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

  1. Paul Huchede
  2. Swann Meyer
  3. Clément Berthelot
  4. Maud Hamadou
  5. Adrien Bertrand-Chapel
  6. Andria Rakotomalala
  7. Line Manceau
  8. Julia Tomine
  9. Nicolas Lespinasse
  10. Paul Lewandowski
  11. Martine Cordier-Bussat
  12. Laura Broutier
  13. Aurélie Dutour
  14. Isabelle Rochet
  15. Jean-Yves Blay
  16. Cyril Degletagne
  17. Valéry Attignon
  18. Angel Montero-Carcaboso
  19. Marion Le Grand
  20. Eddy Pasquier
  21. Alexandre Vasiljevic
  22. Pascale Gilardi-Hebenstreit
  23. Samuel Meignan
  24. Pierre Leblond
  25. Vanessa Ribes
  26. Erika Cosset
  27. Marie Castets

  • Curated by eLife

    eLife logo

    eLife assessment

    This valuable study examines whether the BMP signaling pathway has a role in H3.3K27M DMG tumors, regardless of the presence of ACRVR1 activating mutations. The authors provide solid evidence that BMP2/7 synergizes with H3.3K27M to induce a transcriptomic rewiring associated with a quiescent but invasive cell state. Although this work could be further enhanced by the inclusion of additional models, the study overall points to BMP2/7 as a potential target for future therapies in this deadly cancer.

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Pediatric diffuse midline gliomas (pDMG) are an aggressive type of childhood cancer with a fatal outcome. Their major epigenetic determinism has become clear, notably with the identification of K27M mutations in histone H3. However, the synergistic oncogenic mechanisms that induce and maintain tumor cell phenotype have yet to be deciphered. In 20 to 30% of cases, these tumors have an altered BMP signaling pathway with an oncogenic mutation on the BMP type I receptor ALK2, encoded by ACVR1 . However, the potential impact of the BMP pathway in tumors non-mutated for ACVR1 is less clear. By integrating bulk, single-cell, and spatial transcriptomic data, we show here that the BMP signaling pathway is activated at similar levels between ACVR1 wild-type and mutant tumors and identify BMP2 and BMP7 as putative activators of the pathway in a specific subpopulation of cells. By using both pediatric isogenic glioma lines genetically modified to overexpress H3.3K27M and patients-derived DIPG cell lines, we demonstrate that BMP2/7 synergizes with H3.3K27M to induce a transcriptomic rewiring associated with a quiescent but invasive cell state. These data suggest a generic oncogenic role for the BMP pathway in gliomagenesis of pDMG and pave the way for specific targeting of downstream effectors mediating the K27M/BMP crosstalk.

Article activity feed

  1. Version published to 10.7554/elife.91313.3 on eLife
  2. Version published to 10.7554/elife.91313 on eLife
  3. Version published to 10.7554/elife.91313.2 on eLife
  4. eLife

    eLife assessment

    This valuable study examines whether the BMP signaling pathway has a role in H3.3K27M DMG tumors, regardless of the presence of ACRVR1 activating mutations. The authors provide solid evidence that BMP2/7 synergizes with H3.3K27M to induce a transcriptomic rewiring associated with a quiescent but invasive cell state. Although this work could be further enhanced by the inclusion of additional models, the study overall points to BMP2/7 as a potential target for future therapies in this deadly cancer.

  5. eLife

    Reviewer #1 (Public Review):

    Summary:

    Mutational analysis of diffuse midline glioma (DMG) found that ACVR1 mutations, which up-regulate 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:
    Address an important question in 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 H3.3K27M mutation first.
    (2) Fig. 3. The experiments of BMP2 treatment should be repeated in another H3.3K27M DMG line 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

    Comments on revised version:

    I had three issues listed above on the initial version. The authors did not address my major concerns of #1 and #2, which are re-listed above.

  6. eLife

    Author response:

    The following is the authors’ response to the current reviews.

    Reviewer #1 (Public Review):

    We thank the reviewer for his careful reading, which enabled us to improve the quality of this manuscript. We have addressed some major criticisms, and in particular, we have now included the characterization of the impact of BMP2 on other lines as well as the study of the impact of reversion of the H3.3K27M mutation (Figure 3 - figure supplement 1C-D). This control, judiciously proposed by the reviewer, seems more relevant than using mutant H3.1K27M / ACVR1 lines, given the possibility of BMP2 action via other receptors.

    The following is the authors’ response to the original reviews.

    Reviewer #1

    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.

    We thank the referee for his/her comments that have helped us to strengthen our conclusions. Although we were rather interested in studying how the BMP pathway can participate in installing a particular cell state at the time of expression of the K27M mutation, we have now included the characterization of the native H3.3K27M BT245 and SU-DIPGXIII cell lines, and their counterparts in which the mutation was reverted by CRISPRCas9 (Harutyunyan et al., 2019). As shown in Figure 3-figure supplement D, the growth arrest induced by BMP2 seems indeed to be specific of the K27M epigenetic context, which could also be required to settle a positive regulation loop to activate the BMP pathway, as mentioned in the Discussion.

    (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.

    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. But we have now included 3 different cell lines (HSJD-DIPG-014, BT245 and SU-DIPGXIII), and observed similar impact of BMP2 with growth arrest as a readout (Figure 3-figure supplement C-D)

    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 have corrected this statement.

    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.

    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 inter-patient variability observed in the level of activation of the BMP pathway may indeed be due, at least in part, to different tumor locations. However, we failed to find this information in the publicly available datasets that we used. We however included this element in the Discussion part.

    (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?

    We have now included the outliers present in our datasets in the legends of Figure 1B and Figure 1-figure supplement B and F. From the few samples available to document these outliers in the cohorts that we used, we have not observed major differences regarding the expression levels of BMP2/7 or BMP pathway members and have discussed the fact that it may result from the establishment in all cases of a feedback loop of activation.

    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.

    We thank the reviewer for his/her supportive comments.

  7. Version published to 10.1101/2023.08.09.552628v2 on bioRxiv
  8. eLife

    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.

  9. Version published to 10.7554/elife.91313.1 on eLife
  10. eLife

    eLife assessment

    This important study examined whether the BMP signaling pathway has a role in H3.3K27M DMG tumors, regardless of the presence of ACRVR1 activating mutations. The authors show compelling evidence that BMP2/7 synergizes with H3.3K27M to induce a transcriptomic rewiring associated with a quiescent but invasive cell state. Although this work could be further enhanced by the inclusion of additional models as well as further consideration of elements, such as the location of the tumor, in the datasets used, overall this work suggests that BMP2/7 could be considered as a target for future therapies in this deadly cancer.

  11. eLife

    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.

  12. eLife

    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.

  13. Version published to 10.1101/2023.08.09.552628v1 on bioRxiv