Background: Patients harboring MYCN-amplified neuroblastomas (NBs) have advanced disease and poor outcome. Current therapeutic options fail to cure this patient group and novel therapeutic concepts are urgently needed. Functional screens to identify synthetic lethal combinations with tumor-specific mutations are an exciting new approach to identify novel drug targets and pathways that upon inhibition disclose cancer-specific vulnerabilities.
Material and methods: We performed a genome-wide screen in a MYCN-amplified neuroblastoma cell line, harboring a tet-inducible shRNA against MYCN, using a siRNA library consisting of 3 different siRNAs targeting 11.000 genes. Positive hits were validated in different in vitro systems allowing MYCN modulation. Chromatin immunoprecipitation (ChIP-seq) coupled to sequencing for 6 histone modifications and RNA-sequencing was used to characterize MYCN-related transcriptional and epigenetic changes in cell lines and primary tumors.
Results: From a focused analysis of 334 epigenetic modifiers, we identified inhibition of several methyltransferases, including EZH2, as a synthetic lethal interaction with high MYCN. shRNA mediated knock down of MYCN in MYCN-amplified cells revealed massive transcriptional and epigenetic changes with 865 up-regulated genes and 887 down-regulated genes associated with reduction of H3K27me3 and H3K4me3 histone marks at the transcriptional start site (TSS) of regulated genes, respectively. Tumor ChIP-seq revealed higher abundance of H3K4me3 and H3K27me3 in MYCN amplified as compared to MYCN single-copy NBs. In line with this, several components of the PRC2 complex, such as SUZ12, EED, EZH2 and RBBP7 were up-regulated in MYCN-amplified tumors. Inhibition of EZH2 using the selective EZH2 inhibitor, EPZ-6438, reduces H3K27me3 globally and leads to re-expression of silenced genes and viability reduction.
Conclusion: Selective inhibition is a synthetic lethal interaction with high MYCN in neuroblastoma cells. Target inhibition of EZH2 using small molecules could be new approach to treat high-risk NBs with amplified MYCN.