The transcriptional co-repressor Runx1t1 is essential for N-Myc-driven neuroblastoma tumorigenesis [RNA-Seq 2]

Changes in epigenetic regulation are believed to be a major contributing factor to neuroblastoma development. Using a large-scale in vivo mutagenesis screen in Th-MYCN transgenic mice, we identified a single point mutation in the transcriptional corepressor Runx1t1, that can block N-Myc-driven neuroblastoma tumorigenesis. The loss of function mutation disrupts a highly conserved zinc finger domain (NHR4) within Runx1t1. Crossing an independent Runx1t1 knockout model with Th-MYCN mice, demonstrated that Runx1t1 haploinsufficiency is enough to prevent neuroblastoma development and reverse ganglia hyperplasia. Silencing RUNX1T1 in human neuroblastoma cells resulted in decreased colony formation in vitro, and significant inhibition of tumor growth in vivo. Our results show that RUNX1T1 forms part of a transcriptional LSD1-CoREST3-HDAC repressive complex that regulates the epigenomic landscape and chromatin accessibility, to control neuron-specific pathway genes and maintain an undifferentiated state. Runx1t1 thus represents an entirely novel and highly promising target not previously described in neuroblastoma. Celiac ganglia were dissected from two-week old Th-MYCN homozygote and wild-type mice with 1- or 2-copies of Runx1t1 and cleaned to remove non-ganglion tissue. Total RNA was isolated using the RNeasy Micro kit (50), # 74004 kit and RNA quality was assessed prior to being used (Agilent 2100 Bioanalyzer, Agilent Technologies). Only RNA with an RNA Integrity Number (RIN) of >9.0 was used. Whole transcriptome sequencing was performed at the Ramaciotti Centre for Genomics with SMARTer Stranded Total RNA-seq v2 preparation kit. Prepared libraries were pooled and sequenced on NovaSeq 6000 S1 2x100bp lane generating an average of 60 million reads per sample.