TH-MYCN Mice Research Articles

PRC2-Mediated Transcriptomic Alterations at the Embryonic Stage Govern Tumorigenesis and Clinical Outcome in MYCN-Driven Neuroblastoma.

Pediatric cancers such as neuroblastoma are thought to involve a dysregulation of embryonic development. However, it has been difficult to identify the critical events that trigger tumorigenesis and differentiate them from normal development. In this study, we report the establishment of a spheroid culture method that enriches early-stage tumor cells from TH-MYCN mice, a preclinical model of neuroblastoma. Using this method, we found that tumorigenic cells were evident as early as day E13.5 during embryo development, when the MYC and PRC2 transcriptomes were significantly altered. Ezh2, an essential component of PRC2, was expressed in embryonic and postnatal tumor lesions and physically associated with N-MYC and we observed that H3K27me3 was increased at PRC2 target genes. PRC2 inhibition suppressed in vitro sphere formation, derepressed its target genes, and suppressed in situ tumor growth. In clinical specimens, expression of MYC and PRC2 target genes correlated strongly and predicted survival outcomes. Together, our findings highlighted PRC2-mediated transcriptional control during embryogenesis as a critical step in the development and clinical outcome of neuroblastoma. Cancer Res; 77(19); 5259-71. ©2017 AACR.

Abstract 1940: Prenatal stress increases malignancy of neuroblastoma tumors in TH-MYCN animal model

Abstract Neuroblastoma (NB) is a pediatric malignancy arising due to defects in sympathetic neuron differentiation. NB is a heterogeneous disease, with phenotypes ranging from spontaneously regressing to highly aggressive, incurable tumors. This clinical variability cannot be explained solely by genetic aberrations. Even in families with hereditary NB the penetrance of the disease is incomplete and the same genetic mutation often results in tumors with phenotypes varying from differentiating ganglioneuromas to undifferentiated, highly aggressive NBs. Thus, other, perhaps non-genetic factors can contribute to the disease development and modify its phenotype. Strikingly, the two factors promoting de-differentiation of NB cells and their malignant phenotype, hypoxia and glucocorticoids, are elevated in the fetus during maternal stress, suggesting a role for prenatal stress in NB tumorigenesis. Previously, using TH-MYCN mice as a model of aggressive NB, we have shown that an increase in maternal corticosterone levels during pregnancy attained by inserting slow release pellets resulted in increased tumor frequency in TH-MYCN offspring. The goal of the current study was to determine the effect of prenatal stress on NB metastasis. To this end, pregnant mice carrying TH-MYCN hemizygous offspring were subjected to chronic stress at embryonic days 10-17, the time of sympathetic neuroblast proliferation and differentiation. Two established stress paradigms were used - chronic unpredictable stress, in which mice were subjected daily to various stressors, and chronic cold stress comprising of daily 30 min exposure to cold. The phenotypes of the disease and its dissemination were compared between offspring of control and stressed mothers. The offspring from both prenatally stressed groups presented with more malignant disease, as manifested by the presence of advanced lung metastases disseminating from small primary tumors (<200 mm3). This phenotype was associated with increased mortality in prenatally-stressed TH-MYCN offspring (p<0.01). In contrast, no advanced lung metastases and no disease-related deaths were observed in TH-MYCN offspring of control mothers despite the presence of large primary tumors (>1,000 mm3). Although not common, lung metastases occur preferentially in NB patients with MYCN amplification and are associated with significantly worse prognosis, as compared to patients with metastatic disease, but no pulmonary involvement (14 vs 43% 3-year event-free survival, respectively). Thus, the profound pulmonary dissemination observed in prenatally-stressed TH-MYCN mice mimics one of the most malignant NB phenotypes observed in human disease. Altogether, our data implicate maternal stress during pregnancy as a potential environmental factor modifying the effects of genetic aberrations and promoting malignant phenotype of NB. Citation Format: Sung Hyeok Hong, Larissa Wietlisbach, Susana Galli, Akanksha Mahajan, Shiya Zhu, Jason Tilan, Yichien Lee, Olga Rodriguez, Chris Albanese, Joanna Kitlinska. Prenatal stress increases malignancy of neuroblastoma tumors in TH-MYCN animal model [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 1940. doi:10.1158/1538-7445.AM2017-1940

Neuropeptide Y as a Biomarker and Therapeutic Target for Neuroblastoma

Neuroblastoma (NB) is a pediatric malignant neoplasm of sympathoadrenal origin. Challenges in its management include stratification of this heterogeneous disease and a lack of both adequate treatments for high-risk patients and noninvasive biomarkers of disease progression. Our previous studies have identified neuropeptide Y (NPY), a sympathetic neurotransmitter expressed in NB, as a potential therapeutic target for these tumors by virtue of its Y5 receptor (Y5R)-mediated chemoresistance and Y2 receptor (Y2R)-mediated proliferative and angiogenic activities. The goal of this study was to determine the clinical relevance and utility of these findings. Expression of NPY and its receptors was evaluated in corresponding samples of tumor RNA, tissues, and sera from 87 patients with neuroblastic tumors and in tumor tissues from the TH-MYCN NB mouse model. Elevated serum NPY levels correlated with an adverse clinical presentation, poor survival, metastasis, and relapse, whereas strong Y5R immunoreactivity was a marker of angioinvasive tumor cells. In NB tissues from TH-MYCN mice, high immunoreactivity of both NPY and Y5R marked angioinvasive NB cells. Y2R was uniformly expressed in undifferentiated tumor cells, which supports its previously reported role in NB cell proliferation. Our findings validate NPY as a therapeutic target for advanced NB and implicate the NPY/Y5R axis in disease dissemination. The correlation between elevated systemic NPY and NB progression identifies serum NPY as a novel NB biomarker.

Tumor-homing effect of human mesenchymal stem cells in a TH-MYCN mouse model of neuroblastoma

BackgroundHuman mesenchymal stem cells (hMSCs) are multipotent stem-like cells that are reported to have tumor-suppression effects and migration ability toward damaged tissues or tumors. The aim of this study was to analyze the tumor-homing ability of hMSCs and antitumor potency in a transgenic TH-MYCN mouse model of neuroblastoma (NB). MethodshMSCs (3×106) labeled with DiR, a lipophilic near-infrared dye, were intraperitoneally (i.p.) or intravenously (i.v.) administered to the TH-MYCN mice. hMSC in vivo kinetics were assayed using the IVIS® imaging system for 24h after injection. Immunohistochemistry using human CD90 antibody was also performed to confirm the location of hMSCs in various organs and tumors. Furthermore, the survival curve of TH-MYCN mice treated with hMSCs was compared to a control group administered PBS. Resultsi.p. hMSCs were recognized in the tumors of TH-MYCN mice by IVIS. hMSCs were also located inside the tumor tissue. Conversely, most of the i.v. hMSCs were captured by the lungs, and migration into the tumors was not noted. There was no significant difference in the survival between the hMSC and control groups. ConclusionThe present study suggested that hMSCs may be potential tumor-specific therapeutic delivery vehicles in NB according to their homing potential to tumors.

P53 Loss in MYC-Driven Neuroblastoma Leads to Metabolic Adaptations Supporting Radioresistance.

Neuroblastoma is the most common childhood extracranial solid tumor. In high-risk cases, many of which are characterized by amplification of MYCN, outcome remains poor. Mutations in the p53 (TP53) tumor suppressor are rare at diagnosis, but evidence suggests that p53 function is often impaired in relapsed, treatment-resistant disease. To address the role of p53 loss of function in the development and pathogenesis of high-risk neuroblastoma, we generated a MYCN-driven genetically engineered mouse model in which the tamoxifen-inducible p53ER(TAM) fusion protein was expressed from a knock-in allele (Th-MYCN/Trp53(KI)). We observed no significant differences in tumor-free survival between Th-MYCN mice heterozygous for Trp53(KI) (n = 188) and Th-MYCN mice with wild-type p53 (n = 101). Conversely, the survival of Th-MYCN/Trp53(KI/KI) mice lacking functional p53 (n = 60) was greatly reduced. We found that Th-MYCN/Trp53(KI/KI) tumors were resistant to ionizing radiation (IR), as expected. However, restoration of functional p53ER(TAM) reinstated sensitivity to IR in only 50% of Th-MYCN/Trp53(KI/KI) tumors, indicating the acquisition of additional resistance mechanisms. Gene expression and metabolic analyses indicated that the principal acquired mechanism of resistance to IR in the absence of functional p53 was metabolic adaptation in response to chronic oxidative stress. Tumors exhibited increased antioxidant metabolites and upregulation of glutathione S-transferase pathway genes, including Gstp1 and Gstz1, which are associated with poor outcome in human neuroblastoma. Accordingly, glutathione depletion by buthionine sulfoximine together with restoration of p53 activity resensitized tumors to IR. Our findings highlight the complex pathways operating in relapsed neuroblastomas and the need for combination therapies that target the diverse resistance mechanisms at play. Cancer Res; 76(10); 3025-35. ©2016 AACR.

Glutathione biosynthesis is upregulated at the initiation of MYCN-driven neuroblastoma tumorigenesis

The MYCN gene is amplified and overexpressed in a large proportion of high stage neuroblastoma patients and has been identified as a key driver of tumorigenesis. However, the mechanism by which MYCN promotes tumor initiation is poorly understood. Here we conducted metabolic profiling of pre-malignant sympathetic ganglia and tumors derived from the TH-MYCN mouse model of neuroblastoma, compared to non-malignant ganglia from wildtype littermates. We found that metabolites involved in the biosynthesis of glutathione, the most abundant cellular antioxidant, were the most significantly upregulated metabolic pathway at tumor initiation, and progressively increased to meet the demands of tumorigenesis. A corresponding increase in the expression of genes involved in ribosomal biogenesis suggested that MYCN-driven transactivation of the protein biosynthetic machinery generated the necessary substrates to drive glutathione biosynthesis. Pre-malignant sympathetic ganglia from TH-MYCN mice had higher antioxidant capacity and required glutathione upregulation for cell survival, when compared to wildtype ganglia. Moreover, in vivo administration of inhibitors of glutathione biosynthesis significantly delayed tumorigenesis when administered prophylactically and potentiated the anticancer activity of cytotoxic chemotherapy against established tumors. Together these results identify enhanced glutathione biosynthesis as a selective metabolic adaptation required for initiation of MYCN-driven neuroblastoma, and suggest that glutathione-targeted agents may be used as a potential preventative strategy, or as an adjuvant to existing chemotherapies in established disease.

Immune response modulation by Galectin-1 in a transgenic model of neuroblastoma

ABSTRACTGalectin-1 (Gal-1) has been described to promote tumor growth by inducing angiogenesis and to contribute to tumor immune escape by promoting apoptosis of activated T cells. We had previously identified upregulation of Gal-1 in preclinical models of aggressive neuroblastoma (NB), a solid tumor of childhood. However, the clinical and biological relevance of Gal-1 in this tumor entity is unclear. Here, the effect of Gal-1 on the immune system and tumorigenesis was assessed using modulation of Gal-1 expression in immune effector cells and in a transgenic NB model, designated TH-MYCN. The fraction of CD4+ T cells was decreased in tumor-bearing TH-MYCN mice compared to tumor-free littermates, while both CD4+ T cells as well as CD8+ T cells were less activated, compatible with a reduced immune response in tumor-bearing mice. Tumor incidence was not significantly altered by decreasing Gal-1/LGALS1 gene dosage in TH-MYCN mice, but TH-MYCN/Gal-1−/− double transgenic mice displayed impaired tumor angiogenesis, splenomegaly, and impaired T cell tumor-infiltration with no differences in T cell activation and apoptosis rate. Additionally, a lower migratory capacity of Gal-1 deficient CD4+ T cells toward tumor cells was observed in vitro. Transplantation of TH-MYCN-derived tumor cells into syngeneic mice resulted in significantly reduced tumor growth and elevated immune cell infiltration when Gal-1 was downregulated by shRNA. We therefore conclude that T cell-derived Gal-1 mediates T cell tumor-infiltration, whereas NB-derived Gal-1 promotes tumor growth. This opposing effect of Gal-1 in NB should be considered in therapeutic targeting strategies, as currently being developed for other tumor entities.

Cyclin-Dependent Kinase Inhibitor AT7519 as a Potential Drug for MYCN-Dependent Neuroblastoma.

MYCN-dependent neuroblastomas have low cure rates with current multimodal treatment regimens and novel therapeutic drugs are therefore urgently needed. In previous preclinical studies, we have shown that targeted inhibition of cyclin-dependent kinase 2 (CDK2) resulted in specific killing of MYCN-amplified neuroblastoma cells. This study describes the in vivo preclinical evaluation of the CDK inhibitor AT7519. Preclinical drug testing was performed using a panel of MYCN-amplified and MYCN single copy neuroblastoma cell lines and different MYCN-dependent mouse models of neuroblastoma. AT7519 killed MYCN-amplified neuroblastoma cell lines more potently than MYCN single copy cell lines with a median LC50 value of 1.7 compared to 8.1 μmol/L (P = 0.0053) and a significantly stronger induction of apoptosis. Preclinical studies in female NMRI homozygous (nu/nu) mice with neuroblastoma patient-derived MYCN-amplified AMC711T xenografts revealed dose-dependent growth inhibition, which correlated with intratumoral AT7519 levels. CDK2 target inhibition by AT7519 was confirmed by significant reductions in levels of phosphorylated retinoblastoma (p-Rb) and nucleophosmin (p-NPM). AT7519 treatment of Th-MYCN transgenic mice resulted in improved survival and clinically significant tumor regression (average tumor size reduction of 86% at day 7 after treatment initiation). The improved efficacy of AT7519 observed in Th-MYCN mice correlated with higher tumor exposure to the drug. This study strongly suggests that AT7519 is a promising drug for the treatment of high-risk neuroblastoma patients with MYCN amplification.

Abstract PR09: MYCN and is a therapeutic target in neuroblastoma

Abstract Neuroblastoma is a childhood cancer of the sympathetic nervous system. Approximately 20% of patients present with an aggressive metastatic disease characterized by amplification of the MYCN locus. However, an additional 30% of patients suffer from an equally aggressive disease, yet there are no unified genetic markers available. To identify potential biomarkers of poor prognosis in neuroblastoma, we conducted unsupervised hierarchical clustering on 649 primary neuroblastoma tumors based on a 51 gene MYC core target signature. This demonstrated strong activation within the MYCN amplified cohort as expected, but surprisingly there was a large subset of MYCN non-amplified patients that exhibited high MYC signaling. High expression of this MYC signature proved to be strongly predictive of poor overall survival, in both the full cohort (Hazard ratio (HR): 47.6, p= 2.7x10-35, n=649) and in MYCN non-amplified patients (HR=32.8, p=1.3x10-13, n=554), suggesting that neuroblastoma can be driven by MYC signaling in the absence of MYCN amplification. To evaluate potential therapeutic targets within this MYC signature, we conducted cox regression analysis on the 51 individual members and identified that SPT16 was a particularly potent predictor of poor outcome in these 649 neuroblastoma patients (HR: 7.2, p=2.7x10-17). SPT16 is a subunit of the FACT histone chaperone complex, which together with its heterodimer partner SSRP1, has previously been shown to function in dynamic regulation of chromatin to drive transcription, DNA replication and DNA repair. Similar to SPT16, high SSRP1 expression was a strong predictor of poor prognosis in 649 neuroblastoma patients (HR: 12.5, p=5.812x10-26) implicating FACT as a mediator of MYC driven neuroblastoma. Next we investigated the biological relationship between FACT and MYCN in neuroblastoma. siRNA experiments showed that FACT and MYCN participate in a positive feedback transcriptional loop that is essential for maintaining mutual high expression. Immunohistochemistry in sympathetic ganglia of pre-tumor TH-MYCN mouse model of neuroblastoma identified that FACT was highly expressed at tumor initiation and chemical inhibition of FACT by CBL0137 markedly reduced tumor initiation and subsequent tumorigenesis in vivo. Next we tested CBL0137 against established neuroblastoma in TH-MYCN mice. CBL0137 was highly cytotoxic to tumors either as a single agent or by synergistically promoting clinically used chemotherapeutics. This was accompanied by marked MYCN depletion and prolonged tumor regression in a majority of recipient mice. Pulsed-field electrophoresis showed that CBL0137 impaired FACT dependent DNA damage repair in the presence of genotoxic chemotherapy, thus creating a synthetic lethal environment in MYCN-amplified neuroblastoma cells. Together our data define FACT as a mediator of neuroblastoma tumorigenesis and MYCN-related treatment target. Given the current poor survival rates of children with high-risk neuroblastoma, our findings provide support for the clinical development of CBL0137 as a novel treatment approach for this refractory malignancy. Citation Format: Daniel R. Carter, Jayne Murray, Belamy B. Cheung, Heyam Kalla, Laura Gamble, Joanna Tsang, Selina Sutton, Jessica Koach, Sarah Syed, Andrew Gifford, Natalia Issaeva, Asel Biktasova, Bernard Atmadibrata, Yuting Sun, Nicolas Sokolowski, Dora Ling, Patrick Y. Kim, Hannah Webber, Ashleigh Clark, Michelle Ruhle, Bing Liu, André Oberthuer, Matthias Fischer, Jennifer Byrne, Federica Saletta, Andrei Purmal, David Ziegler, Tao Liu, Katerina V. Gurova, Andrei V. Gudkov, Murray D. Norris, Michelle Haber, Glenn M. Marshall. MYCN and is a therapeutic target in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR09.

Abstract 5384: Prolonged survival of the TH-MYCN murine model of high-risk neuroblastoma, in response to the histone deacetylase inhibitor panobinostat, is the result of both apoptosis and differentiation

Abstract Background/Aims: Deregulated acetylation of histones plays a key role in the pathogenesis of hematological as well as solid tumors by changing the chromatin structure and consequently altering transcription of genes involved in cell cycle control, differentiation and apoptosis. Inhibitors of histone deacetylases (HDACs) can therefore result in multiple cellular responses, including cell death and differentiation. The aim of this study was to investigate the mechanism of major prolongation in survival induced by the HDAC-inhibitor panobinostat, in the TH-MYCN mouse model for neuroblastoma, Methods: Mice homozygous for the TH-MYCN transgene underwent abdominal ultrasounds (US) from five weeks of age until neuroblastomas of between 50mm3 and 200 mm3 were detected. Mice were then randomized to receive the pan-HDAC inhibitor, panobinostat or vehicle daily for nine weeks. Tumor volume was monitored by serial US. One hundred days following cessation of therapy tumors were harvested for immunohistochemical (IHC) analysis of key markers for differentiation. For short-term experiments, tumors were harvested after 24hr for western blot and IHC analysis of key proteins of the apoptosis pathways as well as biomarkers of HDAC inhibition. RNA-seq analysis was performed on tumors harvested at baseline, 14 hr and 24 hr post panobinostat treatment. Gene set enrichment, and pathway analysis was then performed to investigate changes in the expression of genes associated with apoptosis, differentiation and NMYC expression. Results: Treatment with panobinostat significantly improved survival with 100% of TH-MYCN mice alive at day 63 compared with vehicle (0.0%, mean survival 7 days; p<0.0001). One hundred days after the withdrawal of drug 88.9% of panobinostat treated mice remained alive (p<0.0001) with no evidence of disease progression on US. Panobinostat induced rapid tumor regression as determined by US, and significant apoptosis mediated through the caspase-dependent pathway via up-regulation of the BH3 only pro-apoptotic proteins, BMF and BIM. Panobinostat also induced differentiation of malignant neuroblastomas into benign ganglioneuromas as evidenced by H&E and IHC analysis of markers of differentiation including S100 and SSTR2. Correspondingly tumors had reduced expression of NMYC compared with untreated tumors. The findings confirmed up-regulation of genes associated with apoptosis and differentiation, as well as down-regulation of NMYC expression. Conclusions: Treatment of TH-MYCN mice with continuous panobinostat significantly improved survival and reduced tumor burden, primarily via induction of apoptosis and differentiation. These preclinical findings indicate that HDAC inhibition is a promising therapeutic strategy, and supports the further study of panobinostat as a treatment option for high-risk neuroblastoma. Citation Format: Kelly Waldeck, Carleen Cullinane, Jake Shortt, Ben Martin, Kerry Ardley, Richard Tothill, Grant McArthur, Paul J. Wood. Prolonged survival of the TH-MYCN murine model of high-risk neuroblastoma, in response to the histone deacetylase inhibitor panobinostat, is the result of both apoptosis and differentiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5384. doi:10.1158/1538-7445.AM2015-5384

Abstract 3291: Prenatal stress increases neuroblastoma tumorigenesis in TH-MYCN mice model

Abstract Neuroblastomas (NB) are pediatric malignancies with heterogenous phenotypes, ranging from spontaneously regressing to highly aggressive, incurable tumors. Although NB is considered a genetic disease, its etiology and heterogeneity cannot be explained solely by genetic aberrations. NB arises due to defects in sympathetic neuron (SN) differentiation occurring during fetal development. Strikingly, the two factors promoting de-differentiation of NB cells, hypoxia and glucocorticoids, are elevated in the fetus during maternal stress, suggesting a role for prenatal stress in NB tumorigenesis. To test this hypothesis we used mice expressing MYCN oncogene under tyrosine hydroxylase promoter (TH-MYCN mice), which spontaneously develop NBs. To mimic stress, pregnant mothers carrying hemizygous TH-MYCN offspring were implanted with pellets containing either the main rodent glucocorticoid, corticosterone, or placebo at the time of neuroblast proliferation (embryonic days 10-20). Tumor frequency was compared between these two experimental groups and TH-MYCN offspring from intact pregnancies. Surprisingly, in pregnant mothers from the placebo group, physiological stress associated with experimental procedures alone (animal handling, blood collections, pellet insertion and anesthesia) was sufficient to elevate their corticosterone levels and increase tumorigenicity in their hemizygous TH-MYCN offspring from 32 to 64% (p = 0.03). A similar effect was observed in offspring of corticosterone-treated mothers with its levels comparable to mice eliciting a physiological stress response in the placebo group (<1200ng/ml). This stress-induced increase in tumorigenicity was associated with decreased tumor cell apoptosis, suggesting defects in neuroblast elimination as one of the mechanisms underlying this effect. While all female mice presented with ovarian metastases, tumor-bearing prenatally stressed offspring had increased frequency of other distant metastases (0% vs 71% for intact control and stressed groups respectively, p<0.05). No increase in tumorigenicity was observed in offspring of corticosterone-treated mothers having corticosterone levels markedly exceeding those observed in the placebo group (>1200ng/ml), indicating that only physiologically relevant levels of stress mediators can accurately recapitulate the stress response in animal models. Taken together, these findings support the role for prenatal stress in NB development, as well as implicate other pathologies associated with elevated levels of glucocorticoids in its etiology. Citation Format: Sung-Hyeok Hong, David Christian, Emily Trinh, Susana Galli, Meredith Horton, Yichien Lee, Christopher Albanese, Olga Rodriguez, Jason Ulip Tilan, Joanna Kitlinska. Prenatal stress increases neuroblastoma tumorigenesis in TH-MYCN mice model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3291. doi:10.1158/1538-7445.AM2015-3291

Abstract 491: Tumor response to cabozantinib in the TH-MYCN GEM model of neuroblastoma

Abstract Neuroblastoma (NB) is the most common extra-cranial solid tumor in childhood. The proto-oncogene MYCN is amplified in 25% of NB, and is associated with enhanced angiogenesis and poor survival. Vascular endothelial growth factor (VEGF) is the most potent angiogenic growth factor and, with the hepatocyte growth factor (HGF)/c-MET signalling pathway, is implicated in the progression of human NB. Cabozantinib (CBZ) is a small-molecule kinase inhibitor with potent activity against VEGFR2 and MET, currently in clinical trials against NB. In this study, the efficacy of CBZ was evaluated in tumors spontaneously arising in the TH-MYCN GEM model of high-risk NB, previously shown to faithfully recapitulate the radiological and hypervascular characteristics of childhood disease. TH-MYCN mice with spontaneous abdominal tumors were treated daily with either vehicle or 30mg/kg CBZ. Non-invasive T2-weighted anatomical MRI revealed that CBZ induced significant (p<0.001) tumor growth arrest after 48 hours compared to control. Quantitative functional MRI showed this volumetric response was associated with significant decreases in both the native longitudinal MRI relaxation time T1 (p<0.01) a measure of the ratio of bound to free water in tissue reflecting the increased extracellular space in tumors, and transverse MRI relaxation rate R2* (p<0.05), sensitive to paramagnetic deoxyhemoglobin. Histopathological analysis revealed a significant increase in necrosis (p<0.01), and significant decrease in microvessel density (p<0.01) and VEGFR2 scoring (p<0.02) in CBZ-treated tumors compared to control. Significant (p<0.01) tumor growth delay was maintained after 7 days treatment with CBZ compared to control, which translated into a significant improvement in survival rate (p<0.0001). This study demonstrates that CBZ is efficacious against, and conveys a survival benefit for the TH-MYCN GEM model of NB. The data reinforces native T1 as a biomarker of response to anti-vascular therapy due to its sensitivity to therapy-induced damage to erythrocytes and/or cell death-mediated increase in macromolecules into the extracellular space. Relatively fast R2* rates in NB arising in the TH-MYCN model are strongly associated with vascular instability, characterised by numerous regions of aggregated paramagnetic erythrocytes (“blood lakes”), and treatment-induced thrombosis with the pan-VEGFR inhibitor cediranib which increased tumor R2*. The CBZ-induced decrease in R2* is clearly associated with vascular disruption, hence precluding the extravasation of deoxygenated red blood cells. Citation Format: Gilberto S. Almeida, Philippa King, Yann Jamin, Albert Hallsworth, Hannah Webber, Sergey Popov, Andrew D.J. Pearson, Louis Chesler, Simon P. Robinson. Tumor response to cabozantinib in the TH-MYCN GEM model of neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 491. doi:10.1158/1538-7445.AM2015-491

Abstract 1611: The FACT histone chaperone complex is highly expressed in aggressive drug refractory childhood cancers and the anti-FACT compound CBL0137 represents a highly promising therapeutic approach in this setting

Abstract Background: Despite the success of chemotherapy in improving the overall survival rate of childhood cancer, a number of types of children's cancers still have dismal outcomes. Included here are high risk neuroblastomas, Diffuse Intrinsic Pontine Gliomas (DIPG), and infant leukemias with MLL translocations. New treatments for these aggressive childhood cancers are urgently needed. Evidence is emerging of the importance of alterations in chromatin modifier genes in pediatric cancers. In this regard, CBL0137 is a carbazole-based anti-cancer agent with a unique mechanism of action. It is an indirect inhibitor of the chromatin remodeling complex FACT (Facilitates Chromatin Transcription). Inhibition of FACT by CBL0137 modulates the activity of several transcription factors involved in cancer: NF-kB and HSF1 are suppressed, while p53 is activated (Science Transl Med, 2011). We have examined FACT expression in neuroblastoma, DIPG and MLL leukemia, as well as the efficacy of CBL0137 in preclinical models of these diseases. Methods: Expression of the FACT subunits, SSRP1 and SPT16, was examined in neuroblastoma, DIPG and MLL leukemia cells using RT-PCR and Western analysis. The clinical significance of SSRP1 and SPT16 was also analysed using expression array data on 650 primary untreated neuroblastomas. Colony-forming assays were used to study the effect of CBL0137, either alone or combined with chemotherapeutic drugs. Cohorts of neuroblastoma, DIPG and MLL leukemia xenografted mice, as well as neuroblastoma-prone TH-MYCN mice, were treated with CBL0137, alone or combined with chemotherapeutic drugs. Results: High levels of SSRP1 and SPT16 expression were observed in all three types of child cancer. In addition, in neuroblastoma, the two FACT subunits were associated with MYCN amplification, and were strongly predictive of poor outcome (p<0.0001). As a single agent, CBL0137 administered iv had remarkable in vivo anti-tumor activity against neuroblastoma, DIPG and MLL leukemia xenografted mice. CBL0137 also synergized strongly with a variety of chemotherapeutic agents, including cyclophosphamide. Most dramatic results were observed when CBL0137 was combined with cyclophosphamide/topotecan, a highly active therapy for relapsed neuroblastoma that is currently in clinical trial. Cylophosphamide/topotecan plus CBL0137 resulted in cure of 90% of tumor-bearing MYCN-transgenic mice. Conclusions: Targeting FACT offers a highly promising novel therapeutic approach for aggressive childhood cancers. The results for CBL0137 are as good or better than any chemotherapy regimens we have tested in our preclinical models, and a Phase I COG trial of this nongenotoxic agent in refractory pediatric cancer patients is currently being planned. Citation Format: Michelle Haber, Jayne Murray, Laura Gamble, Ashleigh Carnegie-Clark, Hannah Webber, Michelle Ruhle, Michelle J. Henderson, Shiloh Middlemass, Daniel Carter, Maria Tsoli, Anahid Ehteda, Sandy Simon, Andre Oberthuer, Matthias Fischer, Katerina Gurova, Catherine Burkhart, Andrei Purmal, Richard B. Lock, David Ziegler, Glenn M. Marshall, Andrei V. Gudkov, Murray D. Norris. The FACT histone chaperone complex is highly expressed in aggressive drug refractory childhood cancers and the anti-FACT compound CBL0137 represents a highly promising therapeutic approach in this setting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1611. doi:10.1158/1538-7445.AM2015-1611

Ciliary Neurotrophic Factor Reduces the Proliferation and Promotes the Differentiation of TH-MYCN Transformed Sympathoadrenal Progenitors

Neuroblastoma is a childhood cancer caused by the transformation of sympathoadrenal progenitors. By following the formation of tumors in homozygous TH-MYCN mice, an established mouse model of neuroblastoma, we were able to capture transformed cells prior to the formation of large, vascularized tumors in order to determine the responsiveness of cells to neurotrophic factors. We discovered that the ciliary neurotrophic factor (CNTF) receptor is abundantly expressed in tumor cells from these mice. Furthermore, CNTF - but not nerve growth factor, brain-derived nerve growth factor, neurotrophin 3, or glial cell line-derived neurotrophic factor - promoted neuronal differentiation and withdrawal from the cell cycle. Thus, the transformation of sympathoadrenal progenitors by MYCN overexpression differentially affects responsiveness to neurotrophic molecules.

Identification of plasma Complement C3 as a potential biomarker for neuroblastoma using a quantitative proteomic approach

The majority of patients diagnosed with neuroblastoma present with aggressive disease. Improved detection of neuroblastoma cancer cells following initial therapy may help in stratifying patient outcome and monitoring for relapse. To identify potential plasma biomarkers, we utilised a liquid chromatography-tandem mass spectrometry-based proteomics approach to detect differentially-expressed proteins in serum from TH-MYCN mice. TH-MYCN mice carry multiple copies of the human MYCN oncogene in the germline and homozygous mice for the transgene develop neuroblastoma in a manner resembling the human disease. The abundance of plasma proteins was measured over the course of disease initiation and progression. A list of 86 candidate plasma biomarkers was generated. Pathway analysis identified significant association of these proteins with genes involved in the complement system. One candidate, complement C3 protein, was significantly enriched in the plasma of TH-MYCN(+/+) mice at both 4 and 6weeks of age, and was found to be elevated in a cohort of human neuroblastoma plasma samples, compared to healthy subjects. In conclusion, we have demonstrated the suitability of the TH-MYCN(+/+) mouse model of neuroblastoma for identification of novel disease biomarkers in humans, and have identified Complement C3 as a candidate plasma biomarker for measuring disease state in neuroblastoma patients. This study has utilised a unique murine model which develops neuroblastoma tumours that are biologically indistinguishable from human neuroblastoma. This animal model has effectively allowed the identification of plasma proteins which may serve as potential biomarkers of neuroblastoma. Furthermore, the label-free ion count quantitation technique which was used displays significant benefits as it is less labour intensive, feasible and accurate. We have been able to successfully validate this approach by confirming the differential abundance of two different plasma proteins. In addition, we have been able to confirm that the candidate biomarker Complement C3, is more abundant in the plasma of human neuroblastoma patient plasma samples when compared to healthy counterparts. Overall we have demonstrated that this approach can be potentially useful in the identification of biomarker candidates, and that further validation of the candidates may lead to the discovery of novel, clinically useful diagnostic tools in the detection of sub-clinical neuroblastoma.

Abstract 5037: Intrinsic susceptibility magnetic resonance imaging identifies tumors with ALKF1174L mutation in transgenic murine models of high-risk neuroblastoma.

Abstract In neuroblastoma, mutations in the anaplastic lymphoma kinase (ALK) tyrosine kinase gene have been identified in 8-10 % of primary tumors. The most common and potent ALK mutation, ALKF1174L, leads to the constitutive activation of the ALK protein and is associated preferentially with MYCN amplification, a markedly poorer prognosis, and confers resistance to the promising ALK inhibitor crizotinib. The development of more efficacious ALK inhibitors will benefit from non-invasive imaging strategies for the rapid identification of children with high-risk ALK-expressing or mutated neuroblastoma. Intrinsic susceptibility magnetic resonance imaging (IS-MRI) data was acquired from tumors arising in TH-ALKF1174L/TH-MYCN and TH-MYCN mice, two genetically engineered mouse model of high-risk neuroblastoma. The native MRI transverse relaxation rate R2*, an imaging biomarker sensitive to the concentration of paramagnetic deoxyhemoglobin, and changes in R2* induced with 100% oxygen inhalation, were quantified. Tumors in the TH-ALKF1174L/TH-MYCN mice demonstrated significantly (p<0.0001) slower native R2* rates (61 ± 3s−1, n=23) than tumors in the TH-MYCN mice (101 ± 6s−1, n=21). With hyperoxic challenge, the TH-ALKF1174L/TH-MYCN mice demonstrated a significantly (p<0.0001) lower and negligible ΔR2*oxygen-air (-3 ± 1s−1, n=10) compared with tumors in TH-MYCN mice (-26 ± 3s−1, n=12). A sensitivity of 90% and a specificity of 81% for native R2*, and a sensitivity of 90% and a specificity of 94% for hyperoxia-induced ΔR2* was determined. Histological correlates revealed a significantly (p<0.05) higher uptake of the perfusion marker Hoechst 33342, and the presence of large hemorrhagic blood lakes filled with stagnant deoxygenated erythrocytes, in tumors within the TH-MYCN mice, but not the TH-ALKF1174L/TH-MYCN model. Together these corroborate the IS-MRI findings (relatively fast native R2* and significant ΔR2*, indicative of vascular instability, in tumors in the TH-MYCN mice). IS-MRI provides a robust method to discriminate and identify TH-MYCN transgenic mice harboring the ALKF1174L mutation based on a stark differential vascular phenotype, which may impact on impaired drug delivery. IS-MRI is suitable for the scanning of young children, and quantitation of native R2* easily incorporated into existing pediatric clinical imaging protocols. This approach could provide a robust and rapid indicator of ALK genotypic status of the tumor, enabling the early identification of children with ultra high-risk neuroblastoma at the time of diagnosis. Citation Format: Yann Jamin, Laura Glass, Albert Hallsworth, Rani E. George, Dow-Mu Koh, Andrew D.J. Pearson, Louis Chesler, Simon P. Robinson. Intrinsic susceptibility magnetic resonance imaging identifies tumors with ALKF1174L mutation in transgenic murine models of high-risk neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5037. doi:10.1158/1538-7445.AM2013-5037

Abstract 2759: Anticancer compound curaxin CBL0137, that simultaneously suppresses NF-κB and activates p53, is highly effective in two independent mouse models of neuroblastoma.

Abstract Background: We have reported that the anti-malarial drug, quinacrine (QC), has significant in vivo anti-tumor activity (Neznanov et al, Cell Cycle, 2009, 8: 1-11). We have since identified curaxins, a more potent class of compounds with similar mechanism of action, whose anti-cancer activity results from chromatin-trapping of the Facilitates Chromatin Transcription (FACT) complex (Gasparian et al, Science Transl. Med, 2011, 3, 95ra74). These novel nongenotoxic agents, like QC, cause simultaneous p53 activation and NF-kB inhibition and tumor cell death. We have now studied the effects of QC and one of the most active curaxins, CBL0137, in the TH-MYCN mouse model of neuroblastoma as well as a nude mouse xenograft model of the disease. Methods: Cohorts of homozygous TH-MYCN mice with small palpable tumors or of human MYCN-amplified BE(2)-C neuroblastoma tumor-bearing nude mice (n=10) were treated with QC (TH-MYCN mice only) or CBL0137, alone or combined with chemotherapeutic drugs. Results: QC alone had no significant effect on tumor progression and did not enhance cisplatin or VP16 activity. However, strong anti-tumor activity was observed when QC was combined with cyclophosphamide (CPM) (4/10 QC/CPM-treated vs 0/10 CPM-treated long-term survivors, P<0.001). The effect of CBL0137 alone, administered either orally or iv, on tumor progression was more dramatic. Oral CBL0137 significantly extended survival (median survival time from start of treatment = 31.0±9.9 days versus controls = 3.0±0.2 days; P<0.0001), and was as effective as cisplatin or CPM single-agent treatment. Remarkably, CBL0137 administered iv resulted in a significant number of long-term tumor free survivors (7/9 alive at Day 120). When oral CBL0137 treatment was combined with CPM, survival was substantially increased (7/10 long-term tumor-free TH-MYCN mice) compared to either CBL0137 (0/10 alive) or CPM (1/10 alive) alone (P<0.005 in each case). Most dramatic results were observed when CBL0137 was combined with the standard neuroblastoma relapse protocol of CPM plus topotecan (TOPO). CPM/TOPO treatment resulted in 0/10 long-term survivors, however when combined with either oral or iv CBL0137, this combination cured 5/8 or 11/12 TH-MYCN mice, respectively. Moreover, the combination of iv CBL0137/CPM/TOPO resulted in more than a doubling of lifespan in BE(2)-C tumor-bearing mice compared to those treated with CPM/TOPO or CBL0137 alone (P<0.0001). Conclusions: These are the most impressive results that we have ever observed for any therapeutic protocol in either the TH-MYCN homozygous or BE(2)-C xenograft mouse models. The combination of CPM/TOPO with iv CBL0137 appears to be a highly promising new treatment approach for refractory neuroblastoma. Safety and efficacy of CBL0137 is currently being investigated in a Phase I clinical trial in advanced cancer patients. Citation Format: Michelle Haber, Jayne Murray, Ashleigh Carnegie-Clark, Hannah Webber, Glenn M. Marshall, Katerina V. Gurova, Catherine A. Burkhart, Andrei Purmal, Andrei V. Gudkov, Murray D. Norris. Anticancer compound curaxin CBL0137, that simultaneously suppresses NF-κB and activates p53, is highly effective in two independent mouse models of neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2759. doi:10.1158/1538-7445.AM2013-2759

Abstract 5043: The facilitates chromatin transcription (FACT) protein complex promotes neuroblastoma tumor initiation.

Abstract Tumour initiation in the TH-MYCN transgenic mouse model of neuroblastoma is characterised by transient repression of p53 stress responses in embryonal ganglionic neuroblasts, causing them to persist postnatally as cancer-prone lesions 1, 2. p53 re-activation may be a potential therapeutic strategy for removal of these lesions before they later transform in response to oncogenic stimuli. One potential target for p53 activation is the FACT protein complex which is known to inhibit p53 signalling. Here we show that high mRNA expression levels of the two FACT subunits, SSRP1 and SPT16, in human primary neuroblastoma tissue were significantly associated with poor patient outcome (p<0.001) on Kaplan Meier analysis of 650 neuroblastoma patients. Both SSRP1 and SPT16 levels had a strong correlation with the level of MYCN expression, particularly in high risk MYCN-amplified neuroblastoma tissues (p<0.05). Inhibition of MYCN expression by a specific siRNA, lowered SSRP1 and SPT16 expression in two neuroblastoma tumor cell lines (BE(2)C and Kelly). Alamar blue assays revealed that a chemical inhibitor of FACT, Curaxin or CBL137, potently and selectively promoted cytotoxicity in neuroblastoma cell lines as compared to non-malignant MRC5 fibroblasts (p<0.001). To examine the role of FACT in MYCN-driven tumourigenesis we evaluated the effect of Curaxin on tumor initiation in TH-MYCN mice. Low micromolar Curaxin doses effectively restored normal in vitro, p53-dependent death responses to primary ganglion cells deprived of serum from neonatal TH-MYCN mice, while ganglion cultures from wild-type littermate mice were significantly more resistant to Curaxin treatment, with or without serum deprivation (p<0.001). Most importantly, prophylactic Curaxin treatment of 6 day old TH-MYCN mice had a marked inhibitory effect on postnatal ganglion neuroblast persistence in vivo for both hemizygote and homozygote TH-MYCN mice, as compared to vehicle-only treated animals (p=0.01, p=0.035, respectively). Taken together, our data for the first time identifies FACT as a novel effector of MYCN-driven neuroblastoma tumorigenesis. Inhibitors of the FACT complex may be a novel treatment for high risk MYCN-amplified neuroblastoma and a potential strategy for neuroblastoma prevention. 1. Hansford, L. et al. Mechanisms of embryonal tumor initiation: Distinct roles for MycN expression and MYCN amplification. Proceedings of the National Academy of Sciences of the United States of America 101, 12664-12669 (2004). 2. Calao, M. et al. Direct effects of Bmi1 on p53 protein stability inactivates oncoprotein stress responses in embryonal cancer precursor cells at tumor initiation. Oncogene [ahead of print] Citation Format: Daniel R. Carter, Belamy B. Cheung, Tao Liu, Carol Au, Murray D. Norris, Michelle Haber, Jayne Murray, Katerina V. Gurova, Andrei V. Gudkov, Glenn M. Marshall. The facilitates chromatin transcription (FACT) protein complex promotes neuroblastoma tumor initiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5043. doi:10.1158/1538-7445.AM2013-5043

Abstract 2746: Microsomal prostaglandin E2 synthase-1 may provide a novel specific therapeutic target in neuroblastoma.

Abstract Background: Neuroblastoma (NB) cells are enriched in the omega-6 fatty acid arachidonic acid, the substrate for the cyclooxygenase (COX) enzymes and prostaglandin biosynthesis. The inducible isoform, COX-2 is overexpressed in NB and NB cells produce prostaglandin E2 (PGE2) that acts as an autocrine and/or paracrine survival and proliferation factor. Downstream of the COX enzymes, specific synthases are responsible for the production of the respective prostaglandins. Microsomal prostaglandin E2 synthase-1 (mPGES-1) specifically converts PGH2 to PGE2 and is thought to primarily couple to COX-2. The aim of this study was to investigate if inhibition of mPGES-1 could represent an alternative therapeutic approach to COX- inhibition in NB. Methods: Western blot and immunohistochemistry were used for protein detection. Cell viability of seven NB cell-lines treated with the mPGES-1 inhibitor CAY1052 was determined by MTT-assay. Stable mPGES-1 knockdown SK-N-BE2 clones were established using shRNA and the clonogenic capacity was analysed by clonogenic assay. To study the in vivo effect of COX inhibition, four week old homozygous TH-MYCN mice were treated with 10mg/L diclofenac for two weeks. Ex vivo analyses of COX-metabolites in tumors were performed by LC-MS/MS. Results: Expression of mPGES-1 in human NB tumors and in NB cell lines could be detected. Inhibition of mPGES-1 reduced NB cell growth in vitro and knockdown of mPGES-1 significantly reduced the clonogenic capacity. Expression of COX-1, COX-2 and mPGES-1 in TH-MYCN tumors could be detected and treatment with the dual COX-inhibitor diclofenac significantly reduced tumour weight, compared to untreated animals. Ex vivo analysis of tumor tissues from treated animals revealed a significantly decreased level of COX metabolites compared to controls. The expression of mPGES-1was not affected by the treatment. Furthermore, cells staining positive for cleaved caspase-3 were more prevalent in treated tumors indicating apoptosis Induction. Conclusion: We found that mPGES-1 is expressed in NB, with a potential role for PGE2 synthesis and tumor growth. mPGES-1 represents an alternative therapeutic target for inhibiting NB growth through specific PGE2 inhibition and the TH-MYCN model is well suited for in vivo studies with this purpose. Citation Format: Anna Kock, Agnes Rasmuson, Marina Korotkova, Helena Idborg, John Inge Johnsen, Per-Johan Jakobsson, Per Kogner. Microsomal prostaglandin E2 synthase-1 may provide a novel specific therapeutic target in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2746. doi:10.1158/1538-7445.AM2013-2746

Tumor Development, Growth Characteristics and Spectrum of Genetic Aberrations in the TH-MYCN Mouse Model of Neuroblastoma

BackgroundThe TH-MYCN transgenic neuroblastoma model, with targeted MYCN expression to the developing neural crest, has been used to study neuroblastoma development and evaluate novel targeted tumor therapies.MethodsWe followed tumor development in 395 TH-MYCN (129X1/SvJ) mice (125 negative, 206 hemizygous and 64 homozygous mice) by abdominal palpations up to 40 weeks of age. DNA sequencing of MYCN in the original plasmid construct and mouse genomic DNA was done to verify the accuracy. Copy number analysis with Affymetrix® Mouse Diversity Genotyping Arrays was used to characterize acquired genetic aberrations.ResultsDNA sequencing confirmed presence of human MYCN cDNA in genomic TH-MYCN DNA corresponding to the original plasmid construct. Tumor incidence and growth correlated significantly to transgene status with event-free survival for hemizygous mice at 50%, and 0% for homozygous mice. Hemizygous mice developed tumors at 5.6–19 weeks (median 9.1) and homozygous mice at 4.0–6.9 weeks (5.4). The mean treatment window, time from palpable tumor to sacrifice, for hemizygous and homozygous mice was 15 and 5.2 days, respectively. Hemizygous mice developing tumors as early as homozygous mice had a longer treatment window. Age at tumor development did not influence treatment window for hemizygous mice, whereas treatment window in homozygous mice decreased significantly with increasing age. Seven out of 10 analysed tumors had a flat DNA profile with neither segmental nor numerical chromosomal aberrations. Only three tumors from hemizygous mice showed acquired genetic features with one or more numerical aberrations. Of these, one event corresponded to gain on the mouse equivalent of human chromosome 17.ConclusionHemizygous and homozygous TH-MYCN mice have significantly different neuroblastoma incidence, tumor growth characteristics and treatment windows but overlap in age at tumor development making correct early genotyping essential to evaluate therapeutic interventions. Contrasting previous studies, our data show that TH-MYCN tumors have few genetic aberrations.