NB Tumors Research Articles

From the identification of actionable molecular targets to the generation of faithful neuroblastoma patient-derived preclinical models

BackgroundNeuroblastoma (NB) represents the most frequent and aggressive form of extracranial solid tumor of infants. Although the overall survival of patients with NB has improved in the last years, more than 50% of high-risk patients still undergo a relapse. Thus, in the era of precision/personalized medicine, the need for high-risk NB patient-specific therapies is urgent.MethodsWithin the PeRsonalizEd Medicine (PREME) program, patient-derived NB tumors and bone marrow (BM)-infiltrating NB cells, derived from either iliac crests or tumor bone lesions, underwent to histological and to flow cytometry immunophenotyping, respectively. BM samples containing a NB cells infiltration from 1 to 50 percent, underwent to a subsequent NB cells enrichment using immune-magnetic manipulation. Then, NB samples were used for the identification of actionable targets and for the generation of 3D/tumor-spheres and Patient-Derived Xenografts (PDX) and Cell PDX (CPDX) preclinical models.ResultsEighty-four percent of NB-patients showed potentially therapeutically targetable somatic alterations (including point mutations, copy number variations and mRNA over-expression). Sixty-six percent of samples showed alterations, graded as “very high priority”, that are validated to be directly targetable by an approved drug or an investigational agent. A molecular targeted therapy was applied for four patients, while a genetic counseling was suggested to two patients having one pathogenic germline variant in known cancer predisposition genes. Out of eleven samples implanted in mice, five gave rise to (C)PDX, all preserved in a local PDX Bio-bank. Interestingly, comparing all molecular alterations and histological and immunophenotypic features among the original patient’s tumors and PDX/CPDX up to second generation, a high grade of similarity was observed. Notably, also 3D models conserved immunophenotypic features and molecular alterations of the original tumors.ConclusionsPREME confirms the possibility of identifying targetable genomic alterations in NB, indeed, a molecular targeted therapy was applied to four NB patients. PREME paves the way to the creation of clinically relevant repositories of faithful patient-derived (C)PDX and 3D models, on which testing precision, NB standard-of-care and experimental medicines.

The survivin-ran inhibitor LLP-3 decreases oxidative phosphorylation, glycolysis and growth of neuroblastoma cells

BackgroundNeuroblastoma (NB), the most common extracranial solid malignancy in children, carries a poor prognosis in high-risk disease, thus requiring novel therapeutic approaches. Survivin is overexpressed in NB, has pro-mitotic and anti-apoptotic functions, and impacts on oxidative phosphorylation (OXPHOS) and aerobic glycolysis. The subcellular localization and hence function of survivin is directed by the GTPase Ran.AimTo determine efficacy and modes of action of the survivin-Ran inhibitor LLP-3 as a potential novel therapy of NB.MethodsSurvivin and Ran mRNA expression in NB tumors was correlated to patient survival. Response to LLP-3 in NB cell lines was determined by assays for viability, proliferation, apoptosis, clonogenicity and anchorage-independent growth. Interaction of survivin and Ran was assessed by proximity-linked ligation assay and their subcellular distribution by confocal immunofluorescence microscopy. Expression of survivin, Ran and proteins important for OXPHOS and glycolysis was determined by Western blot, hexokinase activity by enzymatic assay, interaction of survivin with HIF-1α by co-IP, and OXPHOS and glycolysis by extracellular flux analyzer.ResultsHigh mRNA expression of survivin and Ran is correlated with poor patient survival. LLP-3 decreases viability, induces apoptosis, and inhibits clonogenic and anchorage-independent growth in NB cell lines, including those with MYCN amplification, and mutations of p53 and ALK. LLP-3 inhibits interaction of survivin with Ran, decreasing their concentration both in the cytoplasm and the nucleus. LLP-3 impairs flexibility of energy metabolism by inhibiting both OXPHOS and glycolysis. Metabolic inhibition is associated with mitochondrial dysfunction and attenuated hexokinase activity but is independent of HIF-1α.ConclusionLLP-3 attenuates interaction and concentration of survivin and Ran in NB cells. It controls NB cells with diverse genetic alterations, associated with inhibition of OXPHOS, aerobic glycolysis, mitochondrial function and HK activity. Thus, LLP-3 warrants further studies as a novel drug against NB.

Serum brain-derived neurotrophic factor (BDNF) as predictors of childhood neuroblastoma relapse

BackgroundNeuroblastoma (NB) is a childhood malignant tumor,50% of high-risk NB children still have recurrence, and the long-term survival rate is very low. NB tumors expressing high levels of BDNF/TrkB are associated with poor survival outcomes.In this study, we show that the trends of serum concentration of BDNF at different growth stages after birth, and explore the relationship with NB replase.MethodsIn experiment 1, 87 subjects were enrolled and divided into four groups, neonates group、 children group、adults group and NB patients. The distribution of serum concentration of BDNF by ELISA. In experiment 2, we studied BDNF in stage 4 NB patients to determine their frequency, correlation with clinical parameters, and prognostic impact.ResultsFirst, we identified that serum BDNF concentration decreased from the newborn to childhood in healthy subjects, while it was relatively high in children(age > 1 year) with NB. In the second phase our studies showed no significant increase in serum BDNF concentration in these NB patients, with adverse pathologic features, large tumor maximum diameter, and MYCN amplification. After comprehensive treatment, levels of BDNF gradually increased in children with recurrence and decreased in the remission group. High serum BDNF concentration was associated with relapse. Of 21 stage 4 neuroblastoma patients, adopted a comprehensive treatment approach including ATO-basic modified chemotherapy, traditional radiotherapy,stem cell transplatation and immunotherapy. 76% of alive patients having > 3 years follow-up. Conclusion:The aim is to show that BDNF is a predictor of recurrence risk of NB.

Abstract 3552: YAP-TEAD2 binding mediates therapy resistance in RAS-driven neuroblastoma

Abstract Background/Objectives: Despite intensive multimodal therapy, greater than 50% of children with high-risk neuroblastoma (HR NB) relapse with incurable disease. Next generation sequencing of primary HR NB tumors identified an increase in activating mutations in the RAS/RAF/MAPK pathway. Moreover, gene set enrichment analyses showed a significant decrease in expression of genes suppressed by the Yes-Associated Protein (YAP) at relapse, suggesting increased YAP transcriptional repression. YAP binds with TEAD family transcription factors to regulate gene expression. We have shown that YAP promotes chemotherapy and MEK inhibitor resistance in RAS-mutated NB tumors in vivo by suppressing the expression of Harakiri (HRK), a BH3-only pro-apoptotic protein activated in response to tumor environmental stress such as serum starvation. Our overall objective is to elucidate how YAP represses HRK and tumor suppressor genes globally, and to enhance MEK inhibitor potency by combining MEK inhibition with agents that inhibit YAP or induce HRK to restore the tumor environmental stress response and apoptosis in RAS-mutated NB. Design/Methods: We used publicly available databases to identify TEAD binding sites on the HRK gene locus in NB. To assess the global state of methylation, we treated NB cells, SK-N-AS (NRASQ61K mutation, MYCN non-amplified) and NLF (NF1 deletion, MYCN amplified), with demethylating agent azacitidine and evaluated HRK expression. To identify the specific TEAD (1-4) binding partner to YAP, we performed siRNA and co-immunoprecipitation studies. We further tested novel YAP-TEAD small molecule inhibitors with varying TEAD1-4 inhibition specificity in SK-N-AS and NLF cells in vitro. Results: We observed that TEAD binds near cis-regulatory regions on the HRK gene locus in NB. We found that HRK expression is restored when SK-N-AS and NLF cells are treated with azacitidine despite YAP expression increasing. We also identified TEAD2 as the specific binding partner to YAP in NB and found that TEAD2 is necessary for HRK regulation. Novel YAP-TEAD small molecule inhibitors affect NB cell viability under serum-deprived conditions in vitro, especially the inhibitor with highest specificity against TEAD2, and affect YAP-TEAD downstream targets. Conclusions: YAP-TEAD2 binding is essential for HRK regulation in RAS-mutated NB and thus is a logical therapeutic target to restore therapy response. Further studies are ongoing to test YAP-TEAD small molecule inhibitors in combination with MEK inhibitors and in vivo. Citation Format: Jenny Shim, Andrew Ho, Hunter C. Jonus, Adeiye A. Pilgrim, Benjamin G. Barwick, Tracy T. Tang, Lawrence H. Boise, Kelly C. Goldsmith. YAP-TEAD2 binding mediates therapy resistance in RAS-driven 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 3552.

Silencing of Circ_0135889 Restrains Proliferation and Tumorigenicity of Human Neuroblastoma Cells

IntroductionNeuroblastoma (NB) is the most common extracranial solid tumor in infants and young children. Circular ribonucleic acid (RNA) hsa_circ_0135889 (circ_0135889; hsa_circ argonaute 2 _001) is highly expressed in multiple cancer tissues, including NB. However, its role in tumor progression of NB was unclear. MethodsReal-time quantitative polymerace chain reaction was used to detect RNA expression, and western blotting, or immunohistochemistry was used to measure protein expression. Functional experiments were performed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, 5-Ethynyl-2’- deoxyuridine, Annexin V-fluorescein isothiocyanate/propidium iodide, and transwell assays, as well as xenograft tumor model. The intermolecular interaction was predicted by online databases and confirmed by dual-luciferase reporter assay and RNA pull-down assay. ResultsCirc_0135889 and neuronal differentiation 1 (NEUROD1) were upregulated whilst microRNA (miR)-127-5p, was downregulated in NB tumors and immortalized NB cells. Silencing of circ_0135889 could suppress cell proliferation, migration and invasion, but enhance apoptosis rate of NB cells in vitro. More importantly, circ_0135889 depletion inhibited xenograft tumor growth of NB cells. Circ_0135889 was a sponge for miR-127-5p, and inhibition of miR-127-5p counteracted the inhibitory impact of circ_0135889 knockdown on the malignant behaviors of NB cells. Moreover, NEUROD1 was a direct target of miR-127-5p, and miR-127-5p exerted the anti-tumor role in NB cells by targeting NEUROD1. Furthermore, circ_0135889 regulated NEUROD1expression by sponging miR-127-5p. ConclusionsCirc_0135889 promoted the tumorigenicity of NB by regulating miR-127-5p/NEUROD1 axis, which might provide a promising therapeutic target for NB.

Abstract 5719: Defining TCF4’s role as a novel key regulator in mediating neuroblastoma cell identity

Abstract Neuroblastomas (NB) are embryonal childhood tumors that derive from the multipotent neural crest cells (NCCs) of the peripheral nervous system. NB accounts for more than 15% of all childhood cancer-related deaths. Despite the most intensive multimodal therapy, more than 50% of patients with high-risk NB relapse with often fatal, resistant disease. Finding novel treatments, especially for relapse disease, is desperately needed for high-risk NB. In cancers, distinct transcription factors TFs networks forming core regulatory circuitries (CRCs) control gene expression programs that drive cell identity. Recent studies reported the presence of two types of identity states in NB tumors: one establishing a more proliferative adrenergic (ADRN) cell state and a second establishing a more invasive, therapy-resistant mesenchymal (MES) cell state. While most studies are investigating the CRC members for targeting MYCN amplified NB (ADRN) or the MES/NCCs cell states, most primary tumors are heterogeneous, comprised of cells with both identities. Thus, our work is focused on identifying universal factors shared across different lineage states to determine whether targeting both identities could be a valuable strategy for NB treatment. NB was identified from a screen of 673 cancer cell lines as highly sensitive to the BET-inhibitor JQ1. In our work, we identified the class I basic helix-loop-helix transcription factor (bHLH) TCF4 as a critical target of JQ1 mediated cell death in NB. TCF4 functions as a transcriptional hub that heterodimerizes with class II (bHLH) TFs including the proneural gene ASCL1 (ADRN), and TWIST1 a master regulator of the MES state. Heterodimers formed between TCF4 and ASCL1, and TWIST1, have been demonstrated to provide lineage-specific differentiation from embryonic stem cells. Here, we hypothesize that TCF4 is a cell dependency gene in NB that is crucial for determining NB identity. Using RNA seq analysis of TCF4 dox-inducible shRNA stable cell lines, we found that the gene expression changes in TCF4-depleted cells indicate a role for TCF4 in differentiation, cell signaling, and neurodevelopment. Interestingly, gene set enrichment analysis (GSEA) indicates that both identity states are suppressed after silencing of TCF4. Our data also shows that inducible decrease of TCF4 protein resulted in a significant inhibition of NB cell growth, and impaired tumor growth in vivo. Furthermore, we identified TCF4 targets in NB by combined analysis of TCF4 ChIP-seq data and gene expression changes following TCF4 knockdown. ENRICHR analysis suggests that differentially expressed genes in TCF4-knockdown cells that also have a TCF4 ChIP-seq peak are targets of the TFs TWIST1 (MES) and HAND1 (ADRN) that interact directly with TCF4. In conclusion, our preliminary data supports the hypothesis that TCF4 is a master transcriptional regulator of NB oncogenic program that is crucial for maintaining NB identity states. Citation Format: Nour Aljouda, Dewan Shrestha, Megan Walker, Satyanarayana Alleboina, Evan Glazer, Yong Cheng, Kevin Freeman. Defining TCF4’s role as a novel key regulator in mediating neuroblastoma cell identity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5719.

Abstract 3901: Modeling and deciphering COJEC resistance in high risk neuroblastoma

Abstract Neuroblastoma (NB) is a pediatric cancer of the sympathetic nervous system that accounts for 15% of childhood cancer deaths. High-risk (HR) NB tumors are treated with an aggressive multimodal therapy, including the COJEC induction protocol which is a combination of five different chemotherapeutic agents. However, around 15% of primary tumors are refractory from the start and over 60% present treatment resistant relapses. In this project we aim to decipher the mechanisms involved in treatment resistance in HR-NB. Using various NB patient-derived xenograft (PDX) models we designed and tested a clinically-relevant protocol that mimics COJEC treatment and surgical resection. RNA-sequencing, SNP analysis, and histology, were used to explore the molecular mechanisms involved in treatment response. With our COJEC-like protocol we were able to replicate the original patient response in our PDX models. NB PDX1 presented upfront progression and a lack of morphological differentiation. At a molecular level it was characterized by genome duplication and high number of chromosomal aberrations, as well as a high expression of early development/mesenchymal and cell cycle genes. In contrast, NB PDX3 presented an overall good response, leading to a 22% relapse rate after surgery. Relapsed tumors displayed high expression of early development/mesenchymal genes, similar to those seen in refractory PDX1. Meanwhile, the PDX3 tumors collected during surgery that did not relapse (cured) showed low MYCN expression, downregulation of cell cycle and DNA repair genes, along with a strong upregulation of nervous system/nor-adrenergic genes. These genetic signatures derived from the cured PDX3 samples also correlated with good prognosis in large cohorts of HR-NB patients. Despite observing a convergent/parallel evolution of specific genetic aberrations across our PDXs, their presence or absence does not predict prognosis. Instead, the high RNA expression levels of those specific genes correlates with treatment response both in our PDX models and in patient cohorts. Finally, we established 3D tumor organoids derived from treated PDX tumors and confirmed they maintain the molecular characteristics of the parental tumor as well as the acquired COJEC resistance and the tumorigenic capacity, making them an excellent tool to test novel therapeutics against HR-NB. In conclusion, we designed a clinically-relevant COJEC-like protocol that can mimic patient response in PDX models. Early developmental gene signatures appear to correlate with treatment resistance in HR-NB, while the expression of nor-adrenergic signatures after chemotherapy are associated with good prognosis, independently of the accumulation of copy number aberrations. Based on our results, we propose that a combination of DNA and RNA analysis at the surgical resection time point after COJEC could be of clinical interest to evaluate COJEC treatment response and predict future relapses. Citation Format: Adriana Mañas, Kristina Aaltonen, Natalie Andersson, Karin Hansson, Alexandra Seger, Hiroaki Yasui, Katarzyna Radke, Javanshir Esfandyari, David Lindgren, David Gisselsson, Daniel Bexell. Modeling and deciphering COJEC resistance in high risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3901.

The Effects of Variable Selection and Dimension Reduction Methods on the Classification Model in the Small Round Blue Cell Tumor Dataset

Objective: The purpose of this study is to investigate and compare the effects of different dimension reduction methods (PCA, ICA, PCA + Forward Selection, ICA + Forward Selection) on the K-NN classifier using open access gene expression data of small round blue cell tumor types.Methods: In this study, open access gene expression data of small round blue cell tumor types was used for investigate and compare the effects of different dimension reduction methods. In the study, PCA, ICA, PCA + Forward Selection, ICA + Forward Selection were used as different dimension reduction methods together with K-NN classification method.Results: Accuracy values obtained from the dimension reduction model made with PCA on K-NN model; for EWS, BL, NB, and RMS type tumors with 93.51%, 91.14%, 92.31%, and 94.74% respectively. Accuracy values obtained from the dimension reduction model made with PCA + Forward Selection on K-NN model; for EWS, BL, NB, and RMS type tumors with 96.25%, 96.25%, 95.06% and 95.47%, respectively. Accuracy values obtained from the dimension reduction model made with ICA on K-NN model; for EWS, BL, NB, and RMS type tumors with 91.89%, 90.67%, 88.31% and 89.47% respectively. Accuracy values obtained from the dimension reduction model made with ICA+ Forward Selection on K-NN model; for EWS, BL, NB, and RMS type tumors with 93.51%, 91.14%, 92.31% and 94.74% respectively.Conclusion: In this study, the model created with PCA gives higher results than the model created with ICA. In addition, according to the results of the models obtained by applying the Forward selection method on these 2 models, the forward selection method has increased the classification performance.

Neuroblastoma xenograft models demonstrate the therapeutic potential of 177Lu-octreotate

BackgroundNeuroblastoma (NB) is one of the most frequently diagnosed tumors in infants. NB is a neuroendocrine tumor type with various characteristics and features, and with diverse outcome. The most malignant NBs have a 5-year survival rate of only 40–50%, indicating the need for novel and improved treatment options. 177Lu-octreotate is routinely administered for treatment of neuroendocrine tumors overexpressing somatostatin receptors (SSTR). The aim of this study was to examine the biodistribution of 177Lu-octreotate in mice bearing aggressive human NB cell lines, in order to evaluate the potential usefulness of 177Lu-octreotate for treatment of NB.MethodsBALB/c nude mice bearing CLB-BAR, CLB-GE or IMR-32 tumor xenografts (n = 5–7/group) were i.v. injected with 0.15 MBq, 1.5 MBq or 15 MBq 177Lu-octreotate and sacrificed 1 h, 24 h, 48 h and 168 h after administration. The radioactivity concentration was determined for collected tissue samples, tumor-to-normal-tissue activity concentration ratios (T/N) and mean absorbed dose for each tissue were calculated. Immunohistochemical (IHC) staining for SSTR1–5, and Ki67 were carried out for tumor xenografts from the three cell lines.ResultsHigh 177Lu concentration levels and T/N values were observed in all NB tumors, with the highest for CLB-GE tumor xenografts (72%IA/g 24 h p.i.; 1.5 MBq 177Lu-octreotate). The mean absorbed dose to the tumor was 6.8 Gy, 54 Gy and 29 Gy for CLB-BAR, CLB-GE and IMR-32, respectively, p.i. of 15 MBq 177Lu-octreotate. Receptor saturation was clearly observed in CLB-BAR, resulting in higher concentration levels in the tumor when lower activity levels where administered. IHC staining demonstrated highest expression of SSTR2 in CLB-GE, followed by CLB-BAR and IMR-32.ConclusionT/N values for all three human NB tumor xenograft types investigated were high relative to previously investigated neuroendocrine tumor types. The results indicate a clear potential of 177Lu-octreotate as a therapeutic alternative for metastatic NB.

Abstract 3036: ALK protein expression and its prognostic significance in MYCN non amplified neuroblastoma

Abstract Anaplastic lymphoma kinase (ALK) point mutations (within the kinase domain), gene amplification and protein overexpression are identified (8-14%) both in familial and sporadic high-risk neuroblastoma (HR-NB). ALK mutations are much more frequent at time of relapse and were associated with poor clinical outcomes for children with HR-NB. Evidently, ALK is near exclusively co-amplified with MYCN, and the cooperative activity of ALK and MYCN are known to drive HR-NB pathogenesis. However, the ALK protein expression and its role in the prognosis of MYCN non-amplified NB remained unexplored. In this study, NB tumors representing varied stages, ages, sites, diagnosis/progressive disease, primary/metastatic site from a cohort MYCN non-amplified NB patients (n=92) were custom archived in TMAs and stained with ALK (immunohistochemistry) antibody. The correlations between ALK protein expression, and clinico-pathological and biological variables of MYCN non-amplified NB tumors were analyzed. Assessing the strong positivity, high expression of ALK protein could be detected in 47 (51%) of the 92 NB tumors. The majority of NBs with evident of ALK protein high expression exhibited undifferentiated or poorly differentiated histology (39/47, 82.9%). ALK protein high expression significantly predicted worse survival outcomes for NB (and HR-NB subset) patients with MYCN non-amplified status. Transcriptional data mining (R2: Genomics Analysis and Visualization Platform) across multiple cohorts of MYCN non-amplified NB patients revealed unique and defined associations of ALK to the adverse clinical prognostic factors (e.g. advanced disease stage, risk) and poor survival outcomes. Our study recognized the prognostic role of ALK protein expression in MYCN non-amplified NB. Since, ALK is indicated to transactivate MYCN, increase MYCN protein expression, and coordinate disease progression; further studies are warranted to define the mechanism(s) of ALK high-expression associated disease evolution in MYCN non-amplified HR-NB.Funding: This work was partially or in full, funded by Oklahoma Center for the Advancement of Science and Technology, OCAST-HR19-04; National Institutes of Health, NIH-P20GM103639 and; NIH-NCI- Cancer Center Support Grant P30CA225520 Citation Format: Natarajan Aravindan, Dinesh Babu Somasundaram, Santny Shanmugarama, Sheeja Aravindan. ALK protein expression and its prognostic significance in MYCN non amplified neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3036.

Cell surface Nucleolin represents a novel cellular target for neuroblastoma therapy

BackgroundNeuroblastoma (NB) represents the most frequent and aggressive form of extracranial solid tumor of infants. Nucleolin (NCL) is a protein overexpressed and partially localized on the cell surface of tumor cells of adult cancers. Little is known about NCL and pediatric tumors and nothing is reported about cell surface NCL and NB.MethodsNB cell lines, Schwannian stroma-poor NB tumors and bone marrow (BM)-infiltrating NB cells were evaluated for the expression of cell surface NCL by Flow Cytometry, Imaging Flow Cytometry and Immunohistochemistry analyses. The cytotoxic activity of doxorubicin (DXR)-loaded nanocarriers decorated with the NCL-recognizing F3 peptide (T-DXR) was evaluated in terms of inhibition of NB cell proliferation and induction of cell death in vitro, whereas metastatic and orthotopic animal models of NB were used to examine their in vivo anti-tumor potential.ResultsNB cell lines, NB tumor cells (including patient-derived and Patient-Derived Xenografts-PDX) and 70% of BM-infiltrating NB cells show cell surface NCL expression. NCL staining was evident on both tumor and endothelial tumor cells in NB xenografts. F3 peptide-targeted nanoparticles, co-localizing with cell surface NCL, strongly associates with NB cells showing selective tumor cell internalization. T-DXR result significantly more effective, in terms of inhibition of cell proliferation and reduction of cell viability in vitro, and in terms of delay of tumor growth in all NB animal model tested, when compared to both control mice and those treated with the untargeted formulation.ConclusionsOur findings demonstrate that NCL could represent an innovative therapeutic cellular target for NB.

BTK Inhibition Reverses MDSC-Mediated Immunosuppression and Enhances Response to Anti-PDL1 Therapy in Neuroblastoma.

Simple SummaryNeuroblastoma (NB) is the most common pediatric malignancy, and patients with the high-risk disease show a worse prognosis despite advanced treatments, including immunotherapy. Myeloid-derived suppressor cells (MDSC) frequently accumulate in NB tumors, where they induce immunosuppression and hamper efficient antitumor immune responses. In the current study, we observed that Bruton’s tyrosine kinase (BTK) is highly expressed in both monocytic and granulocytic MDSCs isolated from spleens of mice bearing NB tumors and administration of BTK inhibitor ibrutinib reduced MDSC-mediated immunosuppression, tumor growth, and enhanced anti-PDL1 checkpoint inhibitor therapy in mice bearing NB tumors. These studies demonstrated that ibrutinib could serve as a promising therapeutic agent to control MDSC-mediated immune suppression in NB.MDSCs are immune cells of myeloid lineage that plays a key role in promoting tumor growth. The expansion of MDSCs in tumor-bearing hosts reduces the efficacy of checkpoint inhibitors and CAR-T therapies, and hence strategies that deplete or block the recruitment of MDSCs have shown benefit in improving responses to immunotherapy in various cancers, including NB. Ibrutinib, an irreversible molecular inhibitor of BTK, has been widely studied in B cell malignancies, and recently, this drug is repurposed for the treatment of solid tumors. Herein we report that BTK is highly expressed in both granulocytic and monocytic murine MDSCs isolated from mice bearing NB tumors, and its increased expression correlates with a poor relapse-free survival probability of NB patients. Moreover, in vitro treatment of murine MDSCs with ibrutinib altered NO production, decreased mRNA expression of Ido, Arg, Tgfβ, and displayed defects in T-cell suppression. Consistent with these findings, in vivo inhibition of BTK with ibrutinib resulted in reduced MDSC-mediated immune suppression, increased CD8+ T cell infiltration, decreased tumor growth, and improved response to anti-PDL1 checkpoint inhibitor therapy in a murine model of NB. These results demonstrate that ibrutinib modulates immunosuppressive functions of MDSC and can be used either alone or in combination with immunotherapy for augmenting antitumor immune responses in NB.

Potent STING activation stimulates immunogenic cell death to enhance antitumor immunity in neuroblastoma

BackgroundNeuroblastoma (NB) is a childhood cancer for which new treatment options are needed. The success of immune checkpoint blockade in the treatment of adult solid tumors has prompted the exploration...

Abstract 2890: Paternal pre-conception stress as a potential risk factor for neuroblastoma

Abstract Neuroblastomas (NBs) are pediatric tumors that lack adequate treatment for their aggressive form, despite many efforts focusing on novel therapies. Although the disease is known to have a genetic background, its etiology cannot be explained solely by genetics. Yet, the role of environment in NB development is understudied. Thus, there are no established risk factors for NB aside from rare genetic predispositions and therefore no preventative measures. NBs arise due to defects in sympathetic neuron (SN) differentiation, which occurs in the fetus. Thus, we hypothesize that prenatal or pre-conception exposures may interfere with this process and promote NB. Maternal stress during pregnancy and paternal pre-conception stress induce neurodevelopmental defects in offspring by direct effects on fetal development or epigenetic changes, respectively. Indeed, we have previously shown that in a mouse model of NB prenatal stress interferes with SN differentiation in offspring, accelerating NB development and leading to its increased frequency and malignancy. The goal of the current study was to determine if a similar increase in risk of NB development can result from paternal pre-conception stress. To test the effect of paternal stress on NB, we subjected TH-MYCN males to 6 weeks of chronic unpredictable stress (CUS) prior to mating with wild type females and assessed the impact of stress on 1) miRNA content in the germline (RNAseq); 2) fertility and fetal survival; 3) NB frequency, latency, growth and metastasis in their hemizygous TH-MYCN offspring (periodic MRI, histological analyses). CUS resulted in significant changes in non-coding RNA content of the germline, including miRNAs and tsRNAs. Of particular interest was a stress-induced down-regulation of sperm miRNAs, including miR-200b and miR-128, which are involved in neuroblast differentiation, inhibit NB invasiveness and are inactivated in NB tumors. Paternal stress decreased the rate of successful pregnancies from 75% in controls to 37.5% and increased frequency of death in utero from 5.5% to 29.2% (p=0.02). Moreover, offspring of stressed fathers exhibited accelerated NB development (60% vs 0% of mice with tumors at 6 weeks of age for CUS and control groups, respectively; p=0.03) and growth (p=0.3), higher tumor frequency (60% vs 25% at 14 weeks of age, p=0.03), as well as a trend toward a more metastatic phenotype. However, these effects on NB formation and phenotype were observed solely in male offspring. In summary, our data provide the first evidence for the role of paternal preconception stress in NB development. If confirmed, our findings may open new avenues for NB prevention and identifying biomarkers of increased NB risk. Citation Format: Sung-Hyeok Hong, Susana Galli, Raquel Santana da Cruz, Lu Jin, Larissa Wetlisbach, Shiya Zhu, Abir Malik, Jason Tilan, Sonia de Assis, Joanna B. Kitlinska. Paternal pre-conception stress as a potential risk factor for neuroblastoma [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 2890.

Abstract 4570: Selective depletion of regulatory T cells suppressed neuroblastoma tumor growth in mice

Abstract Purpose of the study Neuroblastoma (NB) is the most common solid extracranial tumor in childhood. Regulatory T cells (Treg) are known to have immune suppressive capacity and to be involved in tolerance establishment. As they support tumor growth and help some tumors to overcome recognition by the host, they are considered to be an important target. Treg are reportedly increased in blood of NB patients. However, there are no scientific proofs to date that they are involved in neuroblastoma growth. To address this important question we employed Foxp3-DTR mice to selectively deplete Treg upon orthotopic application of syngeneic neuroblastoma cells. Methods Orthotopic NB tumors were generated in mice Foxp3-DTR mice and in C57Bl/6J controls by subcapsular tumor cell injection of NB975A cells into the left kidney. Primary tumor growth was analysed for 23 days by high frequency ultrasound measurement. To deplete Treg, InjectionDepletion of Diphtheria Toxin (DT, 15 ng/g bodyweight) intraperitoneally every 4 days in Foxp3.DTR mice depleted the Treg pool. Control Foxp3.DTR animals received PBS and control C57B/l6 received DT. All mice were sacrificed 23 days after tumor inoculation. Tumor as wells as immune cells harvested from spleen and inguinal lymph nodes were analyzed by Flow Cytometry, Immunofluorescenceimmunohistochemistry and qRT-PCR. Results In control mice bearing tumors, Treg presence was confirmed at the contact zone between the kidney and the growing, inoculated tumor. Mice devoid of Treg (Foxp3-DTR+DT) showed a significantly reduced tumor growth compared to animals with normal Treg frequencies (Foxp3-DTR+PBS). Tumor growth in wild type controls was not affected by the application of DTR. Conclusion Ablation of Treg hindered the growth of NB, strongly suggesting their potential as therapeutic target for this aggressive childhood tumor. Citation Format: Sophie von Lenthe, Christine Weißenborn, Ana Claudia Zenclussen, Stefan Fest, Leonid Metelitsa. Selective depletion of regulatory T cells suppressed neuroblastoma tumor growth in mice [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 4570.

Abstract A18: Netrin-1/Neogenin-1 promotes neuroblastoma cell migration via activation of Integrin β1

Abstract Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. It is derived from the sympatho-adrenal lineage of the neural crest, during development of the sympathetic nervous system, and usually arises in the adrenal gland or sympathetic ganglia. Since >50% of diagnosed cases are metastatic, it is important to know the mechanisms underlying its dissemination. During metastasis, cells migrate, invade, and colonize local and distant organs. Focal Adhesion Kinase (FAK) and Integrin β1 are key regulators of focal adhesion formation and cell migration. FAK induces Integrin β1 activation, focal adhesion turnover, and consequently cell migration, and it has been shown to be upregulated in tumors. FAK binds several upstream receptors including Neogenin-1 (NEO1), which is involved in axonal guidance, angiogenesis, and neuronal cell migration. On the other hand, NEO1 binds to the Netrin family of ligands. Analysis of public databases of NB tumors reveals that high expression of NEO1 correlates with poor patient survival. The aim of this work is to evaluate the role of migrationrelated signaling pathways downstream of NEO1/Netrin-1 (main ligand of the Netrin family). To this end, we focused on FAK and Integrin β1 interaction/activation. Western blot in SK-N-SH (MYCN WT) and LAN-1 (MYCN high) NB cell lines indicated that NEO1 and Netrin-4 are expressed while Netrin-1 is only expressed in LAN-1. Also, NEO1 associated with Netrin-1, as shown by co-immunoprecipitation assays in both cell lines. To evaluate the contribution of NEO1 in cell migration, integrin β1 activation, and metastasis, stable NEO1 knockdown cells (shNEO1) were generated using shRNAs via lentiviral infection. Cell migration was decreased in shNEO1 cells, as shown in transwell assays using Netrin-1 as chemoattractant, which could be due to decreased sensibility to the chemotactic stimulus of the ligand. We also assessed FAK dependency by pharmacologic inhibition with the PF562,271 compound in a spheroid-based cell migration assay. To further evaluate the association between NEO1 with Integrin β1 and FAK, we performed co-immunoprecipitation assays. Our data show that NEO1 associates in a complex with Integrin β1 and FAK in SK-N-SH cells. Immunoprecipitation of activated Integrin β1 with a specific conformational antibody shows that activation of Integrin β1 is less in shNEO1 cells compared to control cells. In an in vivo analysis of metastasis, SK-N-SK shNEO1 and control cells were injected into flank of immunodeficient mice (NSG strain). Metastasis was evaluated via luciferase activity in several organs, revealing that NEO1 is important to NB metastases. Our work suggests that NEO1 is a tumor progression-promoting protein, with an active role in metastasis. We propose a possible mechanism whereby NEO1, via its interaction with Netrin-1, is involved in cell migration and metastasis of NB cells. NEO1 likely plays its role by associating with FAK, allowing its phosphorylation and complex formation with Integrin β1, inducing its activation. Our results provide further information on the potential use of NEO1 as a therapeutic target to reduce metastasis in neuroblastoma. Citation Format: Andrea A. Villanueva, Ernesto Muñoz-Palma, Carlos Cubo, Vicente A. Torres, Pilar Sanchez-Gomez, Veronica Palma. Netrin-1/Neogenin-1 promotes neuroblastoma cell migration via activation of Integrin β1 [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A18.

Harmine, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor induces caspase-mediated apoptosis in neuroblastoma

BackgroundNeuroblastoma (NB) is an early childhood malignancy that arises from the developing sympathetic nervous system. Harmine is a tricyclic β-carboline alkaloid isolated from the harmal plant that exhibits both cytostatic and cytotoxic effects. Harmine is capable of blocking the activities of dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family proteins and mitogen-activated protein kinase. These kinases promote proliferation and inhibit apoptosis.MethodsFour human NB cell lines were used to study the effects of harmine treatment: SKNBE and KELLY (MYCN-amplified) as well as SKNAS and SKNFI (MYCN non-amplified). The anti-cancer properties of harmine were examined by RealTime-Glo MT cell viability assays, caspase activity assays, PARP cleavage using Western blot analysis, and flow cytometry-based Annexin V detection. A molecular interaction model of harmine bound to the DYRK2 family kinase was generated by computational docking using X-ray structures. NB tumors from human patients were profiled for DYRK mRNA expression patterns and clinical correlations using the R2 platform.ResultsThe IC50 values for harmine after 72 h treatment were 169.6, 170.8, and 791.7 μM for SKNBE, KELLY, and SKNFI, respectively. Exposure of these NB cell lines to 100 μM of harmine resulted in caspase-3/7 and caspase-9 activation as well as caspase-mediated PARP cleavage and Annexin V-positive stained cells, as early as 24 h after treatment, clearly suggesting apoptosis induction, especially in MYCN-amplified cell lines. Elevated DYRK2 mRNA levels correlated with poor prognosis in a large cohort of NB tumors.ConclusionHarmine is a known inhibitor of DYRK family kinases. It can induce apoptosis in NB cell lines, which led us to investigate the clinical correlations of DYRK family gene expression in NB tumors. The patient results support our hypothesis that DYRK inhibition by harmine and the subsequent triggering of caspase-mediated apoptosis might present a novel approach to NB therapy.

Abstract 5506: SOX11 acts as part of the MYCN-WEE1 regulatory protein complex implicated in neuroblastoma

Abstract Neuroblastoma (NB) is an aggressive lethal pediatric cancer of the developing sympatho-adrenergic nervous system and characterised by a low mutation burden while exhibiting recurrent DNA copy number alterations including chromosome 2p gain and MYCN amplification. Rare amplifications have previously allowed to identify additional oncogenes implicated in NB such as ALK and LIN28B. Here we report on three NB tumours and two NB cell lines with high level focal gain containing the SRY-related HMG-box transcription factor 11 (SOX11) as only protein-coding gene in the smallest region of overlap. The high expression levels of SOX11 in NB and the developing sympathetic nervous system, and high correlation of SOX11 mRNA and protein levels with survival outcome, suggested a role for SOX11 as oncogene and prompted us to further investigate its role in MYCN driven NB formation. SOX11 knock down in NB cell lines showed reduced colony formation capacity and G1-S cell cycle arrest. We observed increased SOX11 levels upon MYCN induction in NB cell lines, in MYCN overexpressing mouse neuroblastomas compared to normal ganglia as well as a high correlation of SOX11 and MYCN mRNA and protein levels in cell lines and tumors. As these data could indicate direct MYCN regulation of SOX11, we performed MYCN ChIPseq and indeed confirmed binding of MYCN to the SOX11 promotor. Next, we demonstrated MYCN and SOX11 cooperative binding to common targets using SOX11 and H3K27ac ChIP-sequencing. First, we showed localisation of more than half (62%) of the SOX11 binding sites at enhancers. Moreover, 19% of SOX11 targets contained an E-Box motif, typically known to be targeted by MYCN, and we showed overlap of 30% of SOX11 and MYCN binding targets. In a further step to unravel SOX11 function in NB cells, we analysed SOX11 binding partners using IP-MS and identified 9 robust interaction partners in multiple NB cell lines. These included MYCN which was confirmed by reciprocal co-IP, as well as WEE1, a tyrosine kinase critically implicated in G2-M checkpoint control for which multiple potent and specific small molecule inhibitors are available. Remarkably, upon testing of the AZD-1775 WEE1 inhibitor in 10 NB cell lines, we observed the strongest effects in cells with the highest SOX11 protein levels indicating that SOX11 expression levels can serve as predictive biomarker for AZD-1775 treatment response. In conclusion we identified high-level focal gain of SOX11 in NB tumors and cell lines and demonstrated the oncogenic role of SOX11 in MYCN driven neuroblastoma with therapeutic opportunities through interaction with WEE1. Citation Format: Bieke Decaesteker, Sara De Brouwer, Fanny De Vloed, Geertrui Denecker, Suzanne Vanhauwaert, Jolien De Wyn, Geneviève Laureys, Bjorn Menten, Pauline Depuydt, Patrick Reynolds, Jo Vandorpe, Kris Gevaert, Jan Koster, Sara Ek, Frank Speleman, Katleen De Preter. SOX11 acts as part of the MYCN-WEE1 regulatory protein complex implicated in 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 5506. doi:10.1158/1538-7445.AM2017-5506

Abstract 3867: Epigenetic siRNA and chemical screens identify SETD8 inhibition as a therapeutic strategy to reactivate p53 in high-risk neuroblastoma

Abstract Neuroblastoma (NB) is considered a failure of sympathoadrenal differentiation. High-risk neuroblastoma (HR-NB) is an aggressive pediatric tumor accounting for 15% of all pediatric oncology deaths. Less than 50% of HR-NB patients have long-term survival, despite intense multimodality treatment. Given the paucity of druggable mutations and findings that epigenetic drivers contribute to NB tumorigenesis, we undertook a chromatin-focused siRNA screen to uncover epigenetic regulators critical for survival of high-risk NBs. Of the 400 genes analyzed, high-content Opera imaging identified 53 genes whose loss of expression led to significant decreases in NB cell number with 16 also inducing differentiation. A screen with 21 epigenetic compounds in 8 NB cell lines and 2 non-transformed cell lines prioritized those siRNA hits with active tool compounds in the drug development pipeline. This revealed UNC0379 (targets SETD8) inhibited NB cell growth and identified SETD8 as an important and druggable NB target. SETD8 is the H4K20me1 methyltransferase which regulates DNA replication, chromosome condensation and gene expression. Analysis of primary NB revealed that high expression of SETD8 is associated with poor prognosis in NB (R2 platform ex. Kocak; p=1.4e-07). Levels of SETD8 were not significantly different between Stage 4 MYCN-amp compared to MYCN-WT tumors but high SETD8 levels were only associated with poor prognosis in the Stage 4 MYCN-WT(p=0.03). To understand SETD8-mechanism of action, we performed RNA-seq transcriptome analyses after genetic or pharmacological inhibition of SETD8. Ingenuity Pathway Analysis revealed that SETD8 ablation rescued p53 pro-apoptotic and cell-cycle arrest functions by activating the canonical p53 pathway. Functional studies showed SETD8 methylates p53 (K382) leading to its inactivation. Levels of p53K382me1 are higher in MYCN-WT NB cell lines compared to those with MYCN-amp. Less than 2% of NB tumors have p53 mutations but multiple mechanisms have been identified in MYCN-amp NB that functionally inactivate p53. This study identified that SETD8 inactivates p53 in NB and may be an important mechanism to inactivate p53 in MYCN-WT HR-NB. This subgroup represents 60-70% of HR-NB tumors. SETD8 inhibition led to increases in caspase-dependent cell death only in p53-WT but not -mutant or -null NB cells. Genetic rescue experiments confirmed that SETD8-induced cell death is p53 dependent and p53K382 is important for this activity. Our in vivo xenograft NB models, showed that genetic or pharmacologic (UNC0379) inhibition of SETD8 confers a significant survival advantage. This work identifies that SETD8 is a novel therapeutic target and its inhibition may be especially relevant for the subset of high-risk NB tumors with wildtype MYCN. This is the first in vivo preclinical study showing that targeting SETD8 inhibits tumor growth. Citation Format: Veronica Veschi, Zhihui Liu, Ty C. Voss, Laurent Ozbun, Berkley Gryder, Chunhua Yan, Ying Hu, Anqi Ma, Jian Jin, Sharlyn J. Mazur, Norris Lam, Barbara K. Souza, Giuseppe Giannini, Gordon L. Hager, Cheryl H. Arrowsmith, Javed Khan, Ettore Appella, Carol Thiele. Epigenetic siRNA and chemical screens identify SETD8 inhibition as a therapeutic strategy to reactivate p53 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 3867. doi:10.1158/1538-7445.AM2017-3867

Abstract 689: Immune checkpoint targeting to improve immunotherapy in neuroblastoma

Abstract Neuroblastoma (NB) is the most common extracranial solid tumor in children. Current treatments accounts only for at the most 40% of survival rate in high-risk NB patients. Hence, there is a need to develop new strategies that disrupt immunosuppressive signals in the tumor microenvironment in order to achieve robust anti-tumor immune responses. Co-inhibitory signals through receptors such as PD-1 can lead to the inactivation of T cells. On the other hand, co-stimulatory signals through receptors such as 41BB can enhance T cell activity against tumors. These receptors can serve as tools to develop strategies to optimize and enhance T cell anti-tumor functions. In this study, we used 9464D NB tumor mouse model to evaluate antibody treatment as a possible immunotherapy strategy. We further examine the infiltration and function of T cells after targeting checkpoints in 9464D NB tumor bearing mice. Blockade of various receptors in mice bearing 9464D tumor showed that anti-PD1 and anti-41BB delayed tumor growth. Combination therapy with anti-PD1 and anti-41BB antibodies significantly delayed tumor growth compared to single antibody treatment. This combination therapy led to a higher infiltration of CD8+ cells within the tumor. Splenocytes from 9464D tumor bearing mice treated with anti-PD1 and anti-41BB are tumor specific. In conclusion, our findings intend to design the best complement therapy to improve survival of patients with advanced NB. Our data demonstrates that tumor reactive T cells in NB tumors may express different checkpoint receptors including PD1 and 41BB. 9464D tumor bearing mice that received a combination of anti-PD1 and anti-41BB antibodies have smaller tumor size compared to single antibody therapy. Future direction includes examine T cells activation after combination therapy of BMT and immunecheckpoint targeting. Also we want to test adoptive transfer of T cells in tumor bearing mice after BMT. Further we want to measure the infiltration of other immune cells, such as myeloid cells and Tregs, in the tumor environment and possible target immune suppressive populations to see the effects in tumor growth. Citation Format: Myrna L. Ortiz-Ruiz. Immune checkpoint targeting to improve immunotherapy in 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 689. doi:10.1158/1538-7445.AM2017-689