I wrote here about a paper from Cold Spring Harbor labs that invalidated MELK as a cancer target. That was straightforward enough: knocking it out via CRISPR across a whole range of cancer cell lines had no effect on their growth at all, so it’s kind of hard to make the case that it’s an important thing to go after. At the same time, the paper showed that a MELK inhibitor with activity in cancer cells continued to show such activity after the target had been deleted.
Now the same team is back with an even larger look at the CRISPR-validation question, and it’s more food for thought. It’s also a look at the RNA-interference-validation question, because in that earlier work (and in this paper), the targets under discussion had been validated by siRNA techniques. These are HDAC6, MAPK14/p38-alpha, PAK4, PBK, PIM1, and caspase-3 (that last one targeted by activators) and the paper references 180 publications that indicate that these are essential in one or more cancer subtypes. The great majority of these assignments have been made through siRNA or shRNA experiments. Identification of these targets has led to the development of a number of small-molecule drug candidates, which have collectively been the subject of 29 clinical trials.
And they are probably not the targets that we thought they were. Because (as this paper shows) when you use CRISPR instead of the RNA techniques to knock down these proteins, the cancer cells that are supposedly sensitive to their loss (32 different lines!) don’t seem to mind any more. And the compounds – which certainly are ligands for the stated proteins – continue to work in the cells where that target has been deleted. For example, the caspase-3 activators continue to kill cancer cells that don’t even have caspase-3 expressed any more, which is a problem.
The group looked carefully to make sure that homologs of these genes weren’t being upregulated instead in the CRISPR experiments, and that doesn’t seem to be the explanation. Such CRISPR screens do identify targets such as BRAF or Aurora B; but they don’t identify these. Notably, even some earlier RNA screens don’t validate them, either: the team re-analyzed hundreds of reported genome-wide shRNA screens and found that these don’t really point at these targets, either. Switching to the CRISPR interference (CRISPRi) system also showed that you can’t explain the results just by assuming different responses to partial versus complete loss of function. It’s worse than that.
There’s also combination therapy to consider. The paper notes that HDAC6 inhibition is being combined with paclitaxel in trials, since its inhibition is thought to sensitize tumor cells to interference with the microtubule system. But the CRISPRed HDAC6 cell lines don’t show any particular sensitivity to paclitaxel (in contrast to past siRNA cell experiments), and similarly, a whole list of putative p38-alpha interactions doesn’t validate, either.
It comes down to effects of the siRNA protocol, apparently. The paper looks at some of the RNA constructs used for those earlier experiments and shows that when you apply those to cells where their putative target gene has already been knocked out by CRISPR, that you still see effects on proliferation and viability. Now that’s bad news. As the paper says, “Our results therefore suggest that these drug targets have advanced to clinical testing due, at least in part, to promiscuous RNAi constructs“. Oy.
The paper goes on to take a closer look at a reported PBK inhibitor (OTS964), which is indeed on the list of compounds that still work (somehow) when there’s no PBK around any more. Developing several resistance cell lines and doing whole-exome sequencing on them identified CDK11B as likely the real target in such cells, and which also makes OTS964 the first selective ligand known for CDK11. It turns out, on closer inspection, to be a 40 nM inhibitor of that subtype, with good selectivity. Not that anyone realized that until now, of course. Knocking in the mutant form of the kinase into tumor cells instantly made them resistant, too. Further work showed that CDK11 is important for mitotic entry, and does indeed appear to be a valid cancer target in its own right – as opposed, say, to PBK. Or the others on the list above.
“Our results indicate that many cancer drugs in clinical trials kill cells independently of their reported targets“, say the authors, and I’d agree that they’ve backed that statement up pretty thoroughly. This doesn’t mean that these compounds are necessarily going to be ineffective in the clinic – polypharmacology can work, and hitting a specific target that you didn’t know about (as with the CDK11 story) can work, too. After all, these compounds had enough preclinical efficacy to make the case for going into man. It’s just that the reasons that people had attached to them were wrong, and that’s something that we really should know about. A serious disconnect can emerge if the patients picked for the clinical trials are selected based on the status of the wrong target protein, of course!
These results also make an even stronger argument against drawing too many conclusions from RNA-level knockdown experiments in cancer cells. Many researchers have already come to similar conclusions about CRISPR experiments versus siRNA/shRNA ones based on their own experiences, but this is an excellent chapter-and-verse demonstration of just what the problems are and how deep they can run. I would say that any paper proposing a cancer target where the main line of evidence is siRNA/shRNA should be double-checked in just this manner. . .