BMP2 and BMP7 cooperate with H3.3K27M to promote quiescence and invasiveness in pediatric diffuse midline gliomas

Reviewer #1 (Public Review):

Summary:
Mutational analysis of diffuse midline glioma (DMG) found that ACVR1 mutations, which up-regulate the BMP signaling pathway are found in most H3.1K27M, but not H3.3K27M DMG cases. In this manuscript, Huchede et al attempted to determine whether the BMP signaling pathway has any role in H3.3K27M DMG tumors. They found that the BMP signaling is activated to a similar level in H3.3K27M DMG cells with wild-type ACVR1 compared to ACVR1 DMG cells, likely due to the expression of BMP7 or BMP2. They went on to test whether cells treated with BMP7 or BMP2 treatments affected the gene expression and cell fitness of tumor cells with H3.3K27M mutation. They concluded that BMP2/7 synergizes with H3.3K27M to induce a transcriptomic rewiring associated with a quiescent but invasive cell state. The major issue for this conclusion is that the authors did not use the right models/controls to obtain results to support this conclusion as detailed below. Therefore, in order to strengthen the conclusion, the authors need to address the major concerns below.

Strength:
This paper addresses an important question in the DMG field.

Major concerns/weakness:

1. All the results in Fig. 2 utilized two glioma lines SF188 and Res259. The authors should repeat all these experiments in a couple of H3.3K27M DMG lines by deleting the H3.3K27M mutation first.
2. Fig. 3. The experiments of BMP2 treatment should be repeated in other H3.3K27M DMG lines using H3.1K27M ACVR1 mutant tumor lines as controls.

Minor concerns
Fig.2A. BMP2 expression increased in H3.3K27M SF188 cells. Therefore, the statement "whereas BMP2 and BMP4 expressions are not significantly modified (Figure 2A and Figure 2-figure supplement A-B)" is not accurate.

Reviewer #2 (Public Review):

The manuscript by Huchede et al investigates the BMP pathway in H3K27M-mutant gliomas carrying or not activating mutations in ALK2 (ACVR1). Their results in cell lines and in datasets acquired from the literature on patient tumors indicate that the BMP signaling pathway is activated at similar levels between ACVR1 wild-type and mutant tumors. The group further identifies BMP2 and BMP7 as possibly the main activators of the pathway in cells. They then show that BMP2 and 7 crosstalk with the H3 mutation and synergize to induce transcriptomic rewiring leading to an invasive cell state.

The paper is well-written and easy to follow with a robust experimental plan and datasets supporting the claims. While previous work (acknowledged by the authors) indicated activation of BMP in H3K27M tumors, wild type for the ACVR1 mutation this paper is a nice addition and provides further mechanistic cues as to the importance of the BMP pathway and specific members in these deadly brain cancers. The effect of these BMPs in quiescence and invasion is of particular interest.

A few suggestions to clarify the message are provided below
1- In thalamic diffuse midline gliomas, the BMP pathway should not be activated as it is in the pons. The authors should identify thalamic tumors in the datasets they explored and patients-derived cell lines from thalamic tumors available to investigate whether this pathway is active across all H3.3K27M mutants in the brain midline or specifically in tumors from the pons.

2- There are ~20% H3.3K27M tumors that carry an ACVR1 mutation and similar numbers of H3.1K27M that are wild type for this gene. Can the authors identify these outliers in their datasets and assess the activation of BMP2 and 7 or other BMP pathway members in this context?

In all this is an interesting paper that provides meaningful data to pursue clinical targeting of the BMP pathway, which would be a nice addition to the field.

Author Response:

Reviewer #1 (Public Review):

[...] Major concerns/weakness:

1. All the results in Fig. 2 utilized two glioma lines SF188 and Res259. The authors should repeat all these experiments in a couple of H3.3K27M DMG lines by deleting the H3.3K27M mutation first.

We thank the referee for his/her comments that will help us to strengthen our conclusions.

The reviewer's proposal is interesting, but this approach to deletion of the K27M mutation rather answers the question of the role of the BMP pathway in maintaining the phenotype of DMG cells. Our aim in the first part of this article (with Res and SF188) is rather to study how the BMP pathway can participate in installing a particular cellular state at the time of expression of the K27M mutation. In other words, the underlying idea is to define the phenotypic changes specifically associated with activation of the BMP pathway when epigenetic modifications are induced by expression of the K27M mutation. We have chosen the SF188 and Res259 models to remain in a glial context, but it would indeed be interesting to test the effect of this synergy in other models, closer to the cells of origin of DMG. In any case, these models should make it possible to answer the question of the cellular state transition at the moment of K27M expression, even if the reciprocal question of the reversibility of this state proposed by the reviewer is also of interest for understanding the oncogenic synergy between BMP/K27M.

1. Fig. 3. The experiments of BMP2 treatment should be repeated in other H3.3K27M DMG lines using H3.1K27M ACVR1 mutant tumor lines as controls.

We will provide the results of these experiments in a revised version. The use of mutant ACVR1 lines is interesting, but their control status seems questionable, as the addition of BMPs could have a cumulative effect on the effect of the mutation, notably by activating other receptors in the pathway.

Minor concerns:

Fig.2A. BMP2 expression increased in H3.3K27M SF188 cells. Therefore, the statement "whereas BMP2 and BMP4 expressions are not significantly modified (Figure 2A and Figure 2-figure supplement A-B)" is not accurate.

The referee is absolutely right and we will correct this statement in the revised version.

Reviewer #2 (Public Review):

[...] The paper is well-written and easy to follow with a robust experimental plan and datasets supporting the claims. While previous work (acknowledged by the authors) indicated activation of BMP in H3K27M tumors, wild type for the ACVR1 mutation this paper is a nice addition and provides further mechanistic cues as to the importance of the BMP pathway and specific members in these deadly brain cancers. The effect of these BMPs in quiescence and invasion is of particular interest.

We thank the referee for his/her supportive comments.

A few suggestions to clarify the message are provided below:

1- In thalamic diffuse midline gliomas, the BMP pathway should not be activated as it is in the pons. The authors should identify thalamic tumors in the datasets they explored and patients-derived cell lines from thalamic tumors available to investigate whether this pathway is active across all H3.3K27M mutants in the brain midline or specifically in tumors from the pons.

The referee's question is an interesting one, and we will try to see if we can determine tumor’s location from the public data we've used. We will nevertheless try to determine whether the inter-patient variability observed in the level of activation of the BMP pathway may be due, in particular, to different tumor locations.

2 - There are ~20% H3.3K27M tumors that carry an ACVR1 mutation and similar numbers of H3.1K27M that are wild type for this gene. Can the authors identify these outliers in their datasets and assess the activation of BMP2 and 7 or other BMP pathway members in this context?

Indeed, defining the level of activation of the pathway in this type of H3.3K27M ACVR1 mutant or H3.1K27M ACVR1 wt tumors would be extremely interesting, but no samples of this type are a priori included in the datasets analyzed. Instead, we will try to define the phenotype of cell lines of this type in response to BMP.