A neurodevelopmental epigenetic programme mediated by SMARCD3–DAB1–Reelin signalling is hijacked to promote medulloblastoma metastasis

How abnormal neurodevelopment relates to the tumour aggressiveness of medulloblastoma (MB), the most common type of embryonal tumour, remains elusive. Here we uncover a neurodevelopmental epigenomic programme that is hijacked to induce MB metastatic dissemination. Unsupervised analyses of integrated publicly available datasets with our newly generated data reveal that SMARCD3 (also known as BAF60C) regulates Disabled 1 (DAB1)-mediated Reelin signalling in Purkinje cell migration and MB metastasis by orchestrating cis-regulatory elements at the DAB1 locus. We further identify that a core set of transcription factors, enhancer of zeste homologue 2 (EZH2) and nuclear factor I X (NFIX), coordinates with the cis-regulatory elements at the SMARCD3 locus to form a chromatin hub to control SMARCD3 expression in the developing cerebellum and in metastatic MB. Increased SMARCD3 expression activates Reelin–DAB1-mediated Src kinase signalling, which results in a MB response to Src inhibition. These data deepen our understanding of how neurodevelopmental programming influences disease progression and provide a potential therapeutic option for patients with MB.

publication in Nature Cell Biology.
We would like to clarify that although we have engaged a third referee (Reviewer 1) with expertise on SWI/SNF in cancer on the referee panel, the expertise/comments by the other two referees were sufficient for us to form a decision in the absence of this expert's feedback, and we felt a further delay would be counterproductive for the authors. We will send you the third report if/when we receive it.
Nature Cell Biology editors discuss the referee reports in detail within the editorial team, including the chief editor, to identify key referee points that should be addressed with priority, and requests that are overruled as being beyond the scope of the current study. To guide the scope of the revisions, I have listed these points below. We are committed to providing a fair and constructive peer-review process, so please feel free to contact me if you would like to discuss any of the referee comments further.
In particular, it would be essential to: A)Strengthen the in vivo/ex vivo data as requested by Reviewer 3: "A weaker part of the manuscript is the cell biology part, e.g. the definite proof that tumor cell migration/invasion is influenced by the proposed mechanism, and how exactly with respect to the known different modes of tumor cell movement. Timelapse videos of cellular invasion/migration, ideally in an in vivo or at least proper ex vivo setting (tumor/brain slices), would be important." "Moreover, it would also improve the story if genetic medulloblastoma models are included, ideally with dysregulated SMARCD3 signaling, to gain a better understanding how the dysregulation of this pathway influences medulloblatoma development, growth and metastasis -"from the start"." B) Clarify the cohort information as questioned by both reviewers: Reviewer 2 "...However, it remains unclear, whether this was a Group 3 cohort or an overall MB cohort across molecular groups, in which case the survival analysis would probably be of limited value. The same holds true for the IHC results on a TMA. There was also no information provided on a potential correlation with metastatic stage (a trend for which was provided in Figure 1i)..." D) Finally please pay close attention to our guidelines on statistical and methodological reporting (listed below) as failure to do so may delay the reconsideration of the revised manuscript. In particular please provide: -a Supplementary Figure including unprocessed images of all gels/blots in the form of a multi-page pdf file. Please ensure that blots/gels are labeled and the sections presented in the figures are clearly indicated.
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We hope that you will find our referees' comments, and editorial guidance helpful. Please do not hesitate to contact me if there is anything you would like to discuss. Remarks to the Author: The manuscript entitled "Hijacking a neurodevelopmental epigenomic program in metastatic dissemination of medulloblastoma" by Han Zou and coworkers aims at identifying the underpinnings of metastatic dissemination in Group 3 medulloblastoma. The authors start by identifying SMARCD3 as the only epigenetic modulator amongst 77 Group 3 specific DEGs, which in in turn shows (modestly) elevated mRNA and protein expression in Group 3 MB. They also show survival data for SMARCD3 high and low expressing MBs. However, it remains unclear, whether this was a Group 3 cohort or an overall MB cohort across molecular groups, in which case the survival analysis would probably be of limited value. The same holds true for the IHC results on a TMA. There was also no information provided on a potential correlation with metastatic stage (a trend for which was provided in Figure 1i).The authors went on to demonstrate that higher SMARCD3 levels in Group 3 cell lines (n=6) were associated with a higher migratory potential in transwell assays as well as in xenograft models. Knockout of SMARCD3 in two cell lines was associated with less migration in vitro and in vivo (the latter with an n=4 each). This was much further substantiated in gain-and loss-of-function assays in a matched primary-metastatic cell line pair including strong differences in the number of CTCs. Survival differences in these experiments were rather modest. Knockout experiments in two cell lines revealed a downregulation of RELN pathway genes. Of these, DAB1 was also associated with modest overexpression in G3 and correlation with SMARCD3 levels (both mRNA and protein). There also seems to be a slight difference in DAB1 expression between metastatic and non-metastatic tumors. It is not clear, whether this was a group 3 MB cohort or across all molecular groups. Next the authors invesigated the role of RELN pathway members in normal embryonal murine Purkinje cells and their interaction with granule cells. They showed that DAB1 (and others) were highly expressed in developing PCs, whereas RELN was highly expressed in GCs. The conclusions from figure 4 seem quite strong given that they are solely based on correlations. ATAC-Seq in one of the knockout cell lines revelaed decreased chromatin accesibility at the DAB1 locus upon knockout of SMARCD3 indicating a direct regulatory role. This was also associated with other repressive chromatin marks in tumor samples. In a cell line model and a small patient cohort the authors also showed that SMARCD3 was associated with increased enhancer activity in group 3 MB. It is not clear why the author didn´t use published enhancer datasets on primary samples to substantiate this finding. Furthermore, the authors demonstrated that specific CREs were responsible for the regulation of SMARCD3. Using knockout experiments, the authors narrowed down candidate transcription factors to regulate SMARCD3 in Gr 3 MB cell lines to EZH2 and NFIX. Finally, the authors provide evidence that SMARCD3 expression and src phosphorylation were correlated in 10 paired patient samples and increased upon metastatic dissemintion. Dasatinib reduced the migratory propensity in vitro and in vivo, but did not influence cell survival.
Overall, this is a well conducted project with some limitations as indicated, which identifies one pro-metastatic mechanism in group 3 medulloblastoma based on hijacking a developmental program.

Minor:
LGG is the most common brain tumor in children, and even for malignant brain tumors, HGGs are more common than MB.
Reviewer #3: Remarks to the Author: In this manuscript, the authors report that medulloblastoma cells of at least the G3 subtype (this subtype issue needs more clarification) use neurodevelopmental epigenetic programs to promote metastatic dissemination, with SMARCD3 activating Reelin/DAB1/SRC signaling. Tumor biology is mapped to normal neuro/cerebellar development, back-and-forth in a quite compelling way.
While the manuscript is often not an easy read for the non-molecular biologist, the findings are of considerable interest and value for the field. The question how (brain) tumor hijack neurodevelopmental programs to thrive is certainly a very relevant one, and one that bears the promise to gain much deeper understanding of these challenging diseases. The authors might want to stress this point even more, and put their findings into a broader (neuro-)oncology context, not restricted to medulloblastoma.
The experiments are well conducted, and the conclusions are solid. There are a few points that reduce the enthusiasm a little bit at this point of time and which should be addressed: A weaker part of the manuscript is the cell biology part, e.g. the definite proof that tumor cell migration/invasion is influenced by the proposed mechanism, and how exactly with respect to the known different modes of tumor cell movement. Timelapse videos of cellular invasion/migration, ideally in an in vivo or at least proper ex vivo setting (tumor/brain slices), would be important.
Moreover, it would also improve the story if genetic medulloblastoma models are included, ideally with dysregulated SMARCD3 signaling, to gain a better understanding how the dysregulation of this pathway influences medulloblatoma development, growth and metastasis -"from the start".  : the difference of SMARCD3 expression between metastastic and nonmetastatic patients is there and statistically significant, but not extremely strong. What about other genes: how many genes differ between two groups-how many show a stronger difference, how many a smaller difference? Where does SMARCD3 fit into this picture?
It would be important to know whether SMARCD3 KO/lower expressing cells have a general problem with cell viability (e.g., decreased proliferation rate), or whether the effect of low/no expression is specific for cell invasion/migration. The latter would be more compelling when speaking of SMARCD3 as a factor for metastasis.
Minor: the first sentence of the abstract ("how dysregulation...remains elusive") reads a little bold, when considering the high number of neurodevelopmental genes that have been investigated in medulloblastoma. Nevertheless, the authors are certainly correct that a study covering both aspects in depths (and providing new findings for both, tumorigenesis and neurodevelopment at the same time) is very rare in the field. Maybe the authors want to consider to stress this point more.
Minor 2: A better graphical summary/schematic drawing could make the complex story bettter digestible for most readers.

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---------Please don't hesitate to contact NCB@nature.com should you have queries about any of the above requirements ---------Author Rebuttal to Initial comments The authors thank the reviewers for their comments on our manuscript (# NCB-A47467) and have addressed these comments as follows: Reviewer #1: None Reviewer #2: Remarks to the Author: The manuscript entitled "Hijacking a neurodevelopmental epigenomic program in metastatic dissemination of medulloblastoma" by Han Zou and coworkers aims at identifying the underpinnings of metastatic dissemination in Group 3 medulloblastoma. The authors start by identifying SMARCD3 as the only epigenetic modulator amongst 77 Group 3 specific DEGs, which in in turn shows (modestly) elevated mRNA and protein expression in Group 3 MB. They also show survival data for SMARCD3 high and low expressing MBs. However, it remains unclear, whether this was a Group 3 cohort or an overall MB cohort across molecular groups, in which case the survival analysis would probably be of limited value. The same holds true for the IHC results on a TMA. There was also no information provided on a potential correlation with metastatic stage (a trend for which was provided in Figure 1i).

Response:
We appreciate the reviewer for highlighting this issue and we apologize for the point being unclear in the manuscript. First, we clarified that the survival analyses in Fig. 1f and Fig. 1g were based upon all four molecular subgroups of medulloblastoma (MB). We did the overall survival in Group 3 MB only and the result showed a trend of higher levels of SMARCD3 mRNA expression associated with a worse prognosis although this is not statistically significant (Rebuttal Fig. 1a). A similar observation was obtained in Group 3 MB by immunohistochemistry (IHC) analysis of SMARCD3 protein expression on the tissue microarray (TMA) (Rebuttal Fig. 1b). The evidence provided in the manuscript together with these additional data suggest that the elevated level of SMARCD3 is a hallmark feature of Group 3 MB that is the most aggressive subgroup compared to other MB subgroups 1 . Therefore, we believe this is why the significant correlation between SMARCD3 and overall survival in Group 3 only was not easily observed.
Accordingly, we clarified these points in the revised manuscript (Page 5; Line 105-110).
The authors went on to demonstrate that higher SMARCD3 levels in Group 3 cell lines (n=6) were associated with a higher migratory potential in transwell assays as well as in xenograft models. Knockout of SMARCD3 in two cell lines was associated with less migration in vitro and in vivo (the latter with an n=4 each). This was much further substantiated in gain-and loss-of-function assays in a matched primary-metastatic cell line pair including strong differences in the number of CTCs. Survival differences in these experiments were rather modest. Knockout experiments in two cell lines revealed a downregulation of RELN pathway genes. Of these, DAB1 was also associated with modest overexpression in G3 and correlation with SMARCD3 levels (both mRNA and protein). There also seems to be a slight difference in DAB1 expression between metastatic and non-metastatic tumors. It is not clear, whether this was a group 3 MB cohort or across all molecular groups.

Response:
We thank the reviewer for this question. The correlations between DAB1 mRNA expression and tumor metastasis were analyzed using MB patients across all the subgroups. We clarified this point in the revised manuscript (Page 8, Line 194).
Next the authors invesigated the role of RELN pathway members in normal embryonal murine Purkinje cells and their interaction with granule cells. They showed that DAB1 (and others) were highly expressed in developing PCs, whereas RELN was highly expressed in GCs. The conclusions from figure 4 seem quite strong given that they are solely based on correlations. ATAC-Seq in one of the knockout cell lines revelaed decreased chromatin accesibility at the DAB1 locus upon knockout of SMARCD3 indicating a direct regulatory role. This was also associated with other repressive chromatin marks in tumor samples. In a cell line model and a small patient cohort the authors also showed that SMARCD3 was associated with increased enhancer activity in group 3 MB. It is not clear why the author didn´t use published enhancer datasets on primary samples to substantiate this finding.

Response:
We thank the reviewer for asking for this clarification. We analyzed the published datasets, and the results were described in the Extended Data Fig. 6a. The datasets were used from the publication 2 .
Furthermore, the authors demonstrated that specific CREs were responsible for the regulation of SMARCD3.
Using knockout experiments, the authors narrowed down candidate transcription factors to regulate SMARCD3 in Gr 3 MB cell lines to EZH2 and NFIX. Finally, the authors provide evidence that SMARCD3 expression and src phosphorylation were correlated in 10 paired patient samples and increased upon metastatic dissemintion. Dasatinib reduced the migratory propensity in vitro and in vivo, but did not influence cell survival.
Overall, this is a well conducted project with some limitations as indicated, which identifies one prometastatic mechanism in group 3 medulloblastoma based on hijacking a developmental program.

Response:
We truly appreciate the evaluation made by the reviewer and the comments that are very helpful in improving the manuscript.

Minor:
LGG is the most common brain tumor in children, and even for malignant brain tumors, HGGs are more common than MB. Response: We agree. We also thank the reviewer for pointing out this mistake. We edited the sentences in the revised manuscript (Page 3, lines 48-49; page 3, Line 63-64).
Reviewer #3: Remarks to the Author: In this manuscript, the authors report that medulloblastoma cells of at least the G3 subtype (this subtype issue needs more clarification) use neurodevelopmental epigenetic programs to promote metastatic dissemination, with SMARCD3 activating Reelin/DAB1/SRC signaling. Tumor biology is mapped to normal neuro/cerebellar development, back-and-forth in a quite compelling way.
While the manuscript is often not an easy read for the non-molecular biologist, the findings are of considerable interest and value for the field. The question how (brain) tumor hijack neurodevelopmental programs to thrive is certainly a very relevant one, and one that bears the promise to gain much deeper understanding of these challenging diseases. The authors might want to stress this point even more, and put their findings into a broader (neuro-)oncology context, not restricted to medulloblastoma.

Response:
We appreciate the reviewer making these important suggestions. MB is the most common type of embryonal tumor that arises in the cerebellum, indicating a relationship between tumor development and embryonal cell maturation. We are interested in understanding how abnormal brain development influences the tumor biology of medulloblastoma. We believe that SMARCD3-mediated tumor hijacking neurodevelopmental programs could happen in other subgroups of medulloblastoma. Interestingly, we found the enrichment of medulloblastoma in the SMARCD3-associated normal developmental genes based on gene-disease network analysis (DisGeNET) (Extended Data Fig.4f). These results suggest that SMARCD3-regulated Reelin/DAB1 signaling in cell migration and tumor cells hijacking SMARCD3-Reelin/DAB1 signaling for promoting metastasis could be specific to cerebellar development and medulloblastoma progression, respectively. Given activation of the Reelin/DAB1 signaling pathway in other cell types located in different regions of the brain, such as Cajal-Retzius cells in the neocortex and hippocampus 3,4 , we also believe the regulatory network of SMARCD3-Reelin/DAB1 signaling could be happening in other cell types and brain areas under physiological and pathological conditions. We agree with the reviewer's comments and believe that tumor cells hijacking normal neurodevelopmental processes and mechanisms may broadly exist in other brain cancers.
We thank the reviewer again for this interesting discussion and we will focus on experimental validations on these points in our future research. We emphasized this point in the section of Discussion in the revised manuscript (Page 17-18, Line 418-420, and Line 433-435).
The experiments are well conducted, and the conclusions are solid. There are a few points that reduce the enthusiasm a little bit at this point of time and which should be addressed: A weaker part of the manuscript is the cell biology part, e.g. the definite proof that tumor cell migration/invasion is influenced by the proposed mechanism, and how exactly with respect to the known different modes of tumor cell movement. Time-lapse videos of cellular invasion/migration, ideally in an in vivo or at least proper ex vivo setting (tumor/brain slices), would be important.

Response:
We thank the reviewer for this question. We also thank the suggested experiments by the reviewer. To this end, time-lapse image acquisition was first performed in the cell culture condition of the medulloblastoma (MB) Group 3 cell line MED8A with SMARCD3 wildtype (WT) or SMARCD3 knockout (KO) in the scratch-wound healing assays. (Supplementary Video 1). Strikingly, SMARCD3 deletion significantly decreased directional cell migration velocity and non-directional cell motility speed in MED8A cells (Revised Extended Data Fig.2j).
To further examine the effect of SMARCD3 on cell movement in vivo, we set up an ex vivo brain slice model by transplanting GFP-labeled MED8A-WT and MED8A-SMARCD3 KO cells into the cerebellum of SCID mice. A week following cell implantation, a time-lapse confocal microscope was used to examine cell movement under the brain slice culture condition (Supplementary Video 2). Consistent with in vitro results, the GFP+ tumor cell movement in directionality, velocity, and speed was significantly reduced in the cerebellar tissues implanted with MED8A-SMARCD3 KO compared with MED8A-WT cells (Revised Extended Data Fig. 2k). We added these extended data in the revised manuscript (Page 7, Line 150-154).
In this study, we found that mechanistically SMARCD3 upregulated DAB1 expression and activated its downstream signaling. It is known that DAB1 is a crucial cellular adaptor in Reelin signaling-mediated Purkinje cell migration and positioning during the early stages of the development of the cerebellum 5 . Moreover, the activated DAB1 signaling regulates cell skeleton, Cadherin, and integrin functions during neuronal positioning and migration 4,6 . Three modes of tumor cell movement were described previously, including mesenchymal, amoeboid, and collective modes; and cancer cells can adapt their migration strategies to the different tumor microenvironments by switching among these migration modes 7,8 . Based on our findings aligning with these time-lapse videos of cell movement, we believe that SMARCD3-driven MB cell movement is mediated by Reelin/DAB1 signaling-mediated neuronal migration, which increases the directionality in both MB cell movement and normal cerebellar development, together with mesenchymal and/or collective modes during tumor progression.
Moreover, it would also improve the story if genetic medulloblastoma models are included, ideally with dysregulated SMARCD3 signaling, to gain a better understanding how the dysregulation of this pathway influences medulloblastoma development, growth and metastasis -"from the start". SMARCD3 in the malignant transformation of neural stem cells that were considered as the cells of origin of Group 3 MB 9-11 . We employed virus-induced spontaneous tumor formation in postnatal C57BL/6J mice.
[REDACTED] The results revealed that overexpression of MYC S62D alone and MYC S62D + SMARCD3 significantly induced tumor formation, however, overexpression of SMARCD3 alone did not induce tumor formation (Rebuttal Fig. 2a, b). While a statistically significant difference between the two groups was not obtained, there is a reproducible trend of shorter survival in mice bearing SMARCD3+ MYC S62D -induced tumors compared with MYC S62D -induced tumors (Rebuttal Fig. 2b). Furthermore, we observed no obvious differences in tumor sizes between MYC S62D -induced tumors and SMARCD3+ MYC S62D -induced tumors based on GFP fluorescence analysis (Rebuttal Fig. 2c). Consistently, SMARCD3 overexpression promoted tumor spinal metastasis in MYC S62D -induced tumors (4 out of 4, 100% in SMARCD3 + MYC S62D vs 2 out of 5, 40% in MYC S62D alone) (Rebuttal Fig. 2d). Histopathological analysis revealed that both MYC S62D -and SMARCD3+ MYC S62D -induced tumors showed the typical features of Group 3 MB, including large cell/anaplastic (LCA) patterns with nuclear molding and wrapping, focally prominent nucleoli, abundant mitotic cells by H&E staining, high proliferation (Ki67), lack of glial marker glial fibrillary acidic protein (GFAP) expression, and differentiated neuronal lineage marker synaptophysin, but positive staining for neuronal progenitor markers, such as Nestin, β3-tubulin, and Oligo2 (Rebuttal Fig. 2e). Importantly, both MYC S62D -and SMARCD3 + MYC S62D -induced tumors displayed Group 3 specific marker NPR3 expression by IHC staining (Rebuttal Fig. 2e). Of note, no differences in cell proliferation index (Ki67) were observed between these two group tumors (Rebuttal Fig. 2e). Collectively, these results suggest that SMARCD3 plays a pivotal role in tumor metastasis, rather than participating in MB initiation and growth.
We previously reported an approach of in vivo malignant transformation of human neural stem cells to generate a glioblastoma model 12 . We employed this strategy to establish a new MB model by using a human cerebellar neural stem cell line. A previous study showed that this human cerebellar neural stem cell line expressing wildtype MYC alone (hereafter hcNSCs) displayed a low malignant potential for MB formation 13 . To examine if SMARCD3 increases the malignant transformation of hcNSCs, cells were infected with lentivirus carrying SMARCD3 (resulting in overexpression) and subsequently orthotopically implanted into the cerebellum of SCID mice. Notably, we did not observe SMARCD3-induced tumor formation in these SCID mice for up to 90 days, however, overexpression of constitutively active MYC S62D in hcNSCs dramatically increased tumor formation in orthotopic SCID mouse models (Rebuttal Fig. 3a). While we did not observe significant differences in mouse survival and tumor sizes between MYC S62D alone and SMARCD3 + MYC S62D induced tumors (Rebuttal Fig. 3a, b), overexpression of SMARCD3 promoted tumor spinal metastasis in MYC S62D induced tumors based on bioluminescence imaging (BLI) analysis and visualization of the spinal cord by assessing GFP-labeled tumor xenograft mice (Rebuttal Fig. 3c, d). The data using this human-in-mouse MB model further support our conclusion that SMARCD3 significantly influences tumor metastasis rather than tumor development and growth.
The new data align with the results in the manuscript and together strongly indicate that SMARCD3 plays a critical role in promoting cell migration and tumor metastasis, rather than tumor initiation and growth, during MB progression.

Response:
We thank the reviewer for this concern. We did observe a trend of higher levels of SMARCD3 expression associated with a worse prognosis in Group 3 MB only, but not statistically different. Our data in this manuscript indicate a major role of SMARCD3 in driving cell migration and tumor metastasis, which is the hallmark of Group 3 MB, [REDACTED] Fig. 1i: the difference of SMARCD3 expression between metastastic and non-metastatic patients is there and statistically significant, but not extremely strong. What about other genes: how many genes differ between two groups-how many show a stronger difference, how many a smaller difference? Where does SMARCD3 fit into this picture?

Response:
We appreciate the reviewer's concerns and questions. We analyzed gene distributions between tumors with metastasis and tumors without metastasis. Based on gene transcriptomics in the Group 3 subgroup of human medulloblastoma, 1,937 genes are statistically significantly higher (P < 0.05, log2(fold change) > 0) in metastatic tumors and 1,126 genes are statistically significantly lower (P < 0.05, log2(fold change) < 0) in metastatic tumors. SMARCD3 is in the top 7.331% of the 1,937 genes according to log2(fold change) (Rebuttal Fig. 4). Based on gene transcriptomics in all subgroups of human medulloblastoma, 3,984 genes are statistically significantly higher (P < 0.05, log2(fold change) > 0) in metastatic tumors and 3,110 genes are statistically significantly lower (P < 0.05, log2(fold change) < 0) in metastatic tumors. SMARCD3 is on the top 8.584% of the 3,984 genes according to the log2(fold change) (Rebuttal Fig. 4).
It would be important to know whether SMARCD3 KO/lower expressing cells have a general problem with cell viability (e.g., decreased proliferation rate), or whether the effect of low/no expression is specific for cell invasion/migration. The latter would be more compelling when speaking of SMARCD3 as a factor for metastasis.

Rebuttal Fig 4: The distributions of SMARCD3 gene expression in patient MBs with/without metastasis.
Histograms showing the number of differentially expressed genes between patients with metastasis and without metastasis by the log2(fold change). The arrows denote where SMARCD3 is located. The data from Group 3 only (right) and all subgroups of human MBs (left) were analyzed.

Response:
We thank the reviewer for these points . To examine whether SMARCD3 influences cell proliferation or viability, we first performed a BrdU assay in MB cell line MED8A with SMARCD3 deletion or overexpression. We found no significant differences in cell proliferation when SMARCD3 was deleted by CRISPR/Cas9-mediated gene knockout or overexpressed SMARCD3 in MED8A cells (Rebuttal Fig. 5a). Second, we performed an MTS assay to examine cell viability in two MB cell lines. The results showed no significant changes in cell viability after SMARCD3 deletion in MED8A and D458 cells during cell growth (Rebuttal Fig. 5b, c). These additional data suggest that SMARCD3 plays a critical role in cell migration and tumor metastasis, rather than cell proliferation.
Minor: the first sentence of the abstract ("how dysregulation...remains elusive") reads a little bold, when considering the high number of neurodevelopmental genes that have been investigated in medulloblastoma. Nevertheless, the authors are certainly correct that a study covering both aspects in depths (and providing new findings for both, tumorigenesis and neurodevelopment at the same time) is very rare in the field. Maybe the authors want to consider to stress this point more.

Response:
We thank the reviewer's thoughtful concern and suggestion. The sentence of the abstract was altered, and we tried to stress the point of how abnormal neurodevelopment relates to tumor aggressiveness in medulloblastoma (Page 3, Line 48-49).
Minor 2: A better graphical summary/schematic drawing could make the complex story better digestible for most readers.

Response:
We agree and thank the reviewer for this suggestion. A graphical summary was included in the revised manuscript (Revised Extended Data Fig. 7e; Page 17, Line 415-418). Your manuscript, "Hijacking a neurodevelopmental epigenomic program in metastatic dissemination of medulloblastoma", has now been seen by our original referees. As you will see from their comments (attached below) they find this work of interest, but have raised some important points. Although we are also very interested in this study, we believe that their concerns should be addressed before we can consider publication in Nature Cell Biology.

References
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We would like to receive the revision within four weeks. If submitted within this time period, reconsideration of the revised manuscript will not be affected by related studies published elsewhere, or accepted for publication in Nature Cell Biology in the meantime. We would be happy to consider a revision even after this timeframe, but in that case we will consider the published literature at the time of resubmission when assessing the file.
We hope that you will find our referees' comments, and editorial guidance helpful. Please do not hesitate to contact me if there is anything you would like to discuss. The revision provides only partially satisfactory improvements over the first submission. However, the survival analyses re. SMARCD3 RNA experssion and IHC should either be completely removed or the group3 specific analyses should be moved into the main manuscript in order to reansparently provide important information needed to fully interpret the data. Same holds true for the DAB1 mRNA epression correlations. A conclusion on the public enhancer datasets (and how supportive this is (or not)) was not provided in the revised manuscript.
Reviewer #3: Remarks to the Author: The authors have responded well and convincingly to my questions and criticisms.
I would strongly suggest to include the additional and important data that were generated in reponse to my points into the manuscript -and not to keep it as reviewer-only figures. This applies to Rebuttal Fig. 2

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We strongly recommend the presentation of source data for graphical and statistical analyses as a separate Supplementary ---------Please don't hesitate to contact NCB@nature.com should you have queries about any of the above requirements ---------Author Rebuttal, first revision: The authors thank the reviewers for their comments on the revision of our manuscript (# NCB-A47467) and have addressed these comments as follows: Reviewer #2: The revision provides only partially satisfactory improvements over the first submission. However, the survival analyses re. SMARCD3 RNA experssion and IHC should either be completely removed or the group3 specific analyses should be moved into the main manuscript in order to reansparently provide important information needed to fully interpret the data. Same holds true for the DAB1 mRNA epression correlations.

Response:
We appreciate the reviewer for the evaluation of our revision and for making helpful comments to improve the manuscript. Following the reviewer's suggestion, we added additional data examining SMARCD3 mRNA and protein expression using gene profiling and IHC analysis of the G3 medulloblastoma (MB) subgroup (Revised Fig. 1 f and g). To interpret the weak correlation between patient survival and SMARCD3 expression levels in the G3 subgroup, we analyzed the relative dispersion of SMARCD3 mRNA expression levels among patient MB samples. We found that the levels of SMARCD3 mRNA expression are higher but with smaller variation in each sample within the G3 cohort compared with the entire MB subgroup cohorts (4.289 in all MB samples vs 0.433 in the G3) (Revised Extended Data Fig.1d). Statistically, a small variation in the characteristic of samples results in a low correlation coefficient, which might explain our observation of the weak correlations between SMARCD3 and patient survival in the G3 subgroup.
Additionally, we believe that the hypothesis and conclusion in this manuscript, that SMARCD3 regulates tumor metastatic dissemination, can be applied to other MB subgroups, not to the G3 subgroup exclusively. Given that G3 is the most aggressive subgroup with strong metastatic potential compared with other MB subgroups, the G3 subgroup becomes the most significant model to examine the association and regulatory relationship between SMARCD3 and tumor metastatic dissemination. Therefore, besides analysis in the G3 subgroup exclusively, we also used the datasets across all the MB subgroups when we analyzed the correlation between SMARCD3 and other related factors, such as patient outcomes, metastasis, DAB1 expression, and so on. Consistently, the levels of DAB1 mRNA expression are also higher but with smaller variation in each sample within the G3 cohort compared with all MB subgroups (10.422 in all MB samples vs 0.459 in the G3) (Revised Extended Data Fig.4c). Thank you for submitting your revised manuscript "Hijacking a neurodevelopmental epigenomic program in metastatic dissemination of medulloblastoma" (NCB-A47467B). It has now been seen by the original referees and their comments are below. The reviewers find that the paper has improved in revision, and therefore we'll be happy in principle to publish it in Nature Cell Biology, pending minor revisions to comply with our editorial and formatting guidelines.
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