Target In Neuroblastoma Research Articles

CD146 is a potential immunotarget for neuroblastoma.

Neuroblastoma, the most common extracranial solid tumor of childhood, is thought to arise from neural crest‐derived immature cells. The prognosis of patients with high‐risk or recurrent/refractory neuroblastoma remains quite poor despite intensive multimodality therapy; therefore, novel therapeutic interventions are required. We examined the expression of a cell adhesion molecule CD146 (melanoma cell adhesion molecule [MCAM]) by neuroblastoma cell lines and in clinical samples and investigated the anti‐tumor effects of CD146‐targeting treatment for neuroblastoma cells both in vitro and in vivo. CD146 is expressed by 4 cell lines and by most of primary tumors at any stage. Short hairpin RNA‐mediated knockdown of CD146, or treatment with an anti‐CD146 polyclonal antibody, effectively inhibited growth of neuroblastoma cells both in vitro and in vivo, principally due to increased apoptosis via the focal adhesion kinase and/or nuclear factor‐kappa B signaling pathway. Furthermore, the anti‐CD146 polyclonal antibody markedly inhibited tumor growth in immunodeficient mice inoculated with primary neuroblastoma cells. In conclusion, CD146 represents a promising therapeutic target for neuroblastoma.

MEIS2 Is an Adrenergic Core Regulatory Transcription Factor Involved in Early Initiation of TH-MYCN-Driven Neuroblastoma Formation.

Simple SummaryNeuroblastoma is a pediatric tumor originating from the sympathetic nervous system responsible for 10–15% of all childhood cancer deaths. Half of all neuroblastoma patients present with high-risk disease, of which nearly 50% relapse and die of their disease. In addition, standard therapies cause serious lifelong side effects and increased risk for secondary tumors. Further research is crucial to better understand the molecular basis of neuroblastomas and to identify novel druggable targets. Neuroblastoma tumorigenesis has to this end been modeled in both mice and zebrafish. Here, we present a detailed dissection of the gene expression patterns that underlie tumor formation in the murine TH-MYCN-driven neuroblastoma model. We identified key factors that are putatively important for neuroblastoma tumor initiation versus tumor progression, pinpointed crucial regulators of the observed expression patterns during neuroblastoma development and scrutinized which factors could be innovative and vulnerable nodes for therapeutic intervention.Roughly half of all high-risk neuroblastoma patients present with MYCN amplification. The molecular consequences of MYCN overexpression in this aggressive pediatric tumor have been studied for decades, but thus far, our understanding of the early initiating steps of MYCN-driven tumor formation is still enigmatic. We performed a detailed transcriptome landscaping during murine TH-MYCN-driven neuroblastoma tumor formation at different time points. The neuroblastoma dependency factor MEIS2, together with ASCL1, was identified as a candidate tumor-initiating factor and shown to be a novel core regulatory circuit member in adrenergic neuroblastomas. Of further interest, we found a KEOPS complex member (gm6890), implicated in homologous double-strand break repair and telomere maintenance, to be strongly upregulated during tumor formation, as well as the checkpoint adaptor Claspin (CLSPN) and three chromosome 17q loci CBX2, GJC1 and LIMD2. Finally, cross-species master regulator analysis identified FOXM1, together with additional hubs controlling transcriptome profiles of MYCN-driven neuroblastoma. In conclusion, time-resolved transcriptome analysis of early hyperplastic lesions and full-blown MYCN-driven neuroblastomas yielded novel components implicated in both tumor initiation and maintenance, providing putative novel drug targets for MYCN-driven neuroblastoma.

Therapeutic targeting of both dihydroorotate dehydrogenase and nucleoside transport in MYCN-amplified neuroblastoma

Metabolic reprogramming is an integral part of the growth-promoting program driven by the MYC family of oncogenes. However, this reprogramming also imposes metabolic dependencies that could be exploited therapeutically. Here we report that the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is an attractive therapeutic target for MYCN-amplified neuroblastoma, a childhood cancer with poor prognosis. Gene expression profiling and metabolomic analysis reveal that MYCN promotes pyrimidine nucleotide production by transcriptional upregulation of DHODH and other enzymes of the pyrimidine-synthesis pathway. Genetic and pharmacological inhibition of DHODH suppresses the proliferation and tumorigenicity of MYCN-amplified neuroblastoma cell lines. Furthermore, we obtain evidence suggesting that serum uridine is a key factor in determining the efficacy of therapeutic agents that target DHODH. In the presence of physiological concentrations of uridine, neuroblastoma cell lines are highly resistant to DHODH inhibition. This uridine-dependent resistance to DHODH inhibitors can be abrogated by dipyridamole, an FDA-approved drug that blocks nucleoside transport. Importantly, dipyridamole synergizes with DHODH inhibition to suppress neuroblastoma growth in animal models. These findings suggest that a combination of targeting DHODH and nucleoside transport is a promising strategy to overcome intrinsic resistance to DHODH-based cancer therapeutics.

A Shortage of FTH Induces ROS and Sensitizes RAS-Proficient Neuroblastoma N2A Cells to Ferroptosis.

Ferroptosis, an iron-dependent form of programmed cell death, has excellent potential as an anti-cancer therapeutic strategy in different types of tumors, especially in RAS-mutated ones. However, the function of ferroptosis for inhibiting neuroblastoma, a common child malignant tumor with minimal treatment, is unclear. This study investigated the anti-cancer function of ferroptosis inducer Erastin or RSL3 in neuroblastoma N2A cells. Our results show that Erastin or RSL3 induces ROS level and cell death and, therefore, reduces the viability of RAS-proficient N2A cells. Importantly, inhibitors to ferroptosis, but not apoptosis, ameliorate the high ROS level and viability defect in Erastin- or RSL3-treated cells. In addition, our data also show that N2A cells are much more sensitive to ferroptosis inducers than primary mouse cortical neural stem cells (NSCs) or neurons. Moreover, a higher level of ROS and PARylation is evidenced in N2A, but not NSCs. Mechanically, ferritin heavy chain 1 (Fth), the ferroxidase function to oxidate redox-active Fe2+ to redox-inactive Fe3+, is likely responsible for the hypersensitivity of N2A to ferroptosis induction since its expression is lower in N2A compared to NSCs; ectopic expression of Fth reduces ROS levels and cell death, and induces expression of GPX4 and cell viability in N2A cells. Most importantly, neuroblastoma cell lines express a significantly low level of Fth than almost all other types of cancer cell lines. All these data suggest that Erastin or RSL3 induce ferroptosis cell death in neuroblastoma N2A cells, but not normal neural cells, regardless of RAS mutations, due to inadequate FTH. This study, therefore, provides new evidence that ferroptosis could be a promising therapeutic target for neuroblastoma.

How Do Telomere Abnormalities Regulate the Biology of Neuroblastoma?

Telomere maintenance plays important roles in genome stability and cell proliferation. Tumor cells acquire replicative immortality by activating a telomere-maintenance mechanism (TMM), either telomerase, a reverse transcriptase, or the alternative lengthening of telomeres (ALT) mechanism. Recent advances in the genetic and molecular characterization of TMM revealed that telomerase activation and ALT define distinct neuroblastoma (NB) subgroups with adverse outcomes, and represent promising therapeutic targets in high-risk neuroblastoma (HRNB), an aggressive childhood solid tumor that accounts for 15% of all pediatric-cancer deaths. Patients with HRNB frequently present with widely metastatic disease, with tumors harboring recurrent genetic aberrations (MYCN amplification, TERT rearrangements, and ATRX mutations), which are mutually exclusive and capable of promoting TMM. This review provides recent insights into our understanding of TMM in NB tumors, and highlights emerging therapeutic strategies as potential treatments for telomerase- and ALT-positive tumors.

Analysis of SARS-CoV-2 infection associated cell entry proteins ACE2, CD147, PPIA, and PPIB in datasets from non SARS-CoV-2 infected neuroblastoma patients, as potential prognostic and infection biomarkers in neuroblastoma

SARS-CoV-2 viral contagion has given rise to a worldwide pandemic. Although most children experience minor symptoms from SARS-CoV-2 infection, some have severe complications including Multisystem Inflammatory Syndrome in Children. Neuroblastoma patients may be at higher risk of severe infection as treatment requires immunocompromising chemotherapy and SARS-CoV-2 has demonstrated tropism for nervous cells. To date, there is no sufficient epidemiological data on neuroblastoma patients with SARS-CoV-2. Therefore, we evaluated datasets of non-SARS-CoV-2 infected neuroblastoma patients to assess for key genes involved with SARS-CoV-2 infection as possible neuroblastoma prognostic and infection biomarkers. We hypothesized that ACE2, CD147, PPIA and PPIB, which are associated with viral-cell entry, are potential biomarkers for poor prognosis neuroblastoma and SARS-CoV-2 infection.We have analysed three publicly available neuroblastoma gene expression datasets to understand the specific molecular susceptibilities that high-risk neuroblastoma patients have to the virus. Gene Expression Omnibus (GEO) GSE49711 and GEO GSE62564 are the microarray and RNA-Seq data, respectively, from 498 neuroblastoma samples published as part of the Sequencing Quality Control initiative. TARGET, contains microarray data from 249 samples and is part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. ACE2, CD147, PPIA and PPIB were identified through their involvement in both SARS-CoV-2 infection and cancer pathogenesis.In-depth statistical analysis using Kaplan-Meier, differential gene expression, and Cox multivariate regression analysis, demonstrated that overexpression of ACE2, CD147, PPIA and PPIB is significantly associated with poor-prognosis neuroblastoma samples. These results were seen in the presence of amplified MYCN, unfavourable tumour histology and in patients older than 18 months of age. Previously, we have shown that high levels of the nerve growth factor receptor NTRK1 together with low levels of the phosphatase PTPN6 and TP53 are associated with increased relapse-free survival of neuroblastoma patients. Interestingly, low levels of expression of ACE2, CD147, PPIA and PPIB are associated with this NTRK1-PTPN6-TP53 module, suggesting that low expression levels of these genes are associated with good prognosis. These findings have implications for clinical care and therapeutic treatment. The upregulation of ACE2, CD147, PPIA and PPIB in poor-prognosis neuroblastoma samples suggests that these patients may be at higher risk of severe SARS-CoV-2 infection. Importantly, our findings reveal ACE2, CD147, PPIA and PPIB as potential biomarkers and therapeutic targets for neuroblastoma.

MCM6 indicates adverse tumor features and poor outcomes and promotes G1/S cell cycle progression in neuroblastoma

BackgroundMinichromosome maintenance complex component 6 (MCM6), as an important replication permission factor, is involved in the pathogenesis of various tumors. Here we studied the expression of MCM6 in neuroblastoma and its influence on tumor characteristics and prognosis.MethodsPublicly available datasets were used to explore the influence of the differential expression of MCM6 on neuroblastoma tumor stage, risk and prognosis. In cell experiments, human neuroblastoma cell lines SK-N-SH and SK-N-BE [ (2)] were utilized to verify the ability of MCM6 to promote cell proliferation, migration and invasion. We further explored the possible molecular mechanism of MCM6 affecting the phenotype of neuroblastoma cells by mutual verification of RNA-seq and western blotting, and flow cytometry to inquire about its potential specific roles in the cell cycle.ResultsThrough multiple datasets mining, we found that high expression of MCM6 was positively correlated with elevated tumor stage, high risk and poor prognosis in neuroblastoma. At the cellular level, neuroblastoma cell proliferation, migration and invasion were significantly inhibited after MCM6 was interfered by siRNA. Mutual verification of RNA-seq and western blotting suggested that the downstream cell cycle-related genes were differentially expressed after MCM6 interference. Flow cytometric analysis revealed that neuroblastoma cells were blocked in G1/S phase after MCM6 interference.ConclusionMCM6 is considered to be the driving force of G1/S cell cycle progression, and it is also a prognostic marker and a potential novel therapeutic target in neuroblastoma.

Ubiquitination of p21 by E3 Ligase TRIM21 Promotes the Proliferation of Human Neuroblastoma Cells.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood, which shows great clinical and biomolecule heterogeneity. Currently, surgery is still the main method of neuroblastoma treatment and specific therapeutic drugs are lacking, so useful targets are urgently needed. TRIM21 is a RING-type E3 ligase that its overexpression promotes the progression of human glioma, while whose effects on neuroblastoma have not been illustrated. Firstly, the shRNAs targeting TRIM21 were designed and found that the ablation of TRIM21 inhibits the proliferation of human neuroblastoma cells. Then the molecular mechanism study indicated that TRIM21 interacts with, and mediates p21 degradation by ubiquitination modification. Further study demonstrates that TRIM21 regulates the proliferation of neuroblastoma cells in a p21-dependent manner. These results suggest that TRIM21 might be a potential therapeutic target for neuroblastoma.

Selective Modulation of a Pan-Essential Protein as a Therapeutic Strategy in Cancer.

Cancer dependency maps, which use CRISPR/Cas9 depletion screens to profile the landscape of genetic dependencies in hundreds of cancer cell lines, have identified context-specific dependencies that could be therapeutically exploited. An ideal therapy is both lethal and precise, but these depletion screens cannot readily distinguish between gene effects that are cytostatic or cytotoxic. Here, we use a diverse panel of functional genomic screening assays to identify NXT1 as a selective and rapidly lethal in vivo relevant genetic dependency in MYCN-amplified neuroblastoma. NXT1 heterodimerizes with NXF1, and together they form the principal mRNA nuclear export machinery. We describe a previously unrecognized mechanism of synthetic lethality between NXT1 and its paralog NXT2: their common essential binding partner NXF1 is lost only in the absence of both. We propose a potential therapeutic strategy for tumor-selective elimination of a protein that, if targeted directly, is expected to cause widespread toxicity. SIGNIFICANCE: We provide a framework for identifying new therapeutic targets from functional genomic screens. We nominate NXT1 as a selective lethal target in neuroblastoma and propose a therapeutic approach where the essential protein NXF1 can be selectively eliminated in tumor cells by exploiting the NXT1-NXT2 paralog relationship.See related commentary by Wang and Abdel-Wahab, p. 2129.This article is highlighted in the In This Issue feature, p. 2113.

Integrated systems biology approach using gene network analysis to identify the important pathways and new potential drug targets for Neuroblastoma

The complex and heterogeneous nature of neuroblastoma poses a great challenge to the scientific community for developing an effective therapeutic drug. Neuroblastoma tumors show relapse and resistance to available drugs and it is proving to be an obstacle for efficient treatment options. The tumor accounts for about 7% of the malignancies worldwide and is seen mostly in children around one year. Our present study aims to understand various mechanisms underlying Neuroblastoma. The gene interaction network of 92 Neuroblastoma related genes along with a total of 339 functional interactions are used for the gene interaction network construction and analysis. The clustering analysis resulted in four densely interconnected gene clusters. We further performed functional enrichment analysis and found important pathways associated with neuroblastoma in PI3K/AKT pathway, MAPK pathway, Ras pathway, Rap1 pathway, Cytokine- cytokine interaction, Neurotrophin signaling, EGFR tyrosine kinase inhibitor resistance, and VEGF signaling pathways. The predominant genes involved in the enriched pathways are ANGPT2, FGF2, NGFR, NTRK1, NTRK2, PDGFC, TGFA, TP53, VEGFB, VEGFC, BDNF, NRAS, and VEGFA. The topological parameter analysis has shown the genes with more direct interactions are considered as the hub genes and are important in the network. The genes TP53, VEGFA, BDNF, MYCN, and FGF2 with the maximum number of functional interactors, can be used as potential therapeutic targets for Neuroblastoma. The results obtained in our study will be helpful for researchers in better understanding the molecular level associations during the Neuroblastoma progression.

NBAT1/CASC15-003/USP36 control MYCN expression and its downstream pathway genes in neuroblastoma.

Background MYCN has been an attractive therapeutic target in neuroblastoma considering the widespread amplification of the MYCN locus in neuroblastoma, and its established role in neuroblastoma development and progression. Thus, understanding neuroblastoma-specific control of MYCN expression at the transcriptional and post-transcriptional level would lead to identification of novel MYCN-dependent oncogenic pathways and potential therapeutic strategies.MethodsBy performing loss- and gain-of-function experiments of the neuroblastoma hotspot locus 6p22.3 derived lncRNAs CASC15-003 and NBAT1, together with coimmunoprecipitation and immunoblotting of MYCN, we have shown that both lncRNAs post-translationally control the expression of MYCN through regulating a deubiquitinase enzyme USP36. USP36 oncogenic properties were investigated using cancer cell lines and in vivo models. RNA-seq analysis of loss-of-function experiments of CASC15-003/NBAT1/MYCN/USP36 and JQ1-treated neuroblastoma cells uncovered MYCN-dependent oncogenic pathways.ResultsWe show that NBAT1/CASC15-003 control the stability of MYCN protein through their common interacting protein partner USP36. USP36 harbors oncogenic properties and its higher expression in neuroblastoma patients correlates with poor prognosis, and its downregulation significantly reduces tumor growth in neuroblastoma cell lines and xenograft models. Unbiased integration of RNA-seq data from CASC15-003, NBAT1, USP36, and MYCN knockdowns and neuroblastoma cells treated with MYCN inhibitor JQ1, identified genes that are jointly regulated by the NBAT1/CASC15-003/USP36/MYCN pathway. Functional experiments on one of the target genes, COL18A1, revealed its role in the NBAT1/CASC15-003-dependent cell adhesion feature in neuroblastoma cells.ConclusionOur data show post-translational regulation of MYCN by NBAT1/CASC15-003/USP36, which represents a new regulatory layer in the complex multilayered gene regulatory network that controls MYCN expression.

CD276 as a novel CAR NK‐92 therapeutic target for neuroblastoma

CD276 as a novel CAR NK‐92 therapeutic target for neuroblastoma

The D3-GPC2-PBD ADC is Potently Efficacious Against Neuroblastoma and SCLC Via Engagement of a Conformational GPC2 Epitope

The signaling co-receptor heparan sulfate proteoglycan GPC2 is a MYCN-activated, differentially-expressed cell surface oncoprotein and candidate immunotherapeutic target in neuroblastoma. Here we build on GPC2’s attributes as a robust target for immune-based therapies by finding it is highly expressed on the most clinically aggressive human neuroblastomas and small cell lung cancers (SCLCs), with highly enriched expression in the tumor stem cell compartment. A GPC2 directed antibody-drug conjugate (ADC; D3-GPC2-PBD) induces robust and durable tumor regressions in a panel of neuroblastoma and SCLC preclinical models with diverse oncogenic drivers via induction of DNA damage and apoptosis, as well as potent bystander cell killing. Finally, we illustrate that this ADC binds a tumor specific, conformation dependent epitope of the core human and murine GPC2 proteins explaining its tumor specificity and therapeutic tolerability. These studies provide the preclinical data to support the clinical translation of ADCs targeting GPC2.

CEP131 Abrogates CHK1 Inhibitor-Induced Replication Defects and Is Associated with Unfavorable Outcome in Neuroblastoma.

Checkpoint kinase 1 (CHK1) plays a key role in genome surveillance and integrity throughout the cell cycle. Selective inhibitors of CHK1 (CHK1i) are undergoing clinical evaluation for various human malignancies, including neuroblastoma. Recently, we reported that CHK1i, PF-477736, induced a p53-mediated DNA damage response. As a result, the cancer cells were able to repair DNA damage and became less sensitive to CHK1i. In this study, we discovered that PF-477736 increased expression of MDM2 oncogene along with CHK1i-induced replication defects in neuroblastoma NB-39-nu cells. A mass spectrometry analysis of protein binding to MDM2 in the presence of CHK1i identified the centrosome-associated family protein 131 (CEP131), which was correlated with unfavorable prognosis of neuroblastoma patients. We revealed that MDM2 was associated with CEP131 protein degradation, whereas overexpression of CEP131 accelerated neuroblastoma cell growth and exhibited resistance to CHK1i-induced replication defects. Thus, these findings may provide a future therapeutic strategy against centrosome-associated oncogenes involving CEP131 as a target in neuroblastoma.

Abstract 5402: Inhibition of RET via BLU-667 in an in vitro model of neuroblastoma

Abstract Neuroblastoma is the most common extracranial solid tumor of childhood. Despite aggressive treatment, outcomes in patients with high-risk disease remains poor and new therapeutics are needed to address their needs. Rearranged during Transfection (RET) is a tyrosine kinase that is a known oncogenic driver of multiple human tumors. RET is expressed on neural crest derived cells, including neuroblastoma cells, and has been shown to play a role in neuroblastoma proliferation and survival as well. Recent studies have shown RET inhibition to be a promising new therapeutic target in neuroblastoma. BLU-667 is a selective RET inhibitor with a higher potency and specificity for RET than the current inhibitors that have been previously tested. We hypothesize that this increased potency and specificity will result in greater efficacy against neuroblastoma in preclinical models with fewer off target effects. Thus, to investigate this we evaluated BLU-667 at different concentrations against a panel of established human neuroblastoma cell lines to assess its effects on neuroblastoma cell proliferation, differentiation, and viability. We found that inhibition of RET resulted in a significant decrease in cell proliferation and viability in vitro. Further, given the increased specificity of BLU-667 to RET, we hope to use western blot protein analysis to establish the specific downstream pathways through which RET inhibition leads to decreased neuroblastoma cell survival, potentially revealing further therapeutic targets. Citation Format: Jessica Gutierrez, Sean Flynn, Yuchen Huo, Jacqueline Lesperance, Peter Zage. Inhibition of RET via BLU-667 in an in vitro model of 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 5402.

Abstract 5871: Immunogenomic approaches to optimize immunotherapeutic targeting of neuroblastoma

Abstract Background: Neuroblastoma (NB) is the most common extra-cranial solid cancer in children. Although multimodal therapies with differentiating agents and immunotherapy with anti-GD2 antibody and GM-CSF have shown promising results, it remains deadly in approximately 50% of patients with high-risk disease. Chimeric antigen receptor T-cell therapies (CAR-T) have been found to be effective in treating refractory and relapsed leukemia and lymphoma, and two of them have been recently approved by the FDA. However, current CARs frequently lose efficacy due to T cell exhaustion and CARs against solid tumor antigens often lack enough specificity due to a low incidence of somatic mutations resulting in a paucity of tumor neoantigens. There have not been effective CAR-T therapies against other solid cancers to date although many clinical trials are underway. We previously identified 2 cell surface cancer-associated antigens, GPC2 (Glypican-2) and CD276 (B7-H3), both highly expressed in NB tumor cells but expressed at low or undetectable levels in normal organs. Here we attempt to develop a high throughput way of identifying optimal CART cell binders that show activation and expansion in the presence of GPC2 and CD276 but lack of exhaustion. Methods: Binders targeting GPC2 or CD276 were cloned into CAR lentiviral constructs and then were separately transduced into T cells to develop CAR-T cells. Then we analyzed cytotoxicity of these CART cells individually. To identify the most effective GPC2 or CD276-specific targeting CAR-T cells, we utilized a combined proteomics and transcriptomics method for every single CAR-T cell to quantify RNA and protein at the same. All CAR-T cells were pooled and co-cultured with CD276/GPC2-expressing NB cancer cells (target cells) for 24 hr. Co-cultured CAR-T cells were examined for their activation, exhaustion, cytotoxicity state and distinguished different cell types by staining with CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) antibodies, and then molecularly barcoded using 10X Genomics platform for single cell RNA-sequencing (scRNA-seq). Results: We attempted to test the efficacy of CAR-T cells using 14 established and novel binders against GPC2 or CD276. According to cytotoxicity assay of these 14 CART cells, we found CT3 was the most effective GPC2 targeting CART cells but majority of CD276 CART cells showed high cytolytic activity in vitro. Conclusions and Future Directions: Using the CITE-seq and RNAseq analyses, we can identify which of the CARs are enriched and have an activated T cell signature, but lack exhaustion markers. Top candidate binders for each antigen are currently being developed into “AND” or “OR” CARs that are being tested in in-vitro and in-vivo models of NB. Thus, we have developed a high throughput method to identify high affinity functional binders against tumor cell surface antigens and provide a novel immunogenomics methods of CARs optimization for development of highly effective immunotherapies against NB and other cancers. Citation Format: Meijie Tian, Adam Tai-Chi Cheuk, Yong Song, Sivasish Sindiri, Nan Li, Christopher M. Dower, Mitchell Ho, Bradley St Croix, Javed Khan. Immunogenomic approaches to optimize immunotherapeutic targeting of 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 5871.

Abstract A13: Immunogenomic approaches to optimize immunotherapeutic targeting of neuroblastoma

Abstract Neuroblastoma (NB) is the most common extracranial solid cancer in children. Although multimodal therapies with differentiating agents and immunotherapy with anti-GD2 antibody and GM-CSF have shown promising results, it remains deadly in approximately 50% of patients with high-risk disease. Chimeric antigen receptor T-cell therapies (CAR-T) have been found to be effective in treating refractory and relapsed leukemia and lymphoma, and two of them have been recently approved by the FDA. However, current CARs frequently lose efficacy due to T-cell exhaustion, and CARs against solid tumor antigens often lack enough specificity due to a low incidence of somatic mutations resulting in a paucity of tumor neoantigens. There have not been effective CAR-T therapies against other solid cancers to date, although many clinical trials are under way. Therefore, we attempted to develop a high-throughput way of identifying optimal CART cell binders that show activation and expansion in the presence of targets but lack of exhaustion. We previously identified two cell surface cancer-associated antigens, GPC2 (Glypican-2) and CD276 (B7-H3), both highly expressed in NB tumor cells but expressed at low or undetectable levels in normal organs. 14 established binders as well as novel binders targeting these two antigens were cloned into CAR lentiviral constructs and then were separately transduced into T cells to develop 14 CAR-T cells using a 2nd-generation design. All 14 CAR-T cells were pooled and cocultured with CD276/GPC2-expressing NB cancer cells (target cells) for 24 hr. To identify the effective GPC2 or CD276-specific targeting CAR-T cells, we utilized a combined proteomics and transcriptomics method for every single CAR-T cell to quantify RNA and protein at the same. Cocultured CAR-T cells were examined for their activation, exhaustion, cytotoxicity state and distinguished different cell types by staining with CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) antibodies, and then molecularly barcoded using 10X Genomics platform for single-cell RNA-sequencing (scRNA-seq). The data are currently being analyzed and will be presented. Using this method, we will be able to identify which of the CARs are enriched and have an activated T-cell signature, and lack exhaustion marks as determined by the CITE-seq and RNAseq analyses. Finally, top candidate binders for each antigen will be developed into “AND” or “OR” CARs and will be tested in in vitro and in vivo models of NB. Thus, we will develop a high-throughput way to identify high-affinity functional binders against tumor cell surface antigens. This study also will provide novel immunogenomics methods of CARs optimization for development of highly effective immunotherapies against NB and other cancers. Citation Format: Meijie Tian, Adam Tai-Chi Cheuk, Jeetendra Kumar, Young K. Song, Sivasish Sindiri, Nan Li, Christopher M. Dower, Mitchell Ho, Brad St. Croix, Javed Khan. Immunogenomic approaches to optimize immunotherapeutic targeting of neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A13.

Ubiquitination of IGF2BP3 by E3 ligase MKRN2 regulates the proliferation and migration of human neuroblastoma SHSY5Y cells

Neuroblastoma (NB) is a paediatric tumour that shows great biomolecule and clinical heterogeneity, and patients with NB often develop various neurological complications. Currently, the disease is mainly treated by surgery and still lacks specific therapeutic drugs; therefore, targets are urgently needed. Makorin ring finger protein 2 (MKRN2) is an E3 ligase whose effects on neuroblastoma have not been illustrated. shRNAs for MKRN2 have been designed, and MKRN2-knockdown human neuroblastoma SHSY5Y cells were established. MKRN2 knockdown promotes the proliferation and migration of SHSY5Y cells. Because MKRN2 is an E3 ligase, we performed a series of experiments, and Insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3) was identified as a new substrate for MKRN2. IGF2BP3 is an RNA-binding protein that regulates the stability of many mRNAs, including CD44 and PDPN, and our study demonstrated that MKRN2 regulates the expression of CD44 and PDPN in an IGF2BP3-dependent manner. These results suggest that MKRN2 might be a potential therapeutic target for neuroblastoma.

PRMT1 promotes neuroblastoma cell survival through ATF5

Aberrant expression of protein arginine methyltransferases (PRMTs) has been implicated in a number of cancers, making PRMTs potential therapeutic targets. But it remains not well understood how PRMTs impact specific oncogenic pathways. We previously identified PRMTs as important regulators of cell growth in neuroblastoma, a deadly childhood tumor of the sympathetic nervous system. Here, we demonstrate a critical role for PRMT1 in neuroblastoma cell survival. PRMT1 depletion decreased the ability of murine neuroblastoma sphere cells to grow and form spheres, and suppressed proliferation and induced apoptosis of human neuroblastoma cells. Mechanistic studies reveal the prosurvival factor, activating transcription factor 5 (ATF5) as a downstream effector of PRMT1-mediated survival signaling. Furthermore, a diamidine class of PRMT1 inhibitors exhibited anti-neuroblastoma efficacy both in vitro and in vivo. Importantly, overexpression of ATF5 rescued cell apoptosis triggered by PRMT1 inhibition genetically or pharmacologically. Taken together, our findings shed new insights into PRMT1 signaling pathway, and provide evidence for PRMT1 as an actionable therapeutic target in neuroblastoma.

Immunotherapy for solid tumors: Chimeric antigen receptor (CAR) T-cells and the tumor microenvironment

Abstract Patients with solid tumors continue to experience decreased survival rates, with metastasis being the leading cause of mortality in these patients. This has prompted the development of novel immunotherapies, with cellular immunotherapy giving rise to the development of Chimeric Antigen Receptor (CAR) T-cells, an approach combining specificity for tumor antigens and cytotoxicity independent of major histocompatibility complex molecule co-stimulation. This modality has proven efficacious in Acute Lymphoblastic Leukemia and B-cell Lymphoma. However, progress has remained limited in solid tumors despite targeting of various antigens, one of such being disialoganglioside (GD2) which has proven a viable target in neuroblastoma. This study investigates the efficacy of GD2 as an antigen target for CAR T-cell therapy in osteosarcoma. Surface flow cytometric analysis revealed the presence of GD2 on human 143B and Hu09H3 osteosarcoma cell lines. Of note, the tumor microenvironment offers an additional potential therapeutic target, as we observed increases in myeloid-derived suppressor cells alongside decreases in CD4+ T cells in primary tumor and metastatic tissues. In summary, we report the utility of GD2 as a target for cellular immunotherapy in osteosarcoma and the role of the tumor microenvironment in anti-tumor therapeutic modalities, a therapeutic combination that could be applicable to optimizing potent therapeutic modalities for solid tumors.