VGLL2-NCOA2 leverages developmental programs for pediatric sarcomagenesis

Sarah Watson; Collette A. LaVigne; Lin Xu; Didier Surdez; Joanna Cyrta; Delia Calderon; Matthew V. Cannon; Matthew R. Kent; Katherine M. Silvius; Jack P. Kucinski; Emma N. Harrison; Whitney Murchison; Dinesh Rakheja; Franck Tirode; Olivier Delattre; James F Amatruda; Genevieve C. Kendall

doi:10.1016/j.celrep.2023.112013

VGLL2-NCOA2 leverages developmental programs for pediatric sarcomagenesis

Sarah Watson, Collette A. LaVigne, Lin Xu, Didier Surdez, Joanna Cyrta, Delia Calderon, Matthew V. Cannon, Matthew R. Kent, Katherine M. Silvius, Jack P. Kucinski, Emma N. Harrison, Whitney Murchison, Dinesh Rakheja, Franck Tirode, Olivier Delattre, James F Amatruda, Genevieve C. Kendall

Research output: Contribution to journal › Article › peer-review

Abstract

Clinical sequencing efforts are rapidly identifying sarcoma gene fusions that lack functional validation. An example is the fusion of transcriptional coactivators, VGLL2-NCOA2, found in infantile rhabdomyosarcoma. To delineate VGLL2-NCOA2 tumorigenic mechanisms and identify therapeutic vulnerabilities, we implement a cross-species comparative oncology approach with zebrafish, mouse allograft, and patient samples. We find that VGLL2-NCOA2 is sufficient to generate mesenchymal tumors that display features of immature skeletal muscle and recapitulate the human disease. A subset of VGLL2-NCOA2 zebrafish tumors transcriptionally cluster with embryonic somitogenesis and identify VGLL2-NCOA2 developmental programs, including a RAS family GTPase, ARF6. In VGLL2-NCOA2 zebrafish, mouse, and patient tumors, ARF6 is highly expressed. ARF6 knockout suppresses VGLL2-NCOA2 oncogenic activity in cell culture, and, more broadly, ARF6 is overexpressed in adult and pediatric sarcomas. Our data indicate that VGLL2-NCOA2 is an oncogene that leverages developmental programs for tumorigenesis and that reactivation or persistence of ARF6 could represent a therapeutic opportunity.

Original language	English (US)
Article number	112013
Journal	Cell Reports
Volume	42
Issue number	1
DOIs	https://doi.org/10.1016/j.celrep.2023.112013
State	Published - Jan 31 2023

Keywords

CP: Cancer
cross-species comparative oncology
developmental biology
functional genomics
fusion oncogene
pediatric cancer
rhabdomyosarcoma

ASJC Scopus subject areas

Biochemistry, Genetics and Molecular Biology(all)

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10.1016/j.celrep.2023.112013

Cite this

Watson, S., LaVigne, C. A., Xu, L., Surdez, D., Cyrta, J., Calderon, D., Cannon, M. V., Kent, M. R., Silvius, K. M., Kucinski, J. P., Harrison, E. N., Murchison, W., Rakheja, D., Tirode, F., Delattre, O., Amatruda, J. F., & Kendall, G. C. (2023). VGLL2-NCOA2 leverages developmental programs for pediatric sarcomagenesis. Cell Reports, 42(1), [112013]. https://doi.org/10.1016/j.celrep.2023.112013

Watson, S, LaVigne, CA, Xu, L, Surdez, D, Cyrta, J, Calderon, D, Cannon, MV, Kent, MR, Silvius, KM, Kucinski, JP, Harrison, EN, Murchison, W, Rakheja, D, Tirode, F, Delattre, O, Amatruda, JF & Kendall, GC 2023, 'VGLL2-NCOA2 leverages developmental programs for pediatric sarcomagenesis', Cell Reports, vol. 42, no. 1, 112013. https://doi.org/10.1016/j.celrep.2023.112013

@article{f1028c1fff5b4af39a26e1721d6e2d72,

title = "VGLL2-NCOA2 leverages developmental programs for pediatric sarcomagenesis",

abstract = "Clinical sequencing efforts are rapidly identifying sarcoma gene fusions that lack functional validation. An example is the fusion of transcriptional coactivators, VGLL2-NCOA2, found in infantile rhabdomyosarcoma. To delineate VGLL2-NCOA2 tumorigenic mechanisms and identify therapeutic vulnerabilities, we implement a cross-species comparative oncology approach with zebrafish, mouse allograft, and patient samples. We find that VGLL2-NCOA2 is sufficient to generate mesenchymal tumors that display features of immature skeletal muscle and recapitulate the human disease. A subset of VGLL2-NCOA2 zebrafish tumors transcriptionally cluster with embryonic somitogenesis and identify VGLL2-NCOA2 developmental programs, including a RAS family GTPase, ARF6. In VGLL2-NCOA2 zebrafish, mouse, and patient tumors, ARF6 is highly expressed. ARF6 knockout suppresses VGLL2-NCOA2 oncogenic activity in cell culture, and, more broadly, ARF6 is overexpressed in adult and pediatric sarcomas. Our data indicate that VGLL2-NCOA2 is an oncogene that leverages developmental programs for tumorigenesis and that reactivation or persistence of ARF6 could represent a therapeutic opportunity.",

keywords = "CP: Cancer, cross-species comparative oncology, developmental biology, functional genomics, fusion oncogene, pediatric cancer, rhabdomyosarcoma",

author = "Sarah Watson and LaVigne, {Collette A.} and Lin Xu and Didier Surdez and Joanna Cyrta and Delia Calderon and Cannon, {Matthew V.} and Kent, {Matthew R.} and Silvius, {Katherine M.} and Kucinski, {Jack P.} and Harrison, {Emma N.} and Whitney Murchison and Dinesh Rakheja and Franck Tirode and Olivier Delattre and Amatruda, {James F} and Kendall, {Genevieve C.}",

note = "Funding Information: We thank the UT Southwestern Histopathology core for excellent services and for their expertise, and the Nationwide Children's Hospital (NCH) Animal Resources Core for their exceptional zebrafish husbandry. Additional support was provided by the Nationwide Children's Hospital Microscopy Lab in the Center for Gene Therapy for imaging, the NCH Morphology Core for tissue processing, the NCH CRISPR/Gene Editing Core for generating cell line knockouts, and the NCH High-Performance Computing group for assistance maintaining and using the NCH cluster. G.C.K. is supported by an Alex's Lemonade Stand Foundation A Award, a V Foundation for Cancer Research V Scholar Grant, and an R01CA272872 grant through the National Cancer Institute of the National Institutes of Health. J.F.A. was supported by grant RP120685-P3 from the Cancer Prevention and Research Institute of Texas (CPRIT) and by Curing Kids Cancer. L.X. is supported by Rally Foundation, Cancer Center Support Grant P30 CA142543 and RP180805 from CPRIT. F.T. is supported by the Institut National de la Recherche M{\'e}diacale (INSERM). D.R. is supported by grant RP180319 from the Cancer Prevention and Research Institute of Texas (CPRIT) and by the John Lawrence and Patsy Louise Goforth Chair in Pathology. S.W. was supported by a grant from the Fondation Nuovo-Soldati pour la recherche en canc{\'e}rologie. M.K. is supported by a T32CA269052 Training Program in Basic and Translational Pediatric Oncology Research postdoctoral fellowship. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. S.W. F.T. O.D. J.F.A. and G.C.K. conceived and supervised the study. F.T. provided computational analysis, insight, and expertise of human and zebrafish RNA-seq data and their similarity in Figures 2, 6, and S3. L.X. provided computational analysis, insight, and expertise into developmental studies and human and zebrafish RNA-seq data in Figures 3, 7, and S4. M.V.C. provided computational analysis, insight, and expertise in Figures 2, 4, S3, and S6. D.R. and J.C. provided pathological assessment. S.W. C.A.L. D.S. J.C. D.C. M.K. K.S. J.K. E.H. W.M. and G.C.K. performed experiments. C.A.L. J.C. D.C. M.K. K.S. J.K. E.H. and G.C.K. analyzed experimental data. C.A.L. J.F.A. and G.C.K. drafted the manuscript and figures. All authors reviewed and edited the final manuscript. The authors declare no competing interests. One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in their field of research or within their geographical location. One or more of the authors of this paper received support from a program designed to increase minority representation in their field of research. One or more of the authors of this paper self-identifies as living with a disability. We support inclusive, diverse, and equitable conduct of research. Funding Information: We thank the UT Southwestern Histopathology core for excellent services and for their expertise, and the Nationwide Children{\textquoteright}s Hospital (NCH) Animal Resources Core for their exceptional zebrafish husbandry. Additional support was provided by the Nationwide Children's Hospital Microscopy Lab in the Center for Gene Therapy for imaging, the NCH Morphology Core for tissue processing, the NCH CRISPR/Gene Editing Core for generating cell line knockouts, and the NCH High-Performance Computing group for assistance maintaining and using the NCH cluster. G.C.K. is supported by an Alex's Lemonade Stand Foundation A Award, a V Foundation for Cancer Research V Scholar Grant, and an R01CA272872 grant through the National Cancer Institute of the National Institutes of Health . J.F.A. was supported by grant RP120685-P3 from the Cancer Prevention and Research Institute of Texas ( CPRIT ) and by Curing Kids Cancer. L.X. is supported by Rally Foundation , Cancer Center Support Grant P30 CA142543 and RP180805 from CPRIT . F.T. is supported by the Institut National de la Recherche M{\'e}diacale (INSERM). D.R. is supported by grant RP180319 from the Cancer Prevention and Research Institute of Texas (CPRIT) and by the John Lawrence and Patsy Louise Goforth Chair in Pathology. S.W. was supported by a grant from the Fondation Nuovo-Soldati pour la recherche en canc{\'e}rologie . M.K. is supported by a T32CA269052 Training Program in Basic and Translational Pediatric Oncology Research postdoctoral fellowship. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Funding Information: RNA-seq data from VGLL2-NCOA2 tumors is from Watson et al., 2018. 4 RNA-seq data for n = 264 adult sarcomas is from TCGA and represents dedifferentiated liposarcoma, leiomyosarcoma, undifferentiated pleomorphic sarcoma, myxofibrosarcoma, malignant peripheral nerve sheath tumor, and synovial sarcoma. 49 RNA-seq data from n = 96 Ewing sarcoma tumors is from Crompton et al., 2014, 50 RNA-seq data from n = 87 Osteosarcoma (phs000468) and n = 13 Clear Cell Sarcoma of the Kidney (phs000466) are based upon data generated by the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) ( https://ocg.cancer.gov/programs/target ) initiative, phs000468 and phs000466. The data used for this analysis are available at https://portal.gdc.cancer.gov/projects . RNA-seq data from n = 42 fusion-positive and fusion-negative rhabdomyosarcoma is from Chen et al., 2013. 51 RNA-seq data from n = 396 adult skeletal muscle samples are from the Genotype-Tissue Expression Project (GTEx). The GTEx Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. The data used for the analyses described in this manuscript in Figure 7 were obtained from the GTEx Portal in March 2018, and in Figures 2 , 4 , S3 and S6 were obtained from the GTEx portal in April 2022. The same computational analysis steps based on hg19 human reference genome data, as detailed in the RNA-seq and analysis section, were applied to process these tumor and muscle datasets side by side to minimize computational batch effects. In addition, we also applied ComBat 65 to remove batch effects. HTSeq Python package 60 was employed to count reads per gene. edgeR R Bioconductor package 61 , 62 , 63 was used to normalize read counts and calculate FPKM values. Publisher Copyright: {\textcopyright} 2023 The Authors",

year = "2023",

month = jan,

day = "31",

doi = "10.1016/j.celrep.2023.112013",

language = "English (US)",

volume = "42",

journal = "Cell Reports",

issn = "2211-1247",

publisher = "Cell Press",

number = "1",

}

TY - JOUR

T1 - VGLL2-NCOA2 leverages developmental programs for pediatric sarcomagenesis

AU - Watson, Sarah

AU - LaVigne, Collette A.

AU - Xu, Lin

AU - Surdez, Didier

AU - Cyrta, Joanna

AU - Calderon, Delia

AU - Cannon, Matthew V.

AU - Kent, Matthew R.

AU - Silvius, Katherine M.

AU - Kucinski, Jack P.

AU - Harrison, Emma N.

AU - Murchison, Whitney

AU - Rakheja, Dinesh

AU - Tirode, Franck

AU - Delattre, Olivier

AU - Amatruda, James F

AU - Kendall, Genevieve C.

N1 - Funding Information: We thank the UT Southwestern Histopathology core for excellent services and for their expertise, and the Nationwide Children's Hospital (NCH) Animal Resources Core for their exceptional zebrafish husbandry. Additional support was provided by the Nationwide Children's Hospital Microscopy Lab in the Center for Gene Therapy for imaging, the NCH Morphology Core for tissue processing, the NCH CRISPR/Gene Editing Core for generating cell line knockouts, and the NCH High-Performance Computing group for assistance maintaining and using the NCH cluster. G.C.K. is supported by an Alex's Lemonade Stand Foundation A Award, a V Foundation for Cancer Research V Scholar Grant, and an R01CA272872 grant through the National Cancer Institute of the National Institutes of Health. J.F.A. was supported by grant RP120685-P3 from the Cancer Prevention and Research Institute of Texas (CPRIT) and by Curing Kids Cancer. L.X. is supported by Rally Foundation, Cancer Center Support Grant P30 CA142543 and RP180805 from CPRIT. F.T. is supported by the Institut National de la Recherche Médiacale (INSERM). D.R. is supported by grant RP180319 from the Cancer Prevention and Research Institute of Texas (CPRIT) and by the John Lawrence and Patsy Louise Goforth Chair in Pathology. S.W. was supported by a grant from the Fondation Nuovo-Soldati pour la recherche en cancérologie. M.K. is supported by a T32CA269052 Training Program in Basic and Translational Pediatric Oncology Research postdoctoral fellowship. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. S.W. F.T. O.D. J.F.A. and G.C.K. conceived and supervised the study. F.T. provided computational analysis, insight, and expertise of human and zebrafish RNA-seq data and their similarity in Figures 2, 6, and S3. L.X. provided computational analysis, insight, and expertise into developmental studies and human and zebrafish RNA-seq data in Figures 3, 7, and S4. M.V.C. provided computational analysis, insight, and expertise in Figures 2, 4, S3, and S6. D.R. and J.C. provided pathological assessment. S.W. C.A.L. D.S. J.C. D.C. M.K. K.S. J.K. E.H. W.M. and G.C.K. performed experiments. C.A.L. J.C. D.C. M.K. K.S. J.K. E.H. and G.C.K. analyzed experimental data. C.A.L. J.F.A. and G.C.K. drafted the manuscript and figures. All authors reviewed and edited the final manuscript. The authors declare no competing interests. One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in their field of research or within their geographical location. One or more of the authors of this paper received support from a program designed to increase minority representation in their field of research. One or more of the authors of this paper self-identifies as living with a disability. We support inclusive, diverse, and equitable conduct of research. Funding Information: We thank the UT Southwestern Histopathology core for excellent services and for their expertise, and the Nationwide Children’s Hospital (NCH) Animal Resources Core for their exceptional zebrafish husbandry. Additional support was provided by the Nationwide Children's Hospital Microscopy Lab in the Center for Gene Therapy for imaging, the NCH Morphology Core for tissue processing, the NCH CRISPR/Gene Editing Core for generating cell line knockouts, and the NCH High-Performance Computing group for assistance maintaining and using the NCH cluster. G.C.K. is supported by an Alex's Lemonade Stand Foundation A Award, a V Foundation for Cancer Research V Scholar Grant, and an R01CA272872 grant through the National Cancer Institute of the National Institutes of Health . J.F.A. was supported by grant RP120685-P3 from the Cancer Prevention and Research Institute of Texas ( CPRIT ) and by Curing Kids Cancer. L.X. is supported by Rally Foundation , Cancer Center Support Grant P30 CA142543 and RP180805 from CPRIT . F.T. is supported by the Institut National de la Recherche Médiacale (INSERM). D.R. is supported by grant RP180319 from the Cancer Prevention and Research Institute of Texas (CPRIT) and by the John Lawrence and Patsy Louise Goforth Chair in Pathology. S.W. was supported by a grant from the Fondation Nuovo-Soldati pour la recherche en cancérologie . M.K. is supported by a T32CA269052 Training Program in Basic and Translational Pediatric Oncology Research postdoctoral fellowship. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Funding Information: RNA-seq data from VGLL2-NCOA2 tumors is from Watson et al., 2018. 4 RNA-seq data for n = 264 adult sarcomas is from TCGA and represents dedifferentiated liposarcoma, leiomyosarcoma, undifferentiated pleomorphic sarcoma, myxofibrosarcoma, malignant peripheral nerve sheath tumor, and synovial sarcoma. 49 RNA-seq data from n = 96 Ewing sarcoma tumors is from Crompton et al., 2014, 50 RNA-seq data from n = 87 Osteosarcoma (phs000468) and n = 13 Clear Cell Sarcoma of the Kidney (phs000466) are based upon data generated by the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) ( https://ocg.cancer.gov/programs/target ) initiative, phs000468 and phs000466. The data used for this analysis are available at https://portal.gdc.cancer.gov/projects . RNA-seq data from n = 42 fusion-positive and fusion-negative rhabdomyosarcoma is from Chen et al., 2013. 51 RNA-seq data from n = 396 adult skeletal muscle samples are from the Genotype-Tissue Expression Project (GTEx). The GTEx Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. The data used for the analyses described in this manuscript in Figure 7 were obtained from the GTEx Portal in March 2018, and in Figures 2 , 4 , S3 and S6 were obtained from the GTEx portal in April 2022. The same computational analysis steps based on hg19 human reference genome data, as detailed in the RNA-seq and analysis section, were applied to process these tumor and muscle datasets side by side to minimize computational batch effects. In addition, we also applied ComBat 65 to remove batch effects. HTSeq Python package 60 was employed to count reads per gene. edgeR R Bioconductor package 61 , 62 , 63 was used to normalize read counts and calculate FPKM values. Publisher Copyright: © 2023 The Authors

PY - 2023/1/31

Y1 - 2023/1/31

N2 - Clinical sequencing efforts are rapidly identifying sarcoma gene fusions that lack functional validation. An example is the fusion of transcriptional coactivators, VGLL2-NCOA2, found in infantile rhabdomyosarcoma. To delineate VGLL2-NCOA2 tumorigenic mechanisms and identify therapeutic vulnerabilities, we implement a cross-species comparative oncology approach with zebrafish, mouse allograft, and patient samples. We find that VGLL2-NCOA2 is sufficient to generate mesenchymal tumors that display features of immature skeletal muscle and recapitulate the human disease. A subset of VGLL2-NCOA2 zebrafish tumors transcriptionally cluster with embryonic somitogenesis and identify VGLL2-NCOA2 developmental programs, including a RAS family GTPase, ARF6. In VGLL2-NCOA2 zebrafish, mouse, and patient tumors, ARF6 is highly expressed. ARF6 knockout suppresses VGLL2-NCOA2 oncogenic activity in cell culture, and, more broadly, ARF6 is overexpressed in adult and pediatric sarcomas. Our data indicate that VGLL2-NCOA2 is an oncogene that leverages developmental programs for tumorigenesis and that reactivation or persistence of ARF6 could represent a therapeutic opportunity.

AB - Clinical sequencing efforts are rapidly identifying sarcoma gene fusions that lack functional validation. An example is the fusion of transcriptional coactivators, VGLL2-NCOA2, found in infantile rhabdomyosarcoma. To delineate VGLL2-NCOA2 tumorigenic mechanisms and identify therapeutic vulnerabilities, we implement a cross-species comparative oncology approach with zebrafish, mouse allograft, and patient samples. We find that VGLL2-NCOA2 is sufficient to generate mesenchymal tumors that display features of immature skeletal muscle and recapitulate the human disease. A subset of VGLL2-NCOA2 zebrafish tumors transcriptionally cluster with embryonic somitogenesis and identify VGLL2-NCOA2 developmental programs, including a RAS family GTPase, ARF6. In VGLL2-NCOA2 zebrafish, mouse, and patient tumors, ARF6 is highly expressed. ARF6 knockout suppresses VGLL2-NCOA2 oncogenic activity in cell culture, and, more broadly, ARF6 is overexpressed in adult and pediatric sarcomas. Our data indicate that VGLL2-NCOA2 is an oncogene that leverages developmental programs for tumorigenesis and that reactivation or persistence of ARF6 could represent a therapeutic opportunity.

KW - CP: Cancer

KW - cross-species comparative oncology

KW - developmental biology

KW - functional genomics

KW - fusion oncogene

KW - pediatric cancer

KW - rhabdomyosarcoma

UR - http://www.scopus.com/inward/record.url?scp=85146444605&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85146444605&partnerID=8YFLogxK

U2 - 10.1016/j.celrep.2023.112013

DO - 10.1016/j.celrep.2023.112013

M3 - Article

C2 - 36656711

AN - SCOPUS:85146444605

SN - 2211-1247

VL - 42

JO - Cell Reports

JF - Cell Reports

IS - 1

M1 - 112013

ER -

VGLL2-NCOA2 leverages developmental programs for pediatric sarcomagenesis

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