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.