation, DNA replication, damage repair and so on, why it seems to be a pharmacological target for intervention. Perhaps it would be more wise to target something which triggers excessive proteasomal activity rather than targeting proteasome, because who knows what would be the long term effect of proteasome inhibition on sparing the normal cells from adverse impact of cancer treatment.

Keith Robison said...

Most existing chemotherapeutic agents target remarkably central processes -- it is crude but can be effective.

It isn't clear cancer cells have excessive proteasomal activity -- it just may be that they are more sensitive to down-modulating it.

MLNM's NAE inhibitor is an example of an attempt to be more specific by moving upstream of the proteasome and thereby hitting only a subset of proteasome activity.