Ganglioside Profiling Uncovers Distinct Patterns in High-Risk Neuroblastoma.

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.

Neuroblastoma (NB) is a neuroendocrine cancer with great clinical heterogeneity and most prevalent in children. Despite recent improvements in therapy, long term survival of high-risk NB patients is still below 50%. Although amplification of the proto-oncogene MYCN is a well-established risk factor in NB, little is known about prognosis markers for high-risk NB patients without MYCN amplification. On account of being a cancer originated from early sympathetic nerve cells, the majority of NB tumors express the norepinephrine transporter (NET). NET is a solute carrier (SLC) monoamine transporter known to play an important role in targeting radiopharmaceuticals (e.g. 123I- or 131I-mIBG) for NB diagnosis and treatment. However, little is currently known about the expression of other functionally related SLC transporters in NB. Here, we analyzed the expression of MYCN, NET and other monoamine and organic cation transporters in NB patient samples and explored their association with overall survival of those patients. In order to perform this analysis, we mined the data from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) program database. The TARGET database contains gene expression data of tumor samples from multiple NB clinical trials consisting of predominantly high-risk or relapsed NB patients with predetermined MYCN amplification status. To compare transporter expression in NB, we analyzed mRNAseq data of NET, the dopamine and serotonin transporters (DAT and SERT), the plasma membrane monoamine transporter (PMAT), vesicular monoamine transporters (VMAT1/2), organic cation transporters (OCT1-3), and multidrug and toxin extrusion proteins (MATE1/2-K) in NB tumor samples and normalized their expression to GAPDH. The association between transporter expression and the overall survival of patients was then explored using Gene Expression Array data from 243 NB patient samples matched to clinical outcomes. Patient samples were divided into high and low expression for each transporter and survival analysis was performed using Kaplan-Meier curves. Our data revealed that NET, PMAT and VMAT1/2 are highly expressed in NB tumors especially in MYCN non-amplified samples. In contrast, DAT, SERT, OCT1-3, and MATE1/2-K are minimally or not expressed in NB tumor samples. Interestingly, survival analysis using Kaplan-Meier curves and Log-rank test indicates a significant association between low expression of PMAT and VMAT1/2 and poor overall survival, especially in patients without MYCN amplification. Our study suggests that besides NET, PMAT and VMAT1/2 are highly expressed in NB tumor samples and can be explored for targeting therapeutics in NB tumor cells. The apparent association of PMAT and VMAT1/2 with overall survival suggests that these transporters could be further explored as biomarkers for disease progression and risk prediction for NB patients without MYCN amplification.

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.

Immune checkpoint molecules in neuroblastoma: A clinical perspective

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.

Disease recurrence is frequent in high-risk neuroblastoma (NBL) patients even after multi-modality aggressive treatment [a combination of chemotherapy, surgical resection, local radiation therapy, autologous stem cell transplantation, and cis-retinoic acid (CRA)]. Recent clinical studies have explored the use of monoclonal antibodies (mAbs) that bind to disialoganglioside (GD2), highly expressed in NBL, as a means to enable immune effector cells to destroy NBL cells via antibody-dependent cell-mediated cytotoxicity (ADCC). Preclinical data indicate that ADCC can be more effective when appropriate effector cells are activated by cytokines. Clinical studies have pursued this by administering anti-GD2 mAb in combination with ADCC-enhancing cytokines (IL2 and GM-CSF), a regimen that has demonstrated improved cancer-free survival. More recently, early clinical studies have used a fusion protein that consists of the anti-GD2 mAb directly linked to IL2, and anti-tumor responses were seen in the Phase II setting. Analyses of genes that code for receptors that influence ADCC activity and natural killer (NK) cell function [Fc receptor (FcR), killer immunoglublin-like receptor (KIR), and KIR-ligand (KIR-L)] suggest patients with anti-tumor activity are more likely to have certain genotype profiles. Further analyses will need to be conducted to determine whether these genotypes can be used as predictive markers for favorable therapeutic outcome. In this review, we discuss factors that affect response to mAb-based tumor therapies such as hu14.18-IL2. Many of our observations have been made in the context of NBL; however, we will also include some observations made with mAbs targeting other tumor types that are consistent with results in NBL. Therefore, we hypothesize that the NBL observations discussed here may also be relevant to mAb therapy for other cancers, in which ADCC is known to play a role.

Despite the success of multimodal therapies, the mortality and morbidity remains substantial for patients with high-risk neuroblastoma (NBL). Sequencing of paired tumor/normal DNA of NBL has revealed a low somatic mutation burden and few recurrent somatically-mutated genes. Here we hypothesize that whole transcriptome sequencing (WTS) in patients with high-risk NBL tumor will yield valuable insights into the biology of this disease. We performed deep WTS of 150 NBLs (129 high-risk stage 3 and 4; and 21 stage 4S tumors) of which majority of them had whole genome sequencing or whole exome sequencing of case-matched tumor/normal pairs through the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. We identified correlations between gene expression and clinical parameters of patients such as MYCN-amplification and survival to provide understandings of high-risk NBL biology. Consensus clustering using gene expression profiles of tumors revealed 4 subgroups with distinct survival probability (p<0.01). Among them, the group with the best outcome consisted of all stage 4S and 4S-like young patients; whereas the worst survival group was made up mostly of high-risk patients with MYCN-amplification. In order to identify the gene expression patterns correlating with outcome in the high-risk patients without MYCN-amplification, we further examined if any other known gene expression signatures can further stratify these patients using Gene Set Enrichment Analysis (GSEA). Among several molecular signatures, a previously reported 157-gene MYCN activation signature was significantly correlated with the poor outcome in high-risk patients without MYCN-amplification. Of note, we identified CD8, cytolytic, and activated nature killer (NK) cell signatures that were significantly associated with outcome in these patients. These findings were validated in an independent dataset. Our data has an important clinical implication for a subset of high-risk neuroblastoma patients without MYCN-amplification that have an activated MYCN signature may benefit from immune-modulating agents such as immune check point inhibitors which should be tested in context of clinical trials. Citation Format: Jun S. Wei, Shile Zhang, Igor Kuznetsov, Young K. Song, Shahab Asgharzadeh, Sivasish Sindiri, Xinyu Wen, Rajesh Patidar, Jaime M. Guidry Auvil, Daniela S. Gerhard, Robert Seeger, John M. Maris, Javed Khan. RNAseq identified immune signatures associated with adverse outcome in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1744. doi:10.1158/1538-7445.AM2017-1744

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

: Neuroblastoma (NBL) is a highly malignant embryonal tumor that originates from the primordial neural crest cells. NBL is the most common tumor in infants and the most common extracranial solid tumor in children. The tumor is more commonly diagnosed in children of 1-4 years of age. NBL is characterized by enigmatic clinical behavior that ranges from spontaneous regression to an aggressive clinical course leading to frequent relapses and death. Based on the likelihood of progression and relapse, the International Neuroblastoma Risk Group classification system categorized NBL into very low risk, low risk, intermediate risk, and high risk (HR) groups. HR NBL is defined based on the patient's age (> 18 months), disease metastasis, tumor histology, and MYCN gene amplification. HR NBL is diagnosed in nearly 40% of patients, mainly those > 18 months of age, and is associated with aggressive clinical behavior. Treatment strategies involve the use of intensive chemotherapy (CTR), surgical resection, high dose CTR with hematopoietic stem cell support, radiotherapy, biotherapy, and immunotherapy with Anti-ganglioside 2 monoclonal antibodies. Although HR NBL is now better characterized and aggressive multimodal therapy is applied, the outcomes of treatment are still poor, with overall survival and event-free survival of approximately 40% and 30% at 3-years, respectively. The short and long-term side effects of therapy are tremendous. HR NBL carries a high mortality rate accounting for nearly 15% of pediatric cancer deaths. However, most mortalities are attributed to the high frequency of disease relapse (50%) and disease reactiveness to therapy (20%). Newer treatment strategies are therefore urgently needed. Recent discoveries in the field of biology and molecular genetics of NBL have led to the identification of several targets that can improve the treatment results. In this review, we discuss the different aspects of the epidemiology, biology, clinical presentations, diagnosis, and treatment of HR NBL, in addition to the recent developments in the management of the disease.

<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>

We recently showed that relapsed neuroblastoma (NB) tumors harbor an increased somatic mutational burden, enriched for ALK or RAS-MAPK activating lesions, nominating targeted therapeutic strategies for a condition that is largely incurable. We also showed that these mutations can be present in subclonal tumor cell populations at diagnosis, suggesting that they may contribute to acquired chemoradiotherapy resistance. We hypothesize that subclonal mutations in NB oncogenes are common at diagnosis, are biomarkers for treatment failure, and can be targeted in conjunction with standard chemotherapy to improve patient survival. Here, we focus on the first aspect of this broad hypothesis by presenting the subclonal landscape of diagnostic, high-risk neuroblastomas. A meta-analysis of our previously published primary/relapse (N=23), whole exome (N=232) and whole genome (N=159) diagnostic data, and recent sequencing study of 78 diagnostic and 67 relapse cases were used to inform design of a custom amplicon panel for ultra-deep sequencing. The 28-gene panel covers 57 mutations in frequently mutated genes. Here, 250 high-risk primary tumors were sequenced to an average depth of 50,000X. More subclonal mutations were called (mean=36) compared to clonal alterations (mean=3), demonstrating the complex subclonal architecture in these tumors. We validated five clonal ALK mutations we originally discovered and identified 15 additional pathogenic ALK mutations with mutant allele frequencies (MAFs) ranging from 0.03-23%. Known ALK activating mutations were found at codons 1170, 1174, 1196, 1245, 1275 in 15 of 38 diagnostic primary tumors. This 39.5% frequency is much higher than the 14% frequency defined by us using Sanger or other sequencing methods. Improvement of subclonal mutation detection of actionable, relapse-specific driver mutations demonstrates the clinical utility of utilizing this sequencing methodology at diagnosis to enable timely improvement of outcomes for children with high-risk refractory NB. Citation Format: Jo Lynne Harenza, Maura A. Diamond, Derek A. Oldridge, Olivia Padovan-Merher, Pichai Raman, Yael P. Mosse, John M. Maris. Defining the subclonal landscape of high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3000. doi:10.1158/1538-7445.AM2017-3000

Central nervous system (CNS) metastatic spread in neuroblastoma (NB) is rare and occurs more often at relapse/progression. We report on CNS involvement in high risk (HR) NB over 25 years. For this retrospective study, we reviewed the CNS imaging of all the patients treated at Bambino Gesù Children Hospital from 1 July 1996 to 30 June 2022. A total of 128 patients with HR NB were diagnosed over 26 years. Out of 128 patients, CNS metastatic spread occurred in 6 patients: 3 patients presented a metastatic spread at diagnosis, while in 3 patients, CNS was involved at relapse. Overall, the rate of occurrence of CNS spread is 4.7% with the same distribution at diagnosis and at relapse, namely 2.3%. Interestingly, CNS spread at diagnosis was observed only before 2012, whereas CNS was observed at relapse only after 2012, in the immunotherapy era. CNS metastases presented similar imaging features at diagnosis and at relapse, with a peculiar hemorrhagic aspect and mainly hemispheric localization in patients with bone skull involvement at the time of diagnosis. The outcome is dismal, and 3 out of 6 patients died for progressive disease.

Single-cell transcriptomic atlas-guided NRG3-ERBB4 axis enhances the efficacy of chemo-immunotherapy during induction phase in high-risk neuroblastoma.

Background: High-risk neuroblastoma (HRNB) is an aggressive form of the most common extracranial solid tumor cancer in children, often characterized by amplified levels of MYCN. HRNB patients in remission have a high relapse rate, resulting in poor survival. Recent studies have shown that cancer stem cells (CSCs) play a role in the progression, chemoresistance, and relapse of many cancers. Difluoromethylornithine (DFMO) is being evaluated to prevent relapse in clinical trials. Our current study examines the in vitro and in vivo effects of DFMO on tumor-initiating cells through evaluating CSC viability, CSC markers and senescence. Methods: BE(2)-C and SMS-KCNR established HRNB cell lines were injected subcutaneously at limiting cell dilutions into nude mice. DFMO was administered in the drinking water (2%, by volume) for 50 and 75 days for BE(2)-C and SMS-KCNR, respectively. Tumor formation was monitored, and LDA was performed. In a second xenograft model BE(2)-C cells were injected into nude mice, once tumors were palpable, the mice were given either normal drinking water (vehicle) or DFMO (2%)-containing water. Following 7 days of treatment, the tumors were resected and measured for volumes/weights as well as the harvested for Western blot analysis of CSC markers (OCT4, SOX2, NANOG, KLF4). BE(2)-C cells were cultured for 48 and 72h with or without 5mM DFMO and stained for SA-βGal activity as a marker of senescence. NB cell lines treated for 48 and 72h with a range of DFMO concentrations (0, 10μM, 50μM, 100μM, 1mM, 2.5mM, and 5mM) were harvested and labeled with cell surface antibodies for CSCs (CD133, CD114), senescence (CD148), differentiation (CD24), and NB (CD56) for 30min in the dark, and cell viability was determined by 7AAD staining immediately before flow cytometric analysis. Results: Limiting dilution analysis (LDA) on xenograft mice receiving DFMO showed both prevention of tumor formation (p value<0.001) and a decrease in frequency of tumor-initiating cells (p value p<0.04). Tumors harvested from mice treated with DFMO for 7 days showed decrease in volume and weight relative to control (p value <0.001) as well as a decrease in the presence of CSC markers shown by Western blot. Galactosidase (SA-βGal) staining of cultured HRNB cells showed the induction of senescence in HRNB cells when treated with DFMO by 72h (p value <0.001). Flow cytometric analysis determined that DFMO specifically targeted the CSC subpopulation at as low as 50uM concentrations (100uM p value<0.05); CSCs had decreased viability and higher levels of senescence-associated protein expression than the overall NB population (p value<0.05). Conclusions: Our results show the preferential induction of senescence and overall decrease in the CSC population at very low doses of DFMO. This may expose a novel treatment mechanism to inhibit CSC function and prevent relapse in HRNB patients. Citation Format: Tracey Avequin, Austin Goodyke, Joseph Zagorski, Tyler Maser, Giselle L. Saulnier Sholler. DFMO preferentially drives cancer stem cells in neuroblastoma towards senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3190.