While MYCN-amplified neuroblastoma has been the focus of neuroblastoma research in the past three decades, most human neuroblastomas do not harbour MYCN oncogene amplification, and their tumorigenic factors are unknown. Long non-coding RNAs (lncRNAs) regulate tumorigenesis by modulating the expression of molecular targets, however, there is limited literature on therapeutic targeting of lncRNAs with small molecule compounds. To determine the oncogenic mechanism though which the lncRNA lncNB promotes neuroblastoma cell proliferation, survival and tumour progression; and to identify a small molecule compound that inhibits the interaction between lncNB and its binding protein MSI2 as an effective anticancer strategy. Kaplan Meier analysis showed that high levels of lncNB expression in neuroblastoma tissues correlated with poor prognosis in 476 patients. We identified lncNB as the lncRNA most over-expressed in MYCN non-amplified, compared with MYCN-amplified, neuroblastoma cell lines. lncNB expression was controlled by super-enhancers, and lncNB RNA bound to MSI2 protein. RNA immunoprecipitation and sequencing identified BMX mRNA as the transcript most significantly disrupted from binding to MSI2 protein, after lncNB knockdown. lncNB or MSI2 knockdown reduced, while their over-expression enhanced, BMX mRNA stability and expression, ERK protein phosphorylation and MYCN non-amplified neuroblastoma cell proliferation. lncNB knockdown significantly suppressed neuroblastoma progression in mice. AlphaScreen of a compound library identified NSC617570 as an efficient inhibitor of lncNB RNA and MSI2 protein interaction, and NSC617570 reduced BMX expression, ERK protein phosphorylation, neuroblastoma cell proliferation in vitro and tumor progression in mice. Our study demonstrates that lncNB RNA interacts with MSI2 protein to induce neuroblastoma tumorigenesis, and that targeting lncNB and MSI2 interaction with small molecule compounds is an effective anticancer strategy. Sujanna Mondal, Pei Y. Liu, Janith Seneviratne, Antoine De Weck, Pooja Venkat, Chelsea Mayoh, Jing Wu, Jesper Maag, Jingwei Chen, Matthew Wong, Nenad Bartonicek, Poh Khoo, Lei Jin, Louise E. Ludlow, David S. Ziegler, Toby Trahair, Pieter Mestdagh, Belamy B. Cheung, Jinyan Li, Marcel E. Dinger, Ian Street, Xu D. Zhang, Glenn M. Marshall, Tao Liu. The super enhancer-driven long noncoding RNA lncNB promotes neuroblastoma tumorigenesis by interacting with MSI2 protein and is targetable by small molecule compounds [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2608.

Tumorigenic drivers of MYCN gene nonamplified neuroblastoma remain largely uncharacterized. Long noncoding RNAs (lncRNAs) regulate tumorigenesis, however, there is little literature on therapeutic targeting of lncRNAs with small molecule compounds. Here PRKCQ-AS1 is identified as the lncRNA most overexpressed in MYCN nonamplified, compared with MYCN-amplified, neuroblastoma cell lines. PRKCQ-AS1 expression is controlled by super-enhancers, and PRKCQ-AS1 RNA bound to MSI2 protein. RNA immunoprecipitation and sequencing identified BMX mRNA as the transcript most significantly disrupted from binding to MSI2 protein, after PRKCQ-AS1 knockdown. PRKCQ-AS1 or MSI2 knockdown reduces, while its overexpression enhances, BMX mRNA stability and expression, ERK protein phosphorylation and MYCN nonamplified neuroblastoma cell proliferation. PRKCQ-AS1 knockdown significantly suppresses neuroblastoma progression in mice. In human neuroblastoma tissues, high levels of PRKCQ-AS1 and MSI2 expression correlate with poor patient outcomes, independent of current prognostic markers. AlphaScreen of a compound library identifies NSC617570 as an efficient inhibitor of PRKCQ-AS1 RNA and MSI2 protein interaction, and NSC617570 reduces BMX expression, ERK protein phosphorylation, neuroblastoma cell proliferation in vitro and tumor progression in mice. The study demonstrates that PRKCQ-AS1 RNA interacts with MSI2 protein to induce neuroblastoma tumorigenesis, and that targeting PRKCQ-AS1 and MSI2 interaction with small molecule compounds is an effective anticancer strategy.

MYCN-amplified neuroblastoma (NB) is one of the most aggressive and treatment refractory cancers in children. NB has a prenatal origin, and therefore environmental factors in utero may play a critical role in driving NB tumorigenesis. A recent epidemiological study showed that children born to mothers who gained weight more than 30 kg during pregnancy had a 2-fold higher risk of developing NB. Using the Th-MYCN NB transgenic mouse model, we found that high-fat diet (HFD)-induced maternal obesity resulted in accelerated NB tumorigenesis in the Th-MYCN homozygous offspring. Transcriptomic patterns in tumour tissues from Th-MYCN homozygous offspring of HFD-fed mothers showed upregulation of fatty acid (FA) metabolism and mitochondrial oxidative phosphorylation (OXPHOS) pathways. We then used human NB cell lines exposed to excess FA to study mechanisms and therapeutics. In vitro studies showed that excess FA increased growth, proliferation, and colony formation in human NB cell lines, which was reversed by Fatostatin, a FA metabolism inhibitor, with an effect which was partially MYCN-dependent. Fatostatin showed significant synergy in combination with doxorubicin and vincristine in reversing FA-accelerated growth, proliferation, and colony formation in human NB cell lines, with a wide therapeutic window. ATP5G1 and ATP5G2 (genes encoding human mitochondrial ATP synthase membrane c-subunit) were identified as candidate genes in the HFD-activated OXPHOS pathways. siRNA knockdown of both genes significantly reversed FA-accelerated growth and proliferation in human NB cell lines. Chromatin immunoprecipitation (ChIP)-PCR showed that ATP5G1 and ATP5G2 are transcriptional targets of MYCN, and their transcription is significantly increased in the presence of FA. Further, the overall ATP production was significantly increased in the presence of FA in human NB cell lines, but not in human normal fibroblasts. siRNA knockdown of ATP5G1 and ATP5G2 significantly decreased this effect. Together, our data strongly support a role for OXPHOS and FA metabolism pathways in HFD-accelerated NB tumorigenesis and highlight a key role of the mitochondrial ATP synthase membrane c-subunit genes, ATP5G1 and ATP5G2 in HFD-accelerated NB tumorigenesis. Md Jahidul Hasan, Ritu Mittra, Marie Joseph, Mukesh Raipuria, 1 Nisitha Jayatilleke, Daniel R Carter, Margaret J Morris, Belamy B Cheung, Glenn M Marshall. Therapeutically targeting genes and pathways activated by high-fat diet in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1680.

Neuroblastoma is the most common extracranial solid tumor in children worldwide. We have found that high expression of the RNAMT1 gene in tumor tissues is associated with poor prognosis in both MYCN-amplified [Overall survival (OS): p = 0.0409 and event-free survival (EFS): p = 0.0061] and MYCN non-amplified neuroblastoma patients (OS: p < 0.0001 and EFS: p < 0.0001). Multivariable Cox regression analysis for neuroblastoma patient prognosis of RNAMT1 gene expression [OS: hazard ratio (HR) = 1.92, p = 0.003 and EFS: HR = 1.72, p = 0.0001] is independent from current prognostic markers: MYCN amplification (OS: HR = 3.39, p = 5.1E-8 and EFS: HR = 1.77, p = 0.0001), age older than 18 months (OS: HR = 2.73, p = 2.9E-5 and EFS: HR = 1.59, p = 0.01), and International Neuroblastoma Staging System stages 3&4 (OS: HR = 5.68, p = 5.00E-6 and EFS: HR = 3.00, p = 6.83E-8). Doxycycline (DOX)-inducible RNAMT1 knockdown in MYCN-amplified and MYCN non-amplified neuroblastoma cells decreased cell viability and colony formation capability, and induced apoptosis in vitro. DOX-inducible RNAMT1 knockdown delayed tumor growth and improved survival in mice bearing SK-N-BE(2)-C shRNA1 (OS: p = 0.0034) and SK-N-AS shRNA2 (OS: p < 0.0001). RNAMT1 regulates protein translation based on previous literatures, we demonstrated that RNAMT1 did not affect global mRNA translation alteration via puromycin incorporation assay and slightly affected polysome profile. The subset of mRNA transcripts after RNAMT1 knockdown and RNAMT1 inhibitor treatment will be analyzed with translating ribosome affinity purification sequencing (TRAP-Seq). RNAMT1 inhibitor was discovered by AlphaScreen of 2952 small molecule compounds provided by National Cancer Institute (NCI). Unravelling the molecular mechanisms of RNAMT1 in neuroblastoma tumorigenesis and mechanism of actions of RNAMT1 inhibitor, may provide novel insights to develop a more effective treatment for neuroblastoma patients. Kian Chung Chok, Kenny Ip, Jing Wu, Satyanarayana Gadde, Sujanna Mondal, Justin Wong, Belamy Cheung, Tao Liu. The role of the RNA methyltransferase RNAMT1 as a tumorigenic driver and therapeutic target in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2613.

Deregulated expression of MYC family transcription factors is present in more than 70% of human cancers. MYC is an intrinsically disordered protein without known drug binding pockets and is essential for adult cell replication, making clinical development of MYC inhibitors challenging. MYCN amplification and overexpression generate driver signals in the childhood cancers, neuroblastoma (NB), medulloblastoma (MB) and diffuse intrinsic pontine glioma (DIPG). We previously reported a small molecule compound, SE486-11, which reduced MYCN protein levels in combination with suberoylanilide hydroxamic acid. We developed more potent analogues of SE486-11 with improved single agent potency, selectivity towards cancer cells and solubility (UNSW-SC compounds). Our lead compound, UNSW-SC-22 is highly effective in vitro against MYCN-driven NB and MB cells with sub-micromolar IC50 values and displayed a wide therapeutic index. UNSW-SC-22 displayed MYCN- and c-MYC-dependent cytotoxicity and decreased MYCN and c-MYC protein levels in NB and MB cell lines. MYCN and c-MYC protein half-life was decreased ∼2-fold, while K-48-linked ubiquitination of MYCN protein was increased by UNSW-SC-22. We show UNSW-SC-22 bound to full length MYCN and c-MYC proteins directly, with KDs of 62.7 µM and 60.5 µM, respectively, by surface plasmon resonance. In-cell target engagement was further confirmed by drug affinity responsive target stability and cellular thermal shift assays. Total RNA from NB and MB cells treated with UNSW-SC-22 showed down-regulation of MYC target genes on GSEA analyses. On in-vitro pharmacology profiling against 47 intracellular signalling targets (78 assays) known to cause adverse drug reactions in humans, UNSW-SC-22 demonstrated no significant target activation at <20 µM. UNSW-SC-22 freely crossed the blood-brain barrier, reaching a concentration of 10.8 µM in brain tissue, with a half-life of 1.22 hours. UNSW-SC-22 significantly reduced the tumour burden and improved overall survival of MYCN-driven NB and MB mice. MYCN protein expression was downregulated in tumours collected from mice treated with UNSW-SC-22. Collectively, our data suggest that MYCN and c-MYC are the direct molecular targets of UNSW-SC compounds. These compounds have strong potential to serve as specific targeted therapy to treat MYCN/c-MYC driven paediatric and adult cancers. Satyanarayana Gadde, Sin Wi Ng, Natalie Chung, Qian Wang, Larissa Doughty, Tracey Nero, Maria Tsoli, David Ziegler, Louis Chesler, Michael Parker, Naresh Kumar, Belamy Cheung, Glenn Marshall. Development of MYCN/MYC inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6863.

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.

Abstract Background: Medulloblastoma (MB) is the most prevalent malignant childhood brain tumour. Although prognosis has improved over time, 95% of children with high-risk or relapsed MB eventually succumb to the disease. A key driver gene in high-risk MB is the c-MYC oncogene, for which there are currently no approved inhibitors. Histone deacetylases (HDACs) are transcriptional repressors that are dysregulated in many cancers, making them an attractive therapeutic target. Although several HDAC inhibitors have been FDA-approved for cancer treatment, they have been associated with high toxicity and limited efficacy as single agents. Therefore, simultaneously targeting c-MYC and HDACs may result in synergistic effects that could overcome drug resistance in high-risk MB. Methods and Results: We previously reported a novel pyrido-benzimidazole analogue, SE486-11, which enhanced the therapeutic effect of HDAC inhibitors in MYCN-driven cancers. We recently developed analogues (UNSW-SC compounds) with more potent activity (IC50: 0.017 to 3.70 µM). Here, we showed that these compounds significantly reduced cell viability and induced apoptosis in MB cells. MYC status was a key determinant of sensitivity of UNSW-SC compounds, with more than 3-fold higher cytotoxicity in high MYC compared to low MYC MB cells. The lead compound, UNSW-SC-22, was shown to reduce MYC protein expression and was found to be highly synergistic in enhancing the efficacy of HDAC inhibitors in MB cells. Most importantly, we demonstrated that UNSW-SC-22 was able to freely cross the blood brain barrier, reaching a concentration of 10.8 μM, with a half-life of 1.22 hours. UNSW-SC-22 directly bound to c-MYC and MYCN proteins at low micromolar equilibrium dissociation constant (KD) values which was demonstrated by surface plasmon resonance assay. In a MYCN-driven MB mouse model, we found that UNSW-SC-22 as a single agent at 90 mg/kg decreased tumor growth and prolonged survival. Through RNA-sequencing, we identified 512 differentially expressed genes after in vitro treatment with UNSW-SC-22. Importantly, GSEA analysis revealed MYC targets and cell cycle pathways as highly downregulated pathways, and upregulation in the p53 pathway. Several genes, such as MELK and USP1 were validated as down-stream targets of UNSW-SC-22. As UNSW-SC-22 is a first-in-class anticancer drug, which we are currently testing extensively in other MYC-driven MB mice models. Conclusion: Collectively, our data strongly suggest that c-Myc and MYCN are the molecular targets of UNSW-SC compounds, and these compounds have strong potential to serve as specific targeted therapy to treat subgroup 3 and 4 of MB patients with c-Myc and MYCN overexpression and amplification. Citation Format: Sin Wi Ng, Satyanarayana Gadde, Qian Wang, Belamy B. Cheung, Glenn M. Marshall. A novel small molecule inhibitor targeting MYC oncogenic signaling as an enhancer of HDAC inhibitors for the treatment of high-risk medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4599.

MYCN amplification predicts poor prognosis in childhood neuroblastoma. To identify MYCN oncogenic signal dependencies we performed N-ethyl-N-nitrosourea (ENU) mutagenesis on the germline of neuroblastoma-prone TH-MYCN transgenic mice to generate founders which had lost tumorigenesis. Sequencing of the mutant mouse genomes identified the Ring Finger Protein 121 (RNF121WT) gene mutated to RNFM158R associated with heritable loss of tumorigenicity. While the RNF121WT protein localised predominantly to the cis-Golgi Complex, the RNF121M158R mutation in Helix 4 of its transmembrane domain caused reduced RNF121 protein stability and absent Golgi localisation. RNF121WT expression markedly increased during TH-MYCN tumorigenesis, whereas hemizygous RNF121WT gene deletion reduced TH-MYCN tumorigenicity. The RNF121WT-enhanced growth of MYCN-amplified neuroblastoma cells depended on RNF121WT transmembrane Helix 5. RNF121WT directly bound MYCN protein and enhanced its stability. High RNF121 mRNA expression associated with poor prognosis in human neuroblastoma tissues and another MYC-driven malignancy, laryngeal cancer. RNF121 is thus an essential oncogenic cofactor for MYCN and a target for drug development. A chemical mutagenesis screen identifies RNF121 as an oncogenic cofactor for MYCN in neuroblastoma.

The overall prognosis of acute myeloid leukemia (AML) remains dismal, largely because of the inability of current therapies to kill leukemia stem cells (LSCs) with intrinsic resistance. Loss of the stress sensor growth arrest and DNA damage-inducible 45 alpha (GADD45A) is implicated in poor clinical outcomes, but its role in LSCs and AML pathogenesis is unknown. Here, we define GADD45A as a key downstream target of G protein-coupled receptor (LGR)4 pathway and discover a regulatory role for GADD45A loss in promoting leukemia-initiating activity and oxidative resistance in LGR4/HOXA9-dependent AML, a poor prognosis subset of leukemia. Knockout of GADD45A enhances AML progression in murine and patient-derived xenograft (PDX) mouse models. Deletion of GADD45A induces substantial mutations, increases LSC self-renewal and stemness in vivo, and reduces levels of reactive oxygen species (ROS), accompanied by a decreased response to ROS-associated genotoxic agents (eg, ferroptosis inducer RSL3) and acquisition of an increasingly aggressive phenotype on serial transplantation in mice. Our single-cell cellular indexing of transcriptomes and epitopes by sequencing analysis on patient-derived LSCs in PDX mice and subsequent functional studies in murine LSCs and primary AML patient cells show that loss of GADD45A is associated with resistance to ferroptosis (an iron-dependent oxidative cell death caused by ROS accumulation) through aberrant activation of antioxidant pathways related to iron and ROS detoxification, such as FTH1 and PRDX1, upregulation of which correlates with unfavorable outcomes in patients with AML. These results reveal a therapy resistance mechanism contributing to poor prognosis and support a role for GADD45A loss as a critical step for leukemia-initiating activity and as a target to overcome resistance in aggressive leukemia.

BACKGROUND:The child cancer, neuroblastoma (NB), is characterised by a low incidence of mutations and strong oncogenic embryonal driver signals. Many new targeted epigenetic modifier drugs have failed in human trials as monotherapy. METHODS:We performed a high-throughput, combination chromatin-modifier drug screen against NB cells. We screened 13 drug candidates in 78 unique combinations. RESULTS:We found that the combination of two histone methyltransferase (HMT) inhibitors: GSK343, targeting EZH2, and SGC0946, targeting DOT1L, demonstrated the strongest synergy across 8 NB cell lines, with low normal fibroblast toxicity. High mRNA expression of both EZH2 and DOT1L in NB tumour samples correlated with the poorest patient survival. Combination HMT inhibitor treatment caused activation of ATF4-mediated endoplasmic reticulum (ER) stress responses. In addition, glutathione and several amino acids were depleted by HMT inhibitor combination on mass spectrometry analysis. The combination of SGC0946 and GSK343 reduced tumour growth in comparison to single agents. CONCLUSION:Our results support further investigation of HMT inhibitor combinations as a therapeutic approach in NB.

Many of the pro-tumorigenic functions of the oncogene MYCN are attributed to its regulation of global gene expression programs. Alternative splicing is another important regulator of gene expression and has been implicated in neuroblastoma development, however, the molecular mechanisms remain unknown. We found that MYCN up-regulated the expression of the core spliceosomal protein, SNRPD3, in models of neuroblastoma initiation and progression. High mRNA expression of SNRPD3 in human neuroblastoma tissues was a strong, independent prognostic factor for poor patient outcome. Repression of SNRPD3 expression correlated with loss of colony formation in vitro and reduced tumorigenicity in vivo. The effect of SNRPD3 on cell viability was in part dependent on MYCN as an oncogenic co-factor. RNA-sequencing revealed a global increase in the number of genes being differentially spliced when MYCN was overexpressed. Surprisingly, depletion of SNRPD3 in the presence of overexpressed MYCN further increased differential splicing, particularly of cell cycle regulators, such as BIRC5 and CDK10. MYCN directly bound SNRPD3, and the protein arginine methyltransferase, PRMT5, consequently increasing SNRPD3 methylation. Indeed, the PRMT5 inhibitor, JNJ-64619178, reduced cell viability and SNRPD3 methylation in neuroblastoma cells with high SNRPD3 and MYCN expression. Our findings demonstrate a functional relationship between MYCN and SNRPD3, which maintains the fidelity of MYCN-driven alternative splicing in the narrow range required for neuroblastoma cell growth. SNRPD3 methylation and its protein-protein interface with MYCN represent novel therapeutic targets. Hypothetical model for SNRPD3 as a co-factor for MYCN oncogenesis. SNRPD3 and MYCN participate in a regulatory loop to balance splicing fidelity in neuroblastoma cells. First MYCN transactivates SNRPD3 to lead to high-level expression. Second, SNRPD3 and MYCN form a protein complex involving PRMT5. Third, this leads to balanced alterative splicing (AS) activitiy that is favorable to neuroblastoma. Together this forms as a therapeutic vulnerability where SNRPD3 perturbation or PRMT5 inhibitors are selectively toxic to neuroblastoma by conditionally disturbing splicing activity.

Abstract Ewing’s sarcoma (EWS) is an aggressive bone and soft-tissue cancer, characterized by a hallmark fusion oncoprotein comprising the amino terminus of a FET gene and the carboxy terminus of an ETS gene, most commonly (85%), the EWSR1-FLI1 t (11,22) fusion.TK-216 is a first-in-class EWSR1-FLI1 inhibitor currently in phase II clinical trials for EWS. Although TK-216 was designed to target the fusion protein interaction directly, it has demonstrated limited efficacy as a monotherapy. Thus, more effective TK-216 combination therapies are needed. We have used genome wide Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Knockout screening in EWS cells treated with TK-216 to identify novel synthetic lethality. We have identified 97 genes whose deletion potentially enhances the action of TK-216 in EWS cells. We have also identified several druggable targets, such as USP9X and the oxidative phosphorylation (OXPHOS) pathway as synthetic lethal interactions for TK-216. USP9X is known to deubiquitinate and stabilize the EWS-FLI1 fusion protein. USP9X and EWS-FLI1 protein expression levels were found to be positively correlated in EWS clinical specimens. USP9X siRNA-mediated gene silencing reduced EWS cell viability. Most importantly, USP9X siRNA-mediated gene silencing sensitized EWS cells to TK-216 treatment. In addition, inhibition of USP9X with a small-molecule inhibitor WP1130, led to significant cell death in EWS alone and in combination with TK-216 with wide therapeutic indices. The molecular mechanism of synergy and in vivo efficacy of the novel TK-216 combination will be determined in our study for future clinical translation. Citation Format: Daenikka Ravindrarajah, Sukriti Krishan, Kayleen Simpson, Twishi Gulati, Belamy B. Cheung, Glenn M. Marshall. Using genome-wide CRISPR screening to develop novel combination therapies for high-risk Ewing’s sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1080.

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.

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.

Supplemental data 13: Summary of Gene Set Enrichment analysis results obtained on expression profiling data of CLB-­â€GA on different time points (10' - 30'- 60' - 2h - 4h - 6h) after pharmacological inhibition using TAE-­â€684.

Supplementary Figure S7. OTX015 and carfilzomib synergistically improve mouse survival in a PDX model of TERT-rearranged neuroblastoma.

<p>mRNA and miRNA expression in 1 week, 2 week and 6 week wildtype or TH-MYCN+/+ ganglia or tumors</p>

<p>Supplementary Table 2-1 All miRNA-mRNA interactions detected for network presented in Figure 2A, Supplementary Table 2-2 Expression of miRNAs in human adrenal neuroblasts and MYCN-amplified tumours</p>

Supplementary Figure S5. BET bromodomain inhibitors and proteasome inhibitors exert synergistic anticancer effects against TERT-rearranged neuroblastoma cells.

The MYCN oncogene and histone deacetylases (HDACs) are key driver genes in the childhood cancer, neuroblastoma. We recently described a novel pyridobenzimidazole analogue, SE486-11, which enhanced the therapeutic effectiveness of HDAC inhibitors by increasing MYCN ubiquitination through effects on the deubiquitinase, ubiquitin-specific protease 5 (USP5). Here we describe the synthesis of a novel series of pyrimido[1,2-a]benzimidazole derivatives, and an evaluation of their cytopathic effects against non-malignant and human neuroblastoma cell lines. Among the tested analogues, 4-(4-methoxyphenyl)benzo[4,5]imidazo[1,2-a]pyrimidine (3a) was the most active compound against neuroblastoma cells (IC50 ≤ 2 µM), with low cytotoxicity (IC50 ≥ 15 µM) to normal cells. We show compound 3a bound to USP5 protein (Kd = 0.47 µM) in vitro and synergistically enhanced the efficacy of HDAC inhibitors against neuroblastoma cells. Moreover, knockdown of USP5 and MYCN in treated neuroblastoma cells showed that both USP5 and MYCN expression was necessary for the cytopathic activity of compound 3a, thus providing a clinically relevant rationale for further development of this of pyrimido[1,2-a]benzimidazole.