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Selective gene dependencies in MYCN-amplified neuroblastoma include the core transcriptional regulatory circuitry

Nature Genetics volume 50, pages 1240–1246 (2018)Cite this article

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Abstract

Childhood high-risk neuroblastomas with MYCN gene amplification are difficult to treat effectively1. This has focused attention on tumor-specific gene dependencies that underlie tumorigenesis and thus provide valuable targets for the development of novel therapeutics. Using unbiased genome-scale CRISPR–Cas9 approaches to detect genes involved in tumor cell growth and survival2,3,4,5,6, we identified 147 candidate gene dependencies selective for MYCN-amplified neuroblastoma cell lines, compared to over 300 other human cancer cell lines. We then used genome-wide chromatin-immunoprecipitation coupled to high-throughput sequencing analysis to demonstrate that a small number of essential transcription factors—MYCN, HAND2, ISL1, PHOX2B, GATA3, and TBX2—are members of the transcriptional core regulatory circuitry (CRC) that maintains cell state in MYCN-amplified neuroblastoma. To disable the CRC, we tested a combination of BRD4 and CDK7 inhibitors, which act synergistically, in vitro and in vivo, with rapid downregulation of CRC transcription factor gene expression. This study defines a set of critical dependency genes in MYCN-amplified neuroblastoma that are essential for cell state and survival in this tumor.

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Fig. 1: Genome-scale CRISPR–Cas9 screening identifies selective neuroblastoma gene dependencies enriched for transcription factors.
Fig. 2: Transcription factor dependency genes are marked by extensive H3K27ac and are enriched for dependency in human MYCN-amplified neuroblastoma.
Fig. 3: Dependency transcription factors form the CRC in MYCN-amplified neuroblastoma.
Fig. 4: Pharmacological disruption of super-enhancer-mediated transcription selectively suppresses the CRC and neuroblastoma cell growth.

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Acknowledgements

This work was supported by National Institutes of Health (NIH) grants R35-CA210064 (A.T.L.), R01-CA180692 (A.T.L.), R35-CA210030 (K.S.), R01-NS088355 (K.S.), R01-GM123511 (R.A.Y.), and U01-CA176058 (W.C.H.). A.T.L. is supported by an Alex’s Lemonade Stand Foundation Innovation Award. K.S. is supported by a Hyundai Hope Grant, Cookies for Kids Cancer, and Friends for Life Fellowship. A.D.D., M.W.Z. and A.B.I. are Damon Runyon–Sohn Pediatric Fellows supported by the Damon Runyon Cancer Research Foundation, DRG-24-18 (A.D.D.), DRSG-9-14 (M.W.Z.) and DRSG-12-15 (A.B.I.). A.D.D. and M.W.Z are recipients of Alex’s Lemonade Stand Foundation Young Investigator Awards. N.V.D. is supported by NIH grant T32-CA136432. B.J.A. is the Hope Funds for Cancer Research Grillo-Marxuach Family Fellow. We also acknowledge the Jake Wetchler Foundation for their support of this work.

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Author notes

  1. These authors contributed equally: Adam. D. Durbin, Mark W. Zimmerman, Neekesh V. Dharia.

  2. These authors jointly supervised: A. Thomas Look, Kimberly Stegmaier.

Authors and Affiliations

  1. Department of Pediatric Oncology, Dana–Farber Cancer Institute, Boston, MA, USA

    Adam D. Durbin, Mark W. Zimmerman, Neekesh V. Dharia, Amanda Balboni Iniguez, Nina Weichert-Leahey, Shuning He, Todd R. Golub, A. Thomas Look & Kimberly Stegmaier

  2. Division of Pediatric Hematology/Oncology, Boston Children’s Hospital, Boston, MA, USA

    Adam D. Durbin, Neekesh V. Dharia, A. Thomas Look & Kimberly Stegmaier

  3. The Broad Institute, Cambridge, MA, USA

    Adam D. Durbin, Neekesh V. Dharia, Amanda Balboni Iniguez, John M. Krill-Burger, David E. Root, Francisca Vazquez, Aviad Tsherniak, William C. Hahn, Todd R. Golub & Kimberly Stegmaier

  4. Whitehead Institute for Biomedical Research, Cambridge, MA, USA

    Brian J. Abraham & Richard A. Young

  5. Department of Medical Oncology, Dana–Farber Cancer Institute, Boston, MA, USA

    William C. Hahn

  6. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Richard A. Young

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Contributions

A.D.D., M.W.Z., N.V.D., K.S., B.J.A., R.A.Y., and A.T.L. conceived the study and designed the experiments. A.D.D. and M.W.Z. performed genomic meta-analyses, ChIP-seq, ATAC-seq, siRNA, low-throughput CRISPR–Cas9, pharmacologic, and gene expression assays. N.W. and S.H. performed ChIP-seq and ATAC-seq experiments. A.B.I., A.D.D., N.V.D., and K.S. performed and interpreted the drug activity and synergy studies. N.V.D., K.S., J.M.K., F.V., D.E.R., A.T., W.C.H., and T.R.G. performed and analyzed the genome-scale CRISPR–Cas9 screening experiments. N.V.D., B.J.A., and R.A.Y. performed computational analysis. A.D.D., M.W.Z., N.V.D., K.S., and A.T.L. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Richard A. Young, A. Thomas Look or Kimberly Stegmaier.

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Competing interests

R.A.Y. is a founder and shareholder of Syros Pharmaceuticals, which is discovering and developing therapeutics directed at transcriptional pathways in cancer. K.S. and W.C.H. consulted for Novartis Pharmaceuticals as part of the Dana–Farber Cancer Institute/Novartis Drug Discovery Program. B.J.A. is a shareholder of Syros Pharmaceuticals. No other potential conflicts of interest are declared.

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Integrated supplementary information

Supplementary Figure 1 CRISPR–Cas9 screening reveals selective dependencies in neuroblastoma.

Scatterplots showing neuroblastoma relative dependency on MYCN and LDB1 with P < 1 × 10–9, PHOX2B with P = 2 × 10–9, LIN28B with P = 4 × 10–9, TFAP2B with P = 7 × 10–9, GATA3 with P = 1.41 × 10–8, MAB21L1 with P = 3.34 × 10–6 and TBX2 with P = 7.30 × 10–4, by permutation testing with n = 9 MYCN-amplified neuroblastoma cell lines compared to n = 330 non-neuroblastoma cancer cell lines. The y axis shows the gene’s dependency rank in an individual cell line. The x axis shows the gene’s dependency score in the indicated neuroblastoma cell line compared to all other cell lines screened. MYCN-amplified lines are depicted with red dots, while MYCN-non-amplified lines are shown in black.

Supplementary Figure 2 Transient siRNA depletes HAND2, ISL1, PHOX2B, GATA3 and TBX2 proteins.

Western blots demonstrating protein depletion in Kelly and BE2C cells subjected to gene-specific siRNAs as detailed in the Methods. Protein was extracted at 24–48 h after treatment with gene-targeted siRNA. Protein lysates were resolved by SDS–PAGE as detailed. Blot images have been cropped. Numerical values represent protein molecular weight markers. Data are representative of three independent lysates and blots.

Supplementary Figure 3 Phenotypic effects of CRC transcription factor knockdown with siRNA.

Kelly cells were treated with two independent siRNAs to no coding sequence (control) or the specified transcription factor for 24 h before fixation and analysis by flow cytometry. Increases in sub-G0 peaks were present for all cells treated with siRNAs to transcription factors and not non-targeted siRNAs. No differences were seen in the distribution of cells in G1, S and G2/M phases. *P < 1 × 10–3 for all CRC-targeted siRNAs compared to either control siRNA#1 or control siRNA#2 by two-way ANOVA with post hoc Tukey correction, n = 3 independent biological experiments. Boxes represent the 25th–75th centiles, with upper and lower bounds representing 10th–90th centiles.

Supplementary Figure 4 Shared super-enhancer-associated transcription factor dependency genes across five MYCN-amplified neuroblastoma cell lines.

Ranked H3K27ac signal across all enhancers in the MYCN-amplified cell lines SKNBE2, NGP and NB1643. Super-enhancers are the small subset with exceptional enhancer signal on the right side of each plot. Marked are super-enhancer-associated transcription factor CRISPR–Cas9 dependency genes. Two other cell lines, BE2C and Kelly, are described in the main text.

Supplementary Figure 5 Shared super-enhancer-associated dependency gene expression is elevated in primary human neuroblastoma relative to other tumor types.

The R2 genome analysis and browser tool was used to compare across human primary tumor experiments, using normalized u133p2, MAS5.0 microarray data. Data displayed represent genes with annotated ontologies including nucleic acid binding and/or transcription factor activity. Neuroblastoma samples (n = 231) are compared against multiple other tumor types (total n > 10,000 samples) for super-enhancer-associated CRISPR–Cas9 gene dependencies. Neuroblastoma datasets are shown in red; other tumor types are shown in blue. Datasets used are (left to right): (1) neuroblastoma (Versteeg), n = 88; (2) neuroblastic (Delattre), n = 64; (3) neuroblastoma (Hiyama), n = 51; (4) neuroblastoma (Lastowska), n = 30; (5) T-ALL (Pieters), n = 92; (6) T-ALL (Carroll), n = 98; (7) T-ALL (Murphy), n = 207; (8) T-ALL (Meijerink), n = 124; (9) AML (Ley), n = 179; (10) AML, n = 22; (11) AML (Metzeler), n = 79; (12) AML (Verhaak), n = 525; (13) AML (Haferlach), n = 251; (14) AML (denBoer), n = 237; (15) AML (Haferlach), n = 96; (16) AML (Dohner), n = 154; (17) AML (Bohlander), n = 140; (18) AML (Delwel), n = 460; (19) CLL (Rosenwald), n = 18; (20) CLL (Wiestner), n = 62; (21) CLL (Kueppers), n = 46; (22) CLL (Kipps), n = 130; (23) myeloma (Shaughnessy), n = 78; (24) myeloma (Hanamura), n = 542; (25) B cell (Xiao), n = 420; (26) lymphoma (Tsai), n = 44; (27) lymphoma (Roche), n = 498; (28) large B cell lymphoma (Johnsen), n = 33; (29) breast (Iglehart), n = 123; (30) breast (Bos), n = 204; (31) breast (Desmedt), n = 55; (32) breast (Loi), n = 77; (33) breast (Sotiriou), n = 120; (34) breast (Bertucci), n = 266; (35) breast (Quiles), n = 61; (36) breast (Smid), n = 210; (37) breast (Concha), n = 66; (38) breast (Black), n = 107; (39) breast (Prat), n = 156; (40) breast (Brown), n = 198; (41) breast (EXPO), n = 351; (42) colon (Smith), n = 232; (43) colon (Sieber), n = 290; (44) colon (Marra), n = 32; (45) colon (Watanabe), n = 84; (46) colon (Jorissen), n = 155; (47) colon (Siebersmith), n = 355; (48) colon (Hummel), n = 53; (49) colon (Sugihara), n = 148; (50) colon (Yagi), n = 83; (51) rectal (Hashimoto), n = 46; (52) colon (Wessels), n = 62; (53) colon (Olschwang), n = 130; (54) colon (Calon), n = 24; (55) colon (Clary), n = 133; (56) colon (Medema), n = 13; (57) colon (Sieber), n = 59; (58) colon (EXPO), n = 315; (59) colon (EXPO), n = 39; (60) colon (EXPO), n = 38; (61) lung (Massague), n = 29; (62) lung (Muley), n = 100; (63) lung (Pietsch), n = 150; (64) NSCLC, n = 100; (65) lung (Bild), n = 114; (66) lung (EXPO), n = 121; (67) ovarian (Anglesio), n = 90; (68) ovarian (McDonald), n = 45; (69) ovarian (Pamula-Pilat), n = 101; (70) ovarium (Tothill), n = 10; (71) ovarian (Bowtell), n = 285; (72) ovary (EXPO), n = 256; (73) esophageal (Minashi), n = 40; (74) esophageal adenocarcinoma (Bass), n = 14; (75) oral cavity (Holsinger), n = 103; (76) melanoma (Bhardwaj), n = 44; (77) bladder (Riester), n = 93; (78) kidney (EXPO), n = 261; (79) prostate (EXPO), n = 72; (80) endometrium (EXPO), n = 209; (81) cervix (EXPO), n = 36; (82) pancreatic (Wu), n = 32; (83) hepatocellular carcinoma (Llovet), n = 91; (84) thyroid (EXPO), n = 34; (85) glioma (Sun), n = 153; (86) glioma (Gleize), n = 30; (87) glioma (French), n = 284; (88) glioma (Paugh), n = 53; (89) glioma (Paugh), n = 37; (90) glioblastoma (Pfister), n = 46; (91) glioma (Kawaguchi), n = 50; (92) glioblastoma (Loeffler), n = 70; (93) glioblastoma (Hegi), n = 84; (94) pilocytic astrocytomas (Gutman), n = 41; (95) ependymoma (Donson), n = 19; (96) ependymoma (Gilberston), n = 83; (97) ependymoma (Hoffman), n = 65; (98) ependymoma (Pfister), n = 209; (99) medulloblastoma (Gilbertson), n = 76; (100) medulloblastoma (Pfister), n = 73; (101) medulloblastoma (ATRT), n = 31; (102) medulloblastoma (DenBo), n = 51; (103) medulloblastoma (Delattre), n = 57; (104) medulloblastoma (Kool), n = 62; (105) CNS/PNET (Grundy), n = 24; (106) CNS-PNET (Kool), n = 182; (107) ATRT (Birks), n = 18; (108) ATRT (Kool), n = 49; (109) rhabdoid (Hand), n = 54; (110) Ewing sarcoma (Frances), n = 37; (111) Ewing sarcoma (Delattre), n = 117; (112) osteosarcoma (Kobayashi), n = 27; (113) rhabdomyosarcoma (Ba), n = 58; (114) GCT (Wang), n = 13. The histology of tumor samples is displayed along the x axis, with log2 gene expression along the y axis. Boxes represent the 25th–75th percentiles (Q1 and Q3, respectively), with horizontal lines in the boxes representing the median. Whiskers represent data spread from the maximum (Q3 + 1.5 × interquartile range) to minimum (Q1 - 1.5 x interquartile range), and x denotes the presence of outlier data points.

Supplementary Figure 6 CRC members are marked with super-enhancers in a majority of cell lines and demonstrate dependency across MYCN amplification status and cell state categories.

a, Available H3K27ac data for 29 additional neuroblastoma cell lines from the literature, a primary sample previously published and 9 cell lines from our group demonstrates that a majority of neuroblastoma cells have super-enhancers marking multiple members of the CRC. Cell state was categorized into adrenergic/noradrenergic (ADRN/NOR) versus mesenchymal/neural crest cell–like (MES/NCC-like) based on previously published data in van Groningen et al. and Boeva et al. b, No significant difference was seen between the number of CRC members marked by super-enhancers in MYCN-amplified (n = 24) and MYCN-non-amplified (n = 15) lines by Welch’s two-sided t test. The vast majority of adrenergic/noradrenergic lines (n = 27) had super-enhancers associated with all five members of the CRC, while the majority of mesenchymal/neural crest cell–like lines (n = 10) had super-enhancers marking at least two of five members. The distribution of the number of super-enhancer-marked CRC members was statistically significantly lower (P = 0.017) in the MES/NCC-like lines than in the ADRN/NOR lines by Welch’s two-sided t test. c, Cancer Cell Line Encyclopedia (CCLE) RNA-seq expression of the mesenchymal and adrenergic differentially expressed genes identified by van Groningen et al. across the neuroblastoma lines screened with the genome-scale CRISPR–Cas9 screen recapitulates the cell state classifications in van Groningen et al. and Boeva et al. The heat map is hierarchically clustered by column using complete linkage and 1 minus Pearson correlation. d, Expression in log2-transformed reads per kilobase per million (RPKM) for the five CRC genes and MYCN in adrenergic/noradrenergic (ADRN/NOR) neuroblastoma cell lines in CCLE (n = 12), mesenchymal/neural crest cell–like (MES/NCC-like) neuroblastoma cell lines (n = 4) and the remainder of the cancer cell lines in CCLE (n = 1,003). The horizontal line in each box indicates the median of the data, whereas the top and bottom of the box represent the upper and lower quartiles, respectively. The whiskers extend to the most extreme point within 1.5 times the interquartile range (IQR) of the box. Data beyond 1.5 times the IQR are depicted as dots. e, Dependency z-scores for each gene across all 341 cell lines in the CRISPR–Cas9 screen demonstrate stronger dependencies in ADRN/NOR transcription factors (combining van Groningen et al. and Boeva et al.), particularly in the subset of CRC genes identified in the current study, with each CRC member with z-score <–4 in multiple neuroblastoma lines. Demonstrated in this heat map are only neuroblastoma cell lines. In comparison, there are no consistent strong unique dependencies (z-score <–4 in more than one line) across the subset of MES/NCC-like transcription factors (combining van Groningen et al. and Boeva et al.) even in the two lines in the screen (CHP212 and SKNAS) that are classified as MES/NCC-like. Of note, ZNF217 is present twice in this heat map as it is classified differently in van Groningen et al. than in Boeva et al.

Supplementary Figure 7 ChIP-seq occupancy maps showing binding/co-regulation of all six transcription factors in MYCN-amplified neuroblastoma cells.

a,b, ChIP-seq tracks for H3K27ac, MYCN, HAND2, ISL1, PHOX2B, GATA3 and TBX2 at each CRC gene locus were performed in BE2C (a) and Kelly (b) cells. Additionally, assay for transposase-accessible chromatin (ATAC) sequencing was performed to identify chromatin accessibility in the selected regions. These data demonstrate that HAND2, ISL1, PHOX2B, GATA3 and TBX2, together with MYCN, bind at epicentres—overlapping regions within super-enhancers that regulate CRC gene expression. ChIP-seq read densities are normalized to reads per million reads sequenced in each sample. The H3K27ac track represents a combination of two independent experiments in BE2C cells, and all other tracks are representative of an independent experiment performed in BE2C and Kelly cells.

Supplementary Figure 8 Distribution of binding of all six transcription factors in MYCN-amplified neuroblastoma cells.

a,b, Peak calls from ChIP-seq to the noted histone mark and transcription factor were subclassified based on DNA element into super-enhancers, typical enhancers (TE), promoters, gene bodies and other in BE2C (a) and Kelly (b) cells. The y axis displays peak sequencing counts normalized against the size of the entire genome-wide element to provide normalized density of binding in that element.

Supplementary Figure 9 ChIP-seq occupancy maps showing binding of all six super-enhancer-associated dependency transcription factors to the MYCN locus in BE2C cells.

ATAC-seq and ChIP-seq tracks for H3K27ac, MYCN, HAND2, ISL1, PHOX2B, GATA3 and TBX2 at the MYCN gene locus in BE2C cells. These data do not produce significant peak calling for any of the transcription factors binding to the MYCN locus, likely owing to background from high-level gene amplification. The H3K27ac track represents a combination of two independent experiments in BE2C cells, and all other tracks are representative of an independent experiment performed in BE2C and Kelly cells.

Supplementary Figure 10 Transient siRNA results in suppression of target gene expression in BE2C and Kelly cells.

BE2C and Kelly cells were transfected with two independent siRNAs targeting HAND2, ISL1, PHOX2B, GATA3, TBX2 or a non-targeted sequence. RNA was recovered and subjected to qRT–PCR against the shown targets. n = 3 independent biological experiments; all siRNA-treated transcription factor gene expression was significantly different from both control siRNAs at P < 0.05 by two-sided t test. Horizontal bars represent the median with upper and lower boxes representing 25th–75th centiles. Error bars represent 10th–90th centiles.

Supplementary Figure 11 Phenotypic effects of combination JQ1–THZ1 treatment of neuroblastoma cells.

a,b, Cell cycle analysis of Kelly cells treated with DMSO (control), JQ1, THZ1 or combination JQ1–THZ1 for 1 h (a) or 24 h (b). JQ1 was used at 3 µM (all), and THZ1 was used at 125 nM (BE2C cells) and 78 nM (Kelly cells), alone or in combination. DMSO was used as vehicle control. No differences were seen between any treatment and cell cycle phase at 1 h. At 24 h, THZ1 and JQ1+THZ1 induced a larger number of cells in the sub-G0 peak, with reduced numbers of S-phase cells. n = 4 biologically independent experiments. Box plots demonstrate the 25th–75th centile range. Error bars display data spread from the 10th–90th centile. *P = 7.7 × 10–4, **P = 2.5 × 10–4 relative to other treatment groups. c, Western blots for cleaved Caspase-3 and cleaved PARP1 in BE2C and Kelly cells treated with DMSO (control), JQ1, THZ1 or combination JQ1–THZ1 for 24 h. β-actin (ACTB) is shown as a loading control for each sample. Numerical values represent protein molecular weight markers. Blot images have been cropped. Data are representative of four independent biological experiments.

Supplementary Figure 12 JQ1 and THZ1 demonstrate synergy across a panel of MYCN-amplified and MYCN-non-amplified cell lines.

a,b, Chou–Talalay-normalized isobolograms depicting combination index (CI) scores over a range of concentrations of THZ1 and JQ1 in MYCN-amplified (a) and MYCN-non-amplified (b) cells treated for 72 h. CI scores <1, synergy; CI scores >1, antagonism. The red line represents additivity, CI = 1.

Supplementary Figure 13 BE2C xenograft tumors in mice with single and combination JQ1–THZ1 treatment.

a, BE2C xenograft demonstrating significantly prolonged survival with THZ1+JQ1 in combination compared to vehicle (P = 0.0001), while JQ1 alone prolonged survival less significantly (P = 0.017) and THZ1 alone did not achieve significance (n = 8 mice per group, *P = 0.017, ***P = 0.0001 by log-rank (Mantel–Cox) test). Animals were subjected to treatment as outlined in the Methods. b, Combination treatment did not cause weight loss greater than 10% on average compared to vehicle or single drug treatment, suggesting that the combination was well tolerated. Mean percent weight loss is plotted with error bars representing s.d.

Supplementary Figure 14 CRC transcription factor occupancy of genes downregulated by combination JQ1–THZ1 treatment at 4 h.

Number of CRC transcription factors occupying the loci of the top 1% most downregulated genes, relative to expression at 0 h. Most genes that are greatly reduced at 4 h are occupied preferentially by all six dependency CRC transcription factors.

Supplementary Figure 15 CRC gene expression levels in JQ1–THZ1-treated neuroblastoma cells.

a,b, qRT–PCR analysis of transcript levels for CRC and control genes (ACTB, HPRT) in BE2C (a) and Kelly (b) cells. JQ1 was used at 3 µM (all), and THZ1 was used at 125 nM (BE2C cells) and 78 nM (Kelly cells) in combination. DMSO was used as vehicle control. Cells were either untreated (time = 0 h) or treated with combination JQ1 and THZ1 for 1–8 h. n = 3 biologically independent experiments, P < 0.05 by two-sided t test for all CRC members compared to ACTB and/or HPRT at 1, 2, 4, 6 and 8 h. Center values represent the mean, and error bars represent s.d. c,d, qRT–PCR analysis of transcript levels from single and combination treatment with JQ1 and THZ1 for 6 h in BE2C (c) and Kelly (d) cells. JQ1 was used at 3 µM (all), and THZ1 was used at 125 nM (BE2C cells) and 78 nM (Kelly cells) alone or in combination. DMSO was used as vehicle control. n = 3 biologically independent experiments,*P ≤ 0.05 by two-way ANOVA with Tukey post hoc correction relative to DMSO control. Box plots demonstrate the 25th–75th centile range with internal bars representing the median. Error bars display data spread from the 10th–90th centiles.

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Durbin, A.D., Zimmerman, M.W., Dharia, N.V. et al. Selective gene dependencies in MYCN-amplified neuroblastoma include the core transcriptional regulatory circuitry. Nat Genet 50, 1240–1246 (2018). https://doi.org/10.1038/s41588-018-0191-z

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