Specific Aim 1. To identify key differentiation genes We hypothesized that epigenetic dysregulation of neural crest stem cells and/or sympathoadrenal progenitors contributes to neuroblastoma initiation, tumorigenesis and progression. Moreover, by targeting critical chromatin regulators that keep neuroblastoma in a self-renewal state we should be able to suppress growth and induce differentiation. To do this we performed an epigenetic focused siRNA screen to identify genes that control NB cell proliferation and differentiation using a high-throughput, high content imaging screen. We identified 53 candidate genes whose loss of expression results in a decrease in the number of NB cells and of these, 16 also induce morphologic and biochemical evidence of differentiation. A secondary screen using additional siRNAs excluded genes which may have resulted from off-target effects of siRNAs. Four of the candidates had already been shown to affect NB cell growth and differentiation. To prioritize those hits that would be amenable to drug development, we performed an additional screen of a tool compound library of 20 small molecule inhibitors of chromatin regulators. We evaluated the growth and differentiation in 8 NB cell lines and 2 immortal, but not transformed cell lines after exposure for 7days to 8 different drug concentrations. The secondary chemical screen identified EZH2 and SETD8 as druggable NB targets.
Specific Aim 2. To characterize molecular mechanisms of action of growth and differentiation genes SETD8 is a histone methyltransferase which mono-methylates H4K20, and regulates critical cell cycle events during G1 and G2. We discovered high levels of SETD8 in primary NB tumors are associated with poor overall survival. In high-risk, Stage 4 NB patients high-levels of SETD8 are associated with poor prognosis in the MYCN-wt subgroup of high risk patients which comprises 60% of high-risk patients. We determined the mechanism of action to be due to SETD8 mono-methylation of p53 which leads to a decrease in its stability. This was the first report that genetic targeting SETD8 had activity in pre-clinical xenograft tumor models reducing tumor growth and conferring a significant survival advantage. This study was recently published in Cancer Cell 31:50-63, 2017.
|Souza, Bárbara Kunzler; da Costa Lopez, Patrícia Luciana; Menegotto, Pâmela Rossi et al. (2018) Targeting Histone Deacetylase Activity to Arrest Cell Growth and Promote Neural Differentiation in Ewing Sarcoma. Mol Neurobiol 55:7242-7258|
|Hua, Zhongyan; Zhan, Yue; Zhang, Simeng et al. (2018) P53/PUMA are potential targets that mediate the protection of brain-derived neurotrophic factor (BDNF)/TrkB from etoposide-induced cell death in neuroblastoma (NB). Apoptosis 23:408-419|
|Chen, Liying; Alexe, Gabriela; Dharia, Neekesh V et al. (2018) CRISPR-Cas9 screen reveals a MYCN-amplified neuroblastoma dependency on EZH2. J Clin Invest 128:446-462|
|Bhaskaran, Natarajan; Liu, Zhihui; Saravanamuthu, Senthil S et al. (2018) Identification of Casz1 as a Regulatory Protein Controlling T Helper Cell Differentiation, Inflammation, and Immunity. Front Immunol 9:184|
|Liu, Zhihui; Thiele, Carol J (2017) When LMO1 Meets MYCN, Neuroblastoma Is Metastatic. Cancer Cell 32:273-275|
|Liu, Z; Lam, N; Wang, E et al. (2017) Identification of CASZ1 NES reveals potential mechanisms for loss of CASZ1 tumor suppressor activity in neuroblastoma. Oncogene 36:97-109|
|Veschi, Veronica; Thiele, Carol J (2017) High-SETD8 inactivates p53 in neuroblastoma. Oncoscience 4:21-22|
|Veschi, Veronica; Liu, Zhihui; Voss, Ty C et al. (2017) Epigenetic siRNA and Chemical Screens Identify SETD8 Inhibition as a Therapeutic Strategy for p53 Activation in High-Risk Neuroblastoma. Cancer Cell 31:50-63|
|Wylie, Luke A; Hardwick, Laura J A; Papkovskaia, Tatiana D et al. (2015) Ascl1 phospho-status regulates neuronal differentiation in a Xenopus developmental model of neuroblastoma. Dis Model Mech 8:429-41|
|Liu, Zhihui; Li, Wenling; Ma, Xuefei et al. (2014) Essential role of the zinc finger transcription factor Casz1 for mammalian cardiac morphogenesis and development. J Biol Chem 289:29801-16|
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