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Abstract

Glioblastoma is a universally lethal cancer with a median survival time of approximately 15 months1. Despite substantial efforts to define druggable targets, there are no therapeutic options that notably extend the lifespan of patients with glioblastoma. While previous work has largely focused on in vitro cellular models, here we demonstrate a more physiologically relevant approach to target discovery in glioblastoma. We adapted pooled RNA interference (RNAi) screening technology2,3,4 for use in orthotopic patient-derived xenograft models, creating a high-throughput negative-selection screening platform in a functional in vivo tumour microenvironment. Using this approach, we performed parallel in vivo and in vitro screens and discovered that the chromatin and transcriptional regulators needed for cell survival in vivo are non-overlapping with those required in vitro. We identified transcription pause–release and elongation factors as one set of in vivo-specific cancer dependencies, and determined that these factors are necessary for enhancer-mediated transcriptional adaptations that enable cells to survive the tumour microenvironment. Our lead hit, JMJD6, mediates the upregulation of in vivo stress and stimulus response pathways through enhancer-mediated transcriptional pause–release, promoting cell survival specifically in vivo. Targeting JMJD6 or other identified elongation factors extends survival in orthotopic xenograft mouse models, suggesting that targeting transcription elongation machinery may be an effective therapeutic strategy for glioblastoma. More broadly, this study demonstrates the power of in vivo phenotypic screening to identify new classes of ‘cancer dependencies’ not identified by previous in vitro approaches, and could supply new opportunities for therapeutic intervention.

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  • 06 March 2018

    In the HTML version of this Letter, several of the figures were numbered incorrectly: Fig. 4 displayed incorrectly as Extended Data Fig. 2; Extended Data Fig 1 displayed incorrectly as Extended Data Fig. 3; Extended Data Fig. 2 displayed incorrectly as Extended Data Fig. 5; Extended Data Fig. 3 displayed incorrectly as Extended Data Fig. 6; Extended Data Fig. 4 displayed incorrectly as Extended Data Fig. 7; Extended Data Fig. 5 displayed incorrectly as Extended Data Fig. 8; Extended Data Fig. 6 displayed incorrectly as Extended Data Fig. 9; and Extended Data Fig. 10 displayed incorrectly as Extended Data Fig. 4. This has been corrected online.

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Acknowledgements

The authors thank the Hemann, Paddison, and Zuber laboratories, along with K. Alazem for technical help with RNAi screening. We thank B. Kristensen and A. M. Rosager for input on histological analysis and A. Saiakhova, E. R. Chan, M. Squatrito and W. Liu for bioinformatic support. Additional support was provided by the Cytometry & Imaging Microscopy and Genomics core facilities of the Case Comprehensive Cancer Center at Cleveland Clinic and Case Western (P30CA043703), respectively. We also thank members of the Rich, Tesar and Scacheri laboratories for input on the project and discussion about the manuscript. This work was supported by Velosano (J.N.R.); New York Stem Cell Foundation-Robertson Investigator Award and philanthropic support from the Goodman family (P.J.T.); CIHR Banting Fellowship (S.C.M.); National Institutes of Health grants CA183510 (T.E.M.); GM007250 (T.E.M., A.R.M., L.J.Y.K., J.J.M.); CA189647 (C.G.H.); CA154130, CA169117, CA197718, CA171652, NS087913 and NS089272 (J.N.R.).

Author information

Author notes

    • Jeremy N. Rich

    Present address: Division of Regenerative Medicine, Department of Medicine, University of California at San Diego, San Diego, California 92093, USA.

Affiliations

  1. Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA

    • Tyler E. Miller
    • , Lisa C. Wallace
    • , Andrew R. Morton
    • , Qi Xie
    • , Deobrat Dixit
    • , Leo J. Y. Kim
    • , Qiulian Wu
    • , Stephen C. Mack
    • , Christopher G. Hubert
    • , William A. Flavahan
    •  & Jeremy N. Rich
  2. Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, Cleveland, Ohio 44195, USA

    • Tyler E. Miller
    • , Leo J. Y. Kim
    • , Stephen C. Mack
    • , Christopher G. Hubert
    • , William A. Flavahan
    •  & Jeremy N. Rich
  3. Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA

    • Tyler E. Miller
    • , Leo J. Y. Kim
    •  & James J. Morrow
  4. Department of Genetics and Genome Sciences, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA

    • Tyler E. Miller
    • , Andrew R. Morton
    • , Daniel C. Factor
    • , James J. Morrow
    • , Peter C. Scacheri
    •  & Paul J. Tesar
  5. Harvard Medical School, Boston, Massachusetts 02114, USA

    • Brian B. Liau
    • , Shawn M. Gillespie
    • , Rohit Thummalapalli
    •  & Bradley E. Bernstein
  6. Epigenomics Program, Broad Institute, Cambridge, Massachusetts 02142, USA

    • Brian B. Liau
    • , Shawn M. Gillespie
    • , Rohit Thummalapalli
    •  & Bradley E. Bernstein
  7. Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA

    • Brian B. Liau
    • , Shawn M. Gillespie
    • , Rohit Thummalapalli
    •  & Bradley E. Bernstein
  8. Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria

    • Thomas Hoffmann
    •  & Johannes Zuber
  9. The Koch Institute for Integrative Cancer Research at MIT, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Michael T. Hemann
  10. Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA

    • Patrick J. Paddison
  11. Department of Neurological Surgery, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA

    • Craig M. Horbinski
  12. Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60615, USA

    • Craig M. Horbinski

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Contributions

T.E.M., P.J.T. and J.N.R designed the overall screening strategy. T.H., J.Z., P.J.P. and M.T.H provided technical expertise, unpublished reagents and methods for the screen. T.E.M., S.C.M., C.G.H. and Q.W. performed the screen. T.E.M., L.C.W. and S.C.M. produced and sequenced screening libraries. T.E.M., P.J.T. and J.N.R. processed and analysed screening results. T.E.M., L.C.W. and D.C.F. generated and analysed RNA-seq data. T.E.M., B.B.L., S.M.G., R.T., B.E.B., L.C.W. and S.C.M. generated ChIP–seq data. T.E.M., B.B.L, A.R.M., B.E.B., J.J.M. and P.C.S. analysed and interpreted ChIP–seq data. T.E.M., L.J.Y.K. and W.A.F. performed bioinformatic analysis of published expression datasets. T.E.M., D.D., Q.X., L.C.W. and Q.W. performed functional knockdown and CRISPR-knockout studies. C.M.H. created and analysed tissue microarray for JMJD6 expression. T.E.M., P.J.T. and J.N.R. analysed all data and wrote the paper. All authors provided intellectual input, edited and approved the final manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Paul J. Tesar or Jeremy N. Rich.

Reviewer Information Nature thanks K. Adelman, P. Northcott and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Table 1

    PDX Model Characteristics. For each of the three main PDX models used in the manuscript, we provide the molecular subtype based on Veerhaak et. al. 2010 classification, IDH1/2 mutation status, Glioma-CpG island methylation phenotype (G-CIMP) status, mutation status for commonly mutated genes and copy number variations for commonly amplified or deleted genes in glioblastoma. Specific mutations are characterized below.

Excel files

  1. 1.

    Supplementary Table 2

    Primary Screen Results. Worksheet 1 contains the normalized RPM (reads per million) values for each hairpin for each replicate from the in vivo screen (13-14 individual animals per replicate). Median RPM value for each replicate was used for analysis. Signal to Noise of replicates was used to calculate individual shRNA score based on their ability to deplete cells in the induced arm compared to the control arm. Worksheet 2 contains the ranked list of genes from in vivo screen. 2nd-Best shRNA score from each expressed gene was used to rank genes for hits. 2nd-Best shRNA score of 2 or greater was considered a hit. Worksheet 3 and 4 contain the same information for the in vitro screen.

  2. 2.

    Supplementary Table 3

    RNAseq Results. RNA sequencing results for GBM528 and GBM3565 from in vivo and in vitro conditions. All expressed genes listed. FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values for known genes were calculated using Cufflinks. FPKM values were quantile normalized. To generate an expressed genes list, an average of replicates for each condition was calculated and genes with FPKM value >0.25 in either intracranial sample or culture sample were considered expressed. Genes that did not meet this expression cut-off (Replicate average FPKM <0.25 in Intracranial and in Culture conditions) were removed as not expressed. Expressed genes were tabled by converting FPKMs <0.25 to 0.25.

  3. 3.

    Supplementary Table 4

    Gene Ontology Enrichment Values. Raw outputs for gene ontology enrichments. Each worksheet contains program, settings, and files used to generate output. Last worksheet contains the GSEA genesets analyzed when using GSEA.

  4. 4.

    Supplementary Table 5

    Super-Enhancer List for GBM528. Lists of all Super-Enhancers detected in in vivo intracranial condition (IC) (worksheet 1) and in the in vitro culture condition (worksheet 2). In vivo and in vitro SEs were identified using the dynamic Enhancer software (retrieved from https://github.com/BradnerLab/pipeline). H3K27Ac peaks identified as enhancers and separated by less than 12.5 kb were stitched together. All stitched peaks were then ranked by the density of H3K27Ac minus input. Peaks higher than the inflection point on the density curve were designated SEs. Worksheet 3 contains called condition-specific enhancers. To call condition-specific SEs, SEs from in vivo and in vitro conditions were merged and H3K27Ac signals for all merged SEs in each cell line were calculated. To be considered specific to in vivo or in vitro, SEs had to be only called a SE in a single condition and had to have at least 1.5-fold change in H3K27Ac signal between conditions.

  5. 5.

    Supplementary Table 6

    JMJD6 correlation with TCGA genes. List of all gene expression correlations with JMJD6 in TCGA primary tumours. To test correlation of all genes with JMJD6 across glioblastoma tumours, TCGA RNAseqV2 data were downloaded and analysed. All glioblastoma tumours in the TCGA with RNA-seq data available were used.

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https://doi.org/10.1038/nature23000

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