#apaperaday: Blockade of IGF2R improves muscle regeneration and ameliorates Duchenne muscular dystrophy

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Blockade of IGF2R improves muscle regeneration and ameliorates Duchenne muscular dystrophy.

This paper from Bella et al in EMBO Molecular Medicine in 2020 on IGF2R blocking as a potential therapy for Duchenne. Paper will be discussed in our journal club. Feedback afterwards of not just me but the group. Doi 10.15252/emmm.201911019

Duchenne patients lack dystrophin which results in muscle damage and regeneration. Several factors play a role in muscle regeneration, including IGF1, which plays a role in muscle repair and growth. The function of IGF2 is less known.

The printed paper did not have the graphic summary – which was very useful to understand the different pathways. Both IGF1 and IGF2 can bind to the IGF1 receptor. There is also an IGF2 receptor (IGF2R), to which only IGF2 can bind. IGF2R is complexed with CD20, a calcium channel.

First authors aim to show that CD20 phosphorylation is affected by IGF signaling. As a group we were puzzled by multiple things, in this experiment but mostly by Figure 1E. The ratio of phosphorylated CD20/CD20 was calculated (bottom). Highest bar should be lowest bar?

Authors aim to show that CD20 and IGF2R can bind. However, they do not use proper controls in their Co-immunoprecipitation. Also they aim to show the effects of anti CD20 (shRNA) and anti-IGF2R on myoblast differentiation – however, they do not use control shRNA.

The conclusion is that the reduction of CD20 by shRNA and slows down differentiation but this is compared to untreated C2C12 cells. Unclear whether maybe the treatment with antibody and shRNA had an impact – this is why irrelevant antibodies and shRNAs are needed as controls

What we did agree on is that IGF2R is over-expressed in patient muscle and mdx muscle. The problem with more IGF2R is that this prevents IGF2 from binding to the IGF1 receptor to trigger muscle repair. So treatment with anti IGF2R antibodies as a therapeutic approach makes sense

This is indeed what authors did, using a low and a high dose of antibodies. However, no irrelevant antibodies were used but saline control. Furthermore, black 6 wild types were used as a reference rather than black 10 (mdx background).

Finally, mice were treated for 4 or 9 weeks starting at 3 months old. However, only one group of untreated mdx and wild types is included. Are there 4 or 6 months old? This makes a difference…Are we comparing 4 months old treated to 6 months untreated? Or the other way around?

Authors see difference in muscle strength but as we do not know what the age is of the reference mice, interpretation of results is difficult. Also the treated mice appear to become twice as strong as wild type mice – that seems unlikely

Authors study fibrosis and strength in the tibialis anterior and vastus medialis muscle – however, they omit the most affected mdx muscle: the diaphragm. For fibrosis they show representative pictures but it is not clear how and if they quantified when they say less fibrosis

Authors claim that the IGF2R antibody treatment improves calcium homeostasis. Finally, authors also study vascularization, showing that in treated mice there are more functional blood vessels. This is expected since IGF signaling also plays a role in vascularization.

As a group we were (sadly) not convinced about most of the results in this paper, due to lack of controls (in many different ways), large variability in loading controls, and strange interpretations of results. We do agree that IGF2R is increased in Duchenne

We also agree that some of the results authors show suggests there can be therapeutic effects of IGF2R antibody treatment in the mdx mouse model – though we would welcome more results including the diaphragm and proper controls and fibrosis quantification of sections.

Authors suggest that blockade of IGF2R in DMD patients could present a good starting point for new DMD treatment ‘even if side effects are not completely unavoidable’. What side effects could that be? IGF signaling is important not just for muscle but for all cells.

Furthermore, IGF signaling is increased in cancer (makes sense, because the cancer cells need to proliferate and get blood vessels for nutrients to allow growth). In light of this the question is whether you want to block IGF2R, which inhibits IGF signaling.

Also because IGF2R is a tumor suppressor gene. So this inhibition may be beneficial for muscle repair in Duchenne patients, however, if you block IGF2R body wide, this could affect IGF signaling elsewhere and likely there would be an increased risks for tumor formation.

So what we would need is very muscle specific IGF2R blockade. This is something the authors should have pointed out rather than giving vague ‘side effects that are not completely unavoidable’.

Disclaimer: I know that in a journal club setting it is easy to spot errors in papers because there is the combined force of people with different expertise. Having said that, this paper is an example of a blatant absence of many controls – sad the reviewers did not pick this up.

 

Pictures by Annemieke, used with permission.