Monday, January 27, 2014

Blocking the MAPK cascade to treat achondroplasia


When I started this article I thought that I would be going directly to the point: let's talk immediately about the interesting study about MAPK inhibition in mice growth plates. 

Then, I remembered the purpose of this blog, which is to share information about potential therapies for achondroplasia with people that are not always familiar with the technical language. The seventeen readers of this blog may complaint with some reason, oh! no, he is repeating everything again.... I must apologize, there have been eventual visitors reading an article for the first time and I think on them, too. That's why we will begin with a short, basic information about achondroplasia. And finally, I always try to include something new. 

The cause of achondroplasia 

Achondroplasia is caused by a single mistake (a mutation) in the structure of the gene encoding (carrying the information to produce) the protein called fibroblast growth factor receptor 3 (FGFR3). The gene mutation provokes the substitution of a single amino acid in the chain of this protein. This single change, technically called G380R, the replacement of an amino acid called glycine for another called arginine, makes FGFR3 more active than normal and induces the clinical features of achondroplasia. (1)

While having key importance during the development of the new life in utero, after birth, FGFR3 is produced in a relevant way almost by just one kind of cell, the chondrocytes of the growth plates, thin and very specialized zones placed in the extremities of the long bones (Figure 1). These regions are where the bone growth occurs during childhood and puberty. When the individual reaches a specific mark on his or her own biological clock, at the end of puberty, the growth plates close and the individual stops to grow.

Figure 1. Growth plate zones

Naski MC & Ornitz DM. Frontiers of Bioscence 1998
FGFR3 in action

FGFR3 is an active protein, so it is called an enzyme. An enzyme is a kind of electric messenger, carrying signals from and to the cell to let it respond to the environment or to its own needs. FGFR3 is normally activated by a small number of FGFs present in the growth plate environment (the extracellular matrix) and works in chondrocytes by reducing the speed these cells multiply and mature. (1) Without FGFR3 bones grow excessively and development problems also arise. (2,3) The blog contains other articles reviewing FGFR3 mode of action. You just have to visit your preferential language index page by clicking the respective button in the top of this page.

FGFR3 acts in concert with other enzymes to exert its effects, through what we call signaling cascades or pathways. In chondrocytes, FGFR3 can activate several cascades, but it has becoming more evident, according to several studies, that the cascade called mitogen-activated protein kinase (MAPK) pathway is key for the clinical consequences of achondroplasia (Figure 2). (read ref. 1 for a good review)

Figure 2. FGFR3- MAPK (ERK) pathway in chondrocytes

The MAPK paradox

If you are following the blog's article sequence you may have already read about the paradoxical action of MAPK in chondrocytes: while in about all other cells the MAPK cascade is a growth trigger, in chondrocytes it impairs cell growth. (4; visit the last two series' articles of the blog) Here, we could make a stop and ask: if MAPK signaling is responsible for impairing the bone growth in achondroplasia, can we treat achondroplasia using drugs against the MAPK cascade? Let's see if we could do it.

MAPK in achondroplasia 

As I said, there is increasing and compelling evidence showing that the inhibition of the MAPK pathway can restore, at least partially, the bone growth in FGFR3-related bone disorders. Evidence comes, for instance, from several different studies performed with C-type natriuretic peptide (CNP) and its analogue BMN-111.(5-9)

CNP is a natural regulator of FGFR3 activity in chondrocytes, reducing the effects of FGFR3 signaling. CNP works precisely inhibiting the enzyme RAF in the beginning of the MAPK pathway (Figure 3). (5)

Figure 3. FGFR3 and CNP pathways

Nevertheless, CNP is just one possible approach that can be used to inhibit the MAPK pathway in achondroplasia. Another strategy could be to use drugs currently in clinical development or already approved to treat some forms of cancer. Is this feasible?

Inhibiting MAPK in cancer

Taking advantage of the main MAPK pathway signaling (the instruction to grow and survive), several types of cancer use the MAPK pathway to grow through diverse mechanisms, such as:

1. Increasing the production of several different receptor enzymes like FGFR3 that use MAPK to conduct signals. Scientists call this over expression. Having more receptors on the cell wall is an advantage for the cell that wants to multiply;

2. Producing mutated and overactive enzymes. Remember that in the last article I mentioned that some cancers gain the ability to produce a mutated form of FGFR3 associated to tanatophoric dysplasia that makes it always active. Mutations also can happen in any enzyme of the MAPK cascade to make it more active than normal.

One good example of how cancer cells are "smart" comes from melanoma. This type of aggressive skin cancer often has a mutation in RAF, called V600E, which makes easier to melanoma cells to grow faster, because the mutated RAF works without the need of an extracellular stimulus.(10) Researchers started to test several compounds against RAF years ago, and now drugs that target RAF, like vemurafenib, have already been approved to treat melanoma. (11)

However, melanoma is so adaptable that after sometime responding to the treatment with an anti-RAF, it becomes resistant to the drug. That's why, to deal with this situation, there are new strategies adding an anti-MEK to the RAF inhibitor to treat melanoma. (12-13).

Designed to aim components of the MAPK cascade, these new potential medicines are qualified according to their targets, so drugs against RAF are called RAF inhibitors and drugs against MEK are called MEKs inhibitors (Table 1). You will see that those advanced in clinical development or already approved adopt names including their targets, so vemuRAFenib, dabRAFenib and soRAFenib are RAF inhibitors or anti-RAF and traMEtinib and soluMEtinib are MEK inhibitors or anti-MEK.

 Table 1. A list of RAF and MEK inhibitors available for lab tests or in clinical development

The list is not exhaustive. Some of these compounds are not specific to MEK or RAF. 
Source: Santa Cruz Biochemicals, Ref 14
 Inhibiting MAPK to treat achondroplasia 

So, there are drugs approved to treat some kinds of cancer which benefit from the pro-proliferation function of MAPK. Why could we not test them for achondroplasia? Is it possible? What are the risks? What would be the challenges to stimulate a pharma industry to explore this strategy?

Old and new drugs in cancer 

Cancer is a devastating disease. Cancer cells grow without control and invade the surroundings, disrupt an organ or tissue function, compete for nutrients and energy and, without any intervention, they will drive the organism to death.

Under these circumstances, it is not difficult to understand why aggressive and toxic treatments are acceptable to treat cancer. Old drugs used to treat tumors traditionally aim the whole cell proliferation process, called mitosis, so they can impact not only cancer cells but also normal cells performing their normal tasks, which include to multiply in order to keep the tissue where they are healthy. That's why the older drugs cause so many undesired side effects in patients, such as sometimes severe gastrointestinal symptoms: the cells of the digestive system mucosa have a fast cell cycle, always multiplying to keep the tissue intact.

New generation drugs are classified in a new group: they are called targeted therapies. Instead of affecting the cell as a whole they aim just specific target(s), like the enzymes located at the cell membrane, such as FGFRs or in the signaling cascades, like MAPK. With this more specific mode of action, we have seen a significant drop in the severity of undesired effects in patients using those new drugs.

Several types of pediatric cancers also use MAPK cascade properties to induce cell growth but we don't see research with these new drugs in this population. The great question is: what would be the risk of using these kinds of drugs in children, whose bodies are in development? 

Enzymes of the MAPK cascade participate in a vast number of vital cell processes. What would be the consequences of using an anti-MEK in a child with achondroplasia or other genetic condition such as the Rasopathies (14) that could benefit with the use of these drugs? The aversion to take risks has always prevented the pharma industry to test these targeted therapies, like MAPK inhibitors, in children with cancer. Well, at least until recently.

The anti-MEK selumetinib in pediatric cancer 

Things seem to be changing in terms of interpretation of risk. Astazeneca has been developing the anti-MEK selumetinib (AZD6244) for adult cancers where the inhibition of MEK may help fight the disease, but has also started testing selumetinib in some types of pediatric cancers. There are 3 clinical studies registered at (NCT01362803, NCT01386450, NCT01089101). This is a rare and bold initiative, given the real or potential risks involved or perceived.

As part of the rules of the drug development process, drug developers must disclose the results of the studies performed with a candidate drug. In many instances, testing a drug for cancer in a developing organism could sound risky because drugs for cancer work in many aspects of the growth machinery. If one such experimental compound was tested in young animal models, those tests would have to be presented to the regulatory authorities. Tests like these may bring unexpected results that could be perceived as negative for the success of the potential new drug. That's why the recently published study by El-Hoss and coworkers (14) is so special. 

To my knowledge, this is the first study published showing the effects on bone and growth plates of drugs like selumetinib and PD0325901, which are anti-MEKs in clinical development, intended to become commercial drugs. There are several studies with other anti-MEKs, but with drugs that have been withdrawn from the clinical program, such as U0126.

Selumetinib and PD0325901 effects in the growth plate

El-Hoss and coworkers have done a lot of experiments aiming to understand what are the effects of these two anti-MEK compounds in the bone and in the growth plate. Basically, they confirmed that both inhibitors, but mainly PD0325901, inhibited MAPK in chondrocytes from normal mice. MEK inhibition caused enlargement of the hypertrophic zone of the growth plate but showed no effect in the proliferative zone (see figure 1 for the growth plate zones). Moreover, they observed that the growth plates didn't enlarge at all, and for them this was caused by chondrocyte leaving the proliferative state earlier. In other words, they concluded that there were more hypertrophic chondrocytes at the cost of less proliferative ones, an outcome that would not be considered a good one.

These first findings are only partially similar to the experiments with CNP. CNP also increased the hypertrophic zone, but there was an overall increase of the growth plate size, which responded for the bone growth observed in animal models, according to the reports already cited here (5-9). CNP investigators observed that the proliferative zone of the growth plate was marginally stimulated under exposure to CNP or analogues. The absence of more robust effect in proliferative chondrocytes may be explained by the fact that FGFR3 also exerts effects through other signaling pathways, not influenced by CNP at all. 

For instance, there is evidence that FGFR3 signaling inhibits the production of a local protein called parathyroid hormone related protein (PTHrP; some refer to it as a peptide), which is a strong chondrocyte proliferation stimulator, delaying their maturation (hypertrophy). (15) Why exposure to CNP and to those anti-MEKs resulted in different findings remains a good question.

El-Hoss et al. also made tests in mice bones and observed that MAPK inhibition also favored bone density through reduction of osteoclasts activity (the cells that absorb bone tissue). These drugs also influenced the bone healing process in a fracture model, maintaining the cartilage callus longer than in control animals. They found that this was happening because of the inhibition of the release of a type of enzyme that degrade the cartilage matrix to leave space for the new bone.

El-Hoss et al. observed that the effects of selumetinib and PD0325901 in the growth plate were reversible after drug interruption, giving a positive perspective about long term consequences of using those drugs for pediatric cancer.

The authors admitted some study limitations, such as the relative short period of exposure the animals were submitted to, only three weeks. 

It is curious that they didn't mention if they measured the animals to see if there was any difference in the total and limb lengths between control and tested animals, possibly exactly because of the short observation period.


I think that just the publication of the tests made with selumetinib and PD0325901 is a breakthrough advancement and seems to represent a change in the way the industry is dealing with drug development challenges and risks, real or imagined, in the pediatric population. I suspect that this has a connection with the recent change in the regulatory requirements for potential drugs intended to be used in the pediatric segment. In few words, drugs to be used by children must be tested first in the same population.

The results of the study by El-Hoss and coworkers are somehow intriguing and I think they should elicit more studies, specific on growth plates, to allow a better understanding of the kind of influence these anti-MAPK compounds exert in bone development and growth. More importantly, the tests done by El-Hoss et al. were performed in normal mice. What would be the effect of those anti-MEKs in an achondroplasia model?  If there was enough interest, these tests should be done in an appropriate animal model, for an appropriate period of time. I guess results would be different.

The future...

Another aspect worth to mention is that for future therapeutic strategies not targeting directly FGFR3 to rescue bone growth in achondroplasia, we could speculate that it might be necessary to combine different drugs to obtain meaningful therapeutic results. For instance, adding a pro-proliferative drug, such as PTH, to a pro-hypertrophic drug, such as an anti-MEK or a CNP analogue. In an exercise of futurology, drug combinations could allow overall lower doses and lesser risks in a long term therapy for children with achondroplasia. 


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