Pediatric Tumor Cell Lines Research Articles

Evaluating antitumor activity and response determinants to trastuzumab deruxtecan in pediatric solid tumors.

10049 Background: Trastuzumab deruxtecan (T-DXd) is an anti-HER2 antibody-drug conjugate (ADC) linked to a topoisomerase inhibitor FDA-approved for several indications, including the treatment of HER2+ breast and gastric cancer. Although HER2 amplification is uncommon in pediatric cancers, recent demonstration of T-DXd efficacy in HER2-low breast cancer patients prompted us to examine the potential clinical relevance of T-DXd in pediatrics by evaluating HER2 expression and activity of T-DXd in preclinical pediatric solid tumor models. Methods: Cell viability was assessed in a panel of histologically diverse pediatric solid tumor cell lines and 2 HER2-amplified adult cancer cell lines treated with T-DXd, payload (DXd), and a control IgG-ADC. HER2 protein expression in patient-derived xenograft (PDX) tumors was evaluated by immunohistochemistry (IHC) using 5 clinically validated anti-HER2 antibodies. RNAseq was performed on clinical (n=290) and PDX (n=184) tumors to determine relative expression of ERBB2. In vivo activity of T-DXd was evaluated in osteosarcoma (OS, n=10), Wilms tumor (WT, n=1) and malignant rhabdoid tumor (MRT, n=1) PDX models and a desmoplastic small round cell tumor (DSRCT) cell line xenograft model. Differences in tumor volume and time to disease progression was assessed and compared across treatments and models. Responses were correlated with HER2 expression by IHC. Results: In vitro, HER2-amplified control cell lines demonstrated a >30-fold reduction of IC50 when comparing T-DXd to ADC control. In contrast, the ADC control IC50 was nearly identical to T-DXd across all pediatric cancer cell lines, consistent with the absence of HER2 amplification in these models. We observed focal and membranous staining of HER2 by IHC in PDXs but was quite variable across antibodies tested: WT 0-17% HER2+ cases (n=6), MRT 0-40% (n=5), DSRCT 0-33% (n=24), OS 0-7% (n=30). ERBB2 gene expression was highest in DSRCT followed by WT, OS and MRT. In vivo efficacy studies demonstrated complete and partial responses in OS, WT, and DSRCT and improved disease control rates (OS: 67%, p=0.006, Mann-Whitney; WT: 100%; DSRCT: 100%). However, T-DXd and ADC control demonstrated similar activity in all tumor types with no consistent correlation between in vivo response and HER2 expression. Consistent with these preclinical studies, 4 patients with progressive DSRCT were treated with T-DXd via compassionate/off-label access with signs of clinical and radiographic responses. Conclusions: T-DXd shows significant preclinical antitumor activity across multiple pediatric solid tumors but little correlation with HER2 expression suggesting a mechanism of action similar to the clinical activity observed in HER2-low breast cancer. Xenograft efficacy studies and preliminary clinical experience with T-DXd in DSRCT patients warrant formal clinical trial investigation in this largely incurable disease.

Abstract LB403: Suppression of antigen presentation in pediatric rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma that is diagnosed based on the tumor cell's resemblance to embryonic or poorly differentiated skeletal muscle progenitors. Multiple immunotherapy clinical trials have found no evidence of immune reactivity against RMS, which has been attributed to the relatively low tumor mutation burden in these cancers. However, we made an unexpected observation that many patient RMS tumors had a near absence of MHC-I expression, a key requisite for anti-tumor CD8+ T lymphocytes and many immunotherapies. In this study, we explore the etiology of low antigen presentation in RMS and systematically evaluate therapeutic interventions that sensitize RMS to immunotherapy. Methods: Surface MHC-I expression was evaluated on pediatric tumor cell lines (n=65) from five major pediatric tumor types. RNA-seq, whole exome and genome sequencing data were analyzed from RMS tumors (total of n=247) and iPSC models of skeletal muscle development (PMID: 32011235). Epigentic drugs targeting DNA methylation (decitabine), EZH2 (tazemetostat), Class I/IV HDACs (mocetinostat), and LSD1 (GSK-LSD1) were used at sub-cytotoxic doses on RMS cell lines (n=10) and analyzed for surface MHC-I expression with flow cytometry and gene expression. T cell cytotoxicity assays were performed using engineered human T cells expressing an optimized TCR which recognizes an HLA*A2:01 restricted peptide from PRAME. Results: RMS cell line models exhibited a range of MHC-I expression with most having low or undetectable MHC-I expression, similar to that of a known MHC-I negative tumor type MYCN-amplified neuroblastoma. Exome sequencing of RMS tumors showed no evidence of loss-of-function alterations in essential antigen processing and presentation (APP) pathway genes. Gene expression profiling by RNAseq and western blotting, however, revealed an absence of core APP genes in many RMS tumors and cell lines. We uncovered that during a specific window of embryonic myogenesis, APP is suppressed and that RMS tumors mimicking this state of development show the deepest loss of APP. Multiple classes of epigenetic drugs were evaluated to rescue APP in RMS. Treatment of RMS using a DNA demethylating agent or HDAC inhibitor exhibited the strongest MHC-I restoration across RMS models with no changes observed using LSD1 or EZH2 inhibitors. As a proof-of-concept, we demonstrate that targeting the RMS antigen PRAME using TCR therapy is enhanced by the inclusion of epigenetic therapy. Conclusions and Significance: We revealed that epigenetic suppression of APP is a new hallmark of RMS and normal embryonic skeletal muscle development. Pharmacological reversal of APP pathway downregulation could be achieved using drugs targeting DNA methylation or class I/IV HDACs, and enhanced T cell cytotoxicity against RMS when used in combination. Overall, these findings may explain the historical lack of RMS responsiveness to immunotherapy and unlocks new immunotherapy strategies against RMS that rely on targeting MHC-I antigens. Citation Format: David Milewski, Hsien-Chao Chou, Vineela Gangalapudi, Meijie Tian, Yong Kim, Young Song, Jun Wei, Javed Khan. Suppression of antigen presentation in pediatric rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB403.

Cytokine Signaling in Pediatric Kidney Tumor Cell Lines WT-CLS1, WT-3ab and G-401.

Renal tumors comprise ~7% of all malignant pediatric tumors. Approximately 90% of pediatric kidney tumors comprise Wilms tumors, and the remaining 10% include clear cell sarcoma of the kidney, malignant rhabdoid tumor of the kidney, renal cell carcinoma and other rare renal tumors. Over the last 30 years, the role of cytokines and their receptors has been considerably investigated in both cancer progression and anti-cancer therapy. However, more effective immunotherapies require the cytokine profiling of each tumor type and comprehensive understanding of tumor biology. In this study, we aimed to investigate the activation of signaling pathways in response to cytokines in three pediatric kidney tumor cell lines, in WT-CLS1 and WT-3ab cells (both are Wilms tumors), and in G-401 cells (a rhabdoid kidney tumor, formerly classified as Wilms tumor). We observed that interferon-alpha (IFN-α) and interferon-gamma (IFN-γ) very strongly induced the activation of the STAT1 protein, whereas IL-6 and IFN-α activated STAT3 and IL-4 activated STAT6 in all examined tumor cell lines. STAT protein activation was examined by flow cytometry and Western blot using phospho-specific anti-STAT antibodies which recognize only activated (phosphorylated) STAT proteins. Nuclear translocation of phospho-STAT proteins upon activation with specific cytokines was furthermore confirmed by immunofluorescence. Our results also showed that both IFN-α and IFN-γ caused upregulation of major histocompatibility complex (MHC) class I proteins, however, these cytokines did not have any effect on the expression of MHC class II proteins. We also observed that pediatric kidney tumor cell lines exhibit the functional expression of an additional cytokine signaling pathway, the tumor necrosis factor (TNF)-α-mediated activation of nuclear factor kappa B (NF-κB). In summary, our data show that human pediatric renal tumor cell lines are responsive to stimulation with various human cytokines and could be used as in vitro models for profiling cytokine signaling pathways.

SYST-22 INTEGRATING GENE DEPENDENCY INTO A PEDIATRIC-FOCUSED CANCER DATA PORTAL FOR TARGETED THERAPY AND BIOMARKER DISCOVERY

Abstract Pediatric solid cancers, particularly those affecting the central nervous system (CNS), are the leading cause of disease-related deaths in children. Despite significant efforts to sequence pediatric solid tumors, they exhibit a "quiescent genome" with a considerably lower mutational burden than adult cancers. This leads to fewer actionable drug targets and poor response to cancer immunotherapy agents, including checkpoint inhibitors. As a result, it is imperative to move beyond genetic alterations and adopt a combination of functional and integrative approaches to develop novel, targeted treatments that are specifically designed for pediatric cancers. Over the past five years, the Next-Gen Precision Medicine Program team has devoted its efforts to establishing the Childhood Cancer Model Atlas (CCMA) (Sun, Daniel et al. Cancer Cell 2023). The CCMA is the largest collection of high-risk pediatric solid tumor cell lines globally, presenting a tremendous opportunity to integrate molecular features with functional dependencies using state-of-the-art computational approaches. However, the analysis of -omics level data is often limited to laboratories with specialized bioinformatics capabilities. Therefore, the establishment of the CCMA data portal with user-friendly analytical and visualization capabilities is essential to accelerate data-driven discovery and translational research in pediatric cancer on a global scale. The data portal is designed to simplify reporting, automate manual processes, and enhance the quality, usability, and longevity of the CCMA dataset. Its highlighted features concentrate on customized analysis, informative results, and user-friendly data visualization. Furthermore, by connecting the established models and their original tumor with clinical and molecular endpoints, the data portal enables clinical integration within the Australian molecular tumor board framework. This analytical data portal will enable the generation of new research hypotheses, prioritizes therapeutic opportunities, and supports international collaborative efforts in pediatric cancer.

Effect of dual properties of regorafenib in osteosarcoma on tumor progression and bone parameters in mouse preclinical models.

e23501 Background: Osteosarcoma (OS) constitutes the most frequent malignant primary bone tumor in children and young adults. Actual treatments in OS are based in a multimodal therapy that combines pre- and post-surgery polychemotherapies including cisplatin, doxorubicin, ifosfamide and/or methotrexate. Regardless the use of this polychemotherapeutic approach, the five years overall survival for patients has not improved during the last four decades and there is a clinical need to identify new active drugs in OS. An interesting strategy implies the use of compounds that showed therapeutic benefits in other solid tumors. Regorafenib, a small molecule with multi-target tyrosine kinase inhibitory activity against FGFR-1 and -2, KIT, PDGFR, RAF, RET, TIE2 and VEGFR-1, -2 and -3, showed an anti-angiogenic/oncogenic properties in various solid tumors that includes metastatic colorectal cancer, hepatocarcinoma and breast cancer. In addition, regorafenib also inhibits in vitro the proliferation of several pediatric solid tumor cell lines such as rhabdomyosarcoma, Ewing sarcoma and OS. Methods: The present work aims to decipher the in vitro and in vivo effects of regorafenib in OS preclinical models. Results: The treatment of MNNG-HOS, KHOS and 143B osteosarcoma cell lines with Regorafenib resulted in the inhibition of cell proliferation in a dose dependent manner. The gene expression profiles of MNNG-HOS and 143B cells treated or not with regorafenib and analyzed by NanoString technology revealed a specific regulation of molecular pathways by regorafenib in both cell lines. The in vivo effect of regorafenib was then analyzed in immunocompetent (mouse MOS-J cells inoculated in C57Bl/6 mice) and immunodeficient (human MNNG-HOS and 143B OS cells injected in Nude mice) murine osteosarcoma models. In all the cases, a significant reduction of primary tumor growth was observed as well as a significant decrease of lung metastases compared to untreated mice. Interestingly, the cortical and trabecular bone mineral density were significantly increased in regorafenib treated mice compared to untreated groups as demonstrated by X ray microtomodensitometry in all models. Conclusions: Our results support the use of regorafenib as an alternative treatment in metastatic OS and demonstrated for the first time the its bone protective effect.

Development of GPC2-directed chimeric antigen receptors using mRNA for pediatric brain tumors

BackgroundPediatric brain tumors are the leading cause of cancer death in children with an urgent need for innovative therapies. Glypican 2 (GPC2) is a cell surface oncoprotein expressed in neuroblastoma...

MODL-28. Patient-derived, three-dimensional organoid platform for pediatric brain tumor modeling

Brain tumors have become the leading cause of cancer-related death in children. An important hurdle to scientific and clinical progress in the field has been the limited availability of preclinical tumor models. Historically, few pediatric brain tumor cell lines have been established and these often poorly recapitulate the phenotypes of the original tumors. In recent years, the Children’s Brain Tumor Network (CBTN) has accelerated the development of patient-derived cell lines and xenografts, offering these resources to the community through open-source access. While these models are extremely valuable, their development process can be lengthy and result in clonally selected lines which presents a challenge for studying complex tumor biology. To address the need for three-dimensional tissue culture, our group in conjunction with CBTN, utilized organoid culture from fresh tissue specimens obtained directly from surgical resection of various pediatric brain tumor histologies. This resulted in the development and banking of over 30 organoid models, which included ependymoma, high-grade glioma, medulloblastoma, atypical teratoid-rhabdoid tumor, diffuse midline glioma, and low-grade glioma diagnoses. Tissue was processed within an hour post extraction and cultured with universal media composition for each diagnosis. Organoid growth was observed within 2-3 weeks of initiation and continued for up to three months before banking. Banked organoids established growth upon return to culture. Phenotypic analysis revealed organoid cell composition that represented clinical histology. Importantly, organoids returned to culture post-banking demonstrated similar cell composition to those in the original culture, indicating their utility for subsequent preclinical testing. Here we provide a simple and efficient workflow for the generation and characterization of three-dimensional tumor organoids generated from fresh surgical pediatric brain tumor tissue. The platform has the potential to accelerate investigations into tumor biology and empower a diverse array of translational studies for the pediatric brain tumor field.

MODL-05 Metronomic Intrathecal Delivery of CDK4/6 Inhibitors in Preclinical Models of Pediatric Brain Tumors

INTRODUCTION: CDK4/6 inhibitors have shown promise against central nervous system (CNS) tumors in vitro. This class of drugs relies on long-term exposure. Their use in early phase clinical studies in children with CNS tumors has defined dose limitations due to systemic toxicity. We have sought to circumvent these limitations in using CDK4/6 inhibitors for pediatric CNS tumors by first demonstrating enhanced efficiency with long-term administration and exploiting prolonged intrathecal delivery (IT). METHODS: Pediatric CNS tumor cell lines were used for cell viability assays: ATRT (BT-12, BT-16), CPC (CCHE-45), diffuse midline glioma (DIPG-XIII, HSJD-007), and medulloblastoma (DAOY); the assays were conducted at 24h, 72h, and 7d post-administration of CDK4/6 inhibitors (abemaciclib, palbociclib, ribociclib). Half maximal growth inhibitory concentrations (GI50) and areas under the curve (AUC) were compared for short-term (24h, 72h) and long-term (7d) dose-response curves. Toxicity with chronic IT administration was assessed using a neurobehavioral safety profile of 7-day continuous infusion of 2.5mM palbociclib (n = 5) into the mouse lateral ventricle compared with vehicle (n = 4). RESULTS: Our results demonstrate increased CDK4/6 inhibitor potency with longer administration. The greatest reductions in short-term to long-term GI50 were observed in ATRT, CPC, and DIPG across all inhibitors. The most pronounced time-dependent efficacy was observed with palbociclib for ATRT and abemaciclib for CPC and DIPG. AUCs significantly decreased (P < 0.05) with increasing drug exposure time across all inhibitors. 7-day intraventricular palbociclib infusion was equivalent in safety to PBS at doses ranging from 1,000 to 10,000-fold the in vitro GI50. CONCLUSIONS: The efficiency of CDK4/6 inhibitors in pediatric CNS tumors is enhanced with prolonged exposure. Long-term IT administration can achieve high CNS doses without associated systemic toxicities. Translational efforts using a metronomic IT strategy are logical to explore for pediatric CNS tumors which have potential for a leptomeningeal disease pattern.

Pediatric brain tumor cell lines exhibit miRNA-depleted, Y RNA-enriched extracellular vesicles.

Medulloblastoma (MB) and diffuse infiltrative pontine glioma (DIPG) are malignant pediatric tumors. Extracellular vesicles (EVs) and their bioactive cargoes have been implicated in tumorigenesis. Most studies have focused on adult tumors, therefore the role of EVs and the noncoding RNA (ncRNA) landscape in pediatric brain tumors is not fully characterized. The overall aim of this pilot study was to isolate EVs from MB and DIPG patient-derived cell lines and to explore the small ncRNA transcriptome. EVs from 3 DIPG and 4MB patient-derived cell lines were analyzed. High-throughput next generation sequencing interrogated the short non-coding RNA (ncRNA) transcriptome. Known and novel miRNAs were quantified. Differential expression analysis, in silico target prediction, and functional gene enrichment were performed. EV secretomes from MB and DIPG patient-derived cell lines demonstrated discrete ncRNA biotypes. Notably, miRNAs were depleted and Y RNAs were enriched in EV samples. Hierarchical cluster analysis revealed high discrimination in miRNA expression between DIPG and MB cell lines and RNA-Seq identified novel miRNAs not previously implicated in MB or DIPG pathogenesis. Known and putative target genes of dysregulated miRNAs were identified. Functional annotation analysis of the target genes for differentially expressed EV-and parental-derived miRNAs revealed significant cancer-related pathway involvement. This hypothesis-generating study demonstrated that pediatric brain tumor-derived cell lines secrete EVs comprised of various ncRNA cargoes. Validation of these findings in patient samples may provide new insights into the pediatric brain tumor microenvironment and identification of novel therapeutic candidates.

THER-03. IN VITRO EVALUATION OF THE EFFECT OF CANNABIDIOL ON PAEDIATRIC BRAIN TUMOUR CELL LINES USING A PULSED TREATMENT REGIME

Paediatric brain tumours are the second most common cancer after haematological malignancies. Intermittent dosing regimens are typical for chemotherapy drugs in order to avoid excessive damage to organs and avoid the onset of late effects. Cannabidiol (CBD) has been shown to have cytotoxic properties on paediatric brain tumour cell lines. Although CBD is far less toxic and damaging than the classical chemotherapy options which are currently available to children suffering with brain tumours, there are some possible side effects. Given that the half-life of the drug is 24 hours, it was important to establish the nature of the effect of cumulative dosing on top of the remaining drug in the system. The pHGG cell line, SF188 was cultured in different concentrations of CBD with either 1, 2 or 3 doses being given on consecutive days. 24 hours after the last dose the cells were analysed using the resazurin assay. It was observed that the amount of drug required for an EC50 to be obtained decreased; 17.6µM (1 dose), 8µM (2 doses), 5µM (3 doses) and that cell survival was reduced to nearly 0% in those cells which received multiple does of CBD at 17.6µM. In order to mimic the intermittent dosing regime, the cells were returned to the incubator for 4 days before the resazurin assay was repeated. The decrease in viability was maintained over the extended culture period meaning that the ability of even the apparent “healthy” cells to proliferate had been permanently affected.

THER-04. IS THERE A ROLE FOR CANNABIDIOL IN THE TREATMENT OF CHILDHOOD BRAIN TUMOURS?

Brain tumours are the leading cause of cancer related death in children with limited treatment options and high recurrence rates. Recent evidence suggests there may be anti-tumoral properties of cannabinoids, and of cannabidiol (CBD) in particular. We evaluated the effect of CBD on paediatric brain tumour cell lines in 2D and 3D spheroids; pHGG (SF188), ependymoma (BxD-1425EPN) and human astrocytes. At the CBD EC50 concentration, astrocytic cell death was insignificant. 3D spheroids decreased in size by approximately 20% when cultured in CBD compared to cells only after 5-day exposure. Cell death increased with time after a single dose of CBD. Western Blot showed an increase in Lc3b expression (autophagy) after 24 hours incubation (early cell death) with CBD in both BxD-1425EPN and SF188 with PARP expression (apoptosis) increased after 5 days incubation (late cell death). Cell cycle analysis showed a decrease of cells in G1 and no change in G2 indicating cell cycle arrest. In hypoxia, SF188 and BxD-1425EPN cells showed decreased cell death after 24 hours and 5 days when compared to normoxia and an EC50 within acceptable limits could not be achieved. SF188 cells pre-treated with receptor antagonists indicate that CBD was not acting through CB1, CB2, GPR18, PPARα or PPARγ receptors but may act as a partial agonist of the TRPV1 and 5-HT1A receptors and a full agonist of the GPR55 receptor (resazurin assay). This provides evidence that CBD is effective at killing paediatric brain tumour cells and does not have a significant effect on normal astrocytes.

Abstract 4176: Involvement of the lysosome in the cytotoxicity of Gallium Maltolate in human pediatric brain tumor cell lines

Abstract Introduction: Gallium maltolate (GaM) is a novel small molecule metallocompound that acts as an iron mimetic Trojan Horse to disrupt iron-dependent proteins essential in tumor biology. We recently reported that GaM retards the growth of orthotopically implanted adult human glioblastoma (GBM) xenografts in a rodent brain tumor model and is cytotoxic to glioblastoma stem cells (Mol Cancer Ther 17:1240, 2018). GaM's mechanisms of action include inhibition of both mitochondrial function and iron-dependent ribonucleotide reductase. In the present study, we further explored whether GaM has antitumor activity in pediatric brain tumor cells. Experimental procedures: The cytotoxicity of GaM in pediatric GBM and Atypical Teratoid Rhabdoid Tumor (ATRT) cell lines was examined by MTT and clonogenic assays. Apoptotic cell death was assessed by flow cytometry with annexin V (AV) and propidium iodide (PI) staining. Cellular bioenergetics was measured using a Seahorse XF96 Extracellular Flux Analyzer. GaM interaction with lysosomes was analyzed by immunofluorescence and electron microscopy (EM). Results: In a panel of pediatric brain tumor cell lines, CHLA-266 ATRT and GBM cells displayed the greatest sensitivity to GaM. Complete inhibition of colony growth was achieved with 6 - 7 µM GaM over a 96-hour incubation. With a shorter incubation of 48-h, 25 µM GaM induced apoptosis in &amp;gt; 60% of CHLA-266 ATRT cells. In contrast, a longer incubation of 96-h with 25 µM GaM was required to induce apoptosis in &amp;gt; 50% of CHLA-200 GBM, 69% of SF188 GBM and 60% of SJ-GBM2 cell lines. After a 24-h of incubation with GaM, prior to detectable cell death, CHLA-266 and GBM cells displayed a marked reduction in mitochondrial reserve capacity, glycolysis, and glycolytic capacity. Immunofluorescence staining for the lysosomal marker LAMP1 showed that GaM produced an increase in lysosome size and number after a 24-h or 48-h incubation relative to control cells. EM studies demonstrated a gallium signal within the lysosomal compartment as early as 6-h of incubation. EM findings consistent with GaM-induced apoptosis were noted after a 24-h incubation, thus confirming the results of the AV/PI assay. After 24-h however, gallium was no longer detectable in the lysosomes, suggesting that lysosomal disruption led to the release of gallium into the cytoplasm. Conclusions: Our results identify GaM as a novel compound with significant activity against a panel of cell lines representative of pediatric brain tumors. Most of the cell lines used in our study were derived from tumors taken from heavily pre-treated patients and were resistant to conventional chemotherapeutic drugs. Their sensitivity of GaM suggests that GaM does not share cross-resistance with other antineoplastic drugs. Our study also provides new insight into the lysosomes as a target for gallium. The prognosis of adult and pediatric brain tumors is dismal and there is an urgent need to develop newer drugs for this malignancy. Further investigation of GaM as a therapeutic agent for brain tumors is warranted. Citation Format: Salvatore Molino, Christopher R. Chitambar. Involvement of the lysosome in the cytotoxicity of Gallium Maltolate in human pediatric brain tumor cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4176.

Small-Molecule Dual PLK1 and BRD4 Inhibitors are Active Against Preclinical Models of Pediatric Solid Tumors

Simultaneous inhibition of multiple molecular targets is an established strategy to improve the continuance of clinical response to therapy. Here, we screened 49 molecules with dual nanomolar inhibitory activity against BRD4 and PLK1, best classified as dual kinase-bromodomain inhibitors, in pediatric tumor cell lines for their antitumor activity. We identified two candidate dual kinase-bromodomain inhibitors with strong and tumor-specific activity against neuroblastoma, medulloblastoma, and rhabdomyosarcoma tumor cells. Dual PLK1 and BRD4 inhibitor treatment suppressed proliferation and induced apoptosis in pediatric tumor cell lines at low nanomolar concentrations. This was associated with reduced MYCN-driven gene expression as assessed by RNA sequencing. Treatment of patient-derived xenografts with dual inhibitor UMB103 led to significant tumor regression. We demonstrate that concurrent inhibition of two central regulators of MYC protein family of protooncogenes, BRD4, and PLK1, with single small molecules has strong and specific antitumor effects in preclinical pediatric cancer models.

Design, Synthesis, and Targeted Delivery of Fluorescent 1,2,4-Triazole-Peptide Conjugates to Pediatric Brain Tumor Cells.

Most of the chemotherapeutics and drug-delivery models pose serious health problems and several undesirable side effects due to nonspecificity, lack of proper targeting system, and their large sizes. The rational design and synthesis of target-specific chemotherapeutics are highly important. This research work is focused on the rational design, synthesis, and anticancer studies of fluorescent 1,2,4-triazole–peptide conjugates for the development of target-specific anticancer drugs. Three novel 1,2,4-triazole derivatives: 4-(4-fluorobenzylidenamino)-3-hydrazino-5-mercapto-1,2,4-triazole (4FBAHMT, 2a), 4-(3,4,5-trimethoxybenzylidenamino)-3-hydrazino-5-mercapto-1,2,4-triazole (TMOBAHMT, 2b), and 4-(4-benzyloxy-2-methyloxbenzylidenamino)-3-hydrazino-5-mercapto-1,2,4-triazole (4BO2MOBAHMT, 2c) were synthesized after screening through molecular docking procedures. The docking studies were performed between ligand molecules and αvβ6 integrin protein. Fluorescent carbon nanoparticles (CNPs, 3) were conjugated with 1,2,4-triazole derivatives (2a–c) and l-carnosine (LC) dipeptide to get their corresponding conjugates (4a–c). The title double conjugates were characterized by spectroscopic (UV/vis spectroscopy, fluorescence spectroscopy, and FTIR spectroscopy) and microscopic (scanning electron microscopy, transmission electron microscopy, and atomic force microscopy) techniques. In vitro efficacy of fluorescent 1,2,4-triazole–peptide conjugates was investigated against two pediatric brain tumor cell lines (CHLA-200 & SJGBM2) and human embryonic kidney cell line (HEK293 as a control) by employing cell proliferation assay/MTS assay and fluorescence microscopy. 1,2,4-Triazole derivatives and their conjugates showed potent and selective anticancer activity against CHLA-200 and SJGBM2 cell lines. Cell proliferation assay and fluorescence microscopy results revealed that conjugates were more highly selective and cytotoxic than control drug temozolomide (TM) against both cell lines. CNPs are highly biocompatible and the quantum-sized conjugates were nontoxic for normal embryonic kidney cell line (HEK 293). The experimental results of MTS bioactivity assay and fluorescence microscopy were in close agreement with the theoretical results of molecular docking studies.

Preclinical Evaluation of Antitumoral and Cytotoxic Properties of Viscum album Fraxini Extract on Pediatric Tumor Cells.

In Europe, especially in German-speaking countries, administration of mistletoe extracts is the most common and popular complementary and alternative therapy approach reported in oncology. Mistletoe therapy is applied to children with cancer for curative and palliative therapeutic regimes with increasing frequency, but at the same time, there are only a few studies on the effectiveness of this therapy. Therefore, we have investigated the response of various pediatric cell lines (acute myeloid leukemia, Ewing's sarcoma, hepatocellular carcinoma, medulloblastoma, neuroblastoma, and osteosarcoma) to mistletoe extract, abnobaVISCUM Fraxini. Effects on cell proliferation, cell cycle distribution as well as on mitochondrial integrity and caspase-mediated apoptosis were investigated in neuroblastoma cell lines, SH-SY5Y and Kelly. Additionally, in vitro tumor cell migration and invasion were studied. In vivo effects of the mistletoe extract were investigated in a syngeneic neuroblastoma mouse model. We could show that tumor cell lines were from 5- to 640-fold more sensitive to abnobaVISCUM Fraxini treatment than non-tumorigenic fibroblasts, whereby neuroblastoma cell lines were the most sensitive. For two neuroblastoma cell lines, SH-SY5Y and Kelly, induction of caspase-9-mediated apoptosis, a decrease of mitochondrial integrity as well as attenuation of migration and invasion were observed. In vivo experiments revealed a reduction of tumor growth and a prolonged survival of tumor-bearing animals. In summary, we can state that these results provide the first preclinical data for cytotoxic activities of abnobaVISCUM Fraxini for a broad panel of pediatric tumor cell lines, in particular, neuroblastoma cells. Thus, it might be a potential remedy for the supportive treatment of neuroblastoma.

Cell therapy based on the chimeric receptor NKG2D

Cell therapy based on the chimeric receptor NKG2D

Anacolosins A-F and Corymbulosins X and Y, Clerodane Diterpenes from Anacolosa clarkii Exhibiting Cytotoxicity toward Pediatric Cancer Cell Lines.

An extract of the plant Anacolosa clarkii was obtained from the NCI Natural Products Repository, and it showed cytotoxic activity toward several types of pediatric solid tumor cell lines. Bioassay-guided fractionation led to the purification of eight new clerodane diterpenes [anacolosins A-F (1-6) and corymbulosins X and Y (7 and 8)] and two known compounds (9 and 10) that contained an isozuelanin skeleton. The structures of the new natural products were determined using 1D and 2D NMR and HRESIMS data, while the relative and absolute configurations of the compounds were assessed using a combination of 1H NMR coupling constant data, ROESY experiments, ECD (electronic circular dichroism) and VCD (vibrational circular dichroism) spectroscopy, chemical methods (including Mosher and 2-naphthacyl esterification), and chiral HPLC analyses. The purified natural products exhibited a range of cytotoxic activities against cell lines representing four pediatric cancer types (i.e., rhabdomyosarcoma, Ewing sarcoma, medulloblastoma, and hepatoblastoma) with total growth inhibitory (TGI) values in the range 0.2-4.1 μM. The rhabdomyosarcoma and medulloblastoma cell lines showed higher sensitivity to compounds 1-4, which are the first compounds reported to contain an isozuelanin skeleton and feature keto carbonyl groups at the C-6 positions. In contrast, the hepatoblastoma cell line was modestly more sensitive to 7-10, which contained a C-6 hydroxy group moiety.

Preclinical investigation of interaction of mistletoe extract (Viscum album L.) with radio- and chemotherapy in pediatric tumor cell lines

Preclinical investigation of interaction of mistletoe extract (Viscum album L.) with radio- and chemotherapy in pediatric tumor cell lines

P02.14 Tumour Treating Fields (TTFields) demonstrate variable efficacy on high-grade paediatric brain tumour cell lines in a frequency-dependent manner

P02.14 Tumour Treating Fields (TTFields) demonstrate variable efficacy on high-grade paediatric brain tumour cell lines in a frequency-dependent manner

Identification of FDA-Approved Oncology Drugs with Selective Potency in High-Risk Childhood Ependymoma.

Children with ependymoma (EPN) are cured in less than 50% of cases, with little improvement in outcome over the last several decades. Chemotherapy has not affected survival in EPN, due in part to a lack of preclinical models that has precluded comprehensive drug testing. We recently developed two human EPN cell lines harboring high-risk phenotypes which provided us with an opportunity to execute translational studies. EPN and other pediatric brain tumor cell lines were subject to a large-scale comparative drug screen of FDA-approved oncology drugs for rapid clinical application. The results of this in vitro study were combined with in silico prediction of drug sensitivity to identify EPN-selective compounds, which were validated by dose curve and time course modeling. Mechanisms of EPN-selective antitumor effect were further investigated using transcriptome and proteome analyses. We identified three classes of oncology drugs that showed EPN-selective antitumor effect, namely, (i) fluorinated pyrimidines (5-fluorouracil, carmofur, and floxuridine), (ii) retinoids (bexarotene, tretinoin and isotretinoin), and (iii) a subset of small-molecule multireceptor tyrosine kinase inhibitors (axitinib, imatinib, and pazopanib). Axitinib's antitumor mechanism in EPN cell lines involved inhibition of PDGFRα and PDGFRβ and was associated with reduced mitosis-related gene expression and cellular senescence. The clinically available, EPN-selective oncology drugs identified by our study have the potential to critically inform design of upcoming clinical studies in EPN, in particular for those children with recurrent EPN who are in the greatest need of novel therapeutic approaches. Mol Cancer Ther; 17(9); 1984-94. ©2018 AACR.