Drugging MYCN Oncogenic Signaling through the MYCN-PA2G4 Binding Interface

Abstract

MYCN is a major driver for the childhood cancer, neuroblastoma, however, there are no inhibitors of this target. Enhanced MYCN protein stability is a key component of MYCN oncogenesis and is maintained by multiple feedforward expression loops involving MYCN transactivation target genes. Here, we reveal the oncogenic role of a novel MYCN target and binding protein, proliferationassociated 2AG4 (PA2G4). Chromatin immunoprecipitation studies demonstrated that MYCN occupies the PA2G4 gene promoter, stimulating transcription. Direct binding of PA2G4 to MYCN protein blocked proteolysis of MYCN and enhanced colony formation in a MYCNdependent manner. Using molecular modeling, surface plasmon resonance, and mutagenesis studies, we mapped the MYCN-PA2G4 interaction site to a 14 amino acid MYCN sequence and a surface crevice of PA2G4. Competitive chemical inhibition of the MYCN-PA2G4 protein-protein interface had potent inhibitory effects on neuroblastoma tumorigenesis in vivo. Treated tumors showed reduced levels of both MYCN and PA2G4. Our findings demonstrate a critical role for PA2G4 as a cofactor in MYCN-driven neuroblastoma and highlight competitive inhibition of the PA2G4-MYCN protein binding as a novel therapeutic strategy in the disease.Significance: Competitive chemical inhibition of the PA2G4-MYCN protein interface provides a basis for drug design of small molecules targeting MYC and MYCNbinding partners in malignancies driven by MYC family oncoproteins. Characterization of the PA2G4-MYCN protein-protein interface. A, GST pulldown of overexpressed GST-MYCN deletion mutant proteins and a PA2G4-3xFlag expression vector for 24 hours in HEK-293T cells, which were then immunoblotted with an anti-Flag antibody. B, Overlay of the independent representation of the docking solutions of WS6 (green carbons) and the MYCN oligopeptide DHKALST (white carbons) to PA2G4. Both were predicted to bind to the same surface pocket of PA2G4 (gray-filled space). C, A representative SPR (Biacore T200) experiment demonstrating a direct dose-response binding interaction between the bound PA2G4 exposed to increasing concentrations of the MYCN oligopeptide, DHKALST. Each experiment was run in duplicate. Overall this interaction had a calculated K d of 28.3 AE 0.73 mmol/L (n ¼ 5). D, A molecular model of the PA2G4-MYCN protein interface. The addition of two MYCN amino acids at the C-terminus and five at the N-terminus of the DHKALST MYCN oligopeptide resulted in an oligopeptide, GGDHKALSTGEDTL (cyan carbons), which interacted with PA2G4 (white carbons) in this molecular model. A visual analysis of this static dock predicted putative hydrogen bonds (yellow dashes) with residues Ser47, Arg271, and Arg272, which were subsequently targeted for mutagenesis. E, Representative SPR curves showing the concentration-response binding of DHKALST to PA2G4 (solid lines). The addition of 10 mmol/L WS6 resulted in a repression of this binding (dotted lines). This experiment was conducted in duplicate, three times. F, HEK293 cells were transiently transfecte...

A structural view of PA2G4 isoforms with opposing functions in cancer

“…Our studies suggested that an alternate region of PA2G4-p48 might be a potential protein-protein interaction site, specifically interacting with the oncoprotein, MYCN (44).…”

“…In our study, we used structurebased drug design methods to target a binding site boarded by helix α2 (aa S44-K62) (44). We hypothesized that the known PA2G4 inhibitor WS6 (54) bound to the same groove on PA2G4 that MYCN binds to, a groove boarded by helix α2 (44). Further biological assessment of WS6 confirmed that it had an inhibitory effect on PA2G4-p48's oncogenic role in malignant neuroblastoma cells in vitro and in vivo (44).…”

“…To investigate this, we created a series of GST-MYCN deletions and analyzed their ability to bind a FLAG-tagged PA2G4-p48 (44). We identified a 7 residue sequence (aa248-254), DHKALST, was able to bind to PA2G4-p48 in a dose-dependent manner.…”

“…Drugs which interact only with the PA2G4-p48 isoform would have the potential to inhibit the oncogenic role of PA2G4-p48, whilst having little effect on the tumor suppressive role of PA2G4-p42. In our study, we used structurebased drug design methods to target a binding site boarded by helix α2 (aa S44-K62) (44). We hypothesized that the known PA2G4 inhibitor WS6 (54) bound to the same groove on PA2G4 that MYCN binds to, a groove boarded by helix α2 (44).…”

“…We hypothesized that the known PA2G4 inhibitor WS6 (54) bound to the same groove on PA2G4 that MYCN binds to, a groove boarded by helix α2 (44). Further biological assessment of WS6 confirmed that it had an inhibitory effect on PA2G4-p48's oncogenic role in malignant neuroblastoma cells in vitro and in vivo (44). Due to the lack of helix α2 in PA2G4-p42, we now hypothesize that this compound would not be able to bind to the shorter PA2G4-p42 isoform effectively and therefore should have no effect on this isoforms function.…”