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Neuroblastoma is the most common extracranial malignant solid tumor in children, and drug resistance is a major reason for poor outcomes. Elevated proteasome activity plays an important role in neuroblastoma tumor development and resistance to conventional chemotherapy. Ubiquitin-specific protease 14 (USP14), one of three deubiquitinases associated with the regulatory subunit of the proteasome, is emerging as a potential therapeutic target in multiple tumor types. However, the role of USP14 in neuroblastoma is yet to be elucidated. We found that USP14 inhibition in neuroblastoma via knockdown or a specific inhibitor such as b-AP15 suppressed cell proliferation by inducing cell apoptosis. Furthermore, b-AP15 significantly inhibited neuroblastoma tumor growth in NGP and SH-SY5Y xenograft mouse models. For combination treatment, b-AP15 plus conventional chemotherapeutic agents such as doxorubicin or VP-16 resulted in synergistic antitumor effects on neuroblastoma. Our study demonstrates that USP14 is required for cell viability and is a novel therapeutic target in neuroblastoma. Moreover, USP14 inhibition may add value in combination therapy due to its powerful synergistic effects in treating neuroblastoma.

USP14 inhibitor b-AP15 inhibits colony formation in neuroblastoma cells.

Summary of 6 neuroblastoma cell lines

Driver mutations in CTNNB1 are a hallmark of hepatoblastoma and offer a common biomarker for a liquid biopsy approach based on the presence of CTNNB1 circulating tumor DNA (ctDNA). We developed and investigated the utility of a quantitative universal next-generation sequencing (NGS) ctDNA assay for hepatoblastoma (QUENCH) to detect CTNNB1 ctDNA and assessed the links between ctDNA and current clinical indicators/biomarkers in hepatoblastoma. Applied to patients with hepatoblastoma, we demonstrate quantitation of various variants including single base substitutions and deletions down to 0.3% variant allele frequency, with 65% sensitivity and 100% specificity at the patient level, to allow biopsy-free tumor genotyping and sensitive ctDNA quantitation. CtDNA positivity correlates with tumor burden and ctDNA levels correlate with macroscopic residual disease and treatment response, thus providing promising evidence for the utility of quantitative ctDNA detection in hepatoblastoma.

Introduction: Hepatoblastoma (HB) is the most common pediatric primary liver tumor and has the fastest rising incidence of all pediatric solid tumors. Patients with high-risk, treatment refractory, or relapse disease have a survival rate of less than 50%. The development of clinically relevant models of these aggressive tumors will facilitate studies to identify drugs that target these cells.Methods: Fresh, whole primary tumor samples were implanted into the livers of immunocompromised mice. Tumor growth was monitored with MRI and ELISA to measure serum human Alpha-fetoprotein (AFP), which is detectable in the blood of tumor-bearing animals. Tumors were validated with immunohistochemistry (IHC) for HB markers Glypican-3 (GPC3) and Beta-catenin; short tandem repeat (STR) DNA validation; next generation sequencing-based mutation profiling of 124 genes involved in pediatric solid tumors; RNA sequencing (RNA-seq), and single cell RNA-seq (scRNA-seq). Lung metastasis was also detected in models with serial sectioning and H&E staining. Cells derived from tumors were grown in vitro in adherent and spheroid conditions and used for high throughput drug screening of candidate agents. Tumors were serially passaged in animals for further in vivo drug testing of novel targeted agents.Results: Nine patient-derived xenograft (PDX) models were generated that represent low- and high-risk tumors, treatment refractory cases, and relapsed tumors. Passaging of these models showed consistent implantation rates at or above 80% with tumors detectable in 2 to 4 weeks. Eight of nine models secrete human serum AFP. All models mimic gene expression and histological patterns of their primary tumor counterparts as well as identical STR DNA profiles. The models also show gene expression consistent with an HB2/high-risk profile according to the Sumazin HB expression signature. Interestingly, two models represent unique sub-clones of a very aggressive HB relapse with different AFP secretion and transcriptomic expression. scRNA-seq of these two models indicated outgrowth of disparate disease sub-clones. The nine models also demonstrate a range of DNA mutations with three or four mutations per tumor; all variants present in the original clinical samples were conserved in the PDX models. Lung metastasis was evident in six of nine models. Two stable patient-derived cell lines (PDCLs) were developed from models, and these cell lines show expression of HB markers and secrete AFP with growth in culture. Drug screening of adherent and spheroid tumor cells support the efficacy of novel targeted agents and indicate a spectrum of sensitivity to cisplatin, a frontline standard chemotherapy agent. Importantly, the models replicate the chemotherapy responses of the corresponding patients. Additional in vitro and in vivo work showed the efficacy of a histone deacetylase inhibitor, panobinostat.Conclusions: These novel orthotopic PDX models of HB fully recapitulate the primary tumors and represent a platform for clinically relevant drug screening and testing. Citation Format: Sarah E Woodfield, Roma H Patel, Andres F Espinoza, Richard S Whitlock, Jessica Epps, Andrew Badachhape, Samuel R Larson, Rohit K Srivastava, Aayushi P Shah, Saiabhiroop R Govindu, Barry Zorman, Brandon J Mistretta, Kevin E Fisher, Ilavarasi Gandhi, Jacquelyn Reuther, Martin Urbicain, Aryana M Ibarra, Sakuni Rankothgedera, Kimberly R Holloway, Stephen F Sarabia, Andras Heczey, Ketan B Ghaghada, Kalyani R Patel, Dolores Lopez-Terrada, Angshumoy Roy, Preethi H Gunaratne, Pavel Sumazin, Sanjeev A Vasudevan. Patient-derived xenograft mouse models of hepatoblastoma for a personalized medicine pipeline [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO013.

Hepatoblastoma (HB) is the most common pediatric primary liver malignancy, and survival for high-risk disease approaches 50%. Mouse models of HB fail to recapitulate hallmarks of high-risk disease. The aim of this work was to generate murine models that show high-risk features including multifocal tumors, vascular invasion, metastasis, and circulating tumor cells (CTCs). HepT1 cells were injected into the livers or tail veins of mice, and tumor growth was monitored with magnetic resonance and bioluminescent imaging. Blood was analyzed with fluorescence-activated cell sorting to identify CTCs. Intra- and extra-hepatic tumor samples were harvested for immunohistochemistry and RNA and DNA sequencing. Cell lines were grown from tumor samples and profiled with RNA sequencing. With intrahepatic injection of HepT1 cells, 100% of animals grew liver tumors and showed vascular invasion, metastasis, and CTCs. Mutation profiling revealed genetic alterations in seven cancer-related genes, while transcriptomic analyses showed changes in gene expression with cells that invade vessels. Tail vein injection of HepT1 cells resulted in multifocal, metastatic disease. These unique models will facilitate further meaningful studies of high-risk HB. This article has an associated First Person interview with the first author of the paper.

Hepatoblastoma (HB) and hepatocellular carcinoma (HCC) are the predominant liver cancers in children, though their respective treatment options and associated outcomes differ dramatically. Risk stratification using a combination of clinical, histological, and molecular parameters can improve treatment selection, but it is particularly challenging for tumors with mixed histological features, including those in the recently created hepatocellular neoplasm not otherwise specified (HCN NOS) provisional category. We aimed to perform the first molecular characterization of clinically annotated cases of HCN NOS.We tested whether these histological features are associated with genetic alterations, cancer gene dysregulation, and outcomes. Namely, we compared the molecular features of HCN NOS, including copy number alterations, mutations, and gene expression profiles, with those in other pediatric hepatocellular neoplasms, including HBs and HCCs, as well as HBs demonstrating focal atypia or pleomorphism (HB FPAs), and HBs diagnosed in older children (8).Molecular profiles of HCN NOS and HB FPAs revealed common underlying biological features that were previously observed in HCCs. Consequently, we designated these tumor types collectively as HBs with HCC features (HBCs). These tumors were associated with high mutation rates (∼3 somatic mutations/Mb) and were enriched with mutations and alterations in key cancer genes and pathways. In addition, recurrent large-scale chromosomal gains, including gains of chromosomal arms 2q (80%), 6p (70%), and 20p (70%), were observed. Overall, HBCs were associated with poor clinical outcomes.Our study indicates that histological features seen in HBCs are associated with combined molecular features of HB and HCC, that HBCs are associated with poor outcomes irrespective of patient age, and that transplanted patients are more likely to have good outcomes than those treated with chemotherapy and surgery alone. These findings highlight the importance of molecular testing and early therapeutic intervention for aggressive childhood hepatocellular neoplasms.We molecularly characterized a class of histologically aggressive childhood liver cancers and showed that these tumors are clinically aggressive and that their observed histological features are associated with underlying recurrent molecular features. We proposed a diagnostic algorithm to identify these cancers using a combination of histological and molecular features, and our analysis suggested that these cancers may benefit from specialized treatment strategies that may differ from treatment guidelines for other childhood liver cancers.

Hepatoblastoma (HB) is the most common pediatric primary liver malignancy, and survival for high-risk disease approaches 50%. Mouse models of HB fail to recapitulate hallmarks of high-risk disease. The aim of this work was to generate murine models that show high-risk features including multifocal tumors, vascular invasion, metastasis, and circulating tumor cells (CTCs). HepT1 cells were injected into the livers or tail veins of mice, and tumor growth was monitored with magnetic resonance and bioluminescent imaging. Blood was analyzed with fluorescence activated cell sorting to identify CTCs. Intra- and extra-hepatic tumor samples were harvested for immunohistochemistry and RNA and DNA sequencing. Cell lines were grown from tumor samples and profiled with RNA sequencing. With intrahepatic injection of HepT1 cells, 100% of animals grew liver tumors and showed vascular invasion, metastasis, and CTCs. Mutation profiling revealed genetic alterations in seven cancer-related genes, while transcriptomic analyses showed changes in gene expression with cells that invade vessels. Tail vein injection of HepT1 cells resulted in multifocal, metastatic disease. These unique models will facilitate further meaningful studies of high-risk HB.Summary StatementIn this work, we developed and thoroughly characterized several unique models of hepatoblastoma derived from the HepT1 cell line that show high-risk features.

Neuroblastoma (NB) is the most common extracranial solid tumor in early childhood. Despite intensive multimodal therapy, nearly half of children with high-risk disease will relapse with therapy-resistant tumors. Dysregulation of MAPK pathway has been implicated in the pathogenesis of relapsed and refractory NB patients, which underscores the possibility of targeting MAPK signaling cascade as a novel therapeutic strategy. In this study, we found that high expressions of RAF family kinases correlated with advanced tumor stage, high-risk disease, tumor progression, and poor overall survival. Targeted inhibition of RAF family kinases with the novel small molecule inhibitor agerafenib abrogated the activation of ERK MAPK pathway in NB cells. Agerafenib significantly inhibited the cell proliferation and colony formation ability of NB cells in vitro, and its combination with traditional chemotherapy showed a synergistic pro-apoptotic effect. More importantly, agerafenib exhibited a favorable toxicity profile, potently suppressed tumor growth, and prolonged survival in NB mouse models. In conclusion, our preclinical data suggest that agerafenib might be an effective therapeutic agent for NB treatment, both as a single-agent and in combination with chemotherapy.

Introduction: Hepatoblastoma (HB) is the most frequent liver malignancy of childhood with an annual incidence of 1.5 per million and a survival rate less than 50%. Due to significant side effects and ineffectiveness of standard chemotherapy for relapse refractory disease, the treatment options are now shifting towards targeted therapies. Cyclin-dependent kinases (CDKs) are potential cancer therapeutic targets because of their critical role in promoting cell growth and regulating transcriptional processes. This study aims to assess the therapeutic efficacy of dinaciclib, cyclin-dependent kinase inhibitor, with HB in preclinical in vitroand in vivo models. Method: Dinaciclib was tested in patient derived cell lines (PDCL) from different patient derived xenograft (PDX) models using CellTiter-Glo Luminescent Viability assays. Dinaciclib was also assayed in vitro for cytotoxicity (MTT assay) and proliferation (CCK-8 assay). Differential expression of CDKs was checked by microarray analysis between normal liver and HB patients. Results were confirmed by immunoblotting assays. Immunoblotting assays were also used to see the effect of dinaciclib on CDK 1/2/5/9 and to assess the induction of apoptosis (PARP cleavage) in vitro. Dinaciclib was also tested in our orthotopic PDX models of high-risk HB. MRI and alpha-fetoprotein (AFP) ELISA were done to evaluate tumor growth. Tumor weights and volumes, as well as, immunohistochemistry were also used to assess effects of dinaciclib in vivo. Result: Dinaciclib suppressed cell proliferation and viability in a dose-dependent manner in all HB cell lines tested with IC50 in the low micromolar range (HepG2- 3.75; HepT1- 0.007; Huh6- 1.42; HB17- 0.72). Microarray analysis showed significant upregulation of CDK1 (p=0.0003), CDK2 (p=0.00023) and CDK9 (p=0.015) in HB patient samples, however, immunoblotting with dinaciclib-treated cells showed decreased expression of CDKs 1/2/5/9 as well as induction of apoptosis. Dinaciclib also showed in vivo inhibition of tumor growth compared to placebo in both PDX models (HB47: relative tumor volume p = 0.03, T/C ratio= 0.36, tumor weight p = 0.02; HB52: relative tumor volume p = 0.06, T/C ratio= 0.16, tumor weight p = 0.07). Conclusion: Dinaciclib treatment showed promising effects with preclinical HB cell lines and PDX models. Citation Format: Rohit K. Srivastava, Roma Patel, Andres F. Espinoza, Yang Yu, Richard S. Whitlock, Samuel Larson, Andrew Badachhape, Jianhua Yang, Sarah E. Woodfield, Sanjeev A. Vasudevan. Efficacy of dinaciclib in hepatoblastoma through inhibition of cyclin dependent kinases [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 3891.

Hepatoblastoma (HB) is the most common pediatric liver malignancy. High-risk patients have poor survival, and current chemotherapies are associated with significant toxicities. Targeted therapies are needed to improve outcomes and patient quality of life. Most HB cases are TP53 wild-type; therefore, we hypothesized that targeting the p53 regulator Murine double minute 4 (MDM4) to reactivate p53 signaling may show efficacy. MDM4 expression was elevated in HB patient samples, and increased expression was strongly correlated with decreased expression of p53 target genes. Treatment with NSC207895 (XI-006), which inhibits MDM4 expression, or ATSP-7041, a stapled peptide dual inhibitor of MDM2 and MDM4, showed significant cytotoxic and antiproliferative effects in HB cells. Similar phenotypes were seen with short hairpin RNA (shRNA)-mediated inhibition of MDM4. Both NSC207895 and ATSP-7041 caused significant upregulation of p53 targets in HB cells. Knocking-down TP53 with shRNA or overexpressing MDM4 led to resistance to NSC207895-mediated cytotoxicity, suggesting that this phenotype is dependent on the MDM4-p53 axis. MDM4 inhibition also showed efficacy in a murine model of HB with significantly decreased tumor weight and increased apoptosis observed in the treatment group. This study demonstrates that inhibition of MDM4 is efficacious in HB by upregulating p53 tumor suppressor signaling.

Hepatoblastoma (HB) is the most common pediatric liver malignancy. Management of HB requires multidisciplinary efforts. The 5-year overall survival of this disease is about 80% in developed countries. Despite advances in the care of these patients, survival in recurrent or treatment-refractory disease is lower than 50%. This is due to more complex tumor biology, including hepatocellular carcinoma (HCC)-like mutations and expression of aggressive gene signatures leading to chemoresistance, vascular invasion, and metastatic spread. The current treatment protocols for pediatric liver cancer do not incorporate targeted therapies, and the ability to test these therapies is limited due to the inaccessibility of cell lines and mouse models. In this review, we discuss the current status of preclinical animal modeling in pediatric liver cancer, primarily HB. Although HB is a rare cancer, the research community has worked together to develop a range of interesting and relevant mouse models for diverse preclinical studies.

Introduction: Hepatoblastoma (HB) is the most frequent liver malignancy of childhood with an annual incidence of 1.5 per million and a survival rate less than 50% for high-risk, relapse, and treatment refractory patients. The overall goal of this study is to assess the therapeutic efficacies and anti-cancer mechanisms of novel drugs with HB through a drug sensitivity testing pipeline utilizing multiple novel, clinically relevant preclinical patient-derived cell line (PDCL) and patient-derived xenograft (PDX) models. Methods: A patient derived xenograft (PDX) mouse model was generated with intrahepatic implantation of tumor thrombus from a non-metastatic stage 3 patient who recurred after transplantation. A stable PDCL was grown from the murine tumor, and 60 targeted agents were tested for efficacy with the PDCL by using CellTiter-Glo Luminescent Viability assays. Anti-oncogenic effects of the most efficacious agent, dinaciclib (cyclin-dependent kinase (CDK) inhibitor), were further assayed in vitro with HB cell lines (HepG2, HepT1, Huh6, HB17) with phenotypic assays for cell toxicity (MTT assay), proliferation (CCK-8 assay), and anchorage independent colony formation (soft agar assay). Immunoblotting assays were used to assess changes in drug targets and induction of apoptosis (PARP cleavage) with cell lines. Dinaciclib was also tested in vivo in our orthotopic PDX models of high-risk HB. The drug was administered three times a week by intraperitoneal injection (20 mg/kg, 100 µL) for 3 weeks. Magnetic resonance imaging (MRI) and Alpha-fetoprotein (AFP) ELISA were done to evaluate tumor growth throughout the study as tumor-bearing animals show expression of human serum AFP. Tumor weights and volumes, as well as immunohistochemistry for H&E, Ki-67, and TUNEL, were also used to assess effects of drug in vivo. Results: The agent that showed the lowest IC50 value out of 60 tested agents was the CDK inhibitor, dinaciclib (0.7 µM). Dinaciclib suppressed cell proliferation and viability in a dose-dependent manner in all HB cell lines tested with IC50 values in the low micromolar range (HepG2 3.75 µM; HepT1 0.007 µM; Huh-6 1.42 µM; HB17 0.72 µM). Immunoblotting with dinaciclib-treated cells showed induction of PARP cleavage, indicating apoptosis, along with decreased protein expression of CDK1, 2, 5, and 9. Dinaciclib also showed significant in vivo inhibition of tumor growth compared to placebo (relative tumor volume p = 0.03, tumor weight p = 0.02). Conclusions: Drug screening of a novel HB PDCL identified an agent, dinaciclib, that shows preclinical efficacy with refractory disease. Further in vitro and in vivo validation confirmed that dinaciclib is a promising therapeutic agent for the treatment of HB. Such a personalized medicine pipeline of development of PDCLs and testing them with targeted agents will lead to the identification of new drugs that can be used with patients that do not respond to standard therapies. Citation Format: Rohit Kumar Srivastava, Roma Patel, Richard S. Whitlock, Samuel R. Larson, Andrew Badachhape, Sarah E. Woodfield, Sanjeev A. Vasudevan. Avenue to personalized targeted therapy for hepatoblastoma [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 1174.

Introduction: Hepatoblastoma (HB) is the most common pediatric primary liver tumor and has the fastest rising incidence of all pediatric solid tumors. Patients with high-risk, treatment refractory, or relapse disease have a survival rate of less than 50%. The development of clinically relevant models of these aggressive tumors will facilitate studies to identify drugs that target these cells. Methods: Fresh, whole primary tumor samples were implanted into the livers of immunocompromised mice. Tumor growth was monitored with MRI and ELISA to measure serum human Alpha-fetoprotein (AFP), which is detectable in the blood of tumor-bearing animals. Tumors were validated with immunohistochemistry (IHC) for HB markers Glypican-3 (GPC3) and Beta-catenin; short tandem repeat (STR) DNA validation; next generation sequencing-based mutation profiling of 124 genes involved in pediatric solid tumors; and RNA sequencing (RNA-seq). Cells derived from tumors were grown in vitro and used for high throughput drug screening of candidate agents. Tumors were serially passaged in animals for further in vivo drug testing of novel targeted agents. Results: Nine patient-derived xenograft (PDX) models were generated that represent low- and high-risk tumors, treatment refractory cases, and relapsed tumors. Passaging of these models showed consistent implantation rates at or above 80% with tumors detectable in 2 to 4 weeks. Eight of nine models secrete human serum AFP. All models mimic gene expression and histological patterns of their primary tumor counterparts as well as identical STR DNA profiles. The models also show gene expression consistent with an HB2/high-risk profile according to the Sumazin HB expression signature. Interestingly, two models represent unique sub-clones of a very aggressive HB relapse with different AFP secretion and transcriptomic expression. The nine models also demonstrate a range of DNA mutations with three or four mutations per tumor; all variants present in the original clinical samples were conserved in the PDX models. Drug screening of tumor cells support the efficacy of novel targeted agents and indicate that these tumors are resistant to frontline standard chemotherapy, cisplatin and doxorubicin. More extensive in vivo testing showed the efficacy of a dual MDM2/MDM4 inhibitor (ALRN-6924), a cyclin dependent kinase inhibitor (dinaciclib), and a histone deacetylase inhibitor (panobinostat), and use of an orthotopic animal model also revealed drug toxicities associated with compromised liver function. Conclusions: These novel orthotopic PDX models of HB fully recapitulate the primary tumors and represent a platform for clinically relevant drug screening and testing. Further studies of the sub-clones of disease that grow in the animals will yield information about particularly aggressive HBs. Citation Format: Sarah Elizabeth Woodfield, Roma H. Patel, Samuel R. Larson, Andrew Badachhape, Rohit K. Srivastava, Aayushi P. Shah, Brandon Mistretta, Barry Zorman, Kevin Fisher, Ilavarasi Gandhi, Jacquelyn Reuther, Richard S. Whitlock, Aryana M. Ibarra, Sakuni Rankothgedera, Kimberly R. Holloway, Stephen F. Sarabia, Martin Urbicain, Andras Heczey, Dolores Lopez-Terrada, Angshumoy Roy, Preethi Gunaratne, Pavel Sumazin, Sanjeev A. Vasudevan. Novel orthotopic patient-derived xenograft mouse models of hepatoblastoma that replicate tumor heterogeneity, chemoresistance, and refractory disease [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 2997.

Driver mutations in CTNNB1 are a hallmark of hepatoblastoma (HB) and offer a common biomarker for a liquid biopsy approach that is based on the presence of CTNNB1 circulating tumor DNA (ctDNA). Initial results 1 showed an association of CTNNB1 ctDNA variant allele frequency (VAF) identified through digital droplet PCR (ddPCR) and the levels of serum Alpha-fetoprotein (AFP, current HB biomarker) throughout the course of treatment of three patients with HB. However, applying custom probe-designed ddPCR assays may pose challenges for real-time data at diagnosis. Our primary objective is to investigate the utility of a universal next-generation sequencing (NGS) assay to detect CTNNB1 ctDNA in patients with HB. Our secondary objective is to compare the levels of: (1) NGS ctDNA, (2) ddPCR ctDNA, (3) AFP, and verify if they correlate with tumor burden and treatment response. We developed and tested a custom NGS assay covering exons 2 to 4 of CTNNB1 for detection of somatic mutations in plasma. We used the QIASeq platform which: (1) is based on a single primer extension and, thus, captures both single nucleotide variants (SNV) and structural variants (SV), and (2) integrates unique molecular indexing (UMI), thus allowing determination of ctDNA levels from VAF. Our cohort included 18 patients, 13 with Sanger sequencing confirmed CTNNB1 somatic mutations in the matched tumors: 6 missense SNVs and 7 deletions ranging from 96 to 348 base pairs. Our NGS assay was able to accurately detect mutations in 9/13 (69%, 7 SV and 2 SNV) of the Sanger confirmed cases, and SV and missense SNV in 2/5 (40%) cases pending orthogonal Sanger confirmation. Available ddPCR data for 3 patients (8 samples) 1 and corresponding NGS data showed similar ctDNA levels (deviation ranging between 1.2-8.5%). NGS CTNNB1 variants were detected in samples taken at initial diagnosis in 8/9 (89%), following neoadjuvant chemotherapy in 4/9 (44%), post-operatively for fully resected localized disease in 0/4 (0%), and metastatic recurrence in 1/5 (20%), suggesting a positive correlation between CTNNB1 variants and tumor burden. AFP levels were available for 24/27 samples, with abnormal levels detected in 23 samples. The ctDNA levels determined from NGS did not correlate with AFP levels; however, longitudinal plasma samples (n=9) showed similar dynamics of both ctDNA and AFP, reflecting the response to treatment. To conclude, we show that ctDNA detectable with our universal HB NGS assay is a good surrogate marker of tumor burden and shows correlation with treatment response. SV, prevalent in approximately 50% of cases of HB, may be inadequately captured and thereby misrepresented in probe-capture NGS platforms but were highly detectable in our assay. Further work is needed to understand the interplay of both AFP and ctDNA in HB monitoring. Reference: 1. Kahana-Edwin S, McCowage G, Cain L, et al. Exploration of CTNNB1 ctDNA as a putative biomarker for hepatoblastoma. Pediatr Blood Cancer. 2020. doi:10.1002/pbc.28594 Citation Format: Smadar Kahana-Edwin, Andre E. Minoche, Lucy E. Cain, Geoffrey McCowage, Sarah E. Woodfield, Sanjeev A. Vasudevan, Sarah Kummerfeld, Jonathan Karpelowsky. Utility of CTNNB1 ctDNA as a biomarker for hepatoblastoma [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 582.

// Wenjing Sun 1, 5 , Yesenia Rojas 1 , Hao Wang 2, 6 , Yang Yu 1 , Yongfeng Wang 3 , Zhenghu Chen 3 , Kimal Rajapakshe 4 , Xin Xu 3 , Wei Huang 3 , Saurabh Agarwal 3 , Roma H. Patel 1 , Sarah Woodfield 1 , Yanling Zhao 3 , Jingling Jin 1 , Hong Zhang 2 , Angela Major 7 , M. John Hicks 7 , Jason M. Shohet 3 , Sanjeev A. Vasudevan 1 , Cristian Coarfa 4 , Jianhua Yang 3 and Jed G. Nuchtern 1 1 Pediatric Surgery Division, Michael E. Debakey Department of Surgery, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX 77030, USA 2 Department of Pathology, MD Anderson Cancer Center, Houston, TX 77030, USA 3 Department of Pediatrics, Texas Children’s Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA 4 Department of Molecular and Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA 5 Laboratory of Medical Genetics, Harbin Medical University, Harbin, Heilongjiang 150081, China 6 Department of Hepatopancreatobiliary Surgery, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150086, China 7 Department of Pathology, Texas Children’s Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA Correspondence to: Jed G. Nuchtern, email: nuchtern@bcm.edu Keywords: neuroblastoma; YK-4-279; chemotherapy; doxorubicin; EWSR1 Abbreviations: NB, neuroblastoma; RHA, RNA helicase A; Dox, doxorubicin Received: July 12, 2017 Accepted: August 21, 2017 Published: October 19, 2017 ABSTRACT Treatment failure in high risk neuroblastoma (NB) is largely due to the development of chemotherapy resistance. We analyzed the gene expression changes associated with exposure to chemotherapy in six high risk NB tumors with the aid of the Connectivity Map bioinformatics platform. Ten therapeutic agents were predicted to have a high probability of reversing the transcriptome changes associated with neoadjuvant chemotherapy treatment. Among these agents, initial screening showed the EWS-FLI1 and RNA helicase A interaction inhibitor YK-4-279, had obvious cytotoxic effects on NB cell lines. Using a panel of NB cell lines, including MYCN nonamplified (SK-N-AS, SH-SY5Y, and CHLA-255), and MYCN amplified (NB-19, NGP, and IMR-32) cell lines, we found that YK-4-279 had cytotoxic effects on all lines tested. In addition, YK-4-279 also inhibited cell proliferation and anchorage-independent growth and induced cell apoptosis of these cells. YK-4-279 enhanced the cytotoxic effect of doxorubicin (Dox). Moreover, YK-4-279 was able to overcome the established chemoresistance of LA-N-6 NB cells. In an orthotopic xenograft NB mouse model, YK-4-279 inhibited NB tumor growth and induced apoptosis in tumor cells through PARP and Caspase 3 cleavage in vivo . While EWS-FLI1 fusion protein is not frequently found in NB, using the R2 public database of neuroblastoma outcome and gene expression, we found that high expression of EWSR1 was associated with poor patient outcome. Knockdown of EWSR1 inhibited the oncogenic potential of neuroblastoma cell lines. Taken together, our results indicate that YK-4-279 might be a promising agent for treatment of NB that merits further exploration.

Neuroblastoma (NB), the most common extracranial pediatric solid tumor, continues to cause significant cancer-related morbidity and mortality in children. Dysregulation of oncogenic receptor tyrosine kinases (RTKs) has been shown to contribute to tumorigenesis in various human cancers and targeting these RTKs has had therapeutic benefit. RET is an RTK which is commonly expressed in NB, and high expression of RET correlates with poor outcomes in patients with NB. Herein we report that RET is required for NB cell proliferation and that the small molecule inhibitor regorafenib (BAY 73-4506) blocks glial cell derived neurotrophic factor (GDNF)-induced RET signaling in NB cells and inhibits NB growth both in vitro and in vivo. We found that regorafenib significantly inhibited cell proliferation and colony formation ability of NB cells. Moreover, regorafenib suppressed tumor growth in both an orthotopic xenograft NB mouse model and a TH-MYCN transgenic NB mouse model. Finally, regorafenib markedly improved the overall survival of TH-MYCN transgenic tumor-bearing mice. In summary, our study suggests that RET is a potential therapeutic target in NB, and that using a novel RET inhibitor, like regorafenib, should be investigated as a therapeutic treatment option for children with NB.

Activating germline mutations of anaplastic lymphoma kinase (ALK) occur in most cases of hereditary neuroblastoma (NB) and the constitutively active kinase activity of ALK promotes cell proliferation and survival in NB. Therefore, ALK kinase is a potential therapeutic target for NB. In this study, we show that the novel ALK inhibitor alectinib effectively suppressed cell proliferation and induces apoptosis in NB cell lines with either wild-type ALK or mutated ALK (F1174L and D1091N) by blocking ALK-mediated PI3K/Akt/mTOR signaling. In addition, alectinib enhanced doxorubicin-induced cytotoxicity and apoptosis in NB cells. Furthermore, alectinib induced apoptosis in an orthotopic xenograft NB mouse model. Also, in the TH-MYCN transgenic mouse model, alectinib resulted in decreased tumor growth and prolonged survival time. These results indicate that alectinib may be a promising therapeutic agent for the treatment of NB.

// Shan Guan 1, 2 , Jiaxiong Lu 2 , Yanling Zhao 2 , Sarah E. Woodfield 3 , Huiyuan Zhang 2 , Xin Xu 2 , Yang Yu 2 , Jing Zhao 1 , Shayahati Bieerkehazhi 4 , Haoqian Liang 2, 5 , Jianhua Yang 2 , Fuchun Zhang 1 , Surong Sun 1 1 Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi, Xinjiang 830046, China 2 Texas Children’s Cancer Center, Department of Pediatrics, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA 3 Division of Pediatric Surgery, Michael E. DeBakey Department of Surgery, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA 4 Department of Labour Hygiene and Sanitary Science, College of Public Health, Xinjiang Medical University, Urumqi, Xinjiang 830011, China 5 School of Pharmacy, Zhengzhou University, Zhengzhou, Henan 450001, China Correspondence to: Surong Sun, email: sr_sun2005@163.com Fuchun Zhang, email: zfcxju@xju.edu.cn Keywords: cervical cancer, TAK1 inhibitor, 5Z-7-oxozeaenol, chemotherapy, doxorubicin Received: February 22, 2017 Accepted: March 28, 2017 Published: April 06, 2017 ABSTRACT Aberrant activation of nuclear factor-κB (NF-κB) allows cancer cells to escape chemotherapy-induced cell death and acts as one of the major mechanisms of acquired chemoresistance in cervical cancer. TAK1, a crucial mediator that upregulates NF-κB activation in response to cellular genotoxic stress, is required for tumor cell viability and survival. Herein, we examined whether TAK1 inhibition is a potential therapeutic strategy for treating cervical cancer. We found that TAK1 inhibitor 5Z-7-oxozeaenol significantly augmented the cytotoxic effects of Dox in a panel of cervical cancer cell lines. Treatment with 5Z-7-oxozeaenol hindered Dox-induced NF-κB activation and promoted Dox-induced apoptosis in cervical cancer cells. Moreover, 5Z-7-oxozeaenol showed similar effects in both positive and negative human papillomavirus-infected cervical cancer cells. Taken together, our results provide evidence that TAK1 inhibition significantly sensitizes cervical cancer cells to chemotherapy-induced cell death and supports the use of TAK1 inhibitor with current chemotherapies in the clinic for patients with refractory cervical cancer.

Neuroblastoma is the most common extracranial malignant solid tumor in children, and drug resistance is a major reason for poor outcomes. Elevated proteasome activity plays an important role in neuroblastoma tumor development and resistance to conventional chemotherapy. Ubiquitin-specific protease 14 (USP14), one of three deubiquitinases associated with the regulatory subunit of the proteasome, is emerging as a potential therapeutic target in multiple tumor types. However, the role of USP14 in neuroblastoma is yet to be elucidated. We found that USP14 inhibition in neuroblastoma via knockdown or a specific inhibitor such as b-AP15 suppressed cell proliferation by inducing cell apoptosis. Furthermore, b-AP15 significantly inhibited neuroblastoma tumor growth in NGP and SH-SY5Y xenograft mouse models. For combination treatment, b-AP15 plus conventional chemotherapeutic agents such as doxorubicin or VP-16 resulted in synergistic antitumor effects on neuroblastoma. Our study demonstrates that USP14 is required for cell viability and is a novel therapeutic target in neuroblastoma. Moreover, USP14 inhibition may add value in combination therapy due to its powerful synergistic effects in treating neuroblastoma.

// Shayahati Bieerkehazhi 1, 2, * , Zhenghu Chen 3, 4, * , Yanling Zhao 4 , Yang Yu 4 , Huiyuan Zhang 4 , Sanjeev A. Vasudevan 5 , Sarah E. Woodfield 5 , Ling Tao 4 , Joanna S. Yi 4 , Jodi A. Muscal 4 , Jonathan C. Pang 4, 6 , Shan Guan 4 , Hong Zhang 2 , Jed G. Nuchtern 5 , Hui Li 7 , Huiwu Li 8 , Jianhua Yang 4 1 Department of Labour Hygiene and Sanitary Science, College of Public Health, Xinjiang Medical University, Urumqi, Xinjiang 830011, P.R. China 2 Department of Pathology, University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA 3 Department of Ophthalmology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, P. R. China 4 Texas Children's Cancer Center, Department of Pediatrics, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas 77030, USA 5 Division of Pediatric Surgery, Texas Children’s Hospital Department of Surgery, Michael E. DeBakey Department of Surgery, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas 77030, USA 6 Department of Biosciences, Weiss School of Natural Sciences, Rice University, Houston, Texas 77005, USA 7 Central Laboratory of Xinjiang Medical University, Urumqi, Xinjiang 830011, P.R. China 8 Cancer Prevention and Research Institute, The Affiliated Tumor Hospital of Xinjiang Medical University, Urumqi, Xinjiang 830011, P.R. China * These authors have contributed equally to this work and should be considered co-first authors Correspondence to: Huiwu Li, email: huiwuli1234@163.com Jianhua Yang, email: jianhuay@bcm.edu Keywords: neuroblastoma, bosutinib, SKI-606, Bosulif, chemotherapy Received: October 29, 2016 Accepted: November 12, 2016 Published: November 26, 2016 ABSTRACT Neuroblastoma (NB) is the most common extracranial solid tumor in children. Aberrant activation of the non-receptor tyrosine kinases Src and c-Abl contributes to the progression of NB. Thus, targeting these kinases could be a promising strategy for NB therapy. In this paper, we report that the potent dual Src/Abl inhibitor bosutinib exerts anti-tumor effects on NB. Bosutinib inhibited NB cell proliferation in a dose-dependent manner and suppressed colony formation ability of NB cells. Mechanistically, bosutinib effectively decreased the activity of Src/Abl and PI3K/AKT/mTOR, MAPK/ERK, and JAK/STAT3 signaling pathways. In addition, bosutinib enhanced doxorubicin (Dox)- and etoposide (VP-16)-induced cytotoxicity in NB cells. Furthermore, bosutinib demonstrated anti-tumor efficacy in an orthotopic xenograft NB mouse model in a similar mechanism as of that in vitro . In summary, our results reveal that Src and c-Abl are potential therapeutic targets in NB and that the novel Src/Abl inhibitor bosutinib alone or in combination with other chemotherapeutic agents may be a valuable therapeutic option for NB patients.

// Xinfang Mao 1, 2, * , Zhenghu Chen 2, 3, * , Yanling Zhao 2 , Yang Yu 2 , Shan Guan 1, 2 , Sarah E. Woodfield 4 , Sanjeev A. Vasudevan 4 , Ling Tao 2 , Jonathan C. Pang 2 , Jiaxiong Lu 2 , Huiyuan Zhang 2 , Fuchun Zhang 1 , Jianhua Yang 2 1 Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, P. R. China 2 Texas Children’s Cancer Center, Department of Pediatrics, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas 77030, USA 3 Department of Ophthalmology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, P. R. China 4 Division of Pediatric Surgery, Texas Children’s Hospital Department of Surgery, Michael E. DeBakey Department of Surgery, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas 77030, USA * These authors have contributed equally to this work and should be considered co-first authors Correspondence to: Fuchun Zhang, email: zfcxju@qq.com Jianhua Yang, email: jianhuay@bcm.edu Keywords: neuroblastoma, EGFR inhibitor, afatinib, apoptosis, chemotherapy Received: October 12, 2016 Accepted: November 08, 2016 Published: November 26, 2016 ABSTRACT Neuroblastoma is the most common extracranial solid tumor in children. The ErbB family of proteins is a group of receptor tyrosine kinases that promote the progression of various malignant cancers including neuroblastoma. Thus, targeting them with small molecule inhibitors is a promising strategy for neuroblastoma therapy. In this study, we investigated the anti-tumor effect of afatinib, an irreversible inhibitor of members of the ErbB family, on neuroblastoma. We found that afatinib suppressed the proliferation and colony formation ability of neuroblastoma cell lines in a dose-dependent manner. Afatinib also induced apoptosis and blocked EGF-induced activation of PI3K/AKT/mTOR signaling in all neuroblastoma cell lines tested. In addition, afatinib enhanced doxorubicin-induced cytotoxicity in neuroblastoma cells, including the chemoresistant LA-N-6 cell line. Finally, afatinib exhibited antitumor efficacy in vivo by inducing apoptosis in an orthotopic xenograft neuroblastoma mouse model. Taken together, these results show that afatinib inhibits neuroblastoma growth both in vitro and in vivo by suppressing EGFR-mediated PI3K/AKT/mTOR signaling. Our study supports the idea that EGFR is a potential therapeutic target in neuroblastoma. And targeting ErbB family protein kinases with small molecule inhibitors like afatinib alone or in combination with doxorubicin is a viable option for treating neuroblastoma.

Background High-risk neuroblastoma has poor outcomes with high rates of relapse despite aggressive treatment, and novel therapies are needed to improve these outcomes. Ponatinib is a multi-tyrosine kinase inhibitor that targets many pathways implicated in neuroblastoma pathogenesis. We hypothesized that ponatinib would be effective against neuroblastoma in preclinical models. Methods We evaluated the effects of ponatinib on survival and migration of human neuroblastoma cells in vitro. Using orthotopic xenograft mouse models of human neuroblastoma, we analyzed tumors treated with ponatinib for growth, gross and histologic appearance, and vascularity. Results Ponatinib treatment of neuroblastoma cells resulted in decreased cell viability and migration in vitro. In mice with orthotopic xenograft neuroblastoma tumors, treatment with ponatinib resulted in decreased growth and vascularity. Conclusions Ponatinib reduces neuroblastoma cell viability in vitro and reduces tumor growth and vascularity in vivo. The antitumor effects of ponatinib suggest its potential as a novel therapeutic agent for neuroblastoma, and further preclinical testing is warranted.

Author(s): Woodfield, Sarah E; Guo, Rong Jun; Liu, Yin; Major, Angela M; Hollingsworth, Emporia Faith; Indiviglio, Sandra; Whittle, Sarah B; Mo, Qianxing; Bean, Andrew J; Ittmann, Michael; Lopez-Terrada, Dolores; Zage, Peter E | Abstract: BackgroundUBE4B is an E3/E4 ubiquitin ligase whose gene is located in chromosome 1p36.22. We analyzed the associations of UBE4B gene and protein expression with neuroblastoma patient outcomes and with tumor prognostic features and histology.MethodsWe evaluated the association of UBE4B gene expression with neuroblastoma patient outcomes using the R2 Platform. We screened neuroblastoma tumor samples for UBE4B protein expression using immunohistochemistry. FISH for UBE4B and 1p36 deletion was performed on tumor samples. We then evaluated UBE4B expression for associations with prognostic factors and with levels of phosphorylated ERK in neuroblastoma tumors and cell lines.ResultsLow UBE4B gene expression is associated with poor outcomes in patients with neuroblastoma and with worse outcomes in all patient subgroups. UBE4B protein expression was associated with neuroblastoma tumor differentiation, and decreased UBE4B protein levels were associated with high-risk features. UBE4B protein levels were also associated with levels of phosphorylated ERK.ConclusionsWe have demonstrated associations between UBE4B gene expression and neuroblastoma patient outcomes and prognostic features. Reduced UBE4B protein expression in neuroblastoma tumors was associated with high-risk features, a lack of differentiation, and with ERK activation. These results suggest UBE4B may contribute to the poor prognosis of neuroblastoma tumors with 1p36 deletions and that UBE4B expression may mediate neuroblastoma differentiation.

Hepatoblastoma (HB) is the most common pediatric liver malignancy, and patients with high-risk, metastatic disease have a five-year overall survival (OS) of only about 40%. Of note, HB patients with vascular invasion (VI) have a much worse OS. Our hypothesis is that HB tumors with VI possess unique clones that invade the vasculature, resulting in dissemination of disease. We injected the aggressive HepT1 cell line with confirmed CTNNB1 and NFE2L2 mutations in the livers of immunocompromised NSG mice to generate intrahepatic tumors. After 4 weeks, we had 100% take of tumors in animals. Serum human α-fetoprotein (AFP) increased with the growth of tumors. At time of death, we saw clear presence of intrahepatic VI, vena caval tumor thrombus, extrahepatic disease including within periportal lymph nodes, and lung metastasis in animals with tumors. We grew adherent and nonadherent cell lines from tumor samples, including one cell line from intrahepatic VI and one from vena caval tumor thrombus. Cells derived from VI areas had obvious differences in phenotype from cells from the primary tumor (PT). To further pursue these provocative changes between PT and VI areas, we generated two unique patient-derived xenograft (PDX) models of HB with intrahepatic implantation of PT and VI tumor samples from a nonmetastatic pretreatment extent of disease (PRETEXT) stage 4 patient. Mice harboring the VI PDX showed serum elevation of human AFP and obvious tumor with MRI. PT and VI PDX tissues expressed established HB markers nuclear Beta-catenin, Glypican-3, and AFP, resembling the primary patient sample. We also developed a novel cell line with growth of cells from a VI tumor in vitro. With both the HepT1 and PDX models, gene set enrichment transcriptomic analyses of RNA sequencing data showed clear changes in expression in aggressive tissues that invaded vessels, including significant (p Citation Format: Sarah E. Woodfield, Roma H. Patel, Aryana M. Ibarra, Barry Zorman, Andrew Badachhape, Ketan B. Ghaghada, Dolores Lopez-Terrada, Pavel Sumazin, Sanjeev A. Vasudevan. Characterizing vascular invasion in hepatoblastoma [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 B76.

Hepatoblastoma is the most common malignant liver tumor in children. Since it is often unresectable and exhibits drug resistance, the treatment of advanced hepatoblastoma is challenging. The orphan nuclear receptor liver receptor homolog-1 (LRH-1) serves prominent roles in malignancy; however, to the best of our knowledge, the role of LRH-1 in hepatoblastoma remains unknown. In the present study, human hepatoblastoma cell lines were analyzed; the mRNA and protein expression levels of LRH-1 were significantly higher in HepG2 and HuH6 cells compared with those in HepT1 cells and control THLE-2 cells. Knockdown of LRH-1 resulted in decreased HepG2 and HuH6 cell proliferation via downregulation of cyclin D1 (CCND1) and c-Myc. Furthermore, treatment with an LRH-1 antagonist (LRA) inhibited the proliferation and colony formation of cell lines in a dose-dependent manner, and induced cell cycle arrest at G(1) phase through inhibition of CCND1 expression. Finally, LRA treatment enhanced the cytotoxic effects of doxorubicin on hepatoblastoma cells. Collectively, these findings suggested that LRH-1 may have an important role in the progression of hepatoblastoma and implicated LRA as a novel, potential therapeutic agent for the treatment of hepatoblastoma.

Currently, preclinical testing of therapies for hepatoblastoma (HB) is limited to subcutaneous and intrasplenic xenograft models that do not recapitulate the hepatic tumors seen in patients. We hypothesized that injection of HB cell lines into the livers of mice would result in liver tumors that resemble their clinical counterparts. HepG2 and Huh-6 HB cell lines were injected, and tumor growth was monitored with bioluminescence imaging (BLI) and magnetic resonance imaging (MRI). Levels of human α-fetoprotein (AFP) were monitored in the serum of animals. Immunohistochemical and gene expression analyses were also completed on xenograft tumor samples. BLI signal indicative of tumor growth was seen in 55% of HepG2- and Huh-6-injected animals after a period of four to seven weeks. Increased AFP levels correlated with tumor growth. MRI showed large intrahepatic tumors with active neovascularization. HepG2 and Huh-6 xenografts showed expression of β-catenin, AFP, and Glypican-3 (GPC3). HepG2 samples displayed a consistent gene expression profile most similar to human HB tumors. Intrahepatic injection of HB cell lines leads to liver tumors in mice with growth patterns and biologic, histologic, and genetic features similar to human HB tumors. This orthotopic xenograft mouse model will enable clinically relevant testing of novel agents for HB.

BACKGROUND Outcomes for children with high-risk neuroblastoma are poor, and improved understanding of the mechanisms underlying neuroblastoma pathogenesis, recurrence, and treatment resistance will lead to improved outcomes. Aberrant growth factor receptor expression and receptor tyrosine kinase signaling are associated with the pathogenesis of many malignancies. A germline polymorphism in the FGFR4 gene is associated with increased receptor expression and activity and with decreased survival, treatment resistance, and aggressive disease for many malignancies. We therefore investigated the role of this FGFR4 polymorphism in neuroblastoma pathogenesis. MATERIALS AND METHODS Germline DNA from neuroblastoma patients and matched controls was assessed for the FGFR4 Gly/Arg388 polymorphism by RT-PCR. Allele frequencies were assessed for association with neuroblastoma patient outcomes and prognostic features. Degradation rates of the FGFR4 Arg388 and Gly388 receptors and rates of receptor internalization into the late endosomal compartment were measured. RESULTS Frequency of the FGFR4 AA genotype and the prevalence of the A allele were significantly higher in patients with neuroblastoma than in matched controls. The Arg388 receptor demonstrated slower degradation than the Gly388 receptor in neuroblastoma cells and reduced internalization into multivesicular bodies. CONCLUSIONS The FGFR4 Arg388 polymorphism is associated with an increased prevalence of neuroblastoma in children, and this association may be linked to differences in FGFR4 degradation rates. Our study provides the first evidence of a role for FGFR4 in neuroblastoma, suggesting that FGFR4 genotype and the pathways regulating FGFR4 trafficking and degradation may be relevant for neuroblastoma pathogenesis.

Children with high-risk neuroblastoma have poor survival rates, and novel therapies are needed. Previous studies have identified a role for the HGF/c-Met pathway in neuroblastoma pathogenesis. We hypothesized that EMD1214063 would be effective against neuroblastoma tumor cells and tumors in preclinical models via inhibition of HGF/c-Met signaling. Methods We determined the expression of c-Met protein by Western blots in a panel of neuroblastoma tumor cell lines and neuroblastoma cell viability after treatment with EMD1214063 using MTT assays. TUNEL assays and assays for DNA ladder formation, were performed to measure the induction of apoptosis after EMD1214063 treatment. Inhibition of intracellular signaling was measured by Western blot analysis of treated and untreated cells. To investigate the efficacy of EMD1214063 against neuroblastoma tumors in vivo, neuroblastoma cells were injected orthotopically into immunocompromised mice, and mice were treated with oral EMD1214063. Tumors were evaluated for growth, histologic appearance, and induction of apoptosis by immunohistochemistry. Results All neuroblastoma cell lines were sensitive to EMD1214063, and IC50 values ranged from 2.4 to 8.5 μM. EMD1214063 treatment inhibited HGF-mediated c-Met phosphorylation and MEK phosphorylation in neuroblastoma cells. EMD1214063 induced apoptosis in all tested cell lines. In mice with neuroblastoma xenograft tumors, EMD1214063 treatment reduced tumor growth. Conclusions Treatment of neuroblastoma tumor cells with EMD1214063 inhibits HGF-induced c-Met phosphorylation and results in cell death. EMD1214063 treatment is also effective in reducing tumor growth in vivo. EMD1214063 therefore represents a novel therapeutic agent for neuroblastoma, and further preclinical studies of EMD1214063 are warranted.

Introduction: Despite aggressive therapeutic regimens, long-term survival of patients with high risk neuroblastoma (NB) is less than 50%. The p53 tumor suppressor gene is mutated in more than 50% of human cancers, however, unlike in most adult tumors, p53 is wild-type and functional in 99% of NB cases at diagnosis. Reactivation of p53 tumor suppressor function by pharmacological inhibition of MDM4, a negative regulator of p53, is a novel strategy in NB treatment. We hypothesized that targeting MDM4 to reactivate p53 signaling would have effective anti-tumor effects on NB. Methods: Levels of gene expression of MDM4 and its association with event-free and overall survival in NB patients were analyzed using the publically available patient datasets in the R2 Genomics Analysis and Visualization Platform. MDM4 gene and protein expression were also examined in six NB cell lines with qPCR and immunoblotting assays. The effects of MDM4 inhibitor NSC207895 on p53 wild-type (p53wt) and mutated cell lines were analyzed for cytotoxicity, growth in soft agar, and expression of apoptosis markers and p53 downstream targets. Moreover, an orthotopic xenograft NB mouse model was used to test the in vivo efficacy of NSC207895. Results: MDM4 expression is significantly correlated with both event-free and overall survival of NB patients in the Kocak (n=476) and Oberthuer (n=251) datasets in the R2 Genomics Analysis and Visualization Platform, with higher expressions correlated with decreased survival (*** p Conclusion: Novel MDM4 inhibitor NSC207895 shows anti-tumor efficacy in p53 wild-type NB by reactivating p53 tumor suppressor signaling and inducing p53-mediated apoptosis, both in vitro and in vivo. Inhibition of MDM4 by small molecules is a promising treatment strategy for high risk patients with wild-type p53. Citation Format: Zhenghu Chen, Sarah E. Woodfield, Roma H. Patel, Aryana M. Ibarra, Sanjeev A. Vasudevan. Targeting MDM4 to reactivate p53 signaling in 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 386.

Neuroblastoma (NB) is the most common extracranial malignant solid tumor seen in children and continues to lead to the death of many pediatric cancer patients. The poor outcome in high risk NB is largely attributed to the development of chemoresistant tumor cells. Doxorubicin (dox) has been widely employed as a potent anti-cancer agent in chemotherapeutic regimens; however, it also leads to chemoresistance in many cancer types including NB. Thus, developing novel small molecules that can overcome dox-induced chemoresistance is a promising strategy in cancer therapy. Here we show that the second generation proteasome inhibitor ixazomib (MLN9708) not only inhibits NB cell proliferation and induces apoptosis in vitro but also enhances dox-induced cytotoxicity in NB cells. Ixazomib inhibits dox-induced NF-κB activity and sensitizes NB cells to dox-induced apoptosis. More importantly, ixazomib demonstrated potent anti-tumor efficacy in vivo by enhancing dox-induced apoptosis in an orthotopic xenograft NB mouse model. Collectively, our study illustrates the anti-tumor efficacy of ixazomib in NB both alone and in combination with dox, suggesting that combination therapy including ixazomib with traditional therapeutic agents such as dox is a viable strategy that may achieve better outcomes for NB patients.

Children with aggressive neural tumors have poor survival rates and novel therapies are needed. Previous studies have identified nifurtimox and buthionine sulfoximine (BSO) as effective agents in children with neuroblastoma and medulloblastoma. We hypothesized that nifurtimox would be effective against other neural tumor cells and would be synergistic with BSO. We determined neural tumor cell viability before and after treatment with nifurtimox using MTT assays. Assays for DNA ladder formation and poly-ADP ribose polymerase (PARP) cleavage were performed to measure the induction of apoptosis after nifurtimox treatment. Inhibition of intracellular signaling was measured by Western blot analysis of treated and untreated cells. Tumor cells were then treated with combinations of nifurtimox and BSO and evaluated for viability using MTT assays. All neural tumor cell lines were sensitive to nifurtimox and IC50 values ranged from approximately 20 to 210 μM. Nifurtimox treatment inhibited ERK phosphorylation and induced apoptosis in tumor cells. Furthermore, the combination of nifurtimox and BSO demonstrated significant synergistic efficacy in all tested cell lines. Additional preclinical and clinical studies of the combination of nifurtimox and BSO in patients with neural tumors are warranted.

Background Novel therapies are needed for children with high-risk and relapsed neuroblastoma. We hypothesized that MAPK/ERK kinase (MEK) inhibition with the novel MEK1/2 inhibitor binimetinib would be effective in neuroblastoma preclinical models. Methods Levels of total and phosphorylated MEK and extracellular signal-regulated kinase (ERK) were examined in primary neuroblastoma tumor samples and in neuroblastoma cell lines by Western blot. A panel of established neuroblastoma tumor cell lines was treated with increasing concentrations of binimetinib, and their viability was determined using MTT assays. Western blot analyses were performed to examine changes in total and phosphorylated MEK and ERK and to measure apoptosis in neuroblastoma tumor cells after binimetinib treatment. NF1 protein levels in neuroblastoma cell lines were determined using Western blot assays. Gene expression of NF1 and MEK1 was examined in relationship to neuroblastoma patient outcomes. Results Both primary neuroblastoma tumor samples and cell lines showed detectable levels of total and phosphorylated MEK and ERK. IC50 values for cells sensitive to binimetinib ranged from 8 nM to 1.16 μM, while resistant cells did not demonstrate any significant reduction in cell viability with doses exceeding 15 μM. Sensitive cells showed higher endogenous expression of phosphorylated MEK and ERK. Gene expression of NF1, but not MEK1, correlated with patient outcomes in neuroblastoma, and NF1 protein expression also correlated with responses to binimetinib. Conclusions Neuroblastoma tumor cells show a range of sensitivities to the novel MEK inhibitor binimetinib. In response to binimetinib, sensitive cells demonstrated complete loss of phosphorylated ERK, while resistant cells demonstrated either incomplete loss of ERK phosphorylation or minimal effects on MEK phosphorylation, suggesting alternative mechanisms of resistance. NF1 protein expression correlated with responses to binimetinib, supporting the use of NF1 as a biomarker to identify patients that may respond to MEK inhibition. MEK inhibition therefore represents a potential new therapeutic strategy for neuroblastoma. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2199-z) contains supplementary material, which is available to authorized users.

Introduction: Hepatoblastoma is the most common pediatric liver malignancy. Despite aggressive therapeutic regimens, long-term survival of patients with high-risk, metastatic disease is less than 50%. Most cases of hepatoblastoma do not carry mutations in the TP53 tumor suppressor gene; therefore, we hypothesized that targeting the two major regulators of p53, MDM2 and MDM4, with the stapled peptide dual inhibitor, ATSP-7041, to reactivate p53 signaling would be an effective therapeutic strategy for hepatoblastoma. Methods: Levels of MDM2 and MDM4 gene expression were examined in HB cell lines and in a cohort of 18 primary HB patient samples from stage III (n=12) and IV (n=6) patients in comparison to expression in four adjacent uninvolved liver samples with qPCR experiments. The effects of the dual MDM2/MDM4 inhibitor ATSP-7041 (Aileron Therapeutics) on the same cell lines were analyzed with MTT assays for cytotoxicity and qPCR experiments to look at changes in mRNA expression of p53 targets Bax, Puma, CDKN1A, and MDM2. Results: In 16 of 18 samples, MDM4 expression was higher than average in the uninvolved liver samples; in five samples expression was at least 5-fold higher and in four samples expression was at least 10-fold higher. Of note, all four samples with at least 10-fold higher expression were from stage IV patients. In comparison, MDM2 expression was only elevated in one of the 18 patient samples. All three hepatoblastoma cell lines showed expression of both MDM2 and MDM4. Treatment with ATSP-7041 showed cytotoxic (5.7-8.6 μM) effects in all cell lines tested. All cell lines also showed upregulation of p53 targets Bax (1.4- to 2.4-fold), Puma (2.4- to 3.2-fold), CDKN1A (2.4- to 7.2-fold), and MDM2 (6.1- to 8.3-fold) with treatment. Conclusions: Dual inhibition of MDM2 and MDM4 shows efficacy in preclinical models of hepatoblastoma by upregulating p53 tumor-suppressor signaling in TP53 wild-type cells and is a promising therapeutic strategy for high-risk patients with wild-type TP53. Citation Format: Sarah E. Woodfield, Roma H. Patel, Aryana M. Ibarra, Zhenghu Chen, Sanjeev A. Vasudevan. Reactivation of p53 signaling in hepatoblastoma with a stapled peptide dual inhibitor of MDM2 and MDM4 [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 B35.

Introduction: Neuroblastoma is the most common pediatric extracranial solid tumor. Despite aggressive therapeutic regimens, long-term survival of patients with high-risk disease is less than 50%. The TP53 tumor suppressor gene is wild-type and functional in 99% of neuroblastoma cases at diagnosis. Thus, we hypothesized that targeting the p53 regulator MDM4 to reactivate p53 signaling would be effective in neuroblastoma. Methods: Levels of gene expression of MDM4 and its association with event-free and overall survival in neuroblastoma patients were analyzed using the publically available patient datasets in the R2 Genomics Analysis and Visualization Platform. MDM4 gene and protein expression were also examined in five neuroblastoma cell lines (IMR-32, SH-SY5Y, NB-19, SK-N-AS, and LAN-1) with qPCR and immunoblotting assays. The effects of MDM4 inhibitor NSC207895 on TP53 wild-type IMR-32 and SH-SY5Y, TP53 mutant SK-N-AS, and TP53 null LAN-1 cells were analyzed with MTT assays for cytotoxicity and qPCR experiments to look at p53 targets. Results: MDM4 expression is significantly correlated with both event-free and overall survival of neuroblastoma patients in the Kocak (n=476) and Oberthuer (n=251) datasets in the R2 Genomics Analysis and Visualization Platform, with lower expression correlated with increased survival (p Conclusions: Inhibition of MDM4 shows efficacy in preclinical models of neuroblastoma by upregulating p53 tumor suppressor signaling in TP53 wild-type cells and is a promising therapeutic strategy for high-risk patients with wild-type TP53. Citation Format: Sarah E. Woodfield, Roma H. Patel, Aryana M. Ibarra, Zhenghu Chen, Sanjeev A. Vasudevan. Targeting MDM4 in neuroblastoma to reactivate p53 signaling [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 B36.

Background/Objectives: UBE4B is an E3/E4 ubiquitin ligase involved in growth factor receptor (GFR) trafficking. The UBE4B gene is located in the chromosome 1p36 region commonly deleted in high-risk neuroblastoma tumors. We have previously identified associations of UBE4B expression with neuroblastoma patient outcomes. However, the functional roles of UBE4B in neuroblastoma tumor differentiation are not known. Design/Methods: We analyzed the association of UBE4B gene expression with expression of known markers of neuroblastoma tumor differentiation using publicly available databases and screened cell lines and neuroblastoma tumor samples for UBE4B protein expression using Western blot and quantitative immunohistochemistry. We measured UBE4B expression by Western blot in cell lines before and after induction of differentiation with 13-cis-retinoic acid treatment and determined the effects of UBE4B overexpression and depletion on retinoic acid-induced differentiation using continuous live-cell imaging of neurite morphology, immunohistochemistry, and Western blot for markers of differentiation. Effects on signaling through the Ras/MAPK pathway were measured using Western blots. Results: UBE4B expression was associated with neuroblastoma differentiation in patient tumors, and UBE4B gene and protein expression were associated with expression levels of known markers of neuroblastoma differentiation. Retinoic acid treatment resulted in increased UBE4B expression in retinoic acid-sensitive, but not -resistant, neuroblastoma cells, and UBE4B depletion was associated with increased ERK phosphorylation, increased proliferation, and inhibition of retinoic acid-induced neuroblastoma differentiation. Conclusion: We have demonstrated associations between UBE4B expression and tumor differentiation in gene expression databases and in neuroblastoma tumor samples, and our data suggests that UBE4B expression is required for retinoic acid-induced differentiation, potentially through effects on activation of the Ras/MAPK pathway. These associations between UBE4B and neuroblastoma differentiation combined with the established role of UBE4B in GFR trafficking suggest that UBE4B-mediated receptor trafficking may contribute to the responses of neuroblastoma tumors to therapy and to the outcomes of patients with neuroblastoma. Citation Format: Jacqueline Lesperance, Divya Subramonian, Sarah Woodfield, Rongjun Guo, Andrew Bean, Dolores Lopez-Terrada, Michael Ittmann, Sara Hakim, Peter E. Zage. Growth factor receptor trafficking in neuroblastoma differentiation [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 3467.

Children with high-risk neuroblastoma have poor survival rates, and novel therapies are needed. We hypothesized that cabozantinib would be effective against neuroblastoma tumor cells and tumors in preclinical models via inhibition of receptor tyrosine kinase signaling pathways. We determined neuroblastoma cell viability after treatment with cabozantinib alone and in combination with 13-cis-retinoic acid, topotecan, and temozolomide using MTT assays. Inhibition of RET and intracellular signaling was measured by Western blot analysis of treated and untreated cells. To investigate the efficacy of cabozantinib against neuroblastoma tumors in vivo, neuroblastoma cells were injected orthotopically into immunocompromised mice, and mice were treated with oral cabozantinib. Tumors were evaluated for growth by determination of in vivo luminescence and final tumor weights. All neuroblastoma cell lines were sensitive to cabozantinib, and IC50 values ranged from 1.6 to 16.2 μM. Cabozantinib treatment was synergistic with 13-cis-retinoic acid and chemotherapy agents topotecan and temozolomide. Cabozantinib treatment inhibited RET phosphorylation in all cell lines and ERK phosphorylation in more sensitive neuroblastoma cell lines. In mice with neuroblastoma xenograft tumors, cabozantinib treatment significantly reduced tumor growth. Treatment of neuroblastoma tumor cells with cabozantinib inhibits RET and ERK phosphorylation and is effective against neuroblastoma tumor cell lines alone and in combination with 13-cis-retinoic acid, topotecan, and temozolomide. Cabozantinib treatment is also effective in reducing tumor growth in vivo. Cabozantinib therefore represents a novel therapeutic agent for neuroblastoma, and further preclinical and clinical studies are warranted.

Objective: New therapeutic strategies are desperately needed for hepatocellular carcinoma (HCC), the fourth most common cause of cancer-related deaths worldwide. Most patients with HCC lack mutations in the p53 tumor suppressor gene. Therefore, we hypothesized that targeting MDM4 or MDM2 to reactivate p53 signaling could be effective in this disease. Methods: HCC cell lines (SK-Hep1, Huh-7, Hep3B) were profiled for gene and protein expression of MDM4 with qPCR and immunoblotting assays, respectively. The effects of MDM4 inhibitor NSC207895 on the same cell lines were analyzed with MTT assays for cytotoxicity and qPCR experiments to look at changes in expression of p53 downstream targets, in comparison to the effects of MDM2 inhibitor Nutlin-3a. Effects of MDM4 on targets other than p53, including family members p63 and p73, were examined in cells with mutant or null p53 for changes in expression with qPCR experiments. Results: Three HCC cell lines showed expression of MDM4. Inhibition of MDM4 showed cytotoxic (high dose, 0.16-15.0 μM) and anti-proliferative (low dose, 0.05-0.3 μM) effects in all three cell lines, including p53 mutant Huh-7 and p53 null Hep3B cells. The same cell lines were strongly resistant to Nutlin-3a. Gene expression analyses were conducted with cells exposed to NSC207895 to examine effects of MDM4 inhibition on p53 targets Bax, Puma, and p21. In SK-Hep1 cells, NSC207895 treatment led to 2.5-fold overexpression of Bax, 27.4-fold of Puma, and 23.1-fold of p21. Huh-7 showed 11.8-fold upregulation of Puma and 12.4-fold of p21, and Hep3B exhibited a very strong 58.4-fold increase in p21 gene expression with the same treatment. Interestingly, Hep3B showed 5.9-fold overexpression of p63 and 1.99-fold of p73 gene expression with NSC207895 exposure, suggesting novel interactions between MDM4 and p63/p73 in the absence of p53. Conclusions: Inhibition of MDM4 shows efficacy in in vitro models of HCC through upregulation of p53, p63, and p73 to activate p53 tumor suppressive signaling. Thus, targeting MDM4 may be a viable option for all HCC patients, no matter the p53 mutation status. Citation Format: Sarah E. Woodfield, Yan Shi, Roma H. Patel, Zhenghu Chen, Aryana M. Ibarra, Sanjeev A. Vasudevan. Targeting MDM4 in hepatocellular carcinoma to reactivate p53 signaling [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 4127. doi:10.1158/1538-7445.AM2017-4127

In Drosophila imaginal epithelia, cells mutant for the endocytic neoplastic tumor suppressor gene vps25 stimulate nearby untransformed cells to express Drosophila Inhibitor-of-Apoptosis-Protein-1 (DIAP-1), conferring resistance to apoptosis non-cell autonomously. Here, we show that the non-cell autonomous induction of DIAP-1 is mediated by Yorkie, the conserved downstream effector of Hippo signaling. The non-cell autonomous induction of Yorkie is due to Notch signaling from vps25 mutant cells. Moreover, activated Notch in normal cells is sufficient to induce non-cell autonomous Yorkie activity in wing imaginal discs. Our data identify a novel mechanism by which Notch promotes cell survival non-cell autonomously and by which neoplastic tumor cells generate a supportive microenvironment for tumor growth.

Programmed cell death, or apoptosis, is a highly conserved cellular process that has been intensively investigated in nematodes, flies and mammals. The genetic conservation, the low redundancy, the feasibility for high-throughput genetic screens and the identification of temporally and spatially regulated apoptotic responses make Drosophila melanogaster a great model for the study of apoptosis. Here, we review the key players of the cell death pathway in Drosophila and discuss their roles in apoptotic and non-apoptotic processes.

Background Genetic studies in yeast have identified class E vps genes that form the ESCRT complexes required for protein sorting at the early endosome. In Drosophila, mutations of the ESCRT-II component vps25 cause endosomal defects leading to accumulation of Notch protein and increased Notch pathway activity. These endosomal and signaling defects are thought to account for several phenotypes. Depending on the developmental context, two different types of overgrowth can be detected. Tissue predominantly mutant for vps25 displays neoplastic tumor characteristics. In contrast, vps25 mutant clones in a wild-type background trigger hyperplastic overgrowth in a non-autonomous manner. In addition, vps25 mutant clones also promote apoptotic resistance in a non-autonomous manner. Principal Findings Here, we genetically characterize the remaining ESCRT-II components vps22 and vps36. Like vps25, mutants of vps22 and vps36 display endosomal defects, accumulate Notch protein and – when the tissue is predominantly mutant – show neoplastic tumor characteristics. However, despite these common phenotypes, they have distinct non-autonomous phenotypes. While vps22 mutations cause strong non-autonomous overgrowth, they do not affect apoptotic resistance. In contrast, vps36 mutations increase apoptotic resistance, but have little effect on non-autonomous proliferation. Further characterization reveals that although all ESCRT-II mutants accumulate Notch protein, only vps22 and vps25 mutations trigger Notch activity. Conclusions/Significance The ESCRT-II components vps22, vps25 and vps36 display common and distinct genetic properties. Our data redefine the role of Notch for hyperplastic and neoplastic overgrowth in these mutants. While Notch is required for hyperplastic growth, it appears to be dispensable for neoplastic transformation.