SGSS05-NS3, a covalent SETD8 inhibitor that activates p53 pathway in neuroblastoma

High-risk neuroblastoma (NB) is one of the most aggressive pediatric tumors accounting for 15% of all pediatric oncology deaths, and with less than 50% of patients experience long-term survival despite intense multimodal treatment. The tumor suppressor p53 is rarely (2%) mutated in NB but its functions are diminished in the majority of these tumors. Multiple mechanisms have been identified that attenuate the activity of p53 in MYCN-amplified (MYCN-amp) NB cells, but fewer mechanisms of p53 inactivation have been revealed in MYCN-WT NBs. Thus, a major challenge is to identify novel targeted therapies for high-risk NB (HR-NB) patients, specifically for the large fraction (70%) that present with MYCN-WT. Previously, we identified SETD8, the H4K20me1 methyltransferase, as a crucial epigenetic regulator of growth and differentiation in NB. In addition to targeting other non-histone proteins, SETD8 monomethylates p53 on lysine 382 (p53K382me1), attenuating its pro-apoptotic and growth arrest functions. Genetic and pharmacological (UNC0379) inhibition of SETD8 impairs NB growth in vivo. IC50 and IVTI (in vitro therapeutic index) of SGSS05-NS3, a SETD8 inhibitor, were measured in a broad collection of MYCN-WT and MYCN-amp NB cell lines. We took advantage of RNA-seq transcriptome analysis, in vitro functional assays and in vivo preclinical NB models. To identify targeted therapies that are less toxic for HR-NB, we evaluated a more specific SETD8 inhibitor with enhanced activity and selectivity, SGSS05-NS3. Our results indicated that in NB cells in vitro treatment with SGSS05-NS3 rescues the canonical p53 functions leading to increases in p53 protein levels and of its target p21 by decreasing p53K382me1, impairing NB cell viability and inducing caspase-dependent cell death. Gene expression profile (RNA-seq analysis) confirmed that the most significantly upregulated genes upon SGSS05-NS3 treatment were among the p53 pathway targets. Pharmacological and genetic SETD8 inhibition restores p53-mediated DNA damage response. In pre-clinical xenograft NB models, pharmacological SETD8 inhibition by SGSS05-NS3 conferred a significant survival advantage in MYCN-WT NB. Our study provides further evidence for targeting SETD8 as a therapeutic strategy in NB, alone or in combination with Topotecan.