Genetic predisposition to neuroblastoma mediated by a LMO1 super-enhancer polymorphism (original) (raw)

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ChIP-seq data sets are available under Gene Expression Omnibus (GEO) super series GSE65664, and relevant accession numbers are shown in Supplementary Table 2.

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Acknowledgements

This work was supported in part by NIH grants R01-CA124709 (J.M.M.), R01-CA180692 (J.M.M. and A.T.L.), R00-CA151869 (S.J.D.), RC1MD004418 to the TARGET consortium, 1K99CA178189 (S.Z.), T32-HG000046 (D.A.O.), R01-CA109901 (R.A.Y.), the Giulio D’Angio Endowed Chair (J.M.M.), the PressOn Foundation (J.M.M.), Andrew’s Army Foundation (J.M.M.), the Abramson Family Cancer Research Institute (J.M.M.), the Brooke Mulford Foundation (J.M.M.), the University of Pennsylvania Genome Frontiers Institute, an Alex’s Lemonade Stand Foundation Innovation Award (A.T.L.), young investigator awards from Alex’s Lemonade Stand Foundation (S.Z., A.C.W.) and the CureSearch for Children’s Cancer Foundation (S.Z.), grant from the German Cancer Aid 110801 (N.W.-L.), St Baldrick’s Foundation Fellow award (A.C.W.), George L. Ohrstrom Jr foundation (A.C.W.), Wellcome Trust Senior Investigator Award Ref:100210/Z/12/Z (N.R.) and NHS funding to the NIHR Biomedical Research Centre at The Royal Marsden and the ICR (N.R.), Fondazione Italiana per la Lotta al Neuroblastoma (M.C.), Associazione Oncologia Pediatrica e Neuroblastoma (M.C.), and Associazione Italiana per la Ricerca sul Cancro (M.C.). We gratefully acknowledge the Children’s Oncology Group (COG) for providing the specimens and clinical data from neuroblastoma patients and thank patients and families for participating in the COG, the UK-based Factors Associated with Childhood Tumors (FACT), and Italian cooperative group studies. We thank A. Renwick who performed the Taqman analyses and A. Zachariou for recruiting participants to the FACT study. We thank G. Blobel for scientific advice and discussion, and generously providing equipment and reagents for ChIP experiments, N. Saeki and H. Sasaki for providing the LMO1 cDNA clone, and Y. Nakatan for providing the lentiviral vector pOZ-FHN.

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Author notes

1. Derek A. Oldridge and Andrew C. Wood: These authors contributed equally to this work.

Authors and Affiliations

1. Division of Oncology and Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, Philadelphia, 19104, Pennsylvania, USA
   Derek A. Oldridge, Ian Crimmins, Robyn Sussman, Cynthia Winter, Lee D. McDaniel, Maura Diamond, Lori S. Hart, Sharon J. Diskin & John M. Maris
2. Medical Scientist Training Program, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, 19104, Pennsylvania, USA
   Derek A. Oldridge
3. Department of Molecular Medicine and Pathology, University of Auckland, Auckland, 1142, Auckland Region, New Zealand
   Andrew C. Wood
4. Department of Pediatric Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, 02215, Massachusetts, USA
   Nina Weichert-Leahey, Adam D. Durbin & A. Thomas Look
5. Division of Pediatric Hematology/Oncology, Boston Children’s Hospital, Boston, 02115, Massachusetts, USA
   Nina Weichert-Leahey, Adam D. Durbin & A. Thomas Look
6. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, 55905, Minnesota, USA
   Shizhen Zhu
7. Whitehead Institute for Biomedical Research and MIT, Boston, 02142, Massachusetts, USA
   Brian J. Abraham, Lars Anders & Richard A. Young
8. Center for Applied Genomics, Children’s Hospital of Philadelphia, Philadelphia, 19104, Pennsylvania, USA
   Lifeng Tian & Hakon Hakonarson
9. Pediatric Oncology Branch, National Cancer Institute, Bethesda, 20892, Maryland, USA
   Shile Zhang, Jun S. Wei & Javed Khan
10. Thermo Fisher Scientific, Austin, 78744, Texas, USA
    Kelli Bramlett
11. The Institute of Cancer Research, London, SM2 5NG, UK
    Nazneen Rahman
12. University of Naples Federico II, Naples, 80131, Italy
    Mario Capasso & Achille Iolascon
13. CEINGE Biotecnologie Avanzate, Naples, 80131, Italy
    Mario Capasso & Achille Iolascon
14. Office of Cancer Genomics, National Cancer Institute, Bethesda, 20892, Maryland, USA
    Daniela S. Gerhard & Jaime M. Guidry Auvil
15. Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, 19104, Pennsylvania, USA
    Hakon Hakonarson, Sharon J. Diskin & John M. Maris
16. Abramson Family Cancer Research Institute, Philadelphia, 19104, Pennsylvania, USA
    Sharon J. Diskin & John M. Maris

Authors

1. Derek A. Oldridge
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2. Andrew C. Wood
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3. Nina Weichert-Leahey
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4. Ian Crimmins
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5. Robyn Sussman
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6. Cynthia Winter
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7. Lee D. McDaniel
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8. Maura Diamond
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9. Lori S. Hart
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10. Shizhen Zhu
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11. Adam D. Durbin
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12. Brian J. Abraham
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13. Lars Anders
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14. Lifeng Tian
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15. Shile Zhang
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16. Jun S. Wei
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17. Javed Khan
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18. Kelli Bramlett
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19. Nazneen Rahman
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20. Mario Capasso
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21. Achille Iolascon
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22. Daniela S. Gerhard
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23. Jaime M. Guidry Auvil
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24. Richard A. Young
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25. Hakon Hakonarson
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26. Sharon J. Diskin
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27. A. Thomas Look
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28. John M. Maris
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Contributions

J.M.M. and A.T.L. conceived the study, guided interpretation of results and guided preparation of the manuscript. D.A.O. and A.C.W. performed and/or oversaw most of the experiments, computational analyses and data interpretation. I.C., R.S., C.W., L.S.H., S.Z., N.W.-L., A.D.D., B.J.A., L.A., L.T., K.B. and R.A.Y. performed the genomic and epigenetic experiments and data analysis including DNA sequencing and ChIP sequencing. L.D.M., S.J.D. and H.H. performed the fine mapping and association testing. J.S.W. and J.K. performed the tumour RNA sequencing. N.R. and M.C. performed the validation genotyping and association testing. M.C. and A.I. replicated the SNP association in the Italian cohort. D.A.O. and A.C.W. drafted the manuscript, while A.T.L. and J.M.M. and other authors edited the manuscript.

Corresponding author

Correspondence toJohn M. Maris.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 The imputed SNP, rs2168101, is associated with neuroblastoma, and the risk ‘G’ allele is enriched in neuroblastoma cases.

Ternary density plots of genotype probability vectors [P(G/G), P(G/T), P(T/T)] output from IMPUTE2 for rs2168101 in the European-American cohort. Vertices represent ‘perfect’ confidence calls in which P(genotype) = 1; dotted lines represent decision boundaries for genotype calling based on most probable genotype. All plots were normalized by the total number of individuals studied and subjected to 2D Gaussian kernel smoothing. Left, 2,101 cases (red); centre, 4,202 controls (blue); right, difference between cases and controls highlights enrichment of G/G genotype (homozygous risk) in cases and of G/T and T/T genotypes in controls. Validation efforts using PCR-based genotyping in 146 out of 2,101 European-American cases confirmed an 86% concordance with imputation based on most probable genotypes (Supplementary Table 1).

Extended Data Figure 2 Conditional analysis reveals a single neuroblastoma association signal at the LMO1 locus and that rs2168101 is the most associated variant.

a, Imputation-based neuroblastoma association study conditional on rs2168101. No variants remain significant after conditioning on rs2168101 (most significant variant: rs34544683, nominal P = 9.0 × 10−4, Bonferroni P = 1). b, Reciprocal analysis conditioned on each of 27 SNPs with a nominal P < 1 × 10−5. For rs2168101, the maximum (least significant) P value across all non-rs2168101 conditional tests is shown, in order to illustrate the extent to which the signal at rs2168101 can be accounted for by other variants (a similar maximum P value statistic is plotted for other variants). Notably, rs2168101 remained significant (worst-case nominal P = 2.6 × 10−7, Bonferroni P = 0.002) across all tests. These results are consistent with a single underlying signal at the LMO1 locus, and re-affirm that rs2168101 is the single best causal SNP candidate because its association with neuroblastoma cannot be accounted for by other single variants.

Extended Data Figure 3 The risk G allele of rs2168101 is associated with decreased event-free and overall survival in the European-American discovery cohort.

Because genotypes for rs2168101 are imputed within the European-American discovery cohort, the most likely genotype for each neuroblastoma case was called based on the maximum of P(G/G), P(G/T) and P(T/T) from IMPUTE2. P values reflect Cox proportional hazards regressions adjusted for MYCN amplification status and the first 20 MDS components to adjust for population stratification. a, Kaplan–Meier plot for event-free survival. Neuroblastoma cases with rs2168101 = G/G versus rs2168101 = G/T or T/T showed significantly worse event-free survival (P = 0.0004). b, Kaplan–Meier plot for overall survival. Neuroblastoma cases with rs2168101 = G/G versus rs2168101 = G/T or T/T showed significantly worse overall survival (P = 0.0004). Censored data points are shown as black crosses. Number of at risk patients at every time point for both event-free survival and overall survival are plotted below each respective Kaplan–Meier plot.

Extended Data Figure 4 rs2168101 genotype is associated with total and allele-specific LMO1 expression in neuroblastoma cell lines and primary tumours, and allele-specific expression differences are not driven by somatic DNA copy number alterations.

a, Neuroblastoma cell line LMO1 mRNA expression as quantified by Affymetrix U95Av2 oligonucleotide arrays and normalized as described11 was significantly higher in cell lines harbouring homozygous risk alleles (G/G) compared to heterozygous alleles (G/T) (P = 0.047, Mann–Whitney two-tailed). b, Allele-specific expression measured by RNA-seq from primary neuroblastoma tumours. Since rs2168101 is an intronic SNP that is spliced out in mRNA, the synonymous exonic SNP rs3750952 was used as a surrogate for measuring allele-specific expression in 39 primary tumours which are heterozygous for rs3750952 (C/G genotype). The DNA allelic fraction for rs3750952 determined by whole-exome sequencing is plotted on the x axis, whereas the RNA allele fraction for rs3750952 determined by mRNA-seq is plotted on the y axis. The solid line indicates where DNA and RNA allele fractions are equal and dotted lines indicate the boundary where DNA and RNA allele fractions are within 10% of each other. Tumours that are heterozygous for rs2168101 (G/T genotype, red dots) exhibit greater RNA allelic imbalance (P = 5.3 × 10−5) than homozygous controls (rs2168101 = G/G genotype, black dots). By contrast, DNA allelic imbalance is no different between G/T versus G/G tumours (P = 0.79), indicating that a _cis_-acting regulatory mechanism, rather than somatic DNA alterations, drives LMO1 allelic expression differences.

Extended Data Figure 5 Expression of LMO1 and GATA-family transcription factors in neuroblastoma primary tumours and cell lines.

a, RPKM expression measurements from mRNA-seq are summarized via boxplots for 127 primary neuroblastoma tumours for paralogues GATA1 through GATA6. Both GATA2 (median RPKM: 56) and GATA3 (median RPKM: 110) are more highly expressed by 1–4 orders of magnitude on average compared to other members of the GATA family in neuroblastoma. b, Neuroblastoma cell lines were lysed for protein and resolved by SDS–PAGE as previously described21. Jurkat T-ALL cells are shown as a positive control for LMO1 and GATA3 expression. Data are representative of at least three independent blots. The rs2168101 genotype is shown below individual cell lines.

Extended Data Figure 6 The LMO1 super-enhancer is observed in neuroblastoma cell lines containing the G allele of rs2168101 and is highly tissue-specific.

a, H3K27ac signal across all enhancers in SHSY5Y (MYCN not amplified; rs2168101 = G/G), BE2 (_MYCN_-amplified; rs2168101 = G/T) and NGP (_MYCN_-amplified; rs2168101 = G/T) is shown. Enhancers are ranked by their signal of H3K27ac minus input signal and are geometrically divided into two populations (see Methods). Super-enhancers are those at the high end of the population and are associated with key genes in neuroblastoma, highlighted on the curve. _LMO1_-associated super-enhancers were identified in BE2, KELLY and SHSY5Y cells, which all contain the G allele of rs2168101, but not in BE2C cells in which the G allele is absent. b, H3K27ac ChIP-seq in the Jurkat cell line. c, All ENCODE non-neuroblastoma cell lines with H3K27ac ChIP-seq profiling. All non-neuroblastoma cell lines considered showed little to no evidence for an active enhancer element within the first intron of the LMO1 gene locus, consistent with a tissue and disease-specific enhancer overlying the neuroblastoma causal SNP rs2168101.

Extended Data Figure 7 Depletion of GATA3 results in suppression of cell growth that is rescued by forced LMO1 expression in neuroblastoma.

Neuroblastoma cells SHSY5Y, KELLY, KELLY overexpressing control vector (EV) and KELLY with forced LMO1 overexpression (LMO1-1 and LMO1-2) were treated with non-targeted (siControl) or GATA3-targeting (siGATA3-1, siGATA3-2) siRNAs and cells were counted at 24, 48 and 72 h after transfection. Rescue of suppressed cell growth after GATA3 depletion by forced LMO1 expression in LMO1-1 and LMO1-2 after 72 h is shown on the bottom. Growth curves over the time of 72 h are shown (to accompany Fig. 3f). Error bars denote ±s.e.m., n = 9 technical replicates.

Extended Data Table 1 Germline variants from 1000 Genomes Project with P < 1 × 10−5 association with neuroblastoma susceptibility from imputation-based SNPTEST analysis of European-American cohort

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Extended Data Table 2 Clinical characteristics for patients in referenced sequencing data sets

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Extended Data Table 3 Association of rs2168101 with clinical/biological co-variates

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Supplementary information

Supplementary Information

This file contains western blots, supplementary text, Supplementary Tables 1-2 and additional references. (PDF 506 kb)

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Oldridge, D., Wood, A., Weichert-Leahey, N. et al. Genetic predisposition to neuroblastoma mediated by a LMO1 super-enhancer polymorphism.Nature 528, 418–421 (2015). https://doi.org/10.1038/nature15540

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• Received: 07 February 2015
• Accepted: 02 September 2015
• Published: 11 November 2015
• Issue Date: 17 December 2015
• DOI: https://doi.org/10.1038/nature15540