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Transition to a mesenchymal state in neuroblastoma confers resistance to anti-GD2 antibody via reduced expression of ST8SIA1

Nature Cancer volume 3, pages 976–993 (2022)Cite this article

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Abstract

Immunotherapy with anti-GD2 antibodies has advanced the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse, and little is known about mechanisms of resistance to anti-GD2 therapy. Here, we show that reduced GD2 expression was significantly correlated with the mesenchymal cell state in neuroblastoma and that a forced adrenergic-to-mesenchymal transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2-expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1, restoring surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.

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Fig. 1: Low GD2 expression is correlated with developmental lineage in neuroblastoma cell line models.
Fig. 2: AMT induction represses GD2 expression and response to anti-GD2 antibody.
Fig. 3: GD2 downregulation is correlated with reduced GD3S expression.
Fig. 4: GD3S downregulation is correlated with AMT.
Fig. 5: Neuroblastoma tumors with mesenchymal properties express reduced ST8SIA1.
Fig. 6: Epigenetic inhibition results in transcriptional reprogramming and restoration of GD2 expression.
Fig. 7: EZH2 inhibition significantly enhances GD2 expression and response to anti-GD2 in vivo.
Fig. 8: EZH2 inhibition does not significantly upregulate GD2 expression in healthy tissue.

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Data availability

Complete RNA-, ChIP- and ATAC-sequencing data are available online through the Gene Expression Omnibus (GEO) portal under project accession number GSE180516. The human neuroblastoma data were derived from the TARGET and Treehouse datasets downloaded from the Tumor Compendium v11 Public PolyA (April 2020) on UCSC Treehouse genomics website (available at https://treehousegenomics.soe.ucsc.edu/public-data). Additional human neuroblastoma data were derived from tumor sequencing data available from R2 (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi). Source data files for Figs. 1, 2, 3, 4, 5, 6, 7 and 8 and Extended Data Figs. 1, 2, 3, 4, 6, 7, 9 and 10 have been provided as Source Data Files. All other data supporting the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This research was supported by the National Institutes of Health (NIH; P01 CA217959 to K.S. and R.G.M., R35 CA210030 to K.S., R01-CA227942 to C.R.B. and F30 CA232541 to B.A.H.S), the National Institute of Neurological Disorders and Stroke (R01 NS088355 to K.S., UM1 HG012076 to A.T.S. and R01-NS092597 to M.M), an NIH Director’s Pioneer Award (DP1NS111132 to M.M.), the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation (M.M.), Cancer Research UK (M.M), the Waxman Family Research Fund (M.M. and A.C.G.), the Howard Hughes Medical Institute (M.M.), a Stand Up 2 Cancer (SU2C) Phillip A. Sharp Innovation in Collaboration Award (K.S., M.S. and C.S.M.) and a SU2C Convergence 2.0 grant (M.S. and C.S.M.), Alex’s Lemonade Stand (R.G.M.) and the V Foundation (R.G.M.). N.W.M. was supported by the National Cancer Institute under a Ruth L. Kirschstein National Research Service Award (F32 CA261035) and the DFCI Ungerer Fellowship award. R.G.M. is the Taube Distinguished Scholar for Pediatric Immunotherapy at Stanford University School of Medicine. A.T.S. was supported by a Technology Impact Award from the Cancer Research Institute, a Career Award for Medical Scientists from the Burroughs Wellcome Fund and a Pew-Stewart Scholars Award. M.M.M. was supported by the Deutsche Forschungsgemeinshaft (MA 8492/1-1). D.A.S was supported by the Internationales Forschungsstipendium from the Medical Curriculum Munich and the Biomedical Education Program. We thank members of the Program Project Grant and New Approaches to Neuroblastoma Therapy group for their thoughtful comments and suggestions. We thank M. Kararoudi and D. Lee at Nationwide Children’s Hospital for their advice and helpfulness with establishing NK cell culture methods.

Author information

Author notes

  1. These authors contributed equally: Nathaniel W. Mabe, Min Huang.

  2. These authors jointly supervised this work: Kimberly Stegmaier, Robbie G. Majzner.

Authors and Affiliations

  1. Department of Pediatric Oncology, Dana-Farber Cancer Institute and Boston Children’s Hospital, Boston, MA, USA

    Nathaniel W. Mabe, Gabriela Alexe, Daniel A. Schaefer, Amanda L. Robichaud, Amy S. Conway, Delan Khalid, Kenneth N. Ross & Kimberly Stegmaier

  2. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Nathaniel W. Mabe, Gabriela Alexe, Kenneth N. Ross & Kimberly Stegmaier

  3. Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA

    Min Huang, Guillermo N. Dalton, Maria Caterina Rotiroti & Robbie G. Majzner

  4. Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA

    Anna C. Geraghty & Michelle Monje

  5. Institute for Stem Cell Biology and Regenerative Medicine, Stanford, CA, USA

    Marius M. Mader & Marius Wernig

  6. Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA

    Marius M. Mader, Nghi Ly, Winnie Yao, Marius Wernig & Ansuman T. Satpathy

  7. Department of Computer Science, Stanford University School of Medicine, Stanford, CA, USA

    Julia A. Belk

  8. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    Michal Sheffer & Constantine S. Mitsiades

  9. Department of Medicine, Division of Hematology, Stanford University School of Medicine, Stanford, CA, USA

    Miles H. Linde & Ravindra Majeti

  10. Immunology Graduate Program, Stanford University School of Medicine, Stanford, CA, USA

    Miles H. Linde

  11. Department of Chemical & Systems Biology, Stanford University School of Medicine, Stanford, CA, USA

    Benjamin A. H. Smith

  12. Department of Chemistry, Stanford University School of Medicine, Stanford, CA, USA

    Carolyn R. Bertozzi

  13. Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA

    Carolyn R. Bertozzi & Michelle Monje

  14. Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA

    Ansuman T. Satpathy

  15. Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA

    Robbie G. Majzner

  16. Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA

    Robbie G. Majzner

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  1. Nathaniel W. Mabe
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  2. Min Huang
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Contributions

N.W.M., M.H., K.S. and R.G.M. conceived of the project. N.W.M., M.H., N.D., D.A.S., A.C.G., M.M.M., M.L., N.L., W.Y., M.C.R., B.A.H.S., C.M., R.M., A.S., K.S. and R.G.M. generated data. M.S. and C.M. provided NK cell cultures and guidance. N.W.M., M.H., N.D., H.A., A.C.G., M.M.M., J.A.B., B.A.H.S., K.S. and R.G.M. analyzed data. N.W.M., M.H., N.D., A.S.C., M.M.M., M.L., B.A.H.S., M.C.R., M.W., C.R.B., M.M., C.M., R.M., A.T.S., K.S. and R.G.M. helped design experiments. N.D., A.L.R., A.S.C. an D.K. planned and executed in vivo studies. N.W.M., G.A., J.A.B., K.N.R. and A.T.S. provided bioinformatics analysis. N.W.M., K.S. and R.G.M. wrote the manuscript. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Kimberly Stegmaier or Robbie G. Majzner.

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Competing interests

K.S. consults for and has stock options in Auron Therapeutics, received grant funding from Novartis, served as an advisor for KronosBio and consulted for AstraZeneca and Bristol Meyers Squibb on topics unrelated to this manuscript. R.G.M. is a co-founder of and holds equity in Syncopation Life Sciences and Link Cell Therapies and is a consultant for Lyell Immunopharma, Innvervate Radiopharmaceuticals, NKarta, Immunai, Arovella Therapeutics, Zai lab and Aptorum Group. A.T.S. is a co-founder of Immunai and Cartography Biosciences and receives research funding from Arsenal Biosciences, Merck Research Laboratories and Allogene Therapeutics. C.R.B. is a co-founder of Redwood Biosciences (a subsidiary of Catalent), Enable Biosciences, Palleon Pharmaceuticals, InterVenn Bio, Lycia Therapeutics, OliLux Biosciences, Grace Science and Virsti Therapeutics. The remaining authors declare no competing interests.

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Nature Cancer thanks Christian Capitini and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Low GD2 expression is correlated with developmental lineage in neuroblastoma cell culture models.

a, Bar plot showing the percentage of GD2 + cells for each cell line from Fig. 1a (n = 12 GD2-high, n = 11 GD2-low). Data are shown as mean ± s.d. b, Scatterplot comparing adrenergic (ADRN) and mesenchymal (MES) composite scores for neuroblastoma cell lines in the CCLE. ADRN and MES scores were calculated based on average log2(TPM + 1) expression of all genes within each gene set. Mesenchymal cell lines were called based on MES score ≥ 4.1 and ADRN score ≤ 5. c, Heatmap showing median-center, Zz-score normalized RNA sequencing for adrenergic (ADRN, green) and mesenchymal (MES, purple) genes in all neuroblastoma cell lines with RNA-sequencing data available in the CCLE. d, Semi-supervised, hierarchical heatmap showing Zz-score normalized expression data for mesenchymal (purple) and adrenergic (green) gene sets in cell lines analyzed by differential gene expression analysis and for which GD2 status is shown in Fig. 1a. e, Western blot showing expression of mesenchymal markers Vimentin, fibronectin, TAZ and YAP1 and adrenergic marker PHOX2B in SH-EP and SH-SY5Y cell lines. GAPDH is shown as a control. Note that the GAPDH panel is the same bands are shown in Fig. 4a. f, Flow cytometry panels demonstrating CD16 staining in two healthy, donor-derived NK cell cultures. g, Cell viability for SH-SY5Y or SH-EP cell lines co-cultured with NK cells at an E:T ratio of 1:2 for 48 h and in the presence or absence of 1 μg/mL dinutuximab (n = 3 samples per treatment group). Data are shown mean ± s.d. Significance determined by one-way ANOVA and Tukey’s post-hoc test. ns = not significant. Representative data from western blots were confirmed in two independent experiments.

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Extended Data Fig. 2 GD2 density influences response to anti-GD2 antibody.

a, Parental Kelly cells were sorted based on GD2 expression into Kelly-GD2low (red) or Kelly-GD2high (black) isogenic cell lines. b, Kelly-GD2low or Kelly-GD2high were co-cultured with blood-derived macrophages from three healthy donors and measured for phagocytosis in presence or absence of anti-GD2. Data shown is phagocytosis with dinutuximab and normalized to the control condition for that cell line. Triplicates for all three donor cultures were combined. Data are shown mean ± s.d. Significance was determined by two-tailed Mann-Whitney U test. c, Cell viability for Kelly-GD2low or Kelly-GD2high cell lines co-cultured with NK cells at an E:T ratio of 1:2 for 48 h in the presence or absence of 1 μg/mL dinutuximab (n = 3 samples per treatment group). Data are shown mean ± s.d. Significance determined by one-way ANOVA and Tukey’s post-hoc test. d, Flow cytometry for Kelly-GD2low and Kelly-GD2high cells for mesenchymal cell marker CD133 (PROM1). ns = not significant. Representative data from flow cytometry were confirmed in two independent experiments.

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Extended Data Fig. 3 Induced Adrenergic-to-Mesenchymal Transition is associated with GD2 downregulation.

a, Gene Set Enrichment Analysis was performed with the neuroblastoma-specific adrenergic or mesenchymal gene signatures for RNA sequencing from vehicle or doxycycline-treated SK-N-BE(2)-tetON-PRRX1 and KP-N-YN-tetON-PRRX1 cell lines. q-values are shown. b, Cell viability for KP-N-YN induced AMT models co-cultured with NK cells at an E:T ratio of 1:2 in the presence or absence of 1 μg/mL dinutuximab for 8 h (n = 4 samples per treatment group). Data are shown as mean ± s.d. Significance determined by one-way ANOVA and Tukey’s post-hoc test. c, Western blot showing mesenchymal (Vimentin, TAZ, NOTCH3IC) or adrenergic (PHOX2A) markers in parental or NOTCH3IC-expressing SK-N-BE(2) cells. GAPDH is included as a control. d, Representative micrographs (20X) of parental, PRRX1-, or NOTCH3IC-expressing SK-N-BE(2) cells. e, Flow cytometry analysis of GD2 for NOTCH3IC-overexpressing SK-N-BE(2) cells. ns = not significant. Representative data from flow cytometry and western blots were confirmed in two independent experiments.

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Extended Data Fig. 4 Low ST8SIA1 expression correlates with low surface GD2 and mesenchymal features.

a, Schematic showing the complete ganglioside synthesis pathway. Enzymes responsible for conversion of each ganglioside are labeled in bold and branch points are colored. b, Parental SH-EP (top) or CHLA-255 (bottom) cell lines were sorted based on GD2 expression into GD2low (red) or GD2high (black) isogenic cell lines, respectively. c, qPCR analysis comparing expression for ST8SIA1, B4GALNT1 and ST3GAL5 in the SH-EP-GD2high and SH-EP-GD2low (top) or CHLA-255-GD2high and CHLA-255-GD2low (bottom) isogenic cell line pairs. Data derived from a single experiment with 4 technical replicates, experiment was completed once. d, Cell viability for Kelly-GD2low and NB-SD with or without GD3 synthase overexpression and co-cultured with NK cells at an E:T ratio of 1:2 and in the presence of absence of 1 μg/mL dinutuximab for 8 h (n = 4 samples per treatment group). Data are shown as mean ± s.d. Significance determined by one-way ANOVA and Tukey’s post-hoc test. e, Flow cytometry plot showing GD2 expression in Kelly-GD2low, SK-N-AS and NB-SD cell lines with or without constitutive expression of ST3GAL5. f, qPCR analysis measuring ST8SIA1 expression in parental or SK-N-BE(2)-NOTCH3IC cells. Data derived from a single experiment with 4 technical replicates, experiment was repeated twice. ns = not significant. Representative data from flow cytometry were confirmed in two independent experiments.

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Extended Data Fig. 5 Neuroblastoma tumors with mesenchymal properties express reduced GD3 synthase.

UMAP 2D projection plot showing integrated global gene expression for tumors (Treehouse/TARGET) and cell lines (CCLE) from 43 tumor lineages from Celligner. The neuroblastoma lineage is highlighted within the red box.

Extended Data Fig. 6 EZH2 inhibition restores GD2 expression by reversal of epigenetic silencing of GD3 synthase.

a, Flow Cytometry analysis measuring GD2 expression in LAN-5 cells. b, ChIP-seq tracks showing active histone mark H3K27ac and the repressive histone mark H3K27me3 at the ST8SIA1 locus in LAN-5 (GD2-high), NB-69 (GD2-high), SK-N-AS (GD2-low) and SK-N-BE(2)C (GD2-low) cells from GSE138314. c, Flow cytometry panel for GD2 expression in SK-N-AS cells treated with increasing concentrations of tazemetostat. d, Flow cytometry plot showing GD2 expression for CHLA-255-GD2low cells treated for 21 days with 1 μM tazemetostat. e, Mean fluorescence intensity for GD2 in the flow cytometry data shown in panel d (n = 3 samples). Data are shown mean ± s.d. Significance was determined by two-tailed Student’s t-test. f, Flow cytometry panels showing GD2 expression in Ewing sarcoma cell lines SK-PN-DW, EW-8 and TC-32 treated for 21 days with 1 μM tazemetostat or the small cell lung cancer cell lines H-69 and H-82 cell lines treated for 14 days with 1 μM tazemetostat. g, Flow cytometry for GD2 expression in SK-N-AS cells treated for 21 days with 1 μM tazemetostat or treated for 21 days and given a 1-month drug holiday. Representative data from flow cytometry and western blots were confirmed in two independent experiments.

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Extended Data Fig. 7 Integrated analysis of RNA-, ChIP- and ATAC-sequencing following EZH2 inhibition in SK-N-AS cells.

a, Heatmap showing H3K27me3 ChIP-seq signal at all detected H3K27me3 peaks (88,858). Heatmap is separated into genomic regions with H3K27me3 that are decreased (82,122), unchanged (2,862) or increased (3,874) with 21-day treatment with 1 μM tazemetostat in SK-N-AS cells. PC = peak center, ns = not changed, incr = increased. b, Signal enrichment profile plot showing average H3K27me3 enrichment signal for vehicle or tazemetostat treatment groups. Average signal is calculated from 88,858 H3K27me3 peaks. Average signal was normalized to background signal. Values are shown ±5 kb from the peak center. Significance was determined by two-tailed Student’s t-test for the area-under-the-curve. c, Scatterplot correlating the log2 fold change in H3K27me3 area-under-the-curve (AUC) signal with the log2 fold change in RNA expression. The number of significant genes (fold change of RNA ≥ 2 and H3K27me3 ∆ log2 AUC ≤ -0.5) in each quadrant are indicated. Significance for the number of genes within a quadrant was determined by two-tailed Fisher’s exact test. d, Scatterplot correlating the log2 fold change in ATAC area-under-the-curve (AUC) with the log2 fold change in RNA expression. The number of significant genes (fold change of RNA ≥ 2 and H3K27me3 ∆ log2 AUC ≥ 0.5) in each quadrant are indicated. Significance for the number of genes within a quadrant was determined by two-tailed Fisher’s exact test. e, Venn diagram showing the number of overlapping genes for which H3K27me3 signal was lost, ATAC-seq signal was gained, and RNA expression increased. ***P < 0.001.

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Extended Data Fig. 8 Pathways enriched in epigenetically regulated genes from integrated RNA-, ChIP- and ATAC-sequencing following EZH2 inhibition.

Dot plot showing the significant overlap of the 575 genes of interest shown in Extended Data Fig. 7e with C2 and C5 MSigDB libraries. Categories were clustered into the top six most similar pathways. Nervous system development/differentiation-related pathways are indicated in red. Dot size indicates the extent of gene overlap with the indicated gene sets. All gene sets have an FDR of ≤ 0.05 and were calculated by one-tailed Fisher’s exact test based on the hypergeometric distribution of the overlapping 575 genes.

Extended Data Fig. 9 EZH2 inhibition increases GD2 density and response to anti-GD2 in multiple models in vivo.

a, Kelly parental cells were injected into the tail vein of NSG mice (n = 4 vehicle, n = 5 tazemetostat) and treated with 350 mg/kg tazemetostat twice daily or control. Flow cytometry panel showing GD2 expression in representative tumors measured at tumor endpoint. b, Quantification of mean fluorescence intensity for GD2 expression for all parental Kelly tumors treated with control (n = 4) or tazemetostat (n = 5). Data are shown as mean ± s.d. Significance was determined by two-tailed Student’s t-test. c, Representative flow cytometry panel for GD2 from MG63.3 osteosarcoma cells orthotopically injected into the hind leg of NSG mice (n = 4 vehicle, n = 5 tazemetostat) and treated with 350 mg/kg tazemetostat twice daily. d, Quantification of mean fluorescence intensity for GD2 expression in all MG63.3 tumors treated with or without tazemetostat. Data are shown as mean ± s.d. Significance was determined by two-tailed Student’s t-test. e, Flow cytometry panel for SK-N-AS cells injected into the tail vein of NSG mice (n = 5 per treatment group) and treated with 300 μg dinutuximab three times a week or 500 mg/kg tazemetostat twice daily alone or in combination. Note that one representative tumor from untreated and tazemetostat only groups were shown in Fig. 7A. Representative data from flow cytometry are shown for the biological replicates presented in panels a and c.

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Extended Data Fig. 10 EZH2 inhibition increases anti-GD2 response in vivo.

a, Bar plots showing the population of macrophage (MP), granulocyte (Gran), monocyte (Mono) and dendritic cell (DC) populations in each treatment arm (n = 5 per arm) as determined by flow cytometry. Population percentages were determined by the following markers within CD45 + cells: macrophages: CD11b + / F4/80 + ; granulocytes: CD11b + / Ly6G + ; monocytes: CD11b + / Ly6C + ; dendritic cells: CD11c + / MHC-II + . Data are shown as mean ± s.d. Significance was determined by one-way ANOVA and Tukey’s post-hoc test. b, Bar plots showing the percent of M1 macrophages (CD86 + / MHC-II (I-A/I-E) + ) or M2 macrophages (CD163 + /CD206 + ) as a total of the macrophage population (CD11b + / F480 + ) (n = 5 per arm). Data are shown as mean ± s.d. Significance was determined by two-tailed Student’s t-test. c, Flow cytometry showing representative GD2 expression in treatment naïve or tazemetostat pretreated tumors treated with either dinutuximab or anti-GD2 CAR T cells in vivo. d,e, Immunofluorescence staining for GD2 or neuronal marker MAP2 on vehicle or tazemetostat-treated, passaged primary human cortical cells (d) or inducible embryonic stem cells (e). f, Human embryonic stem cells were differentiated into induced neurons and treated for 14 days with 1 μM tazemetostat. GD2 was measured by flow cytometry. ns = not significant. Representative data from flow cytometry were confirmed in two independent experiments.

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Supplementary information

Supplementary Information

Supplemental Figures 1–7 showing representative gating strategies for flow cytometry panels.

Reporting Summary

Supplementary Table 1

GSEA for significant Hallmark gene sets between GD2-high and GD2-low cell lines. Supplementary Table 2. Genes correlating with percentage of GD2+ cells in neuroblastoma cell lines. Supplementary Table 3. Summary master file for integrated RNA, ChIP and ATAC sequencing in vehicle or tazemetostat-treated SK-N-AS cells. P values were calculated by DESeq2 on apeglm settings and multiple corrections testing using false discovery rate. Supplementary Table 4. Extended list of gene ontology categories enriched in the 575 overlapping genes shown in Extended Data Figs. 7e and 8. Significance and adjusted P values were determined by hypergeometric distribution analysis using one-tailed Fisher’s exact test for values greater than the contingency table. Supplementary Table 5. Reagents used for studies.

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Mabe, N.W., Huang, M., Dalton, G.N. et al. Transition to a mesenchymal state in neuroblastoma confers resistance to anti-GD2 antibody via reduced expression of ST8SIA1. Nat Cancer 3, 976–993 (2022). https://doi.org/10.1038/s43018-022-00405-x

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