Pediatric Tumor Cell Research Articles

Taraxacum officinale extract shows antitumor effects on pediatric cancer cells and enhance mistletoe therapy

ObjectivesWhile Taraxacum officinale (dandelion) extracts showed antitumor effects on adult cancer cells, effects on pediatric tumor cells as a single agent or in combination with mistletoe extracts are hitherto unknown. Material and methodsThe anti-proliferative effects of an aqueous fermented Taraxacum officinale extract (Taraxacum) on a pediatric cancer cell line panel were assessed by cell viability assays (MTT). In two neuroblastoma cell lines, SH-SY5Y and Kelly, the effects on cell cycle distribution (PI staining), mitochondrial integrity (MitoTracker staining), invasion (Boyden chamber assay) and migration (Scratch-assay) as well as the synergistic effects of the co-treatment of Taraxacum and mistletoe preparations (Iscucin® Tiliae or Iscucin® Pini) were investigated. ResultsAll tested cancer cell lines were more susceptible to Taraxacum than the normal human fibroblast cell line, NHDF-C. In neuroblastoma cell lines Taraxacum caused apoptosis and loss of mitochondrial integrity as well as an inhibition of invasion and migration. The simultaneous therapy of Taraxacum and the mistletoe extracts revealed synergistic effects. ConclusionThis preclinical data support the use of Taraxacum as a potential adjuvant application in pediatric oncology.

In-Vitro Use of 5-ALA for Photodynamic Therapy in Pediatric Brain Tumors.

Light irradiation (635 nm) of cells containing protoporphyrin IX (PPIX) after 5- aminolevulinic acid (5-ALA) pretreatment causes cell death via different pathways including apoptosis and necrosis, as previously demonstrated for malignant glioma cells. To elucidate whether various malignant pediatric brain tumors, which have been shown to accumulate PPIX, would also be susceptible to photodynamic therapy (PDT). Medulloblastoma (DAOY, UW228), pNET (PFSK-1), and rhabdoid tumor (BT16) cell lines were incubated with 5-ALA in variable concentrations for 4 h. Consequently, cells were irradiated by 635 nm diode laser light. After 12 h, cell viability was measured by WST-1 testing and these results were compared to control cells incubated with 5-ALA without irradiation or irradiation only without prior incubation with 5-ALA. We demonstrated significant cell death in malignant pediatric tumor cells after incubation with 5-ALA and laser irradiation in comparison to control groups. In all cell lines, we noticed significant cell death above a 5-ALA concentration of 50 μg/ml (P < .05). Neither 5-ALA incubation alone nor irradiation alone caused cell death. DAOY and PFSK cell lines were more susceptible than UW228 and BT16 cells. We conclude that PDT causes cell death with higher PPIX concentrations after exposure to 5-ALA in vitro in accordance to similar studies with glioma cells. This indicates that PDT might be feasible for eliminating brain tumor cells in malignant pediatric brain tumors. Additionally, we noticed a dependency between fluorescence intensity and death rates.

Tumour Treating Fields (TTFields) have anti-proliferative effects on paediatric brain tumour cell lines at clinically deliverable field settings

Approximately 400 children in the UK are diagnosed with a CNS tumour each, which represents a quarter of childhood cancer cases in the UK. The overall 5-year survival rate of peadiatric brain tumour patients is roughly 75%, however survival still remains poor for some brain tumours, particularly high grade gliomas. Pre-clinical studies have shown efficacy of TTFields on high grade glioma cell lines, while TTFields have only been approved for adult Glioblastoma multiforme (GBM) patients in combination with the Stupp treatment protocol. The lack of overlapping toxicities associated with OptuneTM has made TTFields an attractive treatment strategy for poor prognosis brain tumours. The InovitroTM system is the laboratory testing system used to develop the clinically approved Optune system. InovitroTM was used to deliver TTFields over 72 hours at a range of clinically relevant frequencies (100-400kHz) and intensities (1-3V/cm) to a panel of paediatric GBM, Medulloblastoma and Ependymoma cell lines. The effects of TTFields on cell viability and cell cycle was assessed using metabolic viability tests (Presto blue), cell proliferation assays and cell cycle analysis using flow cytometry. TTFields treatment reduces cell viability of all paediatric brain tumour cell lines tested within the frequency titration. The optimum frequencies were between 200 and 300kHz for all cell lines tested. TTFields treatment had significant effects on cell cycling relative to control cells. This preliminary study has confirmed the efficacy of electric fields on paediatric brain tumour cell lines. The optimum frequencies for the paediatric GBM cell lines did not uniformly fall into the 200kHz range as previously reported for glioma cell lines. This report warrants further study into the potential for TTFields therapy for paediatric patients.

Abstract B36: A biomarker-guided approach to combining PARP inhibitors with radiotherapy in pediatric sarcomas

Abstract Background: Ewing sarcoma (EWS) expresses high levels of Schlafen-11 (SLFN11). SLFN11 disrupts checkpoint maintenance and may serve as a biomarker to assess sensitivity to Poly (ADP-ribose) polymerase 1 and 2 inhibitors (PARPi). The goal of this study is to evaluate SLFN11 protein expression in a panel of pediatric solid tumors and correlate levels of protein with sensitivity to PARP inhibition combined with ionizing radiation (IR), a component of therapy for many pediatric sarcomas and a potent inducer of DNA damage. Methods: SLFN11 mRNA and protein levels were assessed by quantitative RT-PCR, and immunohistochemistry, Western blot, and immunofluorescence microscopy, respectively. PARPi included talazoparib (TAL), niraparib (NIR), veliparib (VEL), and olaparib (OLA). Approximately 30 minutes after addition of systemic therapy, graded doses of radiation were delivered and viability across a panel of pediatric solid tumor cell lines was measured using the ATP-based Cell TiterGlo assay and confirmed with the colony formation assay. Results: We found that SLFN11 mRNA and protein is expressed at high levels in EWS, and SLFN11 is also variably present in a subset of other pediatric sarcomas, including desmoplastic small round cell tumor, osteosarcoma, and rhabdomyosarcoma. In all tumor cells with detectable SLFN11 expression, viability was reduced by greater than 90% when exposed to 2 Gy IR and 1-10 nM TAL, whereas cells with undetectable levels of SLFN11 were 5-10 times less sensitive. Intriguingly, variation in the potentiation between specific PARPi and IR correlated with the ability to form drug-induced PARP-DNA adducts, with the strong PARP trapper TAL showing ~10-fold higher potency compared to the moderate trapper NIR, and ~300-fold more potency relative to the weak trapper VEL. Consistent with our PARPi findings, the toposiomerase 1 inhibitor irinotecan, which also forms DNA adducts, potentiated with IR similarly to TAL at concentrations &amp;lt;10 nM in tumor cells expressing detectable levels of SLFN11. Conclusion: SLFN11 is present in select pediatric sarcomas and may induce a DNA repair defect that is best exploited by combining low doses of TAL and irinotecan with IR. Citation Format: Anang A. Shelat, Christopher L. Tinkle, Elizabeth Stewart, Sara Michele Federico, Brandon Bianski, Marcia Mellado-Lagarde. A biomarker-guided approach to combining PARP inhibitors with radiotherapy in pediatric sarcomas [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B36.

Abstract A111: The alkyl phosphocholine analog CLR1404 delays radiation-induced dsDNA damage repair in pediatric and adult solid tumors

Abstract Exposure of normal tissues to radiation is a major limitation in cancer radiotherapy, with significant consequences particularly in the pediatric setting. Increasing the selective anticancer effect of radiation on malignant cells utilizing cancer-targeted drugs could provide an option to lessen adverse radiation effects on healthy tissue. We investigated the radiosensitizing properties of 18-(p-iodophenyl) octadecyl phosphocholine (CLR1404), a clinical-grade phospholipid ether analog with selective sequestration in cancer cells, in pediatric and adult human solid tumor cell lines. Preliminary data in our laboratory using a fluorescently-labeled analog indicated that human cancer cells uptake and retain 6- to 10-fold more CLR1404 when compared with normal primary cells. In vitro, the clonogenic survival of tumor cells after irradiation was significantly decreased when cells were pretreated with CLR1404. This radiosensitizing effect was detected also in vivo, in xenograft-bearing mice treated with CLR1404 during fractionated radiation, resulting in decreased tumor volumes and markedly delayed tumor regrowth in CLR1404-treated mice compared to controls. We investigated mechanisms responsible for the radiosensitizing properties of CLR1404. Western blotting of tumor cell lysates indicated increased accumulation and prolonged persistence of γ-H2AX in irradiated tumor cells pretreated with CLR1404 compared to nontreated controls. Expression of select markers of homologous and non-homologous end-joining dsDNA radiation damage repair pathways were markedly decreased and significantly delayed in CLR1404-treated tumor cells after exposure to irradiation compared to non-CLR1404-treated cells. CLR1404 treatment also markedly decreased the activation of p53 after irradiation in p53-functional rhabdomyosarcoma cells compared to cells treated with radiation alone. In conclusion, our data show that, in both pediatric and adult human solid tumor cell lines, the alkyl phosphocholine analog CLR1404 interferes with sensing of dsDNA damage, delays DNA damage repair, and potentially interferes with other cell survival mechanisms, for example involving p53. These mechanisms might be responsible for the observed in vitro and in vivo radiosensitizing properties of CLR1404. Tumor-targeted radiosensitization with CLR1404 could improve the selective antitumor effect of radiation, and provide a distinct therapeutic benefit to cancer patients. Citation Format: Ankita Shahi, Mohamed Y. Elsaid, Dana C. Baiu, Eric A. Armstrong, Jamey P. Weichert, Paul M. Harari, Mario Otto. The alkyl phosphocholine analog CLR1404 delays radiation-induced dsDNA damage repair in pediatric and adult solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A111.

Probing the Function of Oncohistones Using Mutant Transgenes and Knock-In Mutations.

Recently, frequent somatic mutations at histone genes have been detected in high grade pediatric brain tumor, chondroblastoma, and giant cell tumor of bone. These mutant histones are also termed oncohistones. Since oncohistone proteins co-exist with wild type histone proteins in cells, it is critically important to understand how they promote tumorigenesis. Here, we describe two methods to analyze the impact of these oncohistones on histone modification and epigenome, including the expression of oncohistone from a transgene and the utilization of CRISPR/Cas9 system to knock-in specific oncohistone mutations. The methods described are useful for the initial characterization of oncohistones. Other methods such as ChIP-seq and RNA-seq, which analyze the effect of oncohistone mutations genome wide, are not detailed in this protocol.

Pediatric brain tumor cells release exosomes with a miRNA repertoire that differs from exosomes secreted by normal cells.

High-grade gliomas (HGGs) are very aggressive brain tumors with a cancer stem cell component. Cells, including cancer stem cells, release vesicles called exosomes which contain small non-coding RNAs such as microRNAs (miRNAs). These are thought to play an important role in cell-cell communication. However, we have limited knowledge of the types of exosomal miRNAs released by pediatric HGG stem cells; a prerequisite for exploring their potential roles in HGG biology. Here we isolated exosomes released by pediatric glioma stem cells (GSCs) and compared their repertoire of miRNAs to genetically normal neural stem cells (NSCs) exosomes, as well as their respective cellular miRNA content. Whereas cellular miRNAs are similar, we find that the exosomal miRNA profiles differ between normal and tumor cells, and identify several differentially expressed miRNAs. Of particular interest is miR-1290 and miR-1246, which have previously been linked to ‘stemness’ and invasion in other cancers. We demonstrate that GSC-secreted exosomes influence the gene expression of receiving NSCs, particularly targeting genes with a role in cell fate and tumorigenesis. Thus, our study shows that GSCs and NSCs have similar cellular miRNA profiles, yet differ significantly in the repertoire of exosomal miRNAs and these could influence malignant features of HGG.

Inhibition of Heparanase in Pediatric Brain Tumor Cells Attenuates their Proliferation, Invasive Capacity, and In Vivo Tumor Growth.

Curative therapy for medulloblastoma and other pediatric embryonal brain tumors has improved, but the outcome still remains poor and current treatment causes long-term complications. Malignant brain tumors infiltrate the healthy brain tissue and, thus despite resection, cells that have already migrated cause rapid tumor regrowth. Heparan sulfate proteoglycans (HSPG), major components of the extracellular matrix (ECM), modulate the activities of a variety of proteins. The major enzyme that degrades HS, heparanase (HPSE), is an important regulator of the ECM. Here, we report that the levels of HPSE in pediatric brain tumors are higher than in healthy brain tissue and that treatment of pediatric brain tumor cells with HPSE stimulated their growth. In addition, the latent, 65 kDa form of HPSE (that requires intracellular enzymatic processing for activation) enhanced cell viability and rapidly activated the ERK and AKT signaling pathways, before enzymatically active HPSE was detected. The HPSE inhibitor PG545 efficiently killed pediatric brain tumor cells, but not normal human astrocytes, and this compound also reduced tumor cell invasion in vitro and potently reduced the size of flank tumors in vivo Our findings indicate that HPSE in malignant brain tumors affects both the tumor cells themselves and their ECM. In conclusion, HPSE plays a substantial role in childhood brain tumors, by contributing to tumor aggressiveness and thereby represents a potential therapeutic target. Mol Cancer Ther; 16(8); 1705-16. ©2017 AACR.

Beta-hairpin hydrogels as scaffolds for high-throughput drug discovery in three-dimensional cell culture

Automated cell-based high-throughput screening (HTS) is a powerful tool in drug discovery, and it is increasingly being recognized that three-dimensional (3D) models, which more closely mimic in vivo-like conditions, are desirable screening platforms. One limitation hampering the development of 3D HTS is the lack of suitable 3D culture scaffolds that can readily be incorporated into existing HTS infrastructure. We now show that β-hairpin peptide hydrogels can serve as a 3D cell culture platform that is compatible with HTS. MAX8 β-hairpin peptides can physically assemble into a hydrogel with defined porosity, permeability and mechanical stability with encapsulated cells. Most importantly, the hydrogels can then be injected under shear-flow and immediately reheal into a hydrogel with the same properties exhibited prior to injection. The post-injection hydrogels are cell culture compatible at physiological conditions. Using standard HTS equipment and medulloblastoma pediatric brain tumor cells as a model system, we show that automatic distribution of cell-peptide mixtures into 384-well assay plates results in evenly dispensed, viable MAX8-cell constructs suitable for commercially available cell viability assays. Since MAX8 peptides can be functionalized to mimic the microenvironment of cells from a variety of origins, MAX8 peptide gels should have broad applicability for 3D HTS drug discovery.

Targeted Molecular Radiotherapy of Pediatric Solid Tumors Using a Radioiodinated Alkyl-Phospholipid Ether Analog.

External-beam radiotherapy plays a critical role in the treatment of most pediatric solid tumors. Particularly in children, achieving an optimal therapeutic index to avoid damage to normal tissue is extremely important. Consequently, in metastatic disease, the utility of external-beam radiotherapy is limited. Molecular radiotherapy with tumor-targeted radionuclides may overcome some of these challenges, but to date there exists no single cancer-selective agent capable of treating various pediatric malignancies independently of their histopathologic origin. We tested the therapeutic potential of the clinical-grade alkyl-phospholipid ether analog CLR1404, 18-(p-iodophenyl)octadecyl phosphocholine, as a scaffold for tumor-targeted radiotherapy of pediatric malignancies. Methods: Uptake of CLR1404 by pediatric solid tumor cells was tested in vitro by flow cytometry and in vivo by PET/CT imaging and dosimetry. The therapeutic potential of 131I-CLR1404 was evaluated in xenograft models. Results: In vitro, fluorescent CLR1404-BODIPY showed significant selective uptake in a variety of pediatric cancer lines compared with normal controls. In vivo tumor-targeted uptake in mouse xenograft models using 124I-CLR1404 was confirmed by imaging. Single-dose intravenous injection of 131I-CLR1404 significantly delayed tumor growth in all rodent pediatric xenograft models and extended animal survival while demonstrating a favorable side effect profile. Conclusion:131I-CLR1404 has the potential to become a tumor-targeted radiotherapeutic drug with broad applicability in pediatric oncology. Because 131I-CLR1404 has entered clinical trials in adults, our data warrant the development of pediatric clinical trials for this particularly vulnerable patient population.

Abstract 702: A novel cell-penetrating ATF5 antagonist peptide CP-d/n-ATF5 exerts in vitro and in vivo anti-tumor effects in a broad spectrum of pediatric cancers

Abstract Purpose: Activating transcription factor 5 (ATF5), a member of the ATF/CREB family transcription factor, has been implicated in the pathogenesis of glioblastoma and other adult tumors. Recently, a novel cell penetrating (CP-d/n-ATF5) peptide has been developed to antagonize ATF5 function. The goal of the current study is to test the efficacy of CP-d/n-ATF5 in several children tumors including neuroblastoma, hepatoblastoma, Ewing sarcoma, and rhabdoid tumor, in vitro and in vivo. Methods: A panel of neuroblastoma cell lines: SK-N-Be(2)C, SK-N-DZ, NGP, IMR-32, NGP, SHEP-21N, KELLY, CHP-212, CHLA-20, CHLA-15 and SK-N-SH; hepatoblastoma cell lines HUH 6 and Hep-G2; Ewing sarcoma cell lines A673, SKNMC, SKNEP1, and TC32; and the rhabdoid cell G401, were treated with vehicle or 50, 100 and 200 μM of CP-d/n-ATF5. Cell viability and apoptosis were assessed after 72 hr by WST-8 and TUNEL assays, respectively. To test in vivo efficacy, SK-N-Be(2)C kidney xenograft tumors were treated with the peptide at dose 50mg/kg, IP injection once per day for first three days and then twice per week. Tumor growth was monitored by bioluminescence imaging and mice were sacked when flux reached a threshold value. Organ metastases were determined by ex vivo imaging. A patient-derived xenograft (PDX) model of rhabdoid tumors was employed where PDX tumors, at 150-200 mm3 size, were enrolled in the penetratin (control) and CP-d/n-ATF5 treatment (same dose as above). Tumors were measured biweekly with a calipers and mice were sacrificed when tumor volume reached a threshold (1500 mm3). Results: CP-d/n-ATF5 exerted cytotoxicity or apoptosis, in a dose dependent manner, across a wide panel of pediatric tumor cell lines. In vitro, tumor cell viabilities were reduced 40-70% (P&amp;lt;0.05) and apoptosis was increased 50-80% at 200 μM of CP-d/n-ATF5. In vivo, CP-d/n-ATF5 significantly inhibited SK-N-Be(2)C xenograft growth in nude mice, with a median survival of 35 days for control against 21 days for CP-d/n-ATF5, P=0.0013. CP-d/n-ATF5 also reduced SK-N-Be(2)C metastatic burden in the liver (P&amp;lt;0.05) and bone marrow (P&amp;lt;0.01). In the rhabdoid PDX model, there was a significant inhibition of tumors treated with CP-d/n-ATF5 as compared to Penetratin treatment, with a mean tumor volume of control 1283 ± 266.6 mm3 (n=5) vs CP-d/n-ATF5 234.2 ± 50.66 mm3 (n=6), at day 10 post treatment. Penetratin treated tumors showed a median post-treatment time of 13 days to reach threshold volume. None of the CP-d/n-ATF5 treated tumors reached the threshold after 28 days of treatment with some tumors demonstrating regression, indicating a profound anti-tumor effect of the peptide. Conclusion: Our study shows that a novel ATF5-targeting peptide CP-d/n-ATF5 has broad and profound cytotoxic and apoptotic effects in several pediatric tumors in vitro and in vivo. Our study also indicates that CP-d/n-ATF5 has the potential to act as an anti-metastatic agent. Citation Format: Debarshi Banerjee, Shuobo Boboila, Cherease Street, Shunpei Okochi, Filemon S. Dela Cruz, Eileen Connolly, Angela Kadenhe-Chiweshe, Darrell Yamashiro. A novel cell-penetrating ATF5 antagonist peptide CP-d/n-ATF5 exerts in vitro and in vivo anti-tumor effects in a broad spectrum of pediatric cancers [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 702. doi:10.1158/1538-7445.AM2017-702

SOX2 immunity and tissue resident memory in children and young adults with glioma.

Therapies targeting immune checkpoints are effective in tumors with a high mutation burden that express multiple neo-antigens. However, glial tumors including those seen in children carry fewer mutations and there is an unmet need to identify new antigenic targets of anti-tumor immunity. SOX2 is an embryonal stem cell antigen implicated in the biology of glioma initiating cells. Expression of SOX2 by pediatric glial tumors and the capacity of the immune system in these patients to recognize SOX2 has not been previously studied. We examined the expression of SOX2 on archived paraffin-embedded tissue from pediatric glial tumors. The presence of T-cell immunity to SOX2 was examined in both blood and tumor-infiltrating T-cells in children and young adults with glioma. The nature of tumor-infiltrating immune cells was analyzed with a 37-marker panel using single-cell mass cytometry. SOX2 is expressed by tumor cells but not surrounding normal tissue in pediatric gliomas of all grades. T-cells against this antigen can be detected in blood and tumor tissue in glioma patients. Glial tumors are enriched for CD8/CD4 T-cells with tissue resident memory (TRM; CD45RO+, CD69+, CCR7-) phenotype, which co-express multiple inhibitory checkpoints including PD-1, PD-L1 and TIGIT. Tumors also contain natural killer cells with reduced expression of lytic granzyme. Our data demonstrate immunogenicity of SOX2, which is specifically overexpressed on pediatric glial tumor cells. Harnessing tumor immunity in glioma will likely require the combined targeting of multiple inhibitory checkpoints.

DIPG-17. TELOMERE-INDUCED DAMAGE AS A NOVEL APPROACH TO TREAT DIFFUSE INTRINSIC PONTINE GLIOMA

AbstractDiffuse intrinsic pontine glioma (DIPG) is one of the deadliest brain tumors in children. Hence, there is an urgent need to identify actionable therapeutic targets. We have previously shown that over 70% of DIPGs demonstrate telomerase activity and elevated hTERT mRNA levels are associated with shorter overall survival. Thus, telomeres and telomerase represent ideal targets to treat DIPG. We recently validated a novel approach consisting of the incorporation of 6-thio-dG, a telomerase substrate analogue, into telomeres to treat therapy-resistant pediatric brain tumor cells. 6-thio-dG treatment of high-risk group 3 medulloblastoma, pediatric GBM and DIPG patients-derived neurospheres caused telomere damage leading to dose-dependent cell growth inhibition and complete abolition of sphere formation ability. Here, we present our results of the in vivo effectiveness of 6-thio-dG in orthotopic DIPG mouse models. Primary DIPG neurospheres were derived post-autopsy from patients’ tumor tissue. Luciferase positive cells were implanted in the pons of two strains of mice. The generated tumors were highly aggressive and the histopathological characterization of the xenograft showed close similarity with the original patient tumor. Relative to the original patient tissue and the derived neurospheres, telomerase expression and activity were sustained in the xenografts. Tumor bearing mice were treated with 6-thio-dG or the vehicle upon tumor establishment. Molecular analyses of the tumors showed that 6-thio-dG generated significantly higher number of telomere dysfunction-induced foci (TIFs) compared to the untreated tumors, indicating that 6-thio-dG reached the tumor tissue in the pons. Of note, 6-thio-dG treatment had no detectable effect on normal brain tissue. Results regarding tumor regression/stabilization and survival will be discussed. Our data suggest that 6-thio-dG-induced telomere damage is a promising novel approach to treat DIPG.

A biomarker-guided approach to combining PARP inhibitors with radiotherapy in pediatric solid tumors.

10556 Background: Ewing sarcoma (EWS) expresses high levels of Schlafen-11 (SLFN11). SLFN11 disrupts checkpoint maintenance and may serve as a biomarker to assess sensitivity to Poly (ADP-ribose) polymerase 1 and 2 inhibitors (PARPi). The goal of this study is to evaluate SLFN11 protein expression in a panel of pediatric solid tumors and correlate levels of protein with sensitivity to PARP inhibition combined with ionizing radiation (IR), a component of therapy for many pediatric solid tumors and a potent inducer of DNA damage. Methods: SLFN11 mRNA and protein levels were assessed by quantitative RT-PCR, and immunohistochemistry, Western blot, and immunofluorescence microscopy, respectively. PARPi included: talazoparib (TAL), niraparib (NIR), veliparib (VEL), and olaparib (OLA). Approximately 30 minutes after addition of systemic therapy, graded doses of radiation were delivered and viability across a panel of pediatric solid tumor cell lines was measured using the ATP-based Cell TiterGlo assay and confirmed with the colony formation assay. Results: We found that SLFN11 mRNA and protein is expressed at high levels in EWS, and SLFN11 is also variably present in a subset of other pediatric solid tumor lines, including desmoplastic small round cell tumor, osteosarcoma, and rhabdomyosarcoma. In all tumor cells with detectable SLFN11 expression, viability was reduced by greater than 90% when exposed to 2Gy IR and 1-10nM TAL, whereas cells with undetectable levels of SLFN11 were 5-10 times less sensitive. Intriguingly, variation in the potentiation between specific PARPi and IR correlated with the ability to form drug-induced PARP-DNA adducts, with the strong PARP trapper TAL showing ~10-fold higher potency compared to the moderate trapper NIR, and ~300-fold more potency relative to the weak trapper VEL. Consistent with our PARPi findings, the toposiomerase 1 inhibitor irinotecan, which also forms DNA adducts, potentiated with IR similarly to TAL at concentrations &lt; 10nM in tumor cells expressing detectable levels of SLFN11. Conclusions: SLFN11 is present in select pediatric solid tumors and may induce a DNA repair defect that is best exploited by combining low-doses of TAL and irinotecan with IR.

SOX2 as a target for immunotherapy of pediatric gliomas.

e22012 Background: New treatments are needed to improve outcomes for pediatric gliomas. Immune checkpoint inhibitors are effective therapies in tumors with a high mutation burden that express multiple neo-antigens. However, for pediatric tumors that carry few mutations, there is a need to identify new antigenic targets of anti-tumor immunity. SOX2 is an embryonal stem cell antigen implicated in the biology of glioma initiating cells. Expression of SOX2 by pediatric glial tumors, and the capacity of the immune system in these patients to recognize SOX2, has not been studied. Methods: We examined the expression of SOX2 on paraffin-embedded tissue from pediatric glial tumors (n = 30). The presence of T cell immunity to SOX2 was examined in both blood and tumor-infiltrating T cells using antigen-dependent cytokine and T cell proliferation assays (n = 15). The nature of tumor-infiltrating immune cells in glial tumors (n = 4) was analyzed using single cell mass cytometry. Results: SOX2 is expressed by tumor cells but not surrounding normal tissue in all low grade gliomas (n = 15), high grade gliomas (n = 7), ependymomas (n = 3) and in 60% of oligodendrogliomas (n = 5). T cells against SOX2 can be detected in blood and tumor tissue in 33% of patients. CD4 and CD8 tumor infiltrating T-cells display a higher proportion of PD-1 expression compared to circulating T cells (p &lt; 0.05). Glial CD4 and CD8 T cells are enriched for tissue resident memory phenotype (TRM; CD45RO+, CD69+, CCR7-) and the expression of PD-1 is primarily on these TRM cells (p &lt; 0.05). A subset of CD4 and CD8 TRM cells also co-express multiple inhibitory checkpoints including PD-L1 and TIGIT. Glial tumors also contain NK cells with reduced expression of lytic granzyme (p &lt; 0.05). Conclusions: Our data demonstrate in vivo immunogenicity of SOX2, which is specifically overexpressed on pediatric glial tumor cells. Our data also suggest that the TRM subset of tumor-infiltrating T cells may be key targets for immune checkpoint blockade, and harnessing tumor immunity will likely require the combined targeting of multiple inhibitory checkpoints. Future efforts to target SOX2 with dendritic cell vaccines combined with immune checkpoint blockade could provide effective tumor immunity and improve outcomes in pediatric brain tumors.

Silencing of DNA repair sensitizes pediatric brain tumor cells to γ-irradiation using gold nanoparticles

We present a nanoparticle (NP)-mediated delivery vehicle that effectively carries and protects siRNA in pediatric ependymoma (EP) and medulloblastoma (MB) cells. The delivery vehicle consists of gold NPs coated with a polymeric shell comprising polyethylene glycol (PG), chitosan and polyethyleneimine (Au-CP-PEI). NPs loaded with siRNA knocked down Ape1 expression by over 75% in both MB and EP cells. Further, this reduction in Ape1 expression is associated with an increase in DNA damage after irradiation. The results indicate that NP-associated delivery of siApe1 is a feasible approach to circumventing pediatric brain tumor resistance to radiation therapy.

Abstract B12: Establishment of paired primary neurosphere and monolayer cells of mouse xenograft derived from pediatric brain tumors

Abstract Background: Stable and reproducible in vitro cell lines derived from clinical samples are currently considered as the best research tools to study human cancers in vitro. Currently there is very limited availability of tumor cell lines, especially paried neurosphere and nomolayer cells. Patient tumor is difficult to obtain and cannot be harvested by multiple times. Direct use of patient tumor is associated with low success rate in in vitro culture. Here we sought to establish a set of paired primary neurosphere and monolayer cells in vitro by taking advantage of patient derived orthotopic xenograft models we have already established. Materials and Methods: We have established murine xenograft models that biologically and molecularly reciprocate their human counterparts. Those human tumor xenografts are able to provide a sustained and ample supply of tumor cells in ex vivo culture. Primary tumor cells could be continuously harvested from human tumor xenografts and are maintained both in serum-free medium to enrich neurosphere cells and in fetal bovine serum (FBS)-containing DMEM medium where tumor cells grow in monolayer. Results: We have already developed in primary culture a panel of paired neurosphere and monolayer cells derived from pediatric glioblastoma (GBM), medulloblastoma (MB)and ependymoma (EPN) at different culture stage including three different GBM cells over 60 passages and four over 20 passages, one EPN cell over 40 passages and one MB cell at early stage of around 20 passages. In vitro cultures demonstrated typical cell morphology of adherent and sphere cells for all pediatric brain tumor cell lines we cultured so far. These cultured cells are tumorigenic even with small number of tumor cells and the relative abundances of putative cancer stem cells are different between neurospheres and monolayer culture. Furthermore, our preliminary data from one pair of GBM neurospheres and monolayer cells by high throughput-out drug screening employing hundred thousands of already-identified molecules showed a panel of promising target candidates. Neurospheres and monolayer cells do not respond equally toward the same library of chemotherapeutic agents. Conclusion: Patient derived orthotopic xenograft (PDOX) can be a good source of human tumor cells for the establishment of in vitro model systems of pediatric brain tumors. The neurosphere and monolayer cells are morphologically and biologically different, which suggest that in future drug screening for both type of cells should be included. Citation Format: Yuchen Du, Lin Qi, Huiyuan Zhang, Mari Kogiso, Holly B. Lindsay, Sibo Zhao, Frank Braun, Xiaonan Li. Establishment of paired primary neurosphere and monolayer cells of mouse xenograft derived from pediatric brain tumors. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B12.

Abstract 2816: Evaluation of Cdk5 inhibitors in neural crest tumors

Abstract Cyclin-dependent kinase 5 (Cdk5) is a ubiquitously expressed proline-directed serine threonine kinase active mainly in post-mitotic neurons due to abundant expression of its obligate activating partners p35 and/or p39. Cdk5 has been viewed narrowly as a neuron-specific kinase and as an essential regulator of neuronal function and migration. Recently, Cdk5 activity has been implicated in mediating tumor invasion, progression, and metastasis. We have found invariably high expression and activity of Cdk5/p35 in a series of pediatric tumor cell lines including neuroblastoma. To evaluate the potential role of Cdk5 inhibitors in the treatment of neural crest tumors, we evaluated the in vitro kinase activity and growth inhibition of 4 Cdk5 inhibitors (AT7519, dinaciclib, flavopiridol, and seliciclib). We compared the in vitro activity against Cdk2/cyclin E, Cdk5/p25, Cdk5/p35, Cdk7/cyclin H, and Cdk9/cyclin K. Using a DNA-based proliferation assay, we assessed the in vitro growth inhibition in a panel of 10 neural crest-derived tumors (melanoma, neuroblastoma, Ewing sarcoma). All Cdk5 inhibitors demonstrated enzyme and growth inhibition at clinically achievable concentrations. Dinaciclib was the most potent inhibitor of Cdk5/p35 with an enzyme IC50 of 0.0002 μM, compared to 0.003 μM for AT7519, 0.021 μM for flavopiridol, and 0.097 μM for seliciclib. Dinaciclib was also the most potent inhibitor of Cdk2. However, flavopiridol and AT7519 were the most potent inhibitors of Cdk7 and Cdk9, respectively. Across all cell lines, dinaciclib was the most potent inhibitor of growth. The median (range) growth inhibition IC50 for dinaciclib was 0.006 μM (0.004 - 0.14), compared to 0.079 μM (0.045-0.16) for flavopiridol, 0.52 μM (0.14 - &amp;gt;15) for AT7519, and 13.2 μM (7.8 - 22.1) for seliciclib. Interestingly, within the Ewing sarcoma cell lines, there was disparate sensitivity to the 2 most potent Cdk5 inhibitors, dinaciclib and AT7519. Based on the activity of these Cdk inhibitors in neural-crest tumors, we expanded our evaluation of AT7519 in 6 pediatric tumor cell lines (medulloblastoma, rhabdomyosarcoma, osteosarcoma) and found similar in vitro potency with a median (range) IC50 of 0.43 μM (0.20 - 1.26). AT7519, dinaciclib, flavopiridol, and seliciclib demonstrated in vitro growth inhibition in a panel of neural crest and pediatric solid tumor cell lines at clinically achievable concentrations. Further in vivo evaluations and in vitro combination studies including this novel class of agents in models of neural crest and select pediatric cancers are warranted. Citation Format: Robin E. Norris, John J. Pink, Tej Pareek, John L. Letterio. Evaluation of Cdk5 inhibitors in neural crest tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2816.

Abstract 2458: Targeting checkpoint kinase 1 (CHK1) with the small molecule inhibitor LY2606368 mesylate monohydrate in models of high-risk pediatric cancer yields significant antitumor effects

Abstract CHK1 is a serine/threonine protein kinase essential for S-phase and G2/M cell cycle checkpoint regulation following DNA damage. Targeted inhibition of CHK1 in several tumor types increases DNA damage and replication stress, culminating in cell death through mitotic catastrophe. Recent studies have identified CHK1 as a therapeutic target in several pediatric tumor types. We evaluated the antitumor efficacy of LY2606368 mesylate monohydrate (“LY”), a checkpoint kinase 1 (CHK1)/CHK2 inhibitor currently in early phase clinical trials for adult solid cancers, in a panel of pediatric tumor cell lines and mouse models of embryonal tumors and pediatric sarcoma. In vitro effects of LY were assessed via Cell Titer Glo, immunoblotting, and cell cycle analysis by flow cytometry. For in vivo studies, mice bearing cell-derived (CDX) or patient-derived xenografts (PDX) of several pediatric tumor types were treated with four weekly cycles of 10 mg/kg LY BID for 3 consecutive days, followed by a 4 day dosing holiday. Tumor volume and body weight were measured 2x weekly. Xenograft tumor health following LY, chemotherapy, or combination treatment was evaluated by fluorescent immunohistochemistry (IHC) for a panel of markers for cell proliferation (Ki67), apoptosis (TUNEL), and angiogenesis (CD31, smooth muscle actin [SMA], MECA32). Single digit nanomolar sensitivity to LY was observed in the majority of pediatric cancer cell lines evaluated in vitro. A more detailed analysis of LY-treated neuroblastoma and pediatric sarcoma cell lines showed increased DNA damage, CHK1 phosphorylation, and MAPK pathway activation. Significant single agent LY activity was observed in mouse models of neuroblastoma and pediatric sarcoma, but not in models of hepatoblastoma or retinoblastoma. Acquired resistance to LY was observed in the ST162 and SJCRH30 models of alveolar rhabdomyosarcoma. Interestingly, more stroma was observed following LY single agent treatment as measured by CD31, SMA, and MECA32 IHC staining; co-treatment with chemotherapy reduced the amount of SMA expressing-cells. Overall, our data demonstrate that LY is highly effective as a single agent in murine in vivo models of human neuroblastoma and several pediatric sarcoma subtypes. Current studies include further evaluation of the LY mechanism of action; investigation into the mechanism of intrinsic and acquired resistance; and identification of possible biomarkers for LY sensitivity. Citation Format: Caitlin D. May, Richard Beckmann, Wayne Blosser, Michele Dowless, Alle VanWye, Teresa Burke, Gerard Oakley, Jennifer Stephens, Julie Stewart, Beverly Falcon, Louis Stancato. Targeting checkpoint kinase 1 (CHK1) with the small molecule inhibitor LY2606368 mesylate monohydrate in models of high-risk pediatric cancer yields significant antitumor effects. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2458.

Photodynamic Therapy Potentiates the Effects of Curcumin on Pediatric Epithelial Liver Tumor Cells.

Curcumin (CUM) is a promising agent in complementary oncology. The present study analyzed the photoactive properties of curcumin on pediatric epithelial liver tumor cell lines. Hepatoblastoma cell lines (HuH6, HepT1) and hepatocellular carcinoma cell lines (HepG2, HC-AFW1) were treated with curcumin and exposed to blue light (phototherapy, 480 nm, 300 W). Cell viability (MTT tests), cellular oxidative stress (production of reactive oxygen species (ROS)) and cellular uptake/degradation of curcumin were analyzed. Significant loss of viability resulted from 24-48 h incubation with curcumin. With photodynamic therapy (PDT), even short time incubation (1 h) with curcumin resulted in significantly lower half maximal inhibitory concentration (IC50) (p<0.001, two-way ANOVA). Significant ROS production was observed with PDT and curcumin. Phototherapy strongly enhances the anticancer properties of curcumin in pediatric solid liver tumors in vitro.