CRISPR-Cas9 screen reveals a MYCN-amplified neuroblastoma dependency on EZH2
Liying Chen, Gabriela Alexe, Neekesh V. Dharia, Linda Ross, Amanda Balboni Iniguez, Amy Saur Conway, Emily Jue Wang, Veronica Veschi, Norris Lam, Jun Qi, W. Clay Gustafson, Nicole Nasholm, Francisca Vazquez, Barbara A. Weir, Glenn S. Cowley, Levi D. Ali, Sasha Pantel, Guozhi Jiang, William F. Harrington, Yenarae Lee, Amy Goodale, Rakela Lubonja, John M. Krill-Burger, Robin M. Meyers, Aviad Tsherniak, David E. Root, James E. Bradner, Todd R. Golub, Charles W.M. Roberts, William C. Hahn, William A. Weiss, Carol J. Thiele, Kimberly Stegmaier
Liying Chen, Gabriela Alexe, Neekesh V. Dharia, Linda Ross, Amanda Balboni Iniguez, Amy Saur Conway, Emily Jue Wang, Veronica Veschi, Norris Lam, Jun Qi, W. Clay Gustafson, Nicole Nasholm, Francisca Vazquez, Barbara A. Weir, Glenn S. Cowley, Levi D. Ali, Sasha Pantel, Guozhi Jiang, William F. Harrington, Yenarae Lee, Amy Goodale, Rakela Lubonja, John M. Krill-Burger, Robin M. Meyers, Aviad Tsherniak, David E. Root, James E. Bradner, Todd R. Golub, Charles W.M. Roberts, William C. Hahn, William A. Weiss, Carol J. Thiele, Kimberly Stegmaier
View: Text | PDF
Research Article Oncology

CRISPR-Cas9 screen reveals a MYCN-amplified neuroblastoma dependency on EZH2

Abstract

Pharmacologically difficult targets, such as MYC transcription factors, represent a major challenge in cancer therapy. For the childhood cancer neuroblastoma, amplification of the oncogene MYCN is associated with high-risk disease and poor prognosis. Here, we deployed genome-scale CRISPR-Cas9 screening of MYCN-amplified neuroblastoma and found a preferential dependency on genes encoding the polycomb repressive complex 2 (PRC2) components EZH2, EED, and SUZ12. Genetic and pharmacological suppression of EZH2 inhibited neuroblastoma growth in vitro and in vivo. Moreover, compared with neuroblastomas without MYCN amplification, MYCN-amplified neuroblastomas expressed higher levels of EZH2. ChIP analysis showed that MYCN binds at the EZH2 promoter, thereby directly driving expression. Transcriptomic and epigenetic analysis, as well as genetic rescue experiments, revealed that EZH2 represses neuronal differentiation in neuroblastoma in a PRC2-dependent manner. Moreover, MYCN-amplified and high-risk primary tumors from patients with neuroblastoma exhibited strong repression of EZH2-regulated genes. Additionally, overexpression of IGFBP3, a direct EZH2 target, suppressed neuroblastoma growth in vitro and in vivo. We further observed strong synergy between histone deacetylase inhibitors and EZH2 inhibitors. Together, these observations demonstrate that MYCN upregulates EZH2, leading to inactivation of a tumor suppressor program in neuroblastoma, and support testing EZH2 inhibitors in patients with MYCN-amplified neuroblastoma.

Authors

Liying Chen, Gabriela Alexe, Neekesh V. Dharia, Linda Ross, Amanda Balboni Iniguez, Amy Saur Conway, Emily Jue Wang, Veronica Veschi, Norris Lam, Jun Qi, W. Clay Gustafson, Nicole Nasholm, Francisca Vazquez, Barbara A. Weir, Glenn S. Cowley, Levi D. Ali, Sasha Pantel, Guozhi Jiang, William F. Harrington, Yenarae Lee, Amy Goodale, Rakela Lubonja, John M. Krill-Burger, Robin M. Meyers, Aviad Tsherniak, David E. Root, James E. Bradner, Todd R. Golub, Charles W.M. Roberts, William C. Hahn, William A. Weiss, Carol J. Thiele, Kimberly Stegmaier

×

Figure 9

EZH2-regulated gene IGFBP3 functions as a neuroblastoma tumor suppressor.

Options: View larger image (or click on image) Download as PowerPoint
EZH2-regulated gene IGFBP3 functions as a neuroblastoma tumor suppressor...
(A) The protein expression level of IGFBP3, a representative gene in the neuroblastoma EZH2 signature, in human neuroblastoma cell lines with or without MYCN amplification. (B) EZH2, H3K27me3, and H3K4me3 binding signal at the promoter of IGFBP3 in Kelly cells. (C) Immunoblot showing the overexpression of EGFP (negative control), NGFR (positive control), and IGFBP3 in SK-N-BE(2) cell line. (D) Cell viability assay after overexpression of EGFP, NGFR, or IGFBP3 in SK-N-BE(2). Results are representative of 3 independent experiments; mean ± SD of 8 technical replicates is shown. (E) Tumor volume in mouse xenograft model of SK-N-BE(2) with or without IGFBP3 overexpression (n = 10). P calculated with 2-way ANOVA. (F) Kaplan-Meier curves show survival of mice with xenografts of SK-N-BE(2) with or without IGFBP3 overexpression, up to 56 days after injection. P calculated using log-rank (Mantel-Cox) test. (G and H) Cell viability assay (G) and immunoblotting (H) after overexpression of IGFBP3 in CHP-212, LAN-1, ACN, and SH-SY-5Y. Results are representative of 3 independent experiments; data in G represent mean ± SD of 8 technical replicates. (I) Effect of overexpression of IGFBP3 on SK-N-BE(2)’s response to EZH2 inhibitors. Shown is a representative of 2 independent experiments; mean ± SD of 8 technical replicates is shown.