Naturally, it would be a big improvement if we could run clinical trials with a much better idea of which patients will benefit, rather than having to take pot luck. Unfortunately, most researchers agree that many of the animal models used to test cancer drugs are very limited in their capacity to predict the most susceptible human cancer. To be fair, this isn’t just true of cancer drugs targeted at epigenetic enzymes, it’s also true of pretty much all oncology drug discovery.
In an attempt to get around this problem, researchers in both academia and industry are now looking for the next generation of epigenetic targets in oncology. DNMT1 is a relatively broad-acting enzyme. DNA methylation is rather all or nothing – a CpG is methylated or it isn’t. HDACs tend to be pretty non-discriminating too. If they can get access to an acetylated lysine on a histone tail, they’ll take that acetyl group off. There are a lot of lysines on a histone tail – there are are seven on histone H3, just for starters. There are at least ten different HDAC enzymes that SAHA can inhibit. It’s quite likely that each of these ten can deacetylate any of the seven lysines on the H3 tail. This is hardly what we would call fine-tuning.
This is why the field is now moving in the direction of assessing different epigenetic enzymes, which are much more limited in their actions, to see which are important players in different cancers. The rationale is that it will be easier to understand the cellular biology of enzymes with quite limited actions, and this will make it easier to determine which patients are likely to respond best to which drugs.
The first problem in doing this is rather a daunting one. Which proteins should we investigate? There are probably at least a hundred enzymes that add or remove histone modifications (writers and erasers of the epigenetic code). There are probably as many proteins that read the epigenetic code. To make matters worse, many of these writers, erasers and readers interact with each other. How can we begin to identify the most promising candidates for new drug discovery programmes?
We don’t have any useful compounds like 5-azacytidine and SAHA to guide us, so we have to rely on our relatively incomplete knowledge in cancer and in epigenetics. One area that is proving useful is considering how histone and DNA modifications work in tandem.
The most heavily repressed areas of the genome have high levels of DNA methylation and are extremely compacted. The DNA has become very tightly wound up, and is exceptionally inaccessible to enzymes that transcribe genes. But it’s the question of how these regions become so heavily repressed that is really important. The model is shown in Figure 11.3.
Figure 11.3 Schematic to illustrate how different types of epigenetic modifications act together to create an increasingly repressed and tightly condensed chromosome region, making it very difficult for the cell to express genes from this region.
In this model, there is a vicious cycle of events that results in the generation of a more and more repressed state. One of the predictions from this model is that repressive histone modifications attract DNA methyltransferases, which deposit DNA methylation near those histones. This methylation in turn attracts more repressive histone modifying enzymes, creating a self-perpetuating cycle that leads to an increasingly hostile region for gene expression.
Experimental data suggest that in many cases this model seems to be right. Repressive histone modifications can act as the bait to attract DNA methylation to the promoter of a tumour suppressor gene. A key example of this is an epigenetic enzyme we met in the previous chapter, called EZH2. The EZH2 protein adds methyl groups to the lysine amino acid at position 27 on histone H3. This amino acid is known as H3K27. K is the single letter code for lysine (L is the code for a different amino acid called leucine).
This H3K27 methylation itself tends to switch off gene expression. However, in at least some mammalian cell types, this histone methylation recruits DNA methyltransferases to the same region of chromatin[192][193]. The DNA methyltransferases include DNMT3A and DNMT3B. This is important because DNMT3A and DNMT3B can carry out the process known as de novo DNA methylation. That is, they can methylate virgin DNA, and create completely new regions of highly repressed chromatin. As a result, the cell can convert a relatively unstable repressive mark (H3K27 methylation) into the more stable DNA methylation.
Other enzymes are also important. An enzyme called LSD1 takes methyl groups off histones – it’s an eraser of epigenetic modifications[194]. It does this particularly strongly at position 4 on histone H3 (H3K4). H3K4 is the opposite of H3K27, because when H3K4 is clear of methyl groups, genes tend to be switched off.
Unmethylated H3K4 can bind proteins, and one of these is called DNMT3L. Perhaps not surprisingly, this is related to DNMT3A and DNMT3B. DNMT3L doesn’t methylate DNA itself, but it attracts DNMT3A and DNMT3B to the unmethylated H3K4. This provides another way to target stable DNA methylation to virgin territory[195].
In all likelihood, many histones positioned at the promoters of tumour suppressor genes carry both of these repressive histone marks – methylation of H3K27 and non-methylation of H3K4 – and these act together to target the DNA methyltransferases even more strongly.
Both EZH2 and LSD1 are up-regulated in certain cancer types, and their expression correlates with the aggressiveness of the cancer and with poor patient survival[196][197]. Basically, the more active these enzymes, the worse the prognosis for the patient.
So, histone modifications and DNA methylation pathways interact. This may explain, at least in part, one of the mysteries of existing epigenetic therapies. Why are compounds like 5-azacytidine and SAHA only controllers of cancer cells, rather than complete destroyers?
In our model, treatment with 5-azacytidine will drive down the DNA methylation for as long as the patients take the drug. Unfortunately, many cancer drugs have serious side-effects and the DNMT inhibitors are no exception. The side effects may eventually become such a problem that the patient has to stop taking the drug. However, the patient’s cancer cells probably still have histone modifications at the tumour suppressor genes. Once the patient stops taking 5-azacytidine, these histone modifications almost certainly start to attract the DNMT enzymes all over again, re-initiating stable repression of gene expression.
Some researchers are carrying out clinical trials using 5-azacytidine and SAHA together to try to interfere with this cycle, by disrupting both the DNA and histone components of epigenetic silencing. It’s not clear yet if these will be successful. If they aren’t, it might suggest that it’s not low levels of histone acetylation which are most important for re-establishing the DNA methylation. It might be that specific histone modifications, of the types just described, are more important. But we don’t yet have drugs to inhibit any of the other epigenetic enzymes, so we’re stuck with Hobson’s choice at the moment, that is, no choice at all.
In the future, we may not need to use DNMT inhibitors at all. The link between DNA methylation and histone modifications in cancer isn’t absolute. If a CpG island is methylated, the downstream gene is repressed. But there are tumour suppressor genes that are downstream of unmethylated CpG islands and tumour suppressor genes that don’t have a CpG island at all. These genes may still be repressed, but solely thanks to histone modifications[198]. This has been shown by Jean-Pierre Issa at the MD Anderson Cancer Center in Houston, who has been so instrumental in the implementation of epigenetic therapies in the clinic. In these instances, if we can find the right epigenetic enzymes to target with inhibitors, we may be able to drive re-expression of the tumour suppressors without needing to worry about DNA methylation.