p53, how we almost forgot about the guardian of the genome when CRISPRing around

When in 1979 six research groups independently described a 53 kDa protein, none of the participants suspected to which genetic superstar this protein would develop. This protein, which due to its molecular weight was given the not-so-impressive name p53, is perhaps the most important policeman in our cells; but only as long as it works properly. If p53 loses its functionality, it’s getting pretty dangerous. In fact, no other gene is mutated more frequently in tumor cells than p53. So how does normal p53 manage to keep all of our body cells in check and what does it all have to do with CRISPR?

The extraordinary history of p53 research

It seems difficult at first to draw a line between the late seventies, in which p53 was discovered and the two articles of the Nature publishing group of this week, which I want to present at the end of this article. The Pub Med Central database now lists more than 180,000 articles when searching for “p53”; there is not a single person in the world who can oversee all these data. All existing models on p53 functions are therefore necessarily simplified. But now back to the beginning and the first descriptions of this amazing protein.

Simian Virus 40 by Phoebus87 for wikimedia.org, CC BY-SA 3.0

Five of the six studies from 1979 identified the p53 protein as the binding partner of another protein, the “large T antigen” (large T, or LT for short) of simian virus 40 (SV40 for short). SV40 belongs to the group of polyomaviruses that can cause tumors in animal cells. The LT protein plays a key role here: it is able to initiate replication of the DNA as well as to inactivate natural brakes that suppress cell division in healthy cells. It appeared that LT binds to an endogenous protein in infected cells: p53. p53 therefore became known as an oncogene, i.e. as a protein involved in tumorigenesis (together with LT). This idea was also given some tailwind by the observation that p53 is particularly active in many tumor cell lines. Since the cloning protocols from these days were still very inefficient, p53 was initially cloned from these cell lines in which the mRNA of p53 was present in high copy numbers. When these p53 genes were now introduced into normal cell lines, many of them also became tumorigenic. These results promised were so promising when it comes to understanding tumorigenesis that in the 1980s a rapidly increasing number of researchers began working on p53. However, discrepancies slowly accumulated: some variants of p53 did not appear to be able to transform normal cells into cancer cells.

Not until ten years after its discovery, around 1990, a remarkable 180 degree reversal occured in our view on p53. The variants of p53 which had been cloned from the tumor cell lines turned out to be largely mutant forms of p53. Normal, wild-type p53, on the other hand, not only showed no tumor-promoting effect, but instead appeared to act as a tumor suppressor. In contrast, the tumor-suppressing effect becomes inactive when p53 is mutated or when it binds to LT.

p53 at the interface between DNA repair, cell cycle and programmed cell death

Understanding how p53 works in normal cells requires understanding three concepts, which are briefly discussed below: the cell cycle, programmed cell death (apoptosis), and DNA repair mechanisms. In a complex organism, like us humans, billions of cells have to be coordinated in their function. The cell cycle plays an important role in this because it regulates when a cell is allowed to divide and when not. Normal body cells should usually be limited to performing their specific tasks and not divide anymore; they are in the so-called G0 phase and thus actually outside the cell cycle. However, in their genome, which is used in very different ways in these different cells, mutations accumulate that can cause cells to go out of line. For example, they can reenter the cell cycle, i.e. move towards cell division. However, several control mechanisms respond to certain warning signals, an important of which is damaged DNA. p53 is part of this control: when DNA damage is detected, p53 ensures that this cell will not divide under any circumstances. Only when the damage to the DNA can be repaired, p53 releases this brake and the cell has another chance to entert he cell cycle in ordert o divide. If the DNA damage is irreparable, p53 will cause the cell to initiate programmed cell death (apoptosis). Then this single cell must die for the benefit of the big whole, the functioning of the whole organism.

OK, but what has p53 to do with CRISPR now?

It became clear why there is so often a mutated, inoperable version of p53 in cancer cells. The normal variant would not allow the cells to divide as uncontrollably as tumor cells unfortunately do. This feature has earned the p53 protein the nickname “guardian of the genome” and led to the publication of Science p53 as the Molecule of the Year 25 years ago. But since then it is not quiet around p53, certainly not since CRISPR revolutionized biomedicine. Not only because, of course, researchers contemplate to use CRISPR in order to repair mutated p53 versions. In general, we are currently in a very „hot“ phase regarding the implementation of CRISPR into the treatment strategy of many genetic diseases, not just cancer. Every week, there are news that affect the scientific, regulatory, public and economic assessments of the risks and opportunities of CRISPR-based genome editing. After an article in Nature Methods warned about so-called off-target effects in 2017, it was withdrawn in March 2018; CRISPR appeared safer again and several clinical trials were on the starting blocks. However, in early June, the FDA suspended the launch of a Phase I/II study and asked for scrutinized risk assesment. And then, this Monday (11th of June 2018) two articles were published simultaneously that actually warn against a very different risk that has nothing to do with CRISPR’s off-target effects, but with p53.

Approaches using CRISPR to heal genetic defects usually look like this: one takes the body’s own cells from the patient and cultivates them in petri dishes. There they are transfected with components of the CRISPR system in order to introduce a healthy copy of whichever gene is mutated in the patient. However, since this process is far from being 100% efficient, it is necessary to select for those few cells in which genome editing has worked and expand them. In doing so, researchers select those cells with a “repaired”, i.e. altered genome which did not undergo apoptosis. Two research teams, one from Novartis, one from the Karolinska Institute in Stockholm, have now discovered that this selection unintentionally selects cells that have a functionally impaired p53 protein. Actually, this is not so surprising: normal p53 monitors DNA damage and intervenes by either preventing cell division or triggering programmed cell death. The CRISPR system works by first damaging the genome at a defined location. This is usually recognized by functionally active p53 proteins, which then initiate cell death in these cells. However, the more inefficient the p53 protein is, the more efficient CRISPR can introduce alterations into the genome. By selecting for the successfully „CRISPRed“ cells, one also selects for cells that have increased potential for tumor formation. Reintroducing these cells into a patient, could have fatal consequences. At the beginning of the week, the shares of the companies behind the CRISPR studies promptly crashed again. However, they should now be used to uphill and downhill rides.

These recent events illustrate nicely that caution is needed before allowing such a new treatment option. Because often it is not necessarily the obvious dangers, such as the much discussed off-target effects, but also very different mechanisms that can bring about potential hazards. Nevertheless, it is expected that CRISPR-based therapeutic approaches will be implemented in our clinics in the near future; Their potential to give countless people a longer and/or pain-free life is simply too high to stop working hard to make them safer. The insight that we should also test cultured cells for their p53 status in addition to their correct genome editing is already a first important step in this direction.

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