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Neuroblastoma is a deadly pediatric solid tumor with infrequent recurrent somatic mutations. Particularly, the pathophysiology of tumors without MYCN amplification remains poorly defined. Utilizing an unbiased approach, we performed gene set enrichment analysis of RNA-sequencing data from 498 patients with neuroblastoma and revealed a differentially overexpressed gene signature in MYCN nonamplified neuroblastomas with telomerase reverse transcriptase (TERT) gene overexpression and coordinated activation of oncogenic signaling pathways, including E2Fs, Wnt, Myc, and the DNA repair pathway. Promoter rearrangement of the TERT gene juxtaposes the coding sequence to strong enhancer elements, leading to TERT overexpression and poor prognosis in neuroblastoma, but TERT-associated oncogenic signaling remains unclear. ChIP-seq analysis of the human CLB-GA neuroblastoma cells harboring TERT rearrangement uncovered genome-wide chromatin co-occupancy of Brd4 and H3K27Ac and robust enrichment of H3K36me3 in TERT and multiple TERT-associated genes. Brd4 and cyclin-dependent kinases (CDK) had critical regulatory roles in the expression and chromatin activation of TERT and multiple TERT-associated genes. Epigenetically targeting Brd4 or CDKs with their respective inhibitors suppressed the expression of TERT and multiple TERT-associated genes in neuroblastoma with TERT overexpression or MYCN amplification. ChIP-seq and ChIP-qPCR provided evidence that the CDK inhibitor directly inhibited Brd4 recruitment to activate chromatin globally. Therefore, inhibiting Brd4 and CDK concurrently with AZD5153 and dinaciclib would be most effective in tumor growth suppression, which we demonstrated in neuroblastoma cell lines, primary human cells, and xenografts. In summary, we describe a unique mechanism in neuroblastoma with TERT overexpression and an epigenetically targeted novel therapeutic strategy.Significance:Epigenetically cotargeting Brd4 and Cdks suppresses human neuroblastoma with TERT overexpression by inhibiting the TERT-associated gene expression networks.

Supplementary Fig. S1. Elevated mRNA expression of TERT and TERT-associated genes in NB with TERT gene rearrangement. Supplementary Fig. S2. Effects of dinaciclib on genome-wide chromatin occupancy of epigenetic modulators in CLB-GA cells. Supplementary Fig. S3: C-Myc and MYCN protein expression in four NB cell lines. Supplementary Fig. S4: Effects of dinaciclib on the chromatin occupancy of epigenetic modulators at Dvl2, AURKA, and AURKB and in CLB-GA cells. Supplementary Fig. S5. Association of overexpression of FEN1, BIRC5, and UHRF1 with poor prognosis and effects of dinaciclib on chromatin occupancy of epigenetic modulators in CLB-GA cells. Supplementary Fig. S6. JQ1 sensitive Brd4 binding profiles in OCI-AML3 cells. Supplementary Fig. S7. JQ1 and dinaciclib are synergistic or additive in inducing cytotoxicity in NB cell lines. Supplementary Fig. S8. Combination index (CI) values for synergy experiment combining Dinaciclib with Brd4 inhibitor JQ1. Supplementary Fig. S9. Effects of dinaciclib on phosphorylation of Rb in CLB-GA and Kelly cells. Supplementary table S1. Primer sequences used in ChIP-qPCR and real-time RT-PCR analysis. Supplementary table 2. Gene sets enriched in NB patients with TERT gene rearrangement (n=32) versus without TERT gene rearrangement (n=278) within MYCN non-amplified NB of Tumor Neuroblastoma (TERT)-Fischer-394-custom. Supplementary table 3. Gene sets enriched in NB patients with high versus low TERT within Tumor Neuroblastoma Gene-TARGET-161. Supplementary table 4: Information on patient samples used in this study.

Immunotherapy targeting GD2 is a primary treatment for patients with high‐risk neuroblastoma. Dinutuximab is a monoclonal antibody with great clinical promise but is limited by side effects such as severe pain. Local delivery has emerged as a potential mechanism to deliver higher doses of therapeutics into the tumor bed, while limiting systemic toxicity. We aim to deliver dinutuximab locally in a lyophilized silk fibroin foam for the treatment of an orthotopic neuroblastoma mouse model. Dinutuximab‐loaded silk fibroin foams were fabricated through lyophilization. In vitro release profile and bioactivity of the release through complement‐dependent cytotoxicity were characterized. MYCN‐amplified neuroblastoma cells (KELLY) were injected into the left gland of mice to generate an orthotopic neuroblastoma model. Once the tumor volume reached 100 mm3, dinutuximab‐, human IgG‐, or buffer‐loaded foams were implanted into the tumor and growth was monitored using high‐resolution ultrasound. Post‐resection histology was performed on tumors. Dinutuximab‐loaded silk fibroin foams exhibited a burst release, with slow release thereafter in vitro with maintenance of bioactivity. The dinutuximab‐loaded foam significantly inhibited xenograft tumor growth compared to IgG‐ and buffer‐loaded foams. Histological analysis revealed the presence of dinutuximab within the tumor and neutrophils and macrophages infiltrating into dinutuximab‐loaded silk foam. Tumors treated with local dinutuximab had decreased MYCN expression on histology compared to control or IgG‐treated tumors. Silk fibroin foams offer a mechanism for local release of dinutuximab within the neuroblastoma tumor. This local delivery achieved a significant decrease in tumor growth rate in a mouse orthotopic tumor model., Dinutuximab, an immunotherapy targeting GD2, was delivered loacally in a lyophilized silk fibroin foam for the treatment of an orthotopic neuroblastoma mouse model. Silk fibroin foam allows for a sustained release of dinutuximab, causing significant tumor growth inhibition.

Neuroblastoma is a deadly pediatric solid tumor with infrequent recurrent somatic mutations. Particularly, the pathophysiology of tumors without MYCN amplification remains poorly defined. Utilizing an unbiased approach, we performed gene set enrichment analysis of RNA-sequencing data from 498 patients with neuroblastoma and revealed a differentially overexpressed gene signature in MYCN nonamplified neuroblastomas with telomerase reverse transcriptase (TERT) gene overexpression and coordinated activation of oncogenic signaling pathways, including E2Fs, Wnt, Myc, and the DNA repair pathway. Promoter rearrangement of the TERT gene juxtaposes the coding sequence to strong enhancer elements, leading to TERT overexpression and poor prognosis in neuroblastoma, but TERT-associated oncogenic signaling remains unclear. ChIP-seq analysis of the human CLB-GA neuroblastoma cells harboring TERT rearrangement uncovered genome-wide chromatin co-occupancy of Brd4 and H3K27Ac and robust enrichment of H3K36me3 in TERT and multiple TERT-associated genes. Brd4 and cyclin-dependent kinases (CDK) had critical regulatory roles in the expression and chromatin activation of TERT and multiple TERT-associated genes. Epigenetically targeting Brd4 or CDKs with their respective inhibitors suppressed the expression of TERT and multiple TERT-associated genes in neuroblastoma with TERT overexpression or MYCN amplification. ChIP-seq and ChIP-qPCR provided evidence that the CDK inhibitor directly inhibited Brd4 recruitment to activate chromatin globally. Therefore, inhibiting Brd4 and CDK concurrently with AZD5153 and dinaciclib would be most effective in tumor growth suppression, which we demonstrated in neuroblastoma cell lines, primary human cells, and xenografts. In summary, we describe a unique mechanism in neuroblastoma with TERT overexpression and an epigenetically targeted novel therapeutic strategy. Significance: Epigenetically cotargeting Brd4 and Cdks suppresses human neuroblastoma with TERT overexpression by inhibiting the TERT-associated gene expression networks.

Purpose Large cell neuroblastomas (LCN) are frequently seen in recurrent, high-risk neuroblastoma but are rare in primary tumors. LCN, characterized by large nuclei with prominent nucleoli, predict a poor prognosis. We hypothesize that LCN can be created with high-dose intra-tumoral chemotherapy and identified by a digital analysis system. Methods Orthotopic mouse xenografts were created using human neuroblastoma and treated with high-dose chemotherapy delivered locally via sustained-release silk platforms, inducing tumor remission. After recurrence, LCN populations were identified on H&E sections manually. Clusters of typical LCN and non-LCN cells were divided equally into training and test sets for digital analysis. Marker-controlled watershed segmentation was used to identify nuclei and characterize their features. Logistic regression was developed to distinguish LCN from non-LCN. Results Image analysis identified 15,000 nuclei and characterized 70 nuclear features. A 19-feature model provided AUC > 0.90 and 100% accuracy when > 30% nuclei/cluster were predicted as LCN. Overall accuracy was 87%. Conclusions We recreated LCN using high-dose chemotherapy and developed an automated method for defining LCN histologically. Features in the model provide insight into LCN nuclear phenotypic changes that may be related to increased activity. This model could be adapted to identify LCN in human tumors and correlated with clinical outcomes.

Neuroblastoma is the most common extra-cranial childhood tumor and current treatment requires surgical resection and multi-drug chemotherapy. Local, perioperative delivery of chemotherapeutics is a promising treatment method for solid tumors that require surgical removal. In this study we have aimed to develop a controlled release implant system to deliver cisplatin in tumor/tumor resection area. Silk fibroin, a biodegradable, non-immunogenic biopolymer was employed to encapsulate different doses of cisplatin in a reservoir system. The physical integrity of the reservoirs was characterized by evaluating the crystalline structure of silk secondary structure using Fourier Transform Infrared (FTIR) spectroscopy. The in vitro release of cisplatin was evaluated in phosphate buffered saline at 37°C and the reservoirs were able to release the drug up to 30 days. The cytotoxicity of cisplatin and cisplatin reservoirs were tested on KELLY cells. Cytotoxicity data showed 3.2 μg/mL cisplatin was required to kill 50 percent of the cell population and the released cisplatin from the silk reservoirs showed significant cytotoxicity up to 21 days. Intratumoral implantation of silk reservoirs into an orthotopic neuroblastoma mouse model decreased tumor growth significantly when compared to controls. These results suggest that silk reservoirs are promising carriers for cisplatin delivery to the tumor site.

Introduction High-risk neuroblastoma is a deadly disease; poor prognosticators are MYCN-amplification and TERT-overexpression. We hypothesized that Gene Set Enrichment Analysis (GSEA) could identify pathways associated with MYCN-amplification and that inhibition of these pathways could decrease tumor growth. Methods We analyzed the Neuroblastoma-Kocak dataset (GSE45547, n = 649) and identified pathways associated with MYCN-amplification. Inhibitors were selected from upregulated gene sets for in vitro cytotoxicity testing using ST16-patient-derived primary neuroblastoma cells and in vivo testing using orthotopic ST16-patient-derived xenografts (PDX) in mice. Tumor volume was measured with ultrasound and tumor sections examined after H&E staining. Results GSEA identified significantly overexpressed gene sets in MYCN-amplified tumors including MYC targets, cell cycle mitotic genes, TERT associated genes, loss of RB1 gene sets, and E2Fs targets. Several genes were potential Bromodomain-containing protein 4 (Brd4) targets, making Brd4 inhibitors - JQ1, AZD5153 - and cyclin-dependent kinase (Brd4’s binding partner) inhibitors – dinaciclib - potential therapeutic agents. JQ1 and dinaciclib were synergistic in inducing cytotoxicity in vitro. Dinaciclib-AZD5153 in vivo decreased tumor size compared to control, and increased tumor lymphocyte infiltration and necrosis on histology. Conclusions GSEA is a powerful approach to identify upregulated genes and potential therapeutic targets. Dinaciclib-AZD5153 combination therapy can be effective against MYCN-amplified and TERT-overexpressing neuroblastoma tumors.

PURPOSE: Advanced stage neuroblastoma patients require multi-agent chemotherapy. Intra-tumoral implantation of vincristine-loaded silk gel utilizes local diffusion to decrease orthotopic neuroblastoma tumor growth in mice. We hypothesize that injecting vincristine-loaded silk gel into eight locations within the tumor, instead of only centrally, decreases the diffusion distance and improves tumor growth suppression. METHODS: Human neuroblastoma cells, KELLY, were injected into mouse adrenal glands to create orthotopic tumors. After the tumors reached 100mm(3) by ultrasound, silk gels loaded with 50μg vincristine were injected centrally or in eight areas throughout the tumor. Drug-release profile was measured in vitro. Endpoints were tumor size>1000mm(3) and histologic examination. RESULTS: Vincristine-loaded silk gels suppressed tumor growth up to an “inflection point” (458.7±234.4mm(3) for central, 514.3±165.8mm(3) for eight-point injection) before tumor growth accelerated >200mm(3) over 3 days. The time to inflection point was 6.6days for central; 13.3days for eight-point injection(p

High-risk neuroblastoma requires surgical resection and multi-drug chemotherapy. This study aimed to develop an extended release, implantable and degradable delivery system for etoposide, commonly used for neuroblastoma treatment. Different concentrations of silk, a biodegradable, non-toxic, non-immunogenic material were employed to prepare etoposide-loaded wafer formulations. Secondary structure of silk in the formulations was characterized using Fourier Transform Infrared (FTIR) spectroscopy and optimized based on the crystalline structure. Accelerated in vitro degradation studies under different conditions such as acidic, alkaline, oxidizing mediums and high temperature, were performed. The integrity of the silk wafer structure was maintained unless exposed to 0.1 N NaOH for 24 h. In vitro release of etoposide was performed in PBS (phosphate buffered saline) at 37 °C. Silk coated 6% wafers released the drug up to 45 days, while uncoated wafers released the drug for 30 days. Cytotoxicity study was performed on KELLY cells to evaluate the etoposide cytotoxicity (LC50) and the long-term efficacy of the etoposide wafer formulations. The results showed that etoposide killed 50% of the cells at 1 μg/mL concentration and the wafer formulations demonstrated significant cytotoxicity up to 22 days when compared to untreated cells. Using an orthotopic neuroblastoma mouse model, intra-tumoral implantation of the coated 6%, uncoated 6%, or uncoated 3% silk wafers were all effective at decreasing tumor growth. Histological examination revealed tumor cell necrosis adjacent to the drug-loaded silk wafer.

INTRODUCTION: Neuroblastoma is the most common pediatric extracranial solid malignancy with limited effective treatment. We have shown that sustained release single drugs delivered locally through a silk-based biomaterial are effective in decreasing orthotopic neuroblastoma xenograft growth. We further optimized this approach and hypothesized that increasing doses of local chemotherapy or delivering two chemotherapeutic agents simultaneously inhibit additional tumor growth. METHODS: MYCN-amplified (KELLY) and non-MYCN-amplified (SKNAS) neuroblastoma cells were treated with combinations of cisplatin, vincristine, doxorubicin, and etoposide to determine cytotoxicity and synergy. Drug-loaded silk material was created, and the amounts of drug released from the material over time were recorded. Murine orthotopic neuroblastoma xenografts were generated; tumors were implanted with single- or dual-agent chemotherapy-loaded silk. Ultrasound was used to monitor tumor growth, and tumor histology was evaluated. RESULTS: In vitro, vincristine/cisplatin combination was synergistic, significantly decreasing cell viability relative to other combinations. Both drugs loaded into silk could be released effectively for over two weeks. Locally-implanted vincristine/cisplatin silk induced increased tumor growth suppression compared to either agent alone in KELLY tumors (p50μg-500μg for vincristine-cisplatin, respectively. Tumor histology demonstrated tumor cell necrosis adjacent to drug-loaded silk material and presence of large cell neuroblastoma. CONCLUSIONS: Local delivery of sustained release chemotherapy can suppress tumor growth especially at high doses or with two synergistic drugs. Locally delivered dual therapy is a promising approach for future clinical testing.

Purpose Precise excision of neuroblastoma is challenging, especially when tumors adhere to vital structures. Indocyanine green (ICG), an FDA-approved dye with absorption peaking at 800 nm, can absorb the near IR laser energy and release heat in the dyed tissue. We hypothesize that by injecting ICG at tumor sites followed by precise laser application, tumor cell death can be selectively targeted. Methods Orthotopic neuroblastoma tumors were created in the adrenal gland of immunocompromised mice. Tumor, liver, kidney, and muscle tissues were chosen for ICG injection. Intervention variables included presence of tumor capsule, continuous vs. pulsed laser treatment and total energy delivered. Control groups included laser or ICG only. Tissues were stained with hematoxylin/eosin. Results Continuous wave laser generated excessive heat, causing damage in all tissues. When using pulsed laser treatment, liver, kidney, muscle, and intact tumor tissues showed no cell death when treated with laser alone or laser plus ICG. Tumor tissue with the capsule removed, however, showed cell death on histology. Conclusions Pulsed laser treatment combined with ICG causes targeted tumor cell death in neuroblastoma tumor without capsule. No cell death was observed when tumor capsule was present, when only laser was used, or when applied over non-tumor tissues.

Neuroblastomas with a high mitosis-karyorrhexis index (High-MKI) are often associated with MYCN amplification, MYCN protein overexpression and adverse clinical outcome. However, the prognostic effect of MYC-family protein expression on these neuroblastomas is less understood, especially when MYCN is not amplified. To address this, MYCN and MYC protein expression in High-MKI cases (120 MYCN amplified and 121 non-MYCN amplified) was examined by immunohistochemistry. The majority (101) of MYCN-amplified High-MKI tumors were MYCN(+), leaving one MYC(+), 2 both(+), and 16 both(-)/(+/-), whereas non-MYCN-amplified cases appeared heterogeneous, including 7 MYCN(+), 36 MYC(+), 3 both(+), and 75 both(-)/(+/-) tumors. These MYC-family proteins(+), or MYC-family driven tumors, were most likely to have prominent nucleolar (PN) formation (indicative of augmented rRNA synthesis). High-MKI neuroblastoma patients showed a poor survival irrespective of MYCN amplification. However, patients with MYC-family driven High-MKI neuroblastomas had significantly lower survival than those with non-MYC-family driven tumors. MYCN(+), MYC-family protein(+), PN(+), and clinical stage independently predicted poor survival. Specific inhibition of hyperactive rRNA synthesis and protein translation was shown to be an effective way to suppress MYC/MYCN protein expression and neuroblastoma growth. Together, MYC-family protein overexpression and PN formation should be included in new neuroblastoma risk stratification and considered for potential therapeutic targets.

Local drug delivery minimizes systemic toxicity while delivering high-dose chemotherapy for neuroblastoma patients. We hypothesized that varying burst and maintenance dosing of implanted silk platforms would improve survival.Platforms were loaded with vincristine 25μg, 50μg, 100μg, and 200μg varying burst (released 1-4days postimplantation) and maintenance (over the next 20days) dosing. Orthotopic tumors were created in mice using human neuroblastoma KELLY cells. Silk platforms were implanted into tumors when volumewas300mmDrug release ranged from burst dosing of 18.2 to 80.9μg, maintenance of 5.0 to 111.6μg, and cumulative of 23.3 to 177.4μg. Animals treated with 200μg platform died 9-13days postimplantation, corresponding to 128.1-141.2μg released (toxic dose). Animals received 30.2±3.4μgday-one survived longer than those that received 10.1±1.1μg (p=0.03), suggesting10.1μgday-one was insufficient. Tumors treated with 100μg or 50μg silk platform took longer to reach 1000 mmPlatform formulations can be manipulated to vary burst and maintenance dosing, summarized by an equation consisting of these variables.

We have previously reported histologic variants among unfavorable histology neuroblastomas (NB), having large cell appearance with vesicular nuclei and or prominent nucleolar (PN) formation, indicating hyperactive rRNA synthesis and protein translation. These features appear to be associated with dismal outcome and high-level MYC family protein expression. PN formation could provide critical prognostic and risk-stratification information for NB. At the same time, this observation could open up an opportunity for innovative therapy for aggressive NB. To assess this possibility, we examined the effect of two potent and specific small-molecule inhibitors of RNA Pol I activity and an aminoacyl tRNA synthetase (CX-5461 and Halofuginone, respectively) on growth and MYC family protein expression in NB cell lines. These small-molecule inhibitors inhibited growth of NB cell lines at low to submicromolar concentrations in vitro in 48 hours. Moreover, the inhibitors destabilized MYC and MYCN proteins in NB cells. Interestingly, halofuginone showed a rapid effect on the stability of MYC family proteins at 500 nM-1 uM ( Citation Format: Naohiko Ikegaki, Jasmine Zeki, Bill Chiu, Hiroyuki Shimada. Inhibitors of RNA Pol I and an aminoacyl-tRNA synthetase result in MYC and MYCN downregulation in MYC family protein-driven neuroblastoma cells [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 2497.