Poster Presentation Advances in Neuroblastoma Research Congress 2016

Epigenetic siRNA and chemosensitivity screens identify a vulnerability to SETD8 inhibition through reactivation of p53 canonical pathway in Neuroblastoma (#371)

Veronica Veschi 1 , Zhijui Liu 1 , Ty Voss 1 , Laurent Ozbun 1 , Berkley Gryder 1 , Chunhua Yan 2 , Ying Hu 2 , Anqi Ma 3 , Jian Jin 3 , Cheryl Arrowsmith 4 , Javed Khan 1 , Ettore Appella 5 , Gordon Hager 1 , Giuseppe Giannini 6 , Carol Thiele 1
  1. Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda
  2. Center for Cancer Research, National Cancer Institute,Biomedical Informatics and Information Technology, Rockville
  3. Icahn School of Medicine at Mount Sinai, New York, New York
  4. Structural Genomics Consortium, University of Toronto, Toronto
  5. National Cancer Institute, National Institutes of Health, Bethesda
  6. Department of Molecular Medicine, University La Sapienza, Rome

High-risk Neuroblastoma(NB) is considered a failure of sympathoadrenal differentiation and there are a paucity of druggable mutations. To uncover epigenetic regulators critical for survival of undifferentiated high-risk NBs, we undertook a chromatin-focused siRNA screen. Of the 400 genes analyzed, high-content Opera imaging identified 53 genes whose loss of expression led to significant decreases in NB proliferation with 16 also inducing differentiation. We further screened 21 epigenetic compounds in 8 NB cell lines to prioritize siRNA hits that are in the drug developmental pipeline. Integration of genetic and chemical screen data revealed SETD8 as an important and druggable NB target. SETD8 is the H4K20me1 methyltransferase regulating DNA replication, chromosome condensation and gene expression. Analysis of 3 different datasets in R2 showed high expression of SETD8 is associated with poor prognosis in NB(ex. Kocak;p=1.4e-07) as well as in Stage-4-NB-wildtype-MYCN(p=0.03) but not in Stage-4-NB-amplified-MYCN(p=0.96). To determine mechanisms of SETD8-mediated growth inhibition, we performed RNA-seq transcriptome analyses on NB cells after genetic or pharmacological inhibition of SETD8. These revealed that SETD8 ablation rescued p53 proapoptotic and cell-cycle arrest functions by reactivation of p53 canonical pathway(IPA). Functional studies indicate SETD8 methylates p53(K382) leading to its inactivation. Moreover the levels of p53K382me1 are higher in wildtype-MYCN-NB cell lines compared to amplified-MYCN cells. Tumor progression is marked by inactivation of p53 and multiple mechanisms have been identified in amplified-MYCN NB cells. For the first time, this study reveals that inhibition of SETD8 is a novel mechanism to reactivate p53 particularly in high-risk wildtype-MYCN NBs which account for some 60-70% of high-risk NB tumors. Our in vivo xenograft NB models, showed that genetic or pharmacologic(UNC0379) inhibition of SETD8 confers a significant survival advantage. This identifies that SETD8 is a novel therapeutic target and its inhibition may be especially relevant for the subset of high-risk NB tumors with wildtype-MYCN.