FGFR4: a problem for ligand trap therapeutic strategies in achondroplasia?

Introduction

Someone interested in learning about the mechanism of bone growth soon learns that the process through which bones develop is incredibly complex. Most of our bones don’t start as osseous structures but as cartilage templates or scaffolds that, after a very sophisticated series of steps, are substituted by the definitive bone tissue, in a continuous process that last up until the end of puberty, when these cartilage templates disappear. When thinking about bone development, one must have in mind that, although we already have a lot of knowledge about the process, we are still only in the beginning to understand all the biological phenomena that take place within the bones. So, it is not surprising that every other day new pieces of the puzzle are brought by Science, challenging that knowledge, reinforcing the saying by Albert Einstein:

       We still do not know one thousandth of one percent of what nature has revealed to us.
 

I have been thinking about this after reading an interesting study about the effects of fibroblast growth factor receptor 4 (FGFR4) on bone growth (1). FGFR4 is a sister of FGFR3, the enzyme that, when mutated, cause achondroplasia. Well, a new piece of the puzzle? I think it is worth to briefly review the study because it might have some implications for some of the new potential therapies for achondroplasia. But, let’s start slowly to put the results of this study in context.

The intricate bone growth process

With some few exceptions, our bones grow starting from small regions located in their extremities, called growth plates (Figure 1). Inside the growth plates live the cells that lead the growth process, the chondrocytes.

Figure 1. Growth plate diagram showing the several chondrocyte layers

The chondrocyte is the “boss” but at the same time has to accomplish many parallel or serial chemical instructions given by dozens of local and systemic growth agents which in turn may work either as growth promoters or suppressors (Figure 2). Chondrocytes, obeying these many local and systemic agents, undergo a series of transformation which can be detected easily in the microscope (Figure 1), beginning from small, dormant cells (resting zone) passing through a phase of high proliferative rate (proliferative zone), to a state in which they have an impressive increase of their cell volume when they are called hypertrophic chondrocytes (Figure 1). This last phase is accounted to be the main responsible stage for bone growth.

Figure 2. Some of the growth factors regulating the growth plate development (2).

Extracted from Kozhemyakina E et al. A pathway to bone: signaling molecules and transcription factors involved in chondrocyte development and maturation. Development 2015;142(5):817-31. Free access. Reproduced here for educational purposes only.

The FGF/FGFR pathway

One of the chemical processes ruling bone growth is provided by what we call the fibroblast growth factor (FGF) / fibroblast growth factor receptor (FGFR) signaling cascade or pathway, which is familiar to whom is interested in achondroplasia. The FGF family is composed by 23 different growth factors, and the FGFR family has four sister enzymes (and one new that is candidate to become the fifth) (3). We are focused in long bone growth here, so I will not mention all the activities the FGF/FGFR axis have in the body and in the few bones that grow by what is called intramembranous process.

To help understanding what is a signaling cascade, you can review other blog related articles (see the English page in the bar above). In short, as in a series of domino blocks a chemical reaction starts another, which activates a third, and that a fourth, in a chain of chemical reactions. These signaling cascades provide instructions to chondrocytes to how they should react to each of the stimuli received. FGFs are called ligands because initiate the chemical cascade by binding (or linking) with the receptor molecule, one of the FGFRs.

Although the number of FGFs is large, only a few have more intense participation in the long bones’ growth process. Scientists have identified so far FGF9 and FGF18 as the most relevant exerting influence over growth plate chondrocytes (3-5).

After decades of their description, researchers have already identified the main functions of the first three FGFRs in bone growth. FGFR4 has not, to date, been recognized as relevant for this process. Take a look in the Table 1, taken from a recent review by Drs. David Ornitz and Nobuyuki Itoh (3), which lists the effect of the ablation of FGFRs in genetic models. You will see that models exploring the ablation (or loss-of-function mutations) of FGFR4 did not show consequences in the growth plate. Rather than that, it seems that FGFR4 is more linked to metabolic processes, including vitamin D and phosphate, which are crucial for bone development/health, but in a redundant fashion with other FGFRs.

Table 1. Consequences of the genetic inhibition (loss of function) of FGFRs

  
FGFR4 relevant for bone growth?

That’s why the recent study by Cinque et al. (1) is interesting. The researchers worked on the role of FGFR4 in bone growth in several cell and animal models and ended to find that blocking FGFR4 signaling causes impairment of bone growth by interfering in a mechanism that is part of the normal cell balance in tissues, called autophagy.

In their study, autophagy was linked to the modulation of collagen 2, which is the main component of the cartilage matrix. I don't want to make it more complex than it already is. Just take that collagen 2 is very important for the expansion of the growth plate and consequently the bone. If you have less collagen 2 in the matrix, the growth process is impaired.

The effect of the inhibition of FGFR4 seems to be especially seen in more mature chondrocytes (pre-hypertrophy/hypertrophy, see above). The absence of FGFR4 signaling would lead to thinner growth plates, thus to smaller bones. Importantly, the researchers found that the FGF with more influence over FGFR4 is FGF18. When FGF18 activates FGFR4, it triggers the chemical reactions inside the chondrocytes that regulate the expression (production) of collagen 2, allowing the normal expansion of the hypertrophic zone. Either the deficiency of FGF18 or the inhibition of FGFR4 lead to disturbance of the autophagy process.

Well, well, this seems odd

These findings are somewhat surprising because, as we saw, previous studies failed to show specific problems caused by the ablation of FGFR4 within the bones. So, how do we deal with this new information? First, we might ask how the researchers in the previous studies assessed the changes caused by the inhibition of FGFR4 in the growth plates. Perhaps they didn't access this specific process in the growth plates at all, or they used different techniques, or the techniques available then were not sensitive enough to spot any relevant consequence of that inhibition. Second, it would be interesting to see if the results obtained by Cinque et al.(1) are reproducible. Sometimes, experiments cannot be reproduced and the original findings are found to be flawed. However, in this case, the investigators seemed to have repeated the tests with different techniques and ended to find basically the same results. Nevertheless, they admit that other studies must be performed to further understand the role of FGFR4 in bone development.
 
And why these findings are a bit worrisome when thinking about treating achondroplasia?  What kind of questions these new data would bring after all?

A problem for ligand trap strategies?

The thing is that we know that FGF18 is one of the relevant activators of FGFR3 in the growth plate. The activation of FGFR3 per se is not a problem in normal conditions: bones need FGFR3 to regulate their growth velocity. However, when FGFR3 is mutated like in achondroplasia, any activation of it contributes to the growth impairment seen in this skeletal dysplasia. One of the approaches being explored to efficiently treat achondroplasia is exactly by blocking FGFR3 activation, and there are several potential strategies aiming this goal. We have been following with great interest the development of the so called ligand trap or ligand decoy strategies (6-8; read this previous article of the blog). Particularly, the study by the group leaded by Dr Gouze (6) showed striking results regarding the rescue of bone growth using a soluble (free) version of FGFR3. Dr. Gouze's group showed so impressive results with their approach in a mouse model (Figure 3) that sFGFR3 is now in serious development aiming potential clinical trials in the future (9).

Figure 3. Effect of the ligand trap sFGFR3 in a mouse model of achondroplasia (6).

  The concept of the ligand trap approach for achondroplasia is quite simple. With a free form of FGFR3 circulating within the growth plate, we would have "competition" between this free version and the cell-anchored FGFR3 (the one that is driving the chondrocyte to stop growing in achondroplasia) for the local FGFs ( mainly FGF9 and 18), resulting in "trapping" of these FGFs by the soluble form and in less activation of the cell-anchored FGFR3s. If these are not activated, they would exert less activity and the bone growth would not be that impaired as we see in achondroplasia. The picture showed above (Figure 3) let us think the ligand trap strategy is promising.

However, these ligand traps will mainly aim FGF9 and FGF18 (and possibly FGF2). Taking in account that FGF18 would be captured by the traps, there would be less activation of FGFR3 (which is what we want, thinking in achondroplasia), but also there could be consequences of having fewer FGF18 activating FGFR4. You see?

FGF18 is thought to be the lead trigger for FGFR3 (3) and, if we take in account the results by Cinque et al. (1), also for FGFR4 signaling. To treat achondroplasia with a ligand trap we could be preventing the action of FGF18 on FGFR3 (which is fine), but also on FGFR4. In the former case, the result would be positive, but in the later, what would be the consequences?

In the study by Cinque et al.(1), the researchers also noted that both FGFR3 and FGFR4 were activated by FGF18 in their models. Both FGFRs were linked to the autophagy process but the researchers noted that only FGFR4 was significantly linked to the control of this phenomenon. Here, again we see some redundancy of the effect of FGFR4 (with FGFR3), so the level of the relevance of the effect of FGFR4 is still to be addressed, and new confirmatory studies are warranted to understand the role of FGFR4 in the growth process.

Meanwhile, with the impressive results demonstrated by the study with sFGFR3 (6; Figure 3), it is possible that any effect of the reduction of FGF18/FGFR4 activation would be surpassed by the growth rescue promoted by the inhibition of the FGFR3 pathway.

We will keep following these developments. The next article will review new information about controlling the production of FGFR3. No, this is no genetic therapy, no DNA editing at all.
 
References

1. Cinque L et al. FGF signalling regulates bone growth through autophagy. Nature 2015; 528:272–5. doi:10.1038/nature16063. 

2. Kozhemyakina E et al. A pathway to bone: signaling molecules and transcription factors involved in chondrocyte development and maturation. Development 2015;142(5):817-31. Free access.

3. Ornitz DM and Itoh N. The Fibroblast Growth Factor signaling pathway. WIRE Dev Biol 2015;4(3):215–66. Free access.

4. Garofalo S et al.Skeletal dysplasia and defective chondrocyte differentiation by targeted overexpression of fibroblast growth factor 9 in transgenic mice. J Bone Miner Res 1999;14(11):1909-15. Free access. 
 
5. Davidson D et al. Fibroblast growth factor (FGF) 18 signals through FGF Receptor 3 to promote chondrogenesis. J Biol Chem 2005;280:20509-15. Free access.

6. Garcia S et al. Postnatal soluble FGFR3 therapy rescues achondroplasia symptoms and restores bone growth in mice. Sci Transl Med 2013;5:203ra124. doi:10.1126/scitranslmed.3006247.

7. Tolcher A et al. Preliminary results of a dose escalation study of the fibroblast growth factor(FGF) “trap” FP-1039 (FGFR1:Fc) in patients with advanced malignancies. 22nd EORTC-NCI-ACR symposium on molecular targets and cancer therapeutics, November 16-19, 2010. Berlin, Germany. Free access.

8. Ghivizzani SC. Delivery of soluble FGFR3 as a treatment for achondroplasia. National Institute of Arthritis and Musculoskeletal and Skin Diseases. 2013; Project Number: 5R01AR057422-04.

9. Therachon press release 2015. Free access.