Abstract It was recently shown that inhibition of polo-like kinase 4 (PLK4) induces synthetic lethality in cancers with chromosome 17q-encoded TRIM37 copy number gain due to cooperative regulation of centriole duplication and mitotic spindle nucleation. We show here that chromosome 17q/TRIM37 gain is a defining feature of high-risk neuroblastoma and renders patient-derived cell lines hypersensitive to the novel PLK4 inhibitor RP-1664. We demonstrate that centriole amplification at low doses of RP-1664 contributes to this sensitivity in a TRIM37 -independent fashion. CRISPR screens and live cell imaging reveal that upon centriole amplification, neuroblastoma cells succumb to multipolar mitoses due to an inability to cluster or inactivate supernumerary centrosomes. RP-1664 monotherapy showed robust anti-tumor activity in 14/15 human neuroblastoma-derived xenograft models, and significantly extended survival in a transgenic MYCN -driven murine model of neuroblastoma. RP-1664 combined with GD2-directed chemoimmunotherapy resulted in maintained complete responses in 6/9 mice with established MYCN -driven murine neuroblastomas. These data support clinical development of PLK4 inhibitors for high-risk neuroblastoma and other cancers with somatically acquired TRIM37 overexpression.

Neuroblastoma is the most common extracranial solid tumor in children. Survival rates of children with high-risk disease are ∼50%, reducing to 10% for relapsed/refractory disease, highlighting the need for more effective treatments. Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase, highly expressed in cancers including neuroblastoma. In addition to its well-established role in driving cell growth and proliferation, there is increasing evidence suggesting a role in supporting tumor immune evasion. 9-ING-41 (Elraglusib), a GSK-3β inhibitor with clinical activity in adult cancers, is effective as a single agent in neuroblastoma animal models, and is currently in Phase 1/2 clinical trial in pediatric patients with refractory malignancies (NCT04239092). However, the efficacy of 9-ING-41 in combination with current clinically relevant chemotherapy and chemoimmunotherapy neuroblastoma protocols that include anti-GD2 antibody for high-risk patients, has not been established. To establish, in the Th-MYCN mouse model of neuroblastoma, clinically relevant chemoimmunotherapy treatment protocols that include anti-GD2 antibody (14G2a), and to evaluate whether and how 9-ING-41 enhances their efficacy. METHODS: Efficacy and pharmacokinetic studies were performed in Th-MYCN mice. Th-MYCN tumor cells were allografted into fully immunocompetent versus immunocompromised mouse strains. Th-MYCN mice relapsed within 60 days following treatment with 2 consecutive daily doses of temozolomide/irinotecan (TEMIRI) or cyclophosphamide/topotecan (CYCLO/TOPO), in addition to 15µg 14G2a on day 1 and day 5. Pharmacokinetics of radiolabelled 14G2a confirmed that this regimen resulted in equivalent 14G2a serum levels in tumor-bearing Th-MYCN mice as observed for Dinutuximab in neuroblastoma patients. Addition of 9-ING-41 to TEMIRI or 14G2a alone, or combined TEMIRI/14G2a, delayed tumor growth in a dose-dependent manner, resulting in significantly extended survival compared to mice receiving the same treatments without 9-ING-41. 6/10 mice treated with TEMIRI/14G2a/70 mg/kg 9-ING-41 were long-term tumor-free survivors at 1 year of age (P<0.0001). Similar results were obtained for CYCLO/TOPO. TEMIRI/9-ING-41 was significantly more effective against Th-MYCN allografts injected into wild-type littermates than when the same cells were injected into immunocompromised NSG mice, suggesting that a fully functional immune system is required to achieve the highest efficacy of this combination. Together with the observation that 9-ING-41 alone enhanced the efficacy of anti-GD2 antibody, this data also suggests that 9-ING-41 exerts part of its potentiating role through boosting anti-tumour immunity. 9-ING-41 in combination with chemoimmunotherapy is highly potent, resulting in long-term tumor-free survival in the Th-MYCN mouse model of neuroblastoma. Our data suggests that 9-ING-41 at least in part works through modulating anti-tumour immunity, and we are currently investigating this further. Jayne Murray, Klaartje Somers, Crystal Mak, Stephanie Alfred, Jennifer Brand, Evon Poon, Nicholas Fletcher, Pei Li, Kristofer Thurecht, Juliet Gray, Andrew DJ. Pearson, Louis Chesler, Michael D. Hogarty, Francis Giles, Jamie Fletcher, David S. Ziegler, Murray D. Norris, Michelle Haber. The glycogen synthase kinase-3β inhibitor 9-ING-41 in combination with chemoimmunotherapy provides long-term survival in the Th-MYCN mouse model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B079.

Dysregulated amino acid metabolism creates cancer-specific vulnerabilities. Neuroblastoma tumors have dysregulated arginine metabolism that renders them sensitive to systemic arginine deprivation. Arginase therapy has been proposed as a therapeutic approach for neuroblastoma treatment and has a favorable safety profile in pediatric cancer patients, however optimal therapeutic combinations remain unexplored. The anti-tumor effects of BCT-100, a pegylated human arginase, were studied in neuroblastoma cell models by metabolite profiling, proteomics, and viability, clonogenicity, and protein translation assays. BCT-100 efficacy was assessed in the Th-MYCN transgenic neuroblastoma mouse model and in neuroblastoma cell line and patient-derived xenograft models. In vitro, depletion of arginine by BCT-100 arrested protein translation and cellular proliferation, with effects on clonogenicity enhanced in combination with standard-of-care chemotherapeutics SN-38/temozolomide and mafosfamide/topotecan. In vivo, BCT-100 treatment spared liver arginine while significantly depleting plasma and tumor arginine in Th-MYCN mice, and extended tumor latency (> 100 vs. 45.5 days) in mice pre-emptively treated at weaning. In mice with established tumors, BCT-100 prolonged tumor progression delay when combined with standard-of-care chemo- (> 90 vs. 25 days) or chemo-immuno-therapy (49.5 vs. 35.5 days). Tumor progression delay was also observed in cell line and patient-derived xenografts with BCT-100 treatment, including relapsed/refractory disease models. No increased toxicity was observed with the addition of BCT-100 to established therapies. The arginase BCT-100 profoundly disrupts neuroblastoma growth in vitro and in vivo, an effect enhanced in combination with standard-of-care chemo-immuno-therapy. Our data supports further assessment of arginine-depleting combination therapies as a new treatment strategy for neuroblastoma.

It was recently shown that inhibition of polo-like kinase 4 (PLK4) induces TP53-dependent synthetic lethality in cancers with chromosome 17q-encoded TRIM37 copy number gain due to cooperative regulation of centriole duplication and mitotic spindle nucleation. We show here that chromosome 17q/TRIM37 gain is a pathognomonic feature of high-risk neuroblastoma and renders patient-derived cell lines hypersensitive to the novel PLK4 inhibitor RP-1664. We demonstrate that centriole amplification at low doses of RP-1664 contributes to this sensitivity in a TRIM37- and TP53-independent fashion. CRISPR screens and live cell imaging reveal that upon centriole amplification, neuroblastoma cells succumb to multipolar mitoses due to an inability to cluster or inactivate supernumerary centrosomes. RP-1664 showed robust anti-tumor activity in 14/15 neuroblastoma xenograft models and significantly extended survival in a transgenic murine neuroblastoma model. These data support biomarker-directed clinical development of PLK4 inhibitors for high-risk neuroblastoma and other cancers with somatically acquired TRIM37 overexpression.

Despite Myc oncoproteins being major causal factors in human cancer, they remain "undruggable." The MYCN oncogene is one of the most powerful prognostic markers for the childhood cancer neuroblastoma and represents an important target for developing novel therapeutics. Here, we report the finding and characterization of M606, a selective small molecule inhibitor of MYCN, which was identified by screening a diverse chemical library. M606 reduced MYCN protein levels in neuroblastoma cell lines and upregulated hypoxia-inducible factor 1 alpha (HIF1A). Using siRNA-mediated knockdown of MYCN, c-Myc, or HIF1A in HepG2 and BE(2)-C cells followed by M606 treatment, we demonstrated that Myc downregulation and HIF1A upregulation were two independent effects of M606 treatment. M606 selectively targeted neuroblastoma cell lines expressing higher levels of MYCN protein and delayed neuroblastoma development in the TH-MYCN transgenic mouse model. Metabolomic analysis showed that M606 modulated glucose metabolism, consistent with a hypoxic response and iron deprivation. Biochemical characterization of M606 not only confirmed its iron-chelating properties but also revealed its ability to downregulate MYCN promoter activity, which could be rescued by the addition of iron. Luciferase assays identified the minimal MYCN promoter region required for the M606 response, which contained overlapping E2F transcription factor binding sites. Further evaluation defined a key role for E2F3 in the M606-mediated response. The finding of a potent cell-permeable iron chelator that can chelate iron to directly downregulate MYCN transcription via an E2F3-mediated response represents a potentially valuable therapeutic approach in the treatment of cancers overexpressing Myc oncoproteins.

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.

MYCN oncogene amplification is frequently observed in aggressive childhood neuroblastoma. Using an unbiased large-scale mutagenesis screen in neuroblastoma-prone transgenic mice, we identify a single germline point mutation in the transcriptional corepressor Runx1t1, which abolishes MYCN-driven tumorigenesis. This loss-of-function mutation disrupts a highly conserved zinc finger domain within Runx1t1. Deletion of one Runx1t1 allele in an independent Runx1t1 knockout mouse model is also sufficient to prevent MYCN-driven neuroblastoma development, and reverse ganglia hyperplasia, a known pre-requisite for tumorigenesis. Silencing RUNX1T1 in human neuroblastoma cells decreases colony formation in vitro, and inhibits tumor growth in vivo. Moreover, RUNX1T1 knockdown inhibits the viability of PAX3-FOXO1 fusion-driven rhabdomyosarcoma and MYC-driven small cell lung cancer cells. Despite the role of Runx1t1 in MYCN-driven tumorigenesis neither gene directly regulates the other. We show RUNX1T1 forms part of a transcriptional LSD1-CoREST3-HDAC repressive complex recruited by HAND2 to enhancer regions to regulate chromatin accessibility and cell-fate pathway genes.

Anti-disialoganglioside (GD2) antibody therapy has provided clinical benefit to patients with neuroblastoma however efficacy is likely impaired by the immunosuppressive tumor microenvironment. We have previously defined a link between intratumoral copper levels and immune evasion. Here, we report that adjuvant copper chelation potentiates anti-GD2 antibody therapy to confer durable tumor control in immunocompetent models of neuroblastoma. Mechanistic studies reveal copper chelation creates an immune-primed tumor microenvironment through enhanced infiltration and activity of Fc-receptor-bearing cells, specifically neutrophils which are emerging as key effectors of antibody therapy. Moreover, we report copper sequestration by neuroblastoma attenuates neutrophil function which can be successfully reversed using copper chelation to increase pro-inflammatory effector functions. Importantly, we repurpose the clinically approved copper chelating agent Cuprior as a non-toxic, efficacious immunomodulatory strategy. Collectively, our findings provide evidence for the clinical testing of Cuprior as an adjuvant to enhance the activity of anti-GD2 antibody therapy and improve outcomes for patients with neuroblastoma.

Abstract Neuroblastoma is the most frequent extra-cranial pediatric cancer with high-risk disease possessing a dismal five-year survival rate <50%. Antibody-based therapies targeting the surface disialoganglioside GD2 have shown promising anti-tumor activity in patients however efficacy is strongly hampered by immunosuppressive mechanisms present in the tumor microenvironment. Neuroblastomas typically feature low mutational burdens, limited major histocompatibility complex class I expression, increased immune checkpoint markers, sparse immune infiltration, and soluble mediators which drastically limit anti-tumor immunity. Given the emerging link between elevated copper levels and tumoral immune evasion, we explored copper chelation as a therapeutic strategy using the agents tetraethylenepentamine (TEPA) and the clinically approved analogue triethylenetetramine (TETA). Using the immunocompetent TH-MYCN model, we performed single-cell RNA sequencing supported by OPAL multiplex immunohistochemistry and cytokine profiling of resected tumors to assess cellular and molecular changes occurring after one week of TEPA treatment. Therapy was observed to successfully deplete intratumoral copper and reinvigorate anti-tumor immunity with increased infiltration and activity of pro-inflammatory immune cells, and was specifically found to induce egress and N1-polarisation of neutrophils. Further mechanistic studies in vitro revealed the sequestration of copper by neuroblastoma cells attenuated neutrophil function, which could be successfully reversed upon TEPA treatment. Findings propose a novel mechanism of immune evasion, highlighting copper chelation as a therapeutic strategy to counteract immunosuppression. Using the TH-MYCN model, TEPA was employed as a priming agent to effectively potentiate anti-GD2 antibody therapy and achieve durable tumor control. Importantly, this was associated with increased Fc-receptor-bearing natural killer and myeloid cells, which elicit tumor clearance via antibody-dependent cellular cytotoxicity. Currently indicated for the treatment of copper overload disorder Wilson’s disease, TETA was evaluated for potential repurposing as a novel immunomodulatory agent. Using the syngeneic NXS2 model, TETA exhibited an exceptional safety profile and combination therapy achieved durable tumor control with no relapses occurring after treatment cessation. This result was similarly associated with the infiltration of pro-inflammatory immune cells, including N1 neutrophils. Collectively, this study credentials copper chelation as a non-toxic strategy to disrupt the immunosuppressive tumor microenvironment and reinvigorate anti-tumour immunity. Findings provide crucial translational evidence for repurposing TETA to potentiate anti-GD2 antibody therapy and improve neuroblastoma patient responses. Citation Format: Jourdin R. Rouaen, Antonella Salerno, Fabio Luciani, Jayne Murray, Tyler Shai-Hee, Nicodemus Tedla, Michelle Haber, Toby N. Trahair, Orazio Vittorio. Copper chelation overcomes the immunosuppressive neuroblastoma microenvironment to potentiate anti-GD2 antibody therapy [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 2673.

Text file of Figure Legends to Supplementary data

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.

Copper lowering drugs increased NK-mediated cell death in vitro

TEPA increases tumor-infiltrating immune cells in Th-MYCN mice

Supplementary Figure Legends 1-3 from ODC1 Is a Critical Determinant of MYCN Oncogenesis and a Therapeutic Target in Neuroblastoma

Supporting data; Supplementary Figure 6:Nucleolar dispersal of rpl5

Supplementary Table 1: siRNA sequences Supplementary Table 2: qRT-PCR primers Supplementary Table 3: Primers for chromatin immunoprecipitation Supplementary Table 4: Multivariate analysis of event free survival and overall survival of neuroblastoma patients

Supplementary Materials and Methods CLEAN version

Supplementary Figure 1 from ODC1 Is a Critical Determinant of MYCN Oncogenesis and a Therapeutic Target in Neuroblastoma