Search

Supplementary Figure S1. PA2G4 is a MYCN transactivation target gene. A, Quantification of protein expression using anti-PA2G4 and anti-MYCN antibodies against whole cell protein lysates from BE(2)-C and Kelly cells, following MYCN siRNA knockdown for 48 hours. B, mRNA expression of PA2G4 and MYCN in SH-EP MYCN3 overexpression cells treated with 1µg/ml doxycycline for 24-96 hr. C, The effect of doxycycline-induced MYCN overexpression in SHEP-TRE-MYCN cells on c-MYC and PA2G4 protein expression. D, Chromatin immunoprecipitation (ChIP) assay in Kelly cells using an anti-MYCN antibody, and real-time PCR analysis with primers identifying the MYCN DNA binding sites in the PA2G4 gene promoter (500bP upstream of transcription start site [TSS]) or Intron 1a & 1b regions of the PA2G4 gene, with and without MYCN siRNA knockdown. ChIP and real-time PCR analysis using primers against a region 1200bp upstream of TSS was used as a negative control for MYCN chromatin binding. ChIP and real-time PCR analysis using primers against the ornithine decarboxylase (ODC1) gene promoter region was used as a positive control for MYCN chromatin binding. E, Immunoblot analysis of PA2G4, MYCN and MYC protein levels in a panel of human MYCN amplified and non-amplified neuroblastoma, and normal fibroblast, cell lines using antibodies recognising PA2G4, MYCN and MYC.

Supplementary Figure S1. F, Confocal microscopy of SHEP Tet21N cells using anti-PA2G4 (red) and anti-MYCN (green) antibodies. G, Immunoblot analysis of cytoplasmic and nuclear fractions from BE(2)-C and Kelly human neuroblastoma cell lines with antibodies recognizing PA2G4 and MYCN, or GAPDH and topoisomerase as loading controls. H, Confocal microscopy of neuroblastoma (BE(2)-C and Kelly) cells using anti-PA2G4 (red) and anti-MYCN (green) antibodies. Alexafluor 555 anti-rabbit (to detect PA2G4) and Alexafluor 488 anti-mouse (to detect MYCN) were used as the secondary antibodies. I, Real-time PCR mRNA expression of MYCN and PA2G4 in ganglia from homozygote TH-MYCN+/+ mice, compared to wild type littermate control mice, obtained at different postnatal age (weeks).

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.Significance: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.

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.

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.

Supplementary Information

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.

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.

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

Supplementary Figure S6 G-K G, Overall patient survival using Kaplan-Meier survival probability plots from the Cologne data set (http://r2.amc.nl) for PA2G4 mRNA expression subdivided around the median PA2G4 expression level among 477 neuroblastoma patients. H, Kaplan-Meier plot for event-free survival of 649 neuroblastoma patients from the kocak dataset (R2 microarray analysis and visualization platform, http://r2.amc.nl) I, Real-time PCR mRNA expression of PA2G4 among 40 neuroblastoma patient tumors treated at Sydney Children's Hospital, subdivided by MYCN amplification status. J, Multivariate event-free survival analysis using cox regression modelling. The p-values were obtained from the cox-regression analysis. K, Incidence of PA2G4 amplification among a range of different human cancer types within The Cancer Genome Atlas (TCGA). Results were generated using cBioportal for Cancer Genomics (https://cancergenome.nih.gov/). NEPC, neuroendocrine prostate cancer; CS, carcinosarcoma.

Supplementary Figure S5. PA2G4 expression enhances the malignant neuroblastoma phenotype in vitro. A, Neurite formation in SH-SY5Y and BE(2)-C cells transfected with PA2G4 siRNA and then treated with 2 µM 13-cis-retinoic acid. B, Immunoblots assessing PA2G4-p48 and PA2G4-p42 protein expression levels using an anti-PA2G4 antibody against whole cell protein lysates from a panel of neuroblastoma and non-malignant myofibroblast cell lines. C, Immunoblots assessing the effect of PA2G4-p48 knockdown on PA2G4 and MYCN protein levels in BE(2)-C and CHP-134 cells. D, Immunoblots confirming transfection of PA2G4-p48 siRNA and MYCN expression plasmid DNA for 72 hours. E and F Cell proliferation measured by BrdU incorporation (E), and, cell viability measured by the Alamar Blue assay.

Supplementary Figure S3. Competitive chemical inhibition of MYCN-PA2G4 binding. A, Chemical structure of WS6. B, Cycloheximide chase assay measuring the half-life of MYCN protein in BE(2)-C and Kelly cells following 0.8 µM WS6 treatment for 24 hours, then treatment with 100 µg/µl CHX for up to 60 minutes. C, BE(2)-C and Kelly cells treated with 0.2 µM or 0.4 µM WS6, followed by colony formation assessment, compared to untreated cells. D, Immunoblotting with an anti-MYC-Tag antibody to quantify PA2G4 protein expression from SH-SY5Y and Kelly cells transfected with the MYC-tagged PA2G4 vector. E, Densitometric quantification of MYCN and PA2G4 protein levels of immunoblots using tumor tissues of TH-MYCN mice treated with either DMSO (n=5) or WS6 (n=5), and quantified by Image J software. Sample means were compared by students t-test. * Represents p-value

16 p.-7 fig.-1 tab., 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 sodium channel function than those treated with Vinca alkaloids, strongly supportive of continued action potential firing., Conclusions:The N-alkylisatins are able to retain cytotoxicity towards ALL cell lines with functionally distinct drug resistance mechanisms and show potential for reduced neurotoxicity. As such they pose as promising candidates for future implementation into anticancer regimes for ALL. Further in vivo studies are therefore warranted., European Union H2020-MSCA-ITN-ETN/0582 ITN TubInTrain to J.F. Diaz is gratefully acknowledged.

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. Significance: 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.