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Supplementary Figure S4 A-E from Drugging MYCN Oncogenic Signaling through the MYCN-PA2G4 Binding Interface

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posted on 2023-03-31, 03:45 authored by Jessica Koach, Jessica K. Holien, Hassina Massudi, Daniel R. Carter, Olivia C. Ciampa, Mika Herath, Taylor Lim, Janith A. Seneviratne, Giorgio Milazzo, Jayne E. Murray, Joshua A. McCarroll, Bing Liu, Chelsea Mayoh, Bryce Keenan, Brendan W. Stevenson, Michael A. Gorman, Jessica L. Bell, Larissa Doughty, Stefan Hüttelmaier, Andre Oberthuer, Matthias Fischer, Andrew J. Gifford, Tao Liu, Xiaoling Zhang, Shizhen Zhu, W. Clay Gustafson, Michelle Haber, Murray D. Norris, Jamie I. Fletcher, Giovanni Perini, Michael W. Parker, Belamy B. Cheung, Glenn M. Marshall

Supplementary Figure S4. Characterization of the PA2G4-MYCN protein-protein interface. A, BE(2)-C cells were transiently transfected with EV, wildtype PA2G4 or 6 different PA2G4 point mutants for 48 hours, then treated with 100 µg/µl Cycloheximide (CHX) for up to 60 minutes, followed by immunoblot analysis for MYCN protein half-life. B, Differential Scanning Fluorimetry (DSF) showed both the seven amino acid (DHKALST, aa248-254) and large peptide (GGDHKALSTGEDTL, aa246-259) MYCN oligopeptides, along with the MYCN oligopeptide shown not to bind via SPR (DHAALAT) changed the melting temperature of the PA2G4 protein, relative to baseline (i.e. 0mM), in a dose-response manner. Shown are the means of 3 independent experiments {plus minus} SEM. C, An example of the raw data for DHKALST, with the shift to the left correlating to an increase in concentration. D, Raw SPR data for PA2G4 triple mutant (R271A, R272A and S47A) and single mutants (S47A and R272A). Also, raw SPR data for MYCN oligopeptide mutants (DHAALST, DHAALAT and DHKALAT). For the mutants and triple mutations, no binding was observed, thus analysis could not be conducted and is not shown. E, Root Mead Squared Deviation (RMSD) of the peptide over the time of the simulation. After the first 100 frames the peptide is relatively stable. Analysis was only conducted after this time.

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National Health and Medical Research Counci

NHMRC

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ARTICLE 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, proliferation-associated 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 MYCN-dependent 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. Competitive chemical inhibition of the PA2G4–MYCN protein interface provides a basis for drug design of small molecules targeting MYC and MYCN-binding partners in malignancies driven by MYC family oncoproteins.

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    Cancer Research

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    BiomarkersDrug TargetsOncoprotein & tumor suppressor drug targetsOncogenes & Tumor SuppressorsPediatric Cancers

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