High-risk Neuroblastoma Cells Research Articles

CDK4/6 inhibitors display a class effect in inducing differentiation of neuroblastoma cells

Background Neuroblastoma is the most common extracranial solid tumour in infants and children, accounting for approximately 15% of paediatric cancer mortality. These tumours are unique in that a subset, namely stage MS, frequently undergo spontaneous regression or differentiation. Differentiation therapy, where cancer cells are re-routed back down their correct developmental pathway, is therefore a promising therapeutic avenue. We have previously shown that the CDK4/6 inhibitor palbociclib induces both decreased proliferation and enhanced neuronal differentiation of neuroblastoma cells in vitro. When combined with retinoic acid, already used clinically for maintenance therapy, this differentiation is enhanced. Methods Here, we investigate two additional CDK4/6 inhibitors, abemaciclib and ribociclib, to induce differentiation of the relapsed, high-risk MYCN-amplified neuroblastoma cell line SK-N-BE(2)C, with and without retinoic acid. We culture SK-N-BE(2)C cells in both adherent and three-dimensional culture and monitor proliferation and differentiation using readouts including live-imaging, immunocytochemistry, qRT-PCR and EdU incorporation. Results We find the CDK4/6 inhibitors palbociclib, abemaciclib and ribociclib all enhance retinoic acid-induced differentiation in both adherent SK-N-BE(2)C cells and 3D spheroids. Conclusions CDK4/6 inhibitors display a class effect in inducing neuronal differentiation together with retinoic acid, both in adherent neuroblastoma cell lines and three-dimensional tumour spheroids. This is an important consideration for potentially developing CDK inhibitor-induced differentiation as a therapy in the clinic.

Inferring cancer type-specific patterns of metastatic spread.

The metastatic spread of a cancer can be reconstructed from DNA sequencing of primary and metastatic tumours, but doing so requires solving a challenging combinatorial optimization problem. This problem often has multiple solutions that cannot be distinguished based on current maximum parsimony principles alone. Current algorithms use ad hoc criteria to select among these solutions, and decide, a priori, what patterns of metastatic spread are more likely, which is itself a key question posed by studies of metastasis seeking to use these tools. Here we introduce Metient, a freely available open-source tool which proposes multiple possible hypotheses of metastatic spread in a cohort of patients and rescores these hypotheses using independent data on genetic distance of metastasizing clones and organotropism. Metient is more accurate and is up to 50x faster than current state-of-the-art. Given a cohort of patients, Metient can calibrate its parsimony criteria, thereby identifying shared patterns of metastatic dissemination in the cohort. Reanalyzing metastasis in 169 patients based on 490 tumors, Metient automatically identifies cancer type-specific trends of metastatic dissemination in melanoma, high-risk neuroblastoma and non-small cell lung cancer. Metient's reconstructions usually agree with semi-manual expert analysis, however, in many patients, Metient identifies more plausible migration histories than experts, and further finds that polyclonal seeding of metastases is more common than previously reported. By removing the need for hard constraints on what patterns of metastatic spread are most likely, Metient introduces a way to further our understanding of cancer type-specific metastatic spread.

Abstract 5124: A CRISPR-drug perturbational map for identifying compounds to combine with commonly used chemotherapeutics

Abstract Combination chemotherapy is crucial for successfully treating cancer. However, the enormous number of possible drug combinations means discovering safe and effective combinations remains a significant challenge. To improve this process, we conduct large-scale targeted CRISPR knockout screens in drug-treated cells, creating a genetic map of druggable genes that sensitize cells to commonly used chemotherapeutics. We prioritize neuroblastoma, the most common extracranial pediatric solid tumor, where ~50% of high-risk patients do not survive. Our screen examines all druggable gene knockouts in 18 cell lines (10 neuroblastoma, 8 others) treated with 8 widely used drugs, resulting in 94,320 unique combination-cell line perturbations, which is comparable to the largest existing drug combination screens. Using dense drug-drug rescreening, we find that the top CRISPR-nominated drug combinations are more synergistic than standard-of-care combinations, suggesting existing combinations could be improved. As proof of principle, we discover that inhibition of PRKDC, a component of the non-homologous end-joining pathway, sensitizes high-risk neuroblastoma cells to the standard-of-care drug doxorubicin in vitro and in vivo using patient-derived xenograft (PDX) models. Our findings provide a valuable resource and demonstrate the feasibility of using targeted CRISPR knockout to discover combinations with common chemotherapeutics, a methodology with application across all cancers. Citation Format: William C. Wright, Paul Geeleher. A CRISPR-drug perturbational map for identifying compounds to combine with commonly used chemotherapeutics [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 5124.

Targeted Degradation of XIAP is Sufficient and Specific to Induce Apoptosis in MYCN-overexpressing High-risk Neuroblastoma.

XIAP degradation is sufficient to kill MYCN-amplified neuroblastoma which overexpresses and relies on XIAP as a brake against cell death, without affecting normal cells.

A CRISPR-drug perturbational map for identifying compounds to combine with commonly used chemotherapeutics

Combination chemotherapy is crucial for successfully treating cancer. However, the enormous number of possible drug combinations means discovering safe and effective combinations remains a significant challenge. To improve this process, we conduct large-scale targeted CRISPR knockout screens in drug-treated cells, creating a genetic map of druggable genes that sensitize cells to commonly used chemotherapeutics. We prioritize neuroblastoma, the most common extracranial pediatric solid tumor, where ~50% of high-risk patients do not survive. Our screen examines all druggable gene knockouts in 18 cell lines (10 neuroblastoma, 8 others) treated with 8 widely used drugs, resulting in 94,320 unique combination-cell line perturbations, which is comparable to the largest existing drug combination screens. Using dense drug-drug rescreening, we find that the top CRISPR-nominated drug combinations are more synergistic than standard-of-care combinations, suggesting existing combinations could be improved. As proof of principle, we discover that inhibition of PRKDC, a component of the non-homologous end-joining pathway, sensitizes high-risk neuroblastoma cells to the standard-of-care drug doxorubicin in vitro and in vivo using patient-derived xenograft (PDX) models. Our findings provide a valuable resource and demonstrate the feasibility of using targeted CRISPR knockout to discover combinations with common chemotherapeutics, a methodology with application across all cancers.

TAF1D promotes proliferation by transcriptionally activating G2/M phase-related genes in MYCN-amplified neuroblastoma.

High-risk neuroblastoma (HR-NB) is an aggressive childhood cancer that responds poorly to currently available therapies and is associated with only about a 50% 5-year survival rate. MYCN amplification is a critical driver of these aggressive tumors, but so far there have not been any approved treatments to effectively treat HR-NB by targeting MYCN or its downstream effectors. Thus, the identification of novel molecular targets and therapeutic strategies to treat children diagnosed with HR-NB represents an urgent unmet medical need. Here, we conducted a targeted siRNA screening and identified TATA box-binding protein-associated factor RNA polymerase I subunit D, TAF1D, as a critical regulator of the cell cycle and proliferation in HR-NB cells. Analysis of three independent primary NB cohorts determined that high TAF1D expression correlated with MYCN-amplified, high-risk disease and poor clinical outcomes. TAF1D knockdown more robustly inhibited cell proliferation in MYCN-amplified NB cells compared with MYCN-non-amplified NB cells, as well as suppressed colony formation and inhibited tumor growth in a xenograft mouse model of MYCN-amplified NB. RNA-seq analysis revealed that TAF1D knockdown downregulates the expression of genes associated with the G2/M transition, including the master cell-cycle regulator, cell-cycle-dependent kinase 1 (CDK1), resulting in cell-cycle arrest at G2/M. Our findings demonstrate that TAF1D is a key oncogenic regulator of MYCN-amplified HR-NB and suggest that therapeutic targeting of TAF1D may be a viable strategy to treat HR-NB patients by blocking cell-cycle progression and the proliferation of tumor cells.

Abstract 481: Investigating molecular glues as a new therapeutic approach to high-risk neuroblastoma

Abstract High-risk neuroblastoma (NB) is the most common extracranial solid tumor of childhood. Despite extensive study, treatments remain highly toxic and are often insufficient to cure. Thus, there is an urgent need to develop more effective and less toxic treatments for children with NB. NB commonly displays reliance on a relatively small cohort of transcription factors (TFs) and epigenetic proteins to drive cell growth, however, many of these targets are inaccessible with conventional small molecules. In recent years, new modalities such as targeted protein degradation (TPD) have emerged. TPD may permit small molecule degradation of traditionally “undruggable” targets, such as TFs. This makes TPD a particularly attractive modality for defining new therapeutic strategies for high-risk NB. One category of TPD agents are molecular glues (MGs). MGs are monovalent small molecules capable of simultaneously binding to an E3 ligase receptor such as CRBN (cereblon), and a target protein of interest. Importantly, in comparison to conventional protein inhibitors, MGs do not require a conventional binding pocket on the protein of interest to bind to it, and can be utilized in sub-stoichiometric concentrations. Thus, this may result in the recruitment, polyubiquitination, and subsequent proteasomal degradation of proteins previously thought to be “undruggable.” Here, we demonstrate that high-risk NB cells are critically sensitive to a cluster of previously unknown MGs. We performed high-throughput screening of a proprietary library (>4000 unique compounds) of MGs in two MYCN-amplified NB cell lines, Kelly and MHHNB11. These two cell lines demonstrate a strikingly close correlation in Cell-Titer Glo-based growth response to MGs, as compared with an outgroup of other MG-responsive cancer cell lines. We identified a unique subcluster of 112 common MGs that display >70% growth inhibition in both cell lines. To identify the mechanism of cell growth inhibition, we established Kelly cells with stable knockout of CRBN, and coupled these with screening to confirm that the majority of these MGs (84%; 94/112) require CRBN expression. To rule out common off-target effects of MGs, candidate compounds were then tested in cells transgenically expressing either wild-type or degradation-resistant GSPT1 (G575N) constructs, revealing a subset of MGs retaining cytotoxicity without effecting GSPT1 protein levels. Current studies are focused on determining the targets of these high confidence MGs, as a foundation for preclinical in vivo studies. These studies will identify new compounds with defined mechanisms of action, with an ultimate goal of developing new treatments that are more effective and less toxic for children with NB. Citation Format: Ian Delahunty, Gisele Nishiguchi, Yang Zhang, Abigail Fish, Anand Mayasundari, Jun Qi, Brian Abraham, Anang Shelat, Zoran Rankovic, Adam Durbin. Investigating molecular glues as a new therapeutic approach to high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 481.

Serine Hydroxyl-Methyltransferase 2 Inhibition Induces Differentiation and Impairs Tumorigenesis in High-Risk Neuroblastoma Cells

INTRODUCTION: High-risk neuroblastoma (NB) is associated with SHMT2 (serine hydroxyl-methyltransferase 2) expression, which is upregulated in several cancers and is associated with tumor aggressiveness. We have previously shown that SHMT2 silencing impairs cellular proliferation, migration, and colony formation, and treatment with a SHMT2 inhibitor induces differentiation in vitro. The stem cell marker genes CD133, CKIT, and NOTCH1 have been shown to downregulate differentiation in NB. We hypothesize that treatment with SHIN2, a novel SHMT2 inhibitor, will induce differentiation and impair tumorigenesis via downregulation of CD133, CKIT, and NOTCH1. METHODS: Human NB cell lines, BE(2)-C and SK-N-SH, were used for the study. Cells were treated with SHIN2 for 24 hours and quantitative reverse transcription polymerase chain reaction and immunoblotting were performed. Cellular proliferation was assessed using CCK-8 assays at 24 and 72 hours after SHIN2 treatment. Wound healing assays were performed to assess cellular migration at 24 hours after SHIN2 treatment. RESULTS: SHIN2 decreased CD133, CKIT, and NOTCH1 mRNA and protein expression by 1.2- to 2.5-fold and 1.1- to 1.4-fold, respectively, in both cell lines. SHIN2 significantly impaired cellular proliferation in both cell lines at 24 and 72 hours. SHIN2 treatment significantly increased wound gap and closure rate in both cell lines (Figs. 1A–1C).Figure 1CONCLUSION: SHMT2 plays an important role in NB tumorigenesis. Inhibition of SHMT2 with SHIN2 impairs cellular proliferation and cellular migration, and it induces differentiation via downregulation of stem cell genes CD133, CKIT, and NOTCH1, in vitro. The use of SHIN2 may serve as a novel therapeutic strategy in high-risk NB.

259 (PB039) - CDK11 is a selective dependency in neuroblastoma harboring loss of chromosome 1p36

Background: Neuroblastoma is the most common infant cancer and exhibits frequent deletion of chromosome 1p36 in the high-risk setting. We sought to identify key dependencies and drug targets driving high-risk neuroblastoma cell growth. Materials and methods: We used targeted shRNA-mediated gene ablation to identify and validate CDK11 as a drug target in SK-N-BE(2) cells. Used CDK11 nanoBRET assay, radiometric kinase activity assay catalyzed by recombinant kinases, and cDNA complementation using exogenous WT CDK11, inhibitor resistant CDK11G579S and kinase deficient CDK11K467M to determine CDK11 target engagement and activity. Total RNA-seq was deployed to define splicing modulation upon CDK11 perturbation and genome-wide CRISPR-Cas9 screening to define synthetic lethal interactions upon CDK11 perturbation. Results: We discovered that CDK11 (CDC2L1/CDC2L2) is frequently deleted of one copy at chromosome 1p36 and is a deep dependency in high-risk neuroblastoma cells harboring 1p36 Loss-of-heterozygosity (Δ1p36). We show that CDK11 loss-of-function via genetic or chemical inhibition preferentially impedes proliferation of Δ1p36 tumor cells vs WT 1p36 tumor cells. Reconstitution of wild-type CDK11 levels in Δ1p36 tumor cell lines rendered neuroblastoma cells resistant to CDK11 inhibitors whereas kinase-deficient CDK11 did not, suggesting a dosage requirement for CDK11 kinase function in Δ1p36 neuroblastoma cells. Chemical inhibition of CDK11 kinase activity in Δ1p36 tumor cells induces intron-retention splicing defects at mRNA processing and export genes, indicating a core role for CDK11 in RNA metabolism. A genome-wide CRISPR screen for synthetic lethality with CDK11 inhibitors revealed that cognate cyclin CCNL1 and a set of core splicing regulators collaborate with CDK11 to enforce neuroblastoma tumor cell growth. Conclusions: Together, our findings identify CDK11 as a druggable vulnerability and potential therapeutic target for high-risk neuroblastoma patients exhibiting Δ1p36. Conflict of interest: Other Substantive Relationships: All authors are employees of Syros Pharmaceuticals.

KDM5B expression in cisplatin resistant neuroblastoma cell lines.

Chemoresistance is a major problem in successful cancer therapy. Lysine-specific demethylase 5B (KDM5B), is a member of the KDM5 family of histone demethylases, whose dysregulation has been observed in numerous types of cancer and plays a role in drug tolerance. The present study examined KDM5B expression in high risk neuroblastoma cell lines. Its level was markedly reduced in cisplatin-resistant cells, UKF-NB-4CDDP, compared with parental sensitive cells UKF-NB-4. Moreover, KDM5B-silencing did not affect either viability nor the response to CDDP in resistant cells, and led to increase of proliferation and migration in CDDP resistant cells but not in sensitive ones. Compliant with these results, short interfering KDM5B transfection resulted in increased S phase in resistant cells. Overall, these findings suggested that KDM5B may be involved in the survival mechanisms of neuroblastoma cells, which makes KDM5B a promising factor for the prediction of sensitivity to CDDP that should therefore be considered for future research.

Kigelia africana inhibits proliferation and induces cell death in stage 4 Neuroblastoma cell lines

Neuroblastoma (NB) is one of the most common solid pediatric tumors and especially high-risk NBs still account for about 12–15% of cancer related deaths in children. Kigelia africana (KA) is a plant used in traditional African medicine which has already shown its anti-cancer potential in several in vitro and in vivo studies. The aim of this study is to evaluate the effect of KA fruit extract on stage 4 high-risk NB cells. Therefore, NB cell lines with and without MYCN amplification and non-neoplastic cells were treated with KA fruit extract at different concentrations. The effect of KA on cell viability and apoptosis rate were assessed by bioluminescence-/fluorescence-based assays. Several proteins involved in survival, tumor growth, inflammation and metastasis were detected via western blot and immunofluorescence. Secreted cytokines were detected via ELISA. Phytochemical composition of the extract was analyzed by liquid chromatography with tandem mass spectrometry (LC/MS/MS). Our group demonstrates a dose- and time-dependent selective cytotoxic effect of KA fruit extract on NB, especially in MYCN non-amplified tumor cells, by inhibiting cell proliferation and inducing cell death. Western blot and immunofluorescence results demonstrate a regulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), disialoganglioside GD2 and epidermal growth factor receptor (EGFR) in KA-treated tumor cells. Our results evidence striking anti-cancer properties of KA fruit and pave the way for further surveys on the therapeutic properties and mechanisms of action in NB.

RRM2 enhances MYCN-driven neuroblastoma formation and acts as a synergistic target with CHK1 inhibition.

High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential.

NAMPT Inhibition Induces Neuroblastoma Cell Death and Blocks Tumor Growth.

High-risk neuroblastoma (NB) portends very poor prognoses in children. Targeting tumor metabolism has emerged as a novel therapeutic strategy. High levels of nicotinamide-adenine-dinucleotide (NAD+) are required for rapid cell proliferation. Nicotinamide phosphoribosyl transferase (NAMPT) is the rate-limiting enzyme for NAD+ salvage and is overexpressed in several cancers. Here, we determine the potential of NAMPT as a therapeutic target for NB treatment. NAMPT inhibition cytotoxicity was determined by trypan blue exclusion and LDH assays. Neuroblastoma stem cell self-renewal was evaluated by neurosphere assay. Protein expression was evaluated via Western blot. The effect of targeting NAMPT in vivo was determined using an NB1691-xenografted mouse model. Robust NAMPT expression was demonstrated in multiple N-MYC amplified, high-risk neuroblastoma cell lines. NAMPT inhibition with STF-118804 (STF) decreased ATP, induced apoptosis, and reduced NB stem cell neurosphere formation. STF treatment down-regulated N-MYC levels and abrogated AKT activation. AKT and glycolytic pathway inhibitors in combination with NAMPT inhibition induced robust, greater-than-additive neuroblastoma cell death. Lastly, STF treatment blocked neuroblastoma tumor growth in mouse xenograft models. NAMPT is a valid therapeutic target as inhibition promoted neuroblastoma cell death in vitro and prevented tumor growth in vivo. Further investigation is warranted to establish this therapy’s role as an adjunctive modality.

Unraveling the extracellular matrix-tumor cell interactions to aid better targeted therapies for neuroblastoma

Treatment in children with high-risk neuroblastoma remains largely unsuccessful due to the development of metastases and drug resistance. The biological complexity of these tumors and their microenvironment represent one of the many challenges to face. Matrix glycoproteins such as vitronectin act as bridge elements between extracellular matrix and tumor cells and can promote tumor cell spreading. In this study, we established through a clinical cohort and preclinical models that the interaction of vitronectin and its ligands, such as αv integrins, are related to the stiffness of the extracellular matrix in high-risk neuroblastoma. These marked alterations found in the matrix led us to specifically target tumor cells within these altered matrices by employing nanomedicine and combination therapy. Loading the conventional cytotoxic drug etoposide into nanoparticles significantly increased its efficacy in neuroblastoma cells. We noted high synergy between etoposide and cilengitide, a high-affinity cyclic pentapeptide αv integrin antagonist. The results of this study highlight the need to characterize cell-extracellular matrix interactions, to improve patient care in high-risk neuroblastoma.

NLRR1 Is a Potential Therapeutic Target in Neuroblastoma and MYCN-Driven Malignant Cancers.

Receptor tyrosine kinases (RTKs) receive different modulation before transmitting proliferative signals. We previously identified neuronal leucine-rich repeat 1 (NLRR1) as a positive regulator of EGF and IGF-1 signals in high-risk neuroblastoma cells. Here, we show that NLRR1 is up-regulated in various adult cancers and acts as a key regulator of tumor cell proliferation. In the extracellular domains of NLRR1, fibronectin type III (FNIII) domain is responsible for its function to promote cell proliferation. We generated monoclonal antibodies against the extracellular domains of NLRR1 (N1mAb) and screened the positive N1mAbs for growth inhibitory effect. The treatment of N1mAbs reduces tumor cell proliferation in vitro and in vivo, and sensitizes the cells to EGFR inhibitor, suggesting that NLRR1 is a novel regulatory molecule of RTK function. Importantly, epitope mapping analysis has revealed that N1mAbs with growth inhibitory effect recognize immunoglobulin-like and FNIII domains of NLRR1, which also indicates the importance of FNIII domain in the function of NLRR1. Thus, the present study provides a new insight into the development of a cancer therapy by targeting NLRR1 as a modulator of proliferative signals on cellular membrane of tumor cells.

Broad-Spectrum HDAC Inhibitors Promote Autophagy through FOXO Transcription Factors in Neuroblastoma.

Depending on context and tumor stage, deregulation of autophagy can either suppress tumorigenesis or promote chemoresistance and tumor survival. Histone deacetylases (HDACs) can modulate autophagy; however, the exact mechanisms are not fully understood. Here, we analyze the effects of the broad-spectrum HDAC inhibitors (HDACi) panobinostat and vorinostat on the transcriptional regulation of autophagy with respect to autophagy transcription factor activity (Transcription factor EB—TFEB, forkhead boxO—FOXO) and autophagic flux in neuroblastoma cells. In combination with the late-stage autophagic flux inhibitor bafilomycin A1, HDACis increase the number of autophagic vesicles, indicating an increase in autophagic flux. Both HDACi induce nuclear translocation of the transcription factors FOXO1 and FOXO3a, but not TFEB and promote the expression of pro-autophagic FOXO1/3a target genes. Moreover, FOXO1/3a knockdown experiments impaired HDACi treatment mediated expression of autophagy related genes. Combination of panobinostat with the lysosomal inhibitor chloroquine, which blocks autophagic flux, enhances neuroblastoma cell death in culture and hampers tumor growth in vivo in a neuroblastoma zebrafish xenograft model. In conclusion, our results indicate that pan-HDACi treatment induces autophagy in neuroblastoma at a transcriptional level. Combining HDACis with autophagy modulating drugs suppresses tumor growth of high-risk neuroblastoma cells. These experimental data provide novel insights for optimization of treatment strategies in neuroblastoma.

Heterogeneities in Cell Cycle Checkpoint Activation Following Doxorubicin Treatment Reveal Targetable Vulnerabilities in TP53 Mutated Ultra High-Risk Neuroblastoma Cell Lines.

Most chemotherapeutics target DNA integrity and thereby trigger tumour cell death through activation of DNA damage responses that are tightly coupled to the cell cycle. Disturbances in cell cycle regulation can therefore lead to treatment resistance. Here, a comprehensive analysis of cell cycle checkpoint activation following doxorubicin (doxo) treatment was performed using flow cytometry, immunofluorescence and live-cell imaging in a panel of TP53 mutated ultra high-risk neuroblastoma (NB) cell lines, SK-N-DZ, Kelly, SK-N-AS, SK-N-FI, and BE(2)-C. Following treatment, a dose-dependent accumulation in either S- and/or G2/M-phase was observed. This coincided with a heterogeneous increase of cell cycle checkpoint proteins, i.e., phos-ATM, phos-CHK1, phos-CHK2, Wee1, p21Cip1/Waf1, and p27Kip among the cell lines. Combination treatment with doxo and a small-molecule inhibitor of ATM showed a delay in regrowth in SK-N-DZ, of CHK1 in BE(2)-C, of Wee1 in SK-N-FI and BE(2)-C, and of p21 in Kelly and BE(2)-C. Further investigation revealed, in all tested cell lines, a subset of cells arrested in mitosis, indicating independence on the intra-S- and/or G2/M-checkpoints. Taken together, we mapped distinct cell cycle checkpoints in ultra high-risk NB cell lines and identified checkpoint dependent and independent druggable targets.

NEAT1 polyA-modulating antisense oligonucleotides reveal opposing functions for both long non-coding RNA isoforms in neuroblastoma.

Many long non-coding RNAs (lncRNA) are highly dysregulated in cancer and are emerging as therapeutic targets. One example is NEAT1, which consists of two overlapping lncRNA isoforms, NEAT1_1 (3.7kb) and NEAT1_2 (23kb), that are functionally distinct. The longer NEAT1_2 is responsible for scaffolding gene-regulatory nuclear bodies termed paraspeckles, whereas NEAT1_1 is involved in paraspeckle-independent function. The NEAT1 isoform ratio is dependent on the efficient cleavage and polyadenylation of NEAT1_1 at the expense of NEAT1_2. Here, we developed a targeted antisense oligonucleotide (ASO) approach to sterically block NEAT1_1 polyadenylation processing, achieving upregulation of NEAT1_2 and abundant paraspeckles. We have applied these ASOs to cells of the heterogeneous infant cancer, neuroblastoma, as we found higher NEAT1_1:NEAT1_2 ratio and lack of paraspeckles in high-risk neuroblastoma cells. These ASOs decrease NEAT1_1 levels, increase NEAT1_2/paraspeckles and concomitantly reduce cell viability in high-risk neuroblastoma specifically. In contrast, overexpression of NEAT1_1 has the opposite effect, increasing cell proliferation. Transcriptomic analyses of high-risk neuroblastoma cells with altered NEAT1 ratios and increased paraspeckle abundance after ASO treatment showed an upregulation of differentiation pathways, as opposed to the usual aggressive neuroblastic phenotype. Thus, we have developed potential anti-cancer ASO drugs that can transiently increase growth-inhibiting NEAT1_2 RNA at the expense of growth-promoting NEAT1_1 RNA. These ASOs, unlike others that degrade lncRNAs, provide insights into the importance of altering lncRNA polyadenylation events to suppress tumorigenesis as a strategy to combat cancer.

Abstract 1374: Preclinical investigation of ATR inhibition alone and in combination with PARP inhibition in high risk neuroblastoma

Abstract Background:Neuroblastoma (NB) is the commonest extra-cranial malignant solid tumour of childhood and one of the most difficult to cure. DNA damage response (DDR) defects are frequently observed in high risk NB including allelic loss and loss of function mutations in key DDR genes, oncogene induced replication stress (RS) and cell cycle checkpoint dysfunction. Cancer cells with defective cell cycle checkpoint signalling and/or increased oncogene-driven RS are acutely dependent on the DNA damage sensor kinase ATR. This study aims to identify features of NB cell lines leading to ATR inhibitor sensitivity. As PARP inhibition causes RS through unrepaired single strand DNA breaks progressing to replication, we hypothesise that ATR inhibition will increase PARP inhibitor cytotoxicity. Aims: 1) To determine which molecular features lead to sensitivity to VE-821 (ATR inhibitor) in cell lines derived from high-risk NB tumours. 2) To assess synergism between PARP inhibition with olaparib and ATR inhibition in high risk NB cell lines and to measure RS. Materials and Methods: Cell proliferation in response to 72 hours treatment with VE-821 was assessed by XTT assay (Roche) in a panel of 11 NB cell lines and the effect of ATR inhibition on olaparib growth inhibition in 4 cell lines: SHSY5Y, SKNAS, NGP and N20_R1. CHK1S345 and H2AXS129 phosphorylation was assessed using Western blotting to determine ATR activity and RS respectively. RS was also measured by γH2AX foci formation using immunofluorescent microscopy. Results: VE-821 caused significantly more growth inhibition in MYCN amplified cell lines and cell lines with low ATM protein expression by XTT assay (p<0.05 Mann-Whitney U test). Olaparib (5 µM) treatment increased CHK1S345 and H2AXS129 phosphorylation after 24 hours treatment in all cell lines. H2AXS129 phosphorylation and foci number was further increased with the addition of VE-821 (1 µM). ATR inhibition prevented CHK1S345 phosphorylation. In cell proliferation assays, combination index analysis (Calcusyn) showed that ATR inhibition by VE-821 is synergistic with olaparib at sub lethal concentrations (<1 µM) (CI value 0.04-0.89). Conclusion: MCYN amplification and low ATM protein expression are determinants of ATRi sensitivity in NB cell lines. ATR inhibition by VE-821 is synergistic with olaparib at sub lethal concentrations (<1 µM) and further increases the replication stress caused by PARP inhibition. Citation Format: Harriet E. Southgate, Lindi Chen, Nicola J. Curtin, Deborah A. Tweddle. Preclinical investigation of ATR inhibition alone and in combination with PARP inhibition in high risk neuroblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1374.

Preclinical evaluation of the anti-tumor activity of pralatrexate in high-risk neuroblastoma cells.

Introduction: Pralatrexate is a folate analogue inhibitor of dihydrofolate reductase exhibiting high affinity for reduced folate carrier-1 with antineoplastic and immunosuppressive activities, similar to methotrexate. Despite advances in multi-modality treatment strategies, the survival rates for children with high-risk neuroblastoma have failed to improve. Therefore, the intense research continues in order to identify the ideal novel agent or combination of chemotherapy drugs to treat high-risk neuroblastoma.Materials and Methods: Four human neuroblastoma cell lines were used to determine IC50 values of select chemotherapy agents. Antiproliferative effects of pralatrexate were assessed by adherent and non-adherent colony formation assays. Cell cycle arrest and apoptosis were measured by flow cytometry and immunoblotting. PDX tissue culture was used to assess ex vivo efficacy.Results: Treatment with pralatrexate in all four neuroblastoma cell lines blocked cell growth in 2D and 3D culture conditions in a time-dependent manner. The potency of pralatrexate was ten-fold stronger than methotrexate, as measured by IC50. Pralatrexate-induced apoptosis was confirmed by caspase-3 activation and PARP cleavage. MYCN and SLC19A1 mRNA expressions were decreased with pralatrexate in MYCN-amplified neuroblastoma cells.Conclusions: Pralatrexate demonstrated effective inhibition of cell growth and viability. The higher potency of pralatrexate compared to methotrexate, a drug with high levels of toxicity, suggests pralatrexate may be a safer alternative to methotrexate as an effective chemotherapeutic agent in the treatment of patients with high-risk neuroblastoma.