TH-MYCN Mouse Model Research Articles

Abstract B132: Addition of AMXT1501 (polyamine uptake inhibitor) plus DFMO (polyamine synthesis inhibitor) to standard-of-care chemotherapy/anti-GD2 antibody in the TH-MYCN mouse neuroblastoma model, enhances efficacy compared to addition of DFMO alone

Abstract Background: We used the TH-MYCN mouse model of neuroblastoma (NB) to determine the importance of simultaneously inhibiting both polyamine uptake and synthesis when added to standard-of-care chemotherapy/immunotherapy. Polyamines are essential cations frequently upregulated in tumors. We have shown the benefit of adding DFMO, an irreversible polyamine synthesis inhibitor, to standard-of-care chemotherapy in several NB mouse models (Evageliou, Clin Cancer Res 2016). Based on our work, a randomized Phase 2 COG trial of DFMO, added to chemotherapy/anti-GD2 immunotherapy, is ongoing for relapsed NB (NCT03794349). We recently showed in NB mouse models that DFMO efficacy is enhanced by adding the polyamine uptake inhibitor AMXT1501 (Gamble, Sci Transl Med, 2019). DFMO/AMXT1501 is now in adult clinical trial (NCT05500508). Preclinical modelling of polyamine-targeted therapeutics in TH-MYCN mice has been invaluable for supporting clinical trial design but has yet to include anti-GD2 immunotherapy. Methods: TH-MYCN mice were used to optimize DFMO/AMXT1501 therapy combined with temozolomide/irinotecan (TEM/IRI) or cyclophosphamide/topotecan (CYCLO/TOPO), and anti-GD2 (14G2a) antibody. We studied escalating doses of DFMO and AMXT1501 (n=10/group), with or without TEM/IRI or CYCLO/TOPO. 14G2a regimen was titrated toward human-relevant pharmacokinetic exposures to establish suitable conditions for combination therapies. Results: In the absence of chemo-immunotherapy, highest-dose DFMO (1.5%) and AMXT1501 (2.5 mg/kg/d) had greatest efficacy without increased toxicity. Median mouse survival was 2-fold greater compared with lower dose DFMO/AMXT1501 combinations, suggesting that maximising DFMO dose is critical. Similar improved efficacy was observed in combination with TEM/IRI or CYCLO/TOPO. Combining 14G2a (100ug 2x/wk, 18 wks; McNerney, Oncoimmunology, 2022) with either CYCLO/TOPO (single 5-day chemo cycle) or DFMO/AMXT1501 (1.5%/2.5 mg/kg/d) increased survival compared with either treatment alone. Combining all 5 drugs resulted in 100% survival at 1 year, with minimal toxicity. However substantial dose reductions of 14G2a (<25µg, 2 doses) and TEM/IRI or CYCLO/TOPO (single 2-day chemo cycle) were needed to identify clinically relevant backbones which achieved human-relevant 14G2a pharmacokinetics. Addition of DFMO alone to the clinically relevant CYCLO/TOPO/14G2a backbone did not result in benefit compared to backbone alone. In contrast, addition of AMXT1501 plus DFMO to the backbone significantly extended survival (P< 0.001). Conclusions: We have increased the utility of the TH-MYCN mouse model for preclinical modelling of polyamine inhibition, and other investigational treatments, by pharmacokinetics-driven optimization of anti-GD2 therapy with standard-of-care chemotherapy backbones. This approach will improve optimisation for future NB clinical trials. Our data highlight the importance of adding AMXT1501 to DFMO for polyamine inhibition and support a planned international DFMO/AMXT1501/chemotherapy/anti-GD2 trial for refractory NB. Citation Format: Jayne Murray, Ruby Pandher, Lin Xiao, Klaartje Somers, Jennifer Brand, Erin Mosmann, Stephanie Alfred, Frances Kusuma, Adam Kearns, Julie R Park, Lynley V Marshall, Chiara Gorrini, Louis Chesler, Michael D Hogarty, Andrew D J Pearson, Mark Burns, Jamie I Fletcher, David Ziegler, Murray D Norris, Michelle Haber. Addition of AMXT1501 (polyamine uptake inhibitor) plus DFMO (polyamine synthesis inhibitor) to standard-of-care chemotherapy/anti-GD2 antibody in the TH-MYCN mouse neuroblastoma model, enhances efficacy compared to addition of DFMO alone [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B132.

Adaptation of the Th-MYCN Mouse Model of Neuroblastoma for Evaluation of Disseminated Disease.

High-risk neuroblastoma remains a profound clinical challenge that requires eradication of neuroblastoma cells from a variety of organ sites, including bone marrow, liver, and CNS, to achieve a cure. While preclinical modeling is a powerful tool for the development of novel cancer therapies, the lack of widely available models of metastatic neuroblastoma represents a significant barrier to the development of effective treatment strategies. To address this need, we report a novel luciferase-expressing derivative of the widely used Th-MYCN mouse. While our model recapitulates the non-metastatic neuroblastoma development seen in the parental transgenic strain, transplantation of primary tumor cells from disease-bearing mice enables longitudinal monitoring of neuroblastoma growth at distinct sites in immune-deficient or immune-competent recipients. The transplanted tumors retain GD2 expression through many rounds of serial transplantation and are sensitive to GD2-targeted immune therapy. With more diverse tissue localization than is seen with human cell line-derived xenografts, this novel model for high-risk neuroblastoma could contribute to the optimization of immune-based treatments for this deadly disease.

The cyclin dependent kinase inhibitor p21Cip1/Waf1 is a therapeutic target in high-risk neuroblastoma.

Platinum-based chemotherapies such as cisplatin are used as first-line treatment for the paediatric tumour neuroblastoma. Although the majority of neuroblastoma tumours respond to therapy, there is a high fraction of high-risk neuroblastoma patients that eventually relapse with increased resistance. Here, we show that one key determinant of cisplatin sensitivity is phosphorylation of the cyclin-dependent kinase inhibitor p21Cip1/Waf1. A panel of eight neuroblastoma cell lines and a TH-MYCN mouse model were investigated for the expression of p21Cip1/Waf1 using RT-qPCR, Western blot, and immunofluorescence. This was followed by investigation of sensitivity towards cisplatin and the p21Cip1/Waf1 inhibitor UC2288. Whereas the cell lines and the mouse model showed low levels of un-phosphorylated p21Cip1/Waf1, the phosphorylated p21Cip1/Waf1 (Thr145) was highly expressed, which in the cell lines correlated to cisplatin resistance. Furthermore, the neuroblastoma cell lines showed high sensitivity to UC2288, and combination treatment with cisplatin resulted in considerably decreased cell viability and delay in regrowth in the two most resistant cell lines, SK-N-DZ and BE(2)-C. Thus, targeting p21Cip1/Waf1 can offer new treatment strategies and subsequently lead to the design of more efficient combination treatments for high-risk neuroblastoma.

Quantitative Proteomics of Th-MYCN Transgenic Mice Reveals Aurora Kinase Inhibitor Altered Metabolic Pathways and Enhanced ACADM To Suppress Neuroblastoma Progression.

Neuroblastoma is a neural crest-derived embryonal tumor and accounts for about 15% of all cancer deaths in children. MYCN amplification is associated with aggressive and advanced stage of high-risk neuroblastoma, which remains difficult to treat and exhibits poor survival under current multimodality treatment. Here, we analyzed the transcriptomic profiles of neuroblastoma patients and showed that aurora kinases lead to poor survival and had positive correlation with MYCN amplification and high-risk disease. Further, pan-aurora kinase inhibitor (tozasertib) treatment not only induces cell-cycle arrest and suppresses cell proliferation, migration, and invasion ability in MYCN-amplified (MNA) neuroblastoma cell lines, but also inhibits tumor growth and prolongs animal survival in Th-MYCN transgenic mice. Moreover, we performed quantitative proteomics and identified 150 differentially expressed proteins after tozasertib treatment in the Th-MYCN mouse model. The functional and network-based enrichment revealed that tozasertib alters metabolic processes and identified a mitochondrial flavoenzyme in fatty acid β-oxidation, ACADM, which is correlated with aurora kinases and neuroblastoma patient survival. Our findings indicate that the aurora kinase inhibitor could cause metabolic imbalance, possibly by disturbing carbohydrate and fatty acid metabolic pathways, and ACADM may be a potential target in MNA neuroblastoma.

Novel mesenchymal stem cell delivery system as targeted therapy against neuroblastoma using the TH-MYCN mouse model

PurposeMesenchymal stem cells (MSCs) are reported to migrate toward damaged tissues or tumors. We previously reported the in vivo short-term (1 day) tumor-homing effect of xenogeneic human MSCs (hMSCs) using the TH-MYCN mouse neuroblastoma model (MYCN-TgM). In this study, we analyzed the long-term tumor-homing effect of allogeneic mouse MSCs (mMSCs) and explored the antitumor effect and drug delivery function of mMSCs. MethodsmMSCs were administered intraperitoneally (i.p.) to MYCN-TgM and traced by an in vivo imaging system (IVIS). We administered green fluorescent protein (GFP)-transduced mMSCs into MYCN-TgM i.p. and examined the cell survival by immunohistochemistry. We also administered interferon beta-transduced mMSCs (mMSCs-IFN-β) to MYCN-TgM i.p. and measured the concentration of IFN-β in the tumor and organs by an enzyme-linked immunosorbent assay (ELISA). The survival curves of MYCN-TgM administered every week was analyzed. ResultsThe IVIS revealed the accumulation of fluorescence was observed in the tumor both in vivo and after excision. Immunohistochemistry using anti-GFP antibody revealed that the mMSCs existed within the tumor until 14 days but not in the organs. The ELISA showed increased concentrations of IFN-β only in the tumors, with the values gradually diminishing over 14 days. The mMSCs-IFN-β group survived significantly longer than the control group (p < 0.03), while the mMSCs-alone group did not show a survival advantage. ConclusionsAllogeneic mMSCs showed a homing ability for mouse neuroblastoma and existed within the tumor for as long as two weeks. This may be a candidate drug delivery vehicle for antitumor agents against neuroblastoma.Level of evidence

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.

Long term, continuous exposure to panobinostat induces terminal differentiation and long term survival in the TH-MYCN neuroblastoma mouse model.

Neuroblastoma is the most common extra-cranial malignancy in childhood and accounts for ∼15% of childhood cancer deaths. Amplification of MYCN in neuroblastoma is associated with aggressive disease and predicts for poor prognosis. Novel therapeutic approaches are therefore essential to improving patient outcomes in this setting. The histone deacetylases are known to interact with N-Myc and regulate numerous cellular processes via epigenetic modulation, including differentiation. In this study, we used the TH-MYCN mouse model of neuroblastoma to investigate the antitumor activity of the pan-HDAC inhibitor, panobinostat. In particular we sought to explore the impact of long term, continuous panobinostat exposure on the epigenetically driven differentiation process. Continuous treatment of tumor bearing TH-MYCN transgenic mice with panobinostat for nine weeks led to a significant improvement in survival as compared with mice treated with panobinostat for a three-week period. Panobinostat induced rapid tumor regression with no regrowth observed following a nine-week treatment period. Initial tumor response was associated with apoptosis mediated via upregulation of BMF and BIM. The process of terminal differentiation of neuroblastoma into benign ganglioneuroma, with a characteristic increase in S100 expression and reduction of N-Myc expression, occurred following prolonged exposure to the drug. RNA-sequencing analysis of tumors from treated animals confirmed significant upregulation of gene pathways associated with apoptosis and differentiation. Together our data demonstrate the potential of panobinostat as a novel therapeutic strategy for high-risk neuroblastoma patients.

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.

Retracted: PHOX2B Is Associated with Neuroblastoma Cell Differentiation

Neuroblastoma is a common pediatric malignancy that accounts for ∼15% of tumor-related deaths in children. The tumor is generally believed to originate from neural crest cells during early sympathetic neurogenesis. As the degree of neuroblastoma differentiation has been correlated with clinical outcome, clarifying the molecular mechanisms that drive neuroblastoma progression and differentiation is important for increasing the survival of these patients. In a previous study, the authors identified paired-like homeobox 2b (PHOX2B) as a key mediator of neuroblastoma pathogenesis in a TH-MYCN mouse model. In the present study, they aimed to define whether PHOX2B is also associated with proliferation and differentiation of human neuroblastoma cells. PHOX2B expression in neuroblastoma cells was evaluated by immunoblot analyses, and the effects of PHOX2B on the proliferation of neuroblastoma cells in vitro were determined using clonogenic and sphere formation assays. Xenograft experiments in NOD/SCID mice were used to examine the in vivo response to PHOX2B knockdown. Their data demonstrated that PHOX2B acts as a prognostic marker in neuroblastoma and that retinoic acid-induced neuronal differentiation downregulates PHOX2B expression, thereby suppressing the self-renewal capacity of neuroblastoma cells and inhibiting tumorigenicity. These findings confirmed that PHOX2B is a key regulator of neuroblastoma differentiation and stemness maintenance and indicated that PHOX2B might serve as a potential therapeutic target in neuroblastoma patients.

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&amp;lt;0.0001). One hundred days after the withdrawal of drug 88.9% of panobinostat treated mice remained alive (p&amp;lt;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&amp;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

β-blockers increase response to chemotherapy via direct antitumour and anti-angiogenic mechanisms in neuroblastoma

Background:The use of β-blockers for the management of hypertension has been recently associated with significant clinical benefits in cancer patients. Herein, we investigated whether β-blockers could be used in combination with chemotherapy for the treatment of neuroblastoma.Methods:Seven β-blockers were tested for their antiproliferative and anti-angiogenic properties alone, and in combination with chemotherapy in vitro; the most potent drug combinations were evaluated in vivo in the TH-MYCN mouse model of neuroblastoma.Results:Three β-blockers (i.e., carvedilol, nebivolol and propranolol) exhibited potent anticancer properties in vitro and interacted synergistically with vincristine, independently of P-glycoprotein expression. β-blockers potentiated the anti-angiogenic, antimitochondrial, antimitotic and ultimately pro-apoptotic effects of vincristine. In vivo, β-blockers alone transiently slowed tumour growth as compared with vehicle only (P<0.01). More importantly, when used in combination, β-blockers significantly increased the tumour regression induced by vincristine (P<0.05). This effect was associated with an increase in tumour angiogenesis inhibition (P<0.001) and ultimately resulted in a four-fold increase in median survival, as compared with vincristine alone (P<0.01).Conclusion:β-blockers can increase treatment efficacy against neuroblastoma, and their combination with chemotherapy may prove beneficial for the treatment of this disease and other drug-refractory cancers.

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&amp;lt;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&amp;lt;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&amp;lt;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&amp;lt;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

Low-dose aspirin delays an inflammatory tumor progression in vivo in a transgenic mouse model of neuroblastoma

Tumor-associated inflammation is a driving force in several adult cancers and intake of low-dose aspirin has proven to reduce cancer incidence. Little is known about tumor-associated inflammation in pediatric neoplasms and no in vivo data exists on the effectiveness of low-dose aspirin on established tumors. The present study employs the transgenic TH-MYCN mouse model for neuroblastoma (NB) to evaluate inflammatory patterns paralleling tumor growth in vivo and low-dose aspirin as a therapeutic option for high-risk NB. Spontaneously arising abdominal tumors were monitored for tumor-associated inflammation ex vivo at various stages of disease and homozygous mice received daily low-dose aspirin (10mg/kg) using oral gavage or no treatment, from 4.5 to 6 weeks of age. Using flow cytometry, a transition from an adaptive immune response predominated by CD8(+) T cell in early neoplastic lesions, towards enrichment in immature cells of the innate immune system, including myeloid-derived suppressor cells, dendritic cells and tumor-associated macrophages, was detected during tumor progression. An M1 to M2 transition of tumor-associated macrophages was demonstrated, paralleled by a deterioration of dendritic cell status. Treatment with low-dose aspirin to mice homozygous for the TH-MYCN transgene significantly reduced the tumor burden (P < 0.01), the presence of tumor-associated cells of the innate immune system (P < 0.01), as well as the intratumoral expression of transforming growth factor-β, thromboxane A2 (P < 0.05) and prostaglandin D2 (P < 0.01). In conclusion, tumor-associated inflammation appears as a potential therapeutic target in NB and low-dose aspirin reduces tumor burden in the TH-MYCN transgenic mouse model of NB, hence warranting further studies on aspirin in high-risk NB.

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.

Abstract 1420: Microsomal prostaglandin E2 synthase 1 (mPGES-1) is expressed in neuroblastoma and may represent a novel target for therapy

Abstract Neuroblastoma (NB) is an embryonic tumor of the sympathetic nervous system and it is the most common extracranial tumor affecting young children. Despite a very intensive treatment regimen high-risk neuroblastoma patients still have a poor prognosis. Neuroblastoma cells are enriched in arachidonic acid, over express COX-2 and produce prostaglandin E2 (PGE2) that acts as an autocrine or paracrine survival and proliferation factor by induction of Akt signaling. Downstream of the COX enzymes, specific synthases converts PGH2 into the different 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 is to investigate if inhibition of mPGES-1 could represent an alternative therapeutic approach to COX- inhibition in neuroblastoma. We could detect expression of mPGES-1 in a panel of seven human neuroblastoma cell lines. Stable knock down of mPGES-1 expression using shRNA significantly reduced the clonogenic capacity and treatment with the mPGES-1 inhibitor CAY1052 inhibited neuroblastoma cell growth in vitro. By immunohistochemistry and western blot we could also detect expression of COX-1, COX-2 and mPGES-1 in tumors from the transgenic TH-MYCN mouse model of neuroblastoma. Treatment of homozygous TH-MYCN mice with 10 mg/L diclofenac for two weeks, starting at the age of four weeks, significantly reduced tumour weight compared to untreated animals. Ex vivo analysis with LC-MS/MS of tumor tissues from diclofenac treated animals revealed a significant decreased level of COX metabolites compared to control. The expression of mPGES-1was not affected by the treatment. Furthermore the number of cells staining positive for cleaved caspase-3 were higher in the treated tumors indicating induced apoptosis. In conclusion, our results show that mPGES-1 is expressed in neuroblastoma therefore mPGES-1 might represent an alternative way of reducing NB growth through specific PGE2 inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1420. doi:1538-7445.AM2012-1420

Abstract LB-496: Low-dose aspirin targets tumor-associated inflammation and delays neuroblastoma tumor growth in vivo

Abstract ABSTRACT Tumor-associated inflammation is a driving force in several adult cancers, and low doses of non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin have proven to reduce cancer incidence. Little is known about tumor-associated inflammation in pediatric neoplasms and no in vivo data exists on the effectiveness of low-dose aspirin on established tumors. Our study evaluates inflammatory patterns paralleling neuroblastoma (NB) tumor growth in vivo and low-dose aspirin as a therapeutic option for high-risk NB. Using the TH-MYCN mouse model of NB, this study demonstrates the presence and progression of tumor-associated inflammation during NB tumor growth. Ex vivo analysis of tumors revealed a transition from an adaptive immune response predominated by CD8+ T-cells in neoplastic lesions from 5 week old homozygous mice, towards an enrichment in immature cells of the innate immune system, including myeloid-derived suppressor cells (MDSCs), immature dendritic cells (DCs) and M2 tumor-associated macrophages (TAMs) during tumor progression. An ongoing M1 to M2 transition of TAMs was demonstrated, which was paralleled by a gradual deterioration of DC status. Ten days of consecutive anti-inflammatory treatment with low-dose aspirin significantly reduced tumor burden (p&amp;lt;0.01) and the presence of tumor-associated cells of the innate immune system (p&amp;lt;0.01) in homozygous mice. In conclusion, our study depicts how inflammatory pathways assist tumor progression through sculpturing of the tumor microenvironment, and for the first time demonstrates an in vivo effect of low-dose aspirin on established MYCN amplified NB tumors in a transgenic mouse model, suggesting a potential new treatment option for high-risk NB patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-496. doi:1538-7445.AM2012-LB-496