High-Risk Neuroblastoma: Poor Outcomes Despite Aggressive Multimodal Therapy

The era of neuroblastoma immunotherapy began with the proof of the effectiveness of monoclonal antibodies to the disialoganglioside GD2, which is expressed on the membrane of tumor cells. Today, these antibodies are included in maintenance therapy regimens for high-risk neuroblastoma. A promising area of immunotherapy for neuroblastoma is the creation of T cells that carry a chimeric antigen receptor to the membrane structures of the tumor (CAR-T cells). Inhibitors of “immunologic checkpoints” – antibodies that block the PD1 receptor and the corresponding PD-L1 ligand – also have theoretical grounds for immunotherapy of neuroblastoma. The introduction of immunotherapy into traditional multicomponent treatment requires further study to optimize the use of this technology for patients with high-risk neuroblastoma. The introduction of multimodality therapy, including surgery, radiation, and aggressive combination chemotherapy, has improved outcomes for many children with high-risk neuroblastoma, but scientists seem to have reached a plateau in what can be achieved with cytotoxic therapy alone. Most children with cancer, including high-risk neuroblastoma, do not benefit from treatment with immune checkpoint inhibitors (ICIs), which have revolutionized the treatment of many highly immunogenic solid tumors in adults. This likely reflects the low mutation rate of the tumor as well as the downregulation of MHC-I that characterizes most high-risk neuroblastomas. For these reasons, neuroblastoma represents an immunotherapeutic challenge that may serve as a model for the development of effective immunotherapy for other cold tumors in children and adults who do not respond to ICI. The identification of strong expression of the disialoganglioside GD2 on the surface of nearly all neuroblastoma cells provided a target for immune recognition by anti-GD2 mAbs, which recruit Fc-receptor-expressing innate immune cells that mediate cytotoxicity or phagocytosis. The adoption of anti-GDNA antibodies in both preliminary and relapse treatment protocols has dramatically improved survival rates and changed the landscape for children with high-risk neuroblastoma. The author set the goal of this work to analyze the latest publications and basic experience on the mechanisms of innate and acquired immunity against neuroblastoma and the use of immunotherapy in its treatment. The work on the article was accompanied by an analysis of the literature over the past seven years. The author notes that neuroblastoma is currently one of the most difficult malignant tumors in children to diagnose and treat, developing from immature nerve cells, neuroblasts. Neuroblastoma can form throughout the sympathetic nervous system. The clinical and biological behavior of this tumor is heterogeneous. It can both progress rapidly with a fatal outcome and spontaneously regress or differentiate into a mature ganglioneuroma (benign tumor). Neuroblastoma is a malignant extracranial solid tumor that is most commonly found in children. In addition, it is the most common solid tumor in young children. The peak incidence of neuroblastoma occurs in children in the first year of life, and 90% of all cases are diagnosed before the age of 10 (most often in children under 5 years). Malignant tumors that develop in childhood are characterized by a relatively low level of genetic instability and, as a result, somatic mutations compared to “adult” tumors. This leads to their lower immunoreactivity and evasion of immunologic surveillance. At the same time, during the development of neuroblastoma, the most common extracranial solid tumor of childhood, the immune system response can unfold quite early. The low mutation rate of the tumor, as well as the downregulation of MHC-I, which characterizes most high-risk neuroblastomas. This phenomenon opens up additional opportunities in planning the treatment of neuroblastoma and gives hope for a good effect of immunotherapy. Despite quite remarkable successes in the treatment of solid tumors in children over the past 20-30 years, due to the widespread use of cytostatic therapy in combination with surgery and radiation treatment, in recent years, scientists and practitioners have reached a so-called “plateau” in the overall survival rate in children with malignant tumors. Unfortunately, neuroblastoma is no exception to this rule. The creation of modern diagnostic and treatment protocols that include various combinations of chemotherapy drugs and staged surgical care has increased the overall survival rate from this pathology to 60–65%. This is a fairly good result, but in the last 7–10 years, this result has not improved, and the effectiveness of treatment of aggressive resistant forms of the tumor still does not exceed 10–15%. For these reasons, neuroblastoma represents an immunotherapeutic challenge that may serve as a model for the development of effective immunotherapy for other “cold” tumors in children and adults who do not respond to ICI.

CONTEXT: Neuroblastoma is the most common extracranial solid tumor in children. Patients with low- and intermediate-risk neuroblastoma (LIRNB) have favorable prognosis and an excellent five-year survival rate of more than 90%. However, in the case of high-risk neuroblastoma (HRNB; ~50% of cases), the prognosis of treatment remains unfavorable and the five-year survival rate remains under 40% despite aggressive multi modal therapy. QUESTION: We wanted to know if there was a defect in immune surveillance that could explain part of the aggressive phenotype of HRNB. METHODS: We performed a prospective multi-center study on thirty two new cases of neuroblastoma at all stages. We performed a wide phenotyping of immune cells by flow cytometry in fresh whole blood, and fresh whole bone marrow at diagnosis of children diagnosed for neuroblastoma. Cell counts were performed in order to be able to reason in absolute values (G/L) rather than only percentages. RESULTS: Blood and bone marrow from 20 LIRNB and 12 HRNB were prospectively analyzed. We found that specific subsets of circulating immune cells such as myeloid dendritic cells BDCA-1+, plasmacytoid dendritic cells, Teff/Treg CD4+ T-cells, were decreased in HRNB blood compared to LIRNB groups as opposed to other subsets (e.g iNKT, g9d2 T-cells,⋯). More surprisingly, these differences were not due to defects in cell production as there was no difference found for the same cell populations in the bone marrow between LIRNB and HRNB children. CONCLUSION: Specific immune cell defects are found in the blood from High risk neuroblastoma children that are not present in the blood of low & intermediate risk neuroblastoma children. These defects are not due to bone marrow production impairment. These results suggest that high risk neuroblastoma disease generates an impairment in the immune homeostasis of children. Gene expression analysis of cytokine/chemokine from these neuroblastoma tumors is underway (NCT01295762). Citation Format: Sandrine Susini, Isabelle Rochet, Estelle Verronese, Christine Bardin, Chantal Rigal, Cecile Conter, Perrine Marec-Berard, Christophe Bergeron, Audrey Lardy-Cleaud, Severine Neymarc, Severine Metzger, Jean-Louis Stephan, Dominique Plantaz, Christophe Caux, Christine Menetrier-Caux, Aurelien Marabelle. Prospective immune phenotyping of neuroblastoma children at diagnosis reveals specific immune defects related to the aggressiveness of the disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3112.

OBJECTIVES/GOALS: Neuroblastoma (NB) is the most common extra-cranial solid tumor with outcomes varying from spontaneous regression to metastatic with high mortality rates. The tumor immune microenvironment (TIME) may play a significant role in this disease. In this study we analyze the TIME comparing high-risk (HR) and low-risk (LR) NBs using multiplex platforms. METHODS/STUDY POPULATION: Two tissue microarrays (TMAs) with 2mm cores were created from 41 patients treated at Columbia University Irving Medical Center. Five micron TMA slides were stained for Digital Spatial Profiling (DSP, nanoString) and multiplex immunofluorescence (mIF). For DSP, a 24-patient subset including 11 HR, 8 LR and 4 intermediate risk patients was analyzed for 34 proteins. Protein expression among risk groups was compared using Mann-Whitney t-test. For mIF, TMA FFPE slides were stained for DAPI, CD3, CD8, CD68, HLA-DR, PDL1 and Chromogranin A. Whole TMA cores were captured as 9 -20X multispectral images (MSIs) stitched into a 3x3 MSI using Vectra (Akoya). MSIs were processed with inForm and qualitative analysis performed comparing HR and LR tumors. RESULTS/ANTICIPATED RESULTS: With DSP, we find significantly more HLA-DR in HR compared to LR tumors (p = 0.016). When controlling for immune cells with CD45 we find HLA-DR/CD45 to be higher in HR than LR tumors (p = 0.026). We found increased PD1 and PDL1 expression in all groups without significant difference between LR and HR (p = 0.778 and p = 0.310, respectively). Preliminary analysis of mIF on 9 patients (4 HR and 5 LR) finds HR tumors appear to have more immune cells than LR tumors, specifically more CD3+CD8- T cells while total CD8+ cells may be similar. There may be less macrophages in the HR compared to LR tumors. Completion of image processing and quantitative analysis of mIF data is underway. DISCUSSION/SIGNIFICANCE OF IMPACT: Increased expression of immune markers in NB TIME correlates with higher risk, which is unlike many other tumors. We compared TIME in HR and LR NB using multiplex platforms, DSP and mIF. We find that HLA-DR is more expressed in HR NB while PD1 and PDL1 expression is consistently high and not different between risk groups. Further analysis is underway. CONFLICT OF INTEREST DESCRIPTION: Robyn D. Gartrell-Corrado received grant support from nanoString for Digital Spatial Profiling and received honoraria and travel support from Northwest Biotherapeutics and PerkinElmer, respectively.

Background: Neuroblastoma (NB), the most common extra-cranial solid tumor in children, originates from the precursor neuroblasts of the sympathetic nervous system. NB accounts for approximately 7-10% of childhood cancers and 15% of childhood cancer death. Despite an aggressive treatment regimen, the 5-year survival for high risk NB remains less than 50%. The differentiation of NB cells into mature cells represents a promising strategy for NB therapy. Currently, retinoids are the most commonly used differentiating agents. However, their use can be limited due to intrinsic or acquired resistance, as well as toxicity. We sought to evaluate the potential of the natural product withaferin A (WA), a steroidal lactone derived from the medicinal plant Withania somnifera, to induce NB cell differentiation. Methods: For differentiation studies NB cell lines (NB1691, SMS-KCNR, SH-SY5Y and the primary cell line SVBM15) were exposed to WA (100-500nM) for 7-10 days and evaluated by light microscopy, immunocytochemistry and western blot analysis. NB stem-like cell lines were generated by culturing NB1691 and SVBM15 cells in neurosphere media. To determine the IC50 (concentration needed to reduce viability by 50%), NB stem-like cell lines were exposed to increasing concentrations of WA and viability was assessed at 72 hours using MTS assay. Reactive oxygen species (ROS) were detected using CM-H2DFDA and ROS induction was inhibited with N-acetyl-L- Cysteine (NAC). To determine WA effect on neurosphere formation, NB cells were plated in 96-well plates at 50 cells/well. Cells were grown in increasing concentrations of WA and spheres were counted at 14 days. Results: WA promoted morphologic alterations (neurite outgrowth) and growth inhibition in a dose dependent manner. Immunocytochemistry and western blot analysis indicated an increase in neuronal markers including neurofilament, β-tubulin and MAP2, as well as a decrease in the stem cell markers BMi-1 and musashi. WA promoted ROS induction, which could be prevented with NAC pretreatment. NAC prevented WA-induced morphological changes and inhibited WA-induced changes in stem cell and differentiation marker expression. WA induced NB stem-like cell death in a dose dependent manner (IC50 of NB1691=1.05 μM; SVBM15=1.06 μM), and significantly inhibited neurosphere formation at concentrations as low as 50nM, which did not exhibit cytotoxicity in regular cell culture conditions. Conclusion: Withania somnifera has been used for centuries and is commonly used in ayurvedic medicine. WA has been shown to affect multiple pathways important for cancer progression and induce anti-cancer effects in breast, prostate and pancreatic cancers. Our data indicates that WA induces NB stem-like cell death, and promotes ROS-mediated NB cell differentiation. Differentiation therapy aims at reducing the risk of the tumor regrowth following high-dose chemotherapy and stem cell transplant in patients classified as high-risk. WA holds great promise as a novel alternative NB treatment strategy for children with persistent minimal residual disease. Citation Format: Gregor A. Rodriguez, Claudia P. Zapata, Anthony Sanchez, Nicolas A. De Cordoba, Beatriz E. Hawkins, Nadia G. Myrthil, Sarah A. Samuels, Amelia Bahamonde, Steven Vanni, Regina M. Graham. Withaferin A promotes ROS-mediated differentiation of neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B41.

Chemotherapeutic drug resistance is one of the major causes for treatment failure in high-risk neuroblastoma (NB), the most common extra cranial solid tumor in children. Poor prognosis is typically associated with MYCN amplification. Here, we utilized a loss-of-function kinome-wide RNA interference screen to identify genes that cause cisplatin sensitization. We identified fibroblast growth factor receptor 2 (FGFR2) as an important determinant of cisplatin resistance. Pharmacological inhibition of FGFR2 confirmed the importance of this kinase in NB chemoresistance. Silencing of FGFR2 sensitized NB cells to cisplatin-induced apoptosis, which was regulated by the downregulation of the anti-apoptotic proteins BCL2 and BCLXL. Mechanistically, FGFR2 was shown to activate protein kinase C-δ to induce BCL2 expression. FGFR2, as well as the ligand fibroblast growth factor-2, were consistently expressed in primary NB and NB cell lines, indicating the presence of an autocrine loop. Expression analysis revealed that FGFR2 correlates with MYCN amplification and with advanced stage disease, demonstrating the clinical relevance of FGFR2 in NB. These findings suggest a novel role for FGFR2 in chemoresistance and provide a rational to combine pharmacological inhibitors against FGFR2 with chemotherapeutic agents for the treatment of NB.

Background: Neuroblastoma (NB) is a common malignant extracranial solid tumor in children, responsible for 11% of cancer deaths in pediatric patients under 15 (Smith et al, Cancer, 2014). High-risk neuroblastoma is a metastatic disease in children aged 18 months or older with L2, M, or MS disease with amplification of the MYCN oncogene (Chuang Pediatr Blood Cancer 2021). Recent analyses by COG show that HR Neuroblastoma still has a 5-year event free survival of 51% despite an extensive treatment regimen (Steven et al, ASCO, 2022). To address improved tumor targeting and overcome antigen escape, chimeric antigen receptor (CAR) technology to Natural Killer (NK) cell therapy has been adapted (Chu/Cairo, et al, Frontiers in Oncology, 2019). TGFβ is a potent mechanism of the NK cell anti-tumor resistance and is present at elevated levels in the NB tumor microenvironment (TME) (Foltz, Cancers, 2018). Studies demonstrated that blockade of TGF-β signaling in NK cells caused the accumulation of NK cells that produce IFN-γ and neutralization of TGF-β prevented NKG2D downregulation and also restored NK cell anti-tumor reactivity (Nayyar/Chu/Cairo, et al Frontierls in Oncology, 2018). ROR1, a transmembrane protein is highly expressed on the majority of NB (Rebagay/Yan Front Oncol, 2012). Our group has successfully expanded functional and active peripheral blood NK cells (exPBNK) with irradiated feeder cells and electroporated CAR mRNA to exPBNK (Chu/Cairo, et al Cancer Immunol Res, 2015). Objective: To determine in vitro cytotoxicity against neuroblastoma cells by ex vivo expanded TGFβ imprinting (TGFβi) NK cells. Methods: TGFβi NK cells are generated by culturing expanded NK cells with irradiated K562-41BBL-mbIL21 in 50 IU/mL IL-2 plus 10 ng/mL TGFβ every 2-3 days for two weeks. NK and TGFβi NK cells specific in-vitro cytotoxicity were compared via luciferase reporter assay against ROR1+ NB cell lines as we previously described (Chu/Cairo, et al, JITC, 2022). Cytokine/chemokine levels secreted by NK and TGFβi NK cells were compared using ELISA assays (Chu/Cairo, et al, JITC, 2022). Results: We have successfully expanded TGFβi NK cells. These TGFβi NK cells showed significantly enhanced in vitro cytotoxicity against NB cells compared to NK cells under TGFβ environment (20+2.4% vs 0%; p<0.001) at E:T=3:1 and consistently, TGFβ-imprinting NK secreted significantly high level of TNF-alpha (393.7pg/ml+16.77pg/ml) against NB cells (p<0.001) compared to NK cells (79.7pg/ml+14.7pg/ml) under TGFβ environment. Conclusion: Our data indicate TGFβi NK cells may be a promising novel therapy to overcome the immunosuppression of TGFβ in the tumor microenvironment for pediatric NB tumors. We will further engineer these TGFβi NK cells to express anti-ROR1 CAR to enhance the targeting specificity against NB tumors and investigate the anti-tumor effects of anti-ROR1 CAR TGFβi NK cells against NB tumors in-vitro and in-vivo (Funded by U54 CA232561). Citation Format: Jessica A. Ayala-Cuesta, Yaya Chu, Keira Foley, Meijuan Tian, Kayleigh Klose, Alyssa S. Mendelowitz, Timothy P. Cripe, Dean A. Lee, Mitchell S. Cairo. Targeting high-risk neuroblastoma by TGFβ imprinted natural killer cells [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 3615.

Purpose: Patients with high-risk (HR) neuroblastoma (NBL) have a poor prognosis and new treatment strategies are needed. 131I- Meta-Iodobenzylguanidine (MIBG) therapy alone has a significant antitumor efficacy against NBL. Topotecan (TPT) is a radio-enhancer and can potentiate radiation-induced cytotoxicity and up-regulate the uptake of MIBG in vitro. We designed a clinical study with upfront 131I-MIBG therapy and Topotecan for HR-NBL. We evaluated short term clinical response rate and outcome. Clinical response was scored according to International Neuroblastoma response Criteria (INRC) and Curie score (MIBG) after 2 courses of MIBG/TPT and after induction chemotherapy and HD ASCT. Haematological toxicity and the effect on harvesting/ feasibility performing autologous stem cell transplantation (ASCT) was evaluated. Topoisomerase-1 activity (Topo-1) levels were measured. Methods: Multi-centre, prospective (window phase II) study performed between 2000-2003 in HR NBL patients treated with 2 cycles of upfront 131I-MIBG therapy followed by the addition of TPT 0.7 mg/m2 for 5 days after each course. Thereafter, patients received 4 courses of VECI chemotherapy, followed by surgery and HD ASCT. Results: Sixteen patients (10 male), median age 2, 8 yrs. NMYC amplified 8/14 (57%) and LOH1P in 7/15 (47%). Surgery performed in 12/16 (75%) patients. Clinical response after 2x MIBG/TPT 15/16 (94%, VGPR/ PR/ MR), 1 PD; after 4x VECI 11/16 (69%, CR/ VGPR/ PR/MR), 3 PD, 1 death and 1 unknown. More than 50% reduction in MIBG Curie score after 2x MIBG/ TPT and subsequent 4x VECI courses in most cases. Overall outcome 2/16 currently alive. Long term follow up was limited, possibly related to low intensity induction chemotherapy as used in this study. Harvesting of stem cells in 13/16 patients (79%), mean yield 2, 2 CD34 pos/ kg, 9/16 (56%) underwent HD ASCT. Haematological toxicity was limited, median platelet recovery after HD ASCT was 73 days. Topo-1 activity was measured in 10/16 tumors, increased compared to reference level. Conclusion: Upfront treatment of HR-NBL with MIBG/TPT is feasible and highly effective after 2 courses. The toxicity is limited (haematological). Stem cell harvesting and HD ASCT are manageable. Citation Format: Max M. van Noesel, Kathelijne CJM Kraal, Jan de Kraker, Berthe LF van Eck-Smit, Godelieve AM Tytgat, Huib N. Caron. Upfront treatment of high-risk neuroblastoma with 131I-MIBG therapy and Topotecan. A phase II pilot window study (AMRO-NB-HR-2000/01). [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B72.

Immune System Profiling Following Autologous Stem Cell Transplant for High-Risk Neuroblastoma Predicts Disease-Free Survival

Introduction: Neuroblastoma (NB) is the most common cancer in infants and the most common extracranial solid tumor in children, accounting for 15% of all childhood cancer deaths. The overall prognosis for those with high-risk or relapsed disease remains poor despite the standard therapies of surgery, radiation, and high dose chemotherapy. We have previously shown that altering levels of specific microRNAs (miRNAs) can improve cellular response to microtubule-targeting agents (MTAs) and that exposure to MTAs results in dramatic changes in microRNA expression. Methods: Libraries of chemically synthesized ncRNA mimics and inhibitors, including both miRNAs and lncRNAs, are screened to identify ncRNAs that regulate neuroblastoma cell viability or sensitize neuroblastoma cells to specific microtubule-targeting agents. Candidate targets are validated using qRT-PCR, protein quantification, and luciferase reporter assays. The response of neuroblastoma cells to perturbations in candidate ncRNA levels is assessed through flow cytometric analysis of cell cycle phase distribution, density and anisotropy of microtubule bundles, and through colony formation and caspase activation assays, and validated in mouse xenograft models. Networks of miRNAs, mRNAs and lncRNAs are derived from combining complementary cellular response with potential regulatory interactions. Results: We previously reported the identification of miRNAs and lncRNAs that significantly decrease or increase neuroblastoma cell viability. Here, we extend those observations to drug response. We first demonstrate that the microtubule-targeting agent eribulin has both short-term and long-term effects, as reflected by a significant alteration in microtubule structure in response to high doses for short times and arrest at the G(2)/M phase of the cell cycle in response to low doses for long times. We next show that altering intracellular levels of ncRNAs that sensitize cells to eribulin both alters microtubule density and anisotropy and recapitulates the effect of eribulin treatment. Finally, we show that complementarity of effect between miRNAs and lncRNAs is accompanied by potential regulatory interaction. Conclusions: Taken together, our results suggest that the response of neuroblastoma cells to eribulin is mediated by a regulatory network that includes different coding and non-coding RNA species. While these RNAs may have intrinsic value as biomarkers or therapeutic agents, either individually or in combination, the vulnerabilities that they uncover may be exploited with pathway-specific perturbations. This project was supported by NIH R01 CA129632 and CPRIT Training Grant RP140105. Citation Format: Xiuye Ma, Xiaojie Yu, Harsh Patolia, Zhenze Zhao, Liqin Du, Alexander Pertsemlidis. Non-coding RNA regulation of eribulin response in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1940.

185: Feasibility of autologous stem cell transplant followed by reduced intensity allogeneic stem cell transplantation for high risk neuroblastoma: A single institution pilot study

Achieving a cure for high-risk neuroblastoma, the most common extracranial solid tumor in children, remains a formidable task despite the recent addition of antibody-mediated anti-GD2 immunotherapy to established multimodality therapy. The PI3K/Akt pathway is a pivotal signaling pathway utilized by a plethora of receptor tyrosine kinases that contribute to the aggressive phenotype of high-risk neuroblastoma. Akt is aberrantly activated in high-risk neuroblastoma and is therefore an attractive therapeutic target. Perifosine is the best-characterized Akt inhibitor in preclinical studies and in clinical trials in adults, although safety in children is not yet confirmed. It is a synthetic third-generation alkylphospholipid with good oral bioavailability and modest side effects. Perifosine targets the lipid-binding PH domain of Akt and inhibits the translocation of Akt to the cell membrane, an essential step for Akt activation. It decreases Akt phosphorylation and increases caspase-dependent apoptosis in neuroblastoma cell lines, inhibits growth of neuroblastoma xenografts, and overcomes RTK/ligand-mediated chemoresistance. It is currently being studied in two Phase I clinical trials in children with recurrent or refractory solid tumors including neuroblastoma. In the single agent trial (ClinicalTrials.gov identifier NCT00776867), maximum tolerated dose has not yet been reached and pharmacokinetic data has been accrued. In the second study (ClinicalTrials.gov identifier NCT01049841), patients are treated with a combination of perifosine and the mTOR-inhibitor temsirolimus based on preclinical data showing synergy of the two agents, and the premise that direct Akt inhibition may overcome Akt activation secondary to mTOR inhibition. Based on results from adult trials, it is unlikely that perifosine alone will produce dramatic therapeutic effects against high-risk neuroblastoma. However, given the recent encouraging early-phase combination therapy results in adults with multiple myeloma and colorectal carcinoma, rational perifosine-containing combination regimens hold promise for neuroblastoma therapy. These will be explored after safety in children is established in Phase I studies.

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.

High-risk (HR) neuroblastoma (NBL) patients often receive standardized treatment despite wide variations in clinical outcomes, underscoring the need for improved stratification tools. A distinguishing feature of NBL is the patient-specific expression of gangliosides (GGs), particularly GD2, which may serve as biomarkers. We analyzed GG profiles in 18 patient-derived tumors and 11 NBL cell lines using thin-layer chromatography and mass spectrometry. Expression of 0-, a-, and b-series GGs was examined and correlated with clinical risk, outcome, and gene expression data. Low-risk (LR) tumors expressed higher levels of complex b-series GGs. In HR tumors, five GG profiles (A-E) were identified. Profile A featured complex b-series GGs; B showed GD2 dominance; C showed synthesis arrest at GM3 or GD3 due to low expression of the GM2/GD2 synthase, encoded by the B4GALNT1 gene; D included complex a- and b-series GGs; and E was marked by GM2 and GD1a prevalence. B4GALNT1 expression served as a prognostic marker. Relapsed tumors following anti-GD2 therapy typically exhibited reduced GD2 levels, except for one profile A tumor that displayed a ceramide anchor shorter than those found in LR tumors. Astonishingly, the ceramide anchor composition of GD2 itself appears to separate LR and HR NBL, hinting at a role of ceramide synthases in NBL biology. All cell lines expressed GM2, but exhibited very low levels of complex b-series GGs. Profile C was found only in cell lines of the mesenchymal subtype. These findings support further investigation of GG composition and associated enzyme expression as potential biomarkers for risk stratification and treatment response in NBL.

<div>Abstract<p>Gene expression is regulated by promoters and enhancers marked by histone H3 lysine 27 acetylation (H3K27ac), which is established by the paralogous histone acetyltransferases (HAT) EP300 and CBP. These enzymes display overlapping regulatory roles in untransformed cells, but less characterized roles in cancer cells. We demonstrate that the majority of high-risk pediatric neuroblastoma (NB) depends on EP300, whereas CBP has a limited role. EP300 controls enhancer acetylation by interacting with TFAP2β, a transcription factor member of the lineage-defining transcriptional core regulatory circuitry (CRC) in NB. To disrupt EP300, we developed a proteolysis-targeting chimera (PROTAC) compound termed “JQAD1” that selectively targets EP300 for degradation. JQAD1 treatment causes loss of H3K27ac at CRC enhancers and rapid NB apoptosis, with limited toxicity to untransformed cells where CBP may compensate. Furthermore, JQAD1 activity is critically determined by cereblon (<i>CRBN</i>) expression across NB cells.</p>Significance:<p>EP300, but not CBP, controls oncogenic CRC-driven transcription in high-risk NB by binding TFAP2β. We developed JQAD1, a CRBN-dependent PROTAC degrader with preferential activity against EP300 and demonstrated its activity in NB. JQAD1 has limited toxicity to untransformed cells and is effective <i>in vivo</i> in a CRBN-dependent manner.</p><p><i>This article is highlighted in the In This Issue feature, p. 587</i></p></div>

<div>Abstract<p>Gene expression is regulated by promoters and enhancers marked by histone H3 lysine 27 acetylation (H3K27ac), which is established by the paralogous histone acetyltransferases (HAT) EP300 and CBP. These enzymes display overlapping regulatory roles in untransformed cells, but less characterized roles in cancer cells. We demonstrate that the majority of high-risk pediatric neuroblastoma (NB) depends on EP300, whereas CBP has a limited role. EP300 controls enhancer acetylation by interacting with TFAP2β, a transcription factor member of the lineage-defining transcriptional core regulatory circuitry (CRC) in NB. To disrupt EP300, we developed a proteolysis-targeting chimera (PROTAC) compound termed “JQAD1” that selectively targets EP300 for degradation. JQAD1 treatment causes loss of H3K27ac at CRC enhancers and rapid NB apoptosis, with limited toxicity to untransformed cells where CBP may compensate. Furthermore, JQAD1 activity is critically determined by cereblon (<i>CRBN</i>) expression across NB cells.</p>Significance:<p>EP300, but not CBP, controls oncogenic CRC-driven transcription in high-risk NB by binding TFAP2β. We developed JQAD1, a CRBN-dependent PROTAC degrader with preferential activity against EP300 and demonstrated its activity in NB. JQAD1 has limited toxicity to untransformed cells and is effective <i>in vivo</i> in a CRBN-dependent manner.</p><p><i>This article is highlighted in the In This Issue feature, p. 587</i></p></div>