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...

Background: Drug resistance and chemotherapy-induced peripheral neuropathy continue to be significant problems in the successful treatment of acute lymphoblastic leukemia (ALL). 5,7-Dibromo-N-alkylisatins, a class of potent microtubule destabilizers, are a promising alternative to traditionally used antimitotics with previous demonstrated efficacy against solid tumours in vivo and ability to overcome P-glycoprotein (P-gp) mediated drug resistance in lymphoma and sarcoma cell lines in vitro. In this study, three di-brominated N-alkylisatins were assessed for their ability to retain potency in vincristine (VCR) and 2-methoxyestradiol (2ME2) resistant ALL cell lines. For the first time, in vitro neurotoxicity was also investigated in order to establish their suitability as candidate drugs for future use in ALL treatment. Methods: Vincristine resistant (CEM-VCR R) and 2-methoxyestradiol resistant (CEM/2ME2-28.8R) ALL cell lines were used to investigate the ability of N-alkylisatins to overcome chemoresistance. Interaction of N-alkylisatins with tubulin at the the colchicine-binding site was studied by competitive assay using the fluorescent colchicine analogue MTC. Human neuroblastoma SH-SY5Y cells differentiated into a morphological and functional dopaminergic-like neurotransmitter phenotype were used for neurotoxicity and neurofunctional assays. Two-way ANOVA followed by a Tukey's post hoc test or a two-tailed paired t test was used to determine statistical significance. Results: CEM-VCR R and CEM/2ME2-28.8R cells displayed resistance indices of > 100 to VCR and 2-ME2, respectively. CEM-VCR R cells additionally displayed a multi-drug resistant phenotype with significant cross resistance to vinblastine, 2ME2, colchicine and paclitaxel consistent with P-gp overexpression. Despite differences in resistance mechanisms observed between the two cell lines, the N-alkylisatins displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell line. The N-alkylisatins proved to be significantly less neurotoxic towards differentiated SH-SY5Y cells than VCR and vinblastine, evidenced by increased neurite length and number of neurite branch points. Neuronal cells treated with 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin showed significantly higher voltage-gated

<p>Supplementary Figure S6. PA2G4 increases neuroblastoma tumorigenicity. A, Representative images of athymic nude mice inoculated with neuroblastoma (SH-EP) cells stably transfected with either EV control or a PA2G4 expression vector at 10 weeks post-injection. B, Images of tumor formation after mice were culled 12 weeks post-injection. C, Real-time PCR analysis of PA2G4 mRNA expression level in tumors from mice injected with either SH-EP cells overexpressing PA2G4, or SH-EP EV control cells. β2-microglobulin was used as a reference gene for total RNA loading. D, Protein was extracted from SH-EP tumor xenografts overexpressing PA2G4 and control vectors, then analysed for PA2G4-Flag and MYCN expression by immunoblotting using anti-MYCN and anti-PA2G4 antibodies, using a Vinculin loading control. E, Immunoblots of three tumor samples from each siRNA-treated cohort showing the levels of PA2G4 and MYCN protein expression, using a Vinculin loading control. F, Real-time PCR analysis of PA2G4 and MYCN mRNA expression in tumor samples taken from tumour-bearing mice xenografted with BE(2)-C neuroblastoma cells, which had been treated with either nano-particle encapsulated siRNA control, PA2G4 siRNA or PA2G4-p48 siRNA. Data are shown as means and SD derived from 6 mice per group, P-values were calculated by t-test.</p>

<p>Supplementary Information</p>

<p>Supplementary Information</p>

<p>Supplementary Figure S3 F-G F, Left panel: Histopathologic and immunohistochemical analyses of MYCN;GFP tumors treated with vehicle (left) or WS6 (right). Left Panel, Top to Bottom: Neuroblastoma tumour sections immunohistochemically stained for Haematoxylin & Eosin (H&E), Proliferating Cell Nuclear Antigen (pCNA), Neural Hu protein C (Hu-C), MYCN, PA2G4 and Tyrosine Hydroxylase. Scale bar, 50 μm. Right Panel: Histograms illustrating the staining intensity of cells of vehicle (left) or WS6 treated neuroblastoma tumours expressing either MYCN, PA2G4 or Tyrosine Hydroxylase as measured by Image J software. Sample means (horizontal bars) were compared by students t-test (two-tailed). *** represents p-value <0.0001. ns represents p-value of no significance. error bars represent SEM. G, IC50 value of WS6, compared to other MYCN oncogenic signal inhibitors, after treatment of MYCN amplified Kelly neuroblastoma cells. IC50 value for CD532 is the average IC50 values for 169 cancer cell lines.</p>

<p>Supplementary Figure S2. PA2G4 increases MYCN protein stability. A and B, Representative immunoblots from cycloheximide (CHX) chase assays measuring the half-life of MYCN protein after PA2G4 knockdown (A) or overexpression (B) in BE(2)-C and Kelly cells for 48 hours. Cells were then treated with 100 µg/µl CHX for up to 60 minutes followed by immunoblotting. C, Co-IP of total protein from BE(2)-C cells using IgG, anti-MYCN, anti-PA2G4 antibodies, followed by immunoblotting with anti-MYCN, anti-PA2G4, anti-AURKA, anti-Fbxw7 or anti-vinculin antibodies.</p>

<p>Supplementary Figure S5 F-H F), at 72 hours post-transfection of EV or a PA2G4 expression vector. G, Cell viability measured by the Alamar Blue assay and immunoblot analyses using an antibody identifying MYC-tagged PA2G4-p42 protein expression in Kelly and SH-SY5Y cells transfected with EV or the MYC-tagged PA2G4-p42 expression vector at 48 and 72 hours post-transfection. Vinculin was used as loading control. H, Colony formation in vitro by Kelly and SH-SY5Y cells following transfection with either EV or MYC-tagged PA2G4-p42.</p>