MYCN is a major oncogenic driver for neuroblastoma tumorigenesis, yet there are no direct MYCN inhibitors. We have previously identified PA2G4 as a direct protein-binding partner of MYCN and drive neuroblastoma tumorigenesis. A small molecule known to bind PA2G4, WS6, significantly decreased tumorigenicity in TH-MYCN neuroblastoma mice, along with the inhibition of PA2G4 and MYCN interactions. Here, we identified a number of novel WS6 analogues, with 80% structural similarity, and used surface plasmon resonance assays to determine their binding affinity. Analogues #5333 and #5338 showed direct binding towards human recombinant PA2G4. Importantly, #5333 and #5338 demonstrated a 70-fold lower toxicity for normal human myofibroblasts compared to WS6. Structure-activity relationship analysis showed that a 2,3 dimethylphenol was the most suitable substituent at the R¹ position. Replacing the trifluoromethyl group on the phenyl ring at the R² position, with a bromine or hydrogen atom, increased the difference between efficacy against neuroblastoma cells and normal myofibroblast toxicity. The WS6 analogues inhibited neuroblastoma cell phenotype in vitro, in part through effects on apoptosis, while their anti-cancer effects required both PA2G4 and MYCN expression. Collectively, chemical inhibition of PA2G4-MYCN binding by WS6 analogues represents a first-in-class drug discovery which may have implications for other MYCN-driven cancers.

MYCN and c-MYC are critical driver oncogenes in several childhood cancers, including neuroblastoma. Currently, the clinical development of MYC inhibitors has been hindered by the intrinsically disordered structure of MYC proteins, which lack well-defined ligand-binding pockets. Proliferation-associated protein 2G4 (PA2G4) directly binds to and stabilizes MYCN protein, leading to markedly increased MYCN levels in neuroblastoma cells. Here, we demonstrate that PA2G4 is essential for MYCN-driven tumor growth in neuroblastoma in vivo. Moreover, PA2G4 elevates c-MYC protein levels in neuroblastoma cells by inhibiting its ubiquitin-mediated degradation. In turn, c-MYC upregulates the transcription and protein expression of PA2G4, creating an oncogenic feed-forward expression loop. A small molecule PA2G4 inhibitor, WS6, directly disrupts the PA2G4-c-MYC protein–protein interaction, resulting in decreased levels of both PA2G4 and c-MYC. WS6 exhibited selective cytotoxicity in c-MYC-overexpressing cell lines. Together, these findings identify PA2G4 as a shared cofactor for both the c-MYC and MYCN oncoproteins and highlight its interaction with MYC family oncoproteins as a promising therapeutic vulnerability in MYC-driven cancers.