Generation of Orthotopic and Subcutaneous Patient-Derived Xenograft Models from Diverse Clinical Tissue Samples of Pediatric Extracranial Solid Tumors.

Realistic and renewable laboratory models that accurately reflect the distinct clinical features of childhood cancers have enormous potential to speed research progress. These models help us to understand disease biology, develop new research methods, advance new therapies to clinical trial, and implement personalized medicine. This chapter describes methods to generate patient-derived xenograft models of neuroblastoma and rhabdomyosarcoma, two tumor types for which children with high-risk disease have abysmal survival outcomes and survivors have lifelong-debilitating effects from treatment. Further, this protocol addresses model development from diverse clinical tumor tissue samples, subcutaneous and orthotopic engraftment, and approaches to avoid model loss.

High-Throughput Drug Screening of Primary Tumor Cells Identifies Therapeutic Strategies for Treating Children with High-Risk Cancer.

For one-third of patients with pediatric cancer enrolled in precision medicine programs, molecular profiling does not result in a therapeutic recommendation. To identify potential strategies for treating these high-risk pediatric patients, we performed in vitro screening of 125 patient-derived samples against a library of 126 anticancer drugs. Tumor cell expansion did not influence drug responses, and 82% of the screens on expanded tumor cells were completed while the patients were still under clinical care. High-throughput drug screening (HTS) confirmed known associations between activating genomic alterations in NTRK, BRAF, and ALK and responses to matching targeted drugs. The in vitro results were further validated in patient-derived xenograft models in vivo and were consistent with clinical responses in treated patients. In addition, effective combinations could be predicted by correlating sensitivity profiles between drugs. Furthermore, molecular integration with HTS identified biomarkers of sensitivity to WEE1 and MEK inhibition. Incorporating HTS into precision medicine programs is a powerful tool to accelerate the improved identification of effective biomarker-driven therapeutic strategies for treating high-risk pediatric cancers. SIGNIFICANCE: Integrating HTS with molecular profiling is a powerful tool for expanding precision medicine to support drug treatment recommendations and broaden the therapeutic options available to high-risk pediatric cancers.

Memory of stochastic single-cell apoptotic signaling promotes chemoresistance in neuroblastoma.

Gene expression noise is known to promote stochastic drug resistance through the elevated expression of individual genes in rare cancer cells. However, we now demonstrate that chemoresistant neuroblastoma cells emerge at a much higher frequency when the influence of noise is integrated across multiple components of an apoptotic signaling network. Using a JNK activity biosensor with longitudinal high-content and in vivo intravital imaging, we identify a population of stochastic, JNK-impaired, chemoresistant cells that exist because of noise within this signaling network. Furthermore, we reveal that the memory of this initially random state is retained following chemotherapy treatment across a series of in vitro, in vivo, and patient models. Using matched PDX models established at diagnosis and relapse from individual patients, we show that HDAC inhibitor priming cannot erase the memory of this resistant state within relapsed neuroblastomas but improves response in the first-line setting by restoring drug-induced JNK activity within the chemoresistant population of treatment-naïve tumors.

Suppression of the ABCA1 Cholesterol Transporter Impairs the Growth and Migration of Epithelial Ovarian Cancer.

BACKGROUND: Epithelial ovarian cancer (EOC) is the most lethal gynaecological malignancy with over 80% of cases already disseminated at diagnosis and facing a dismal five-year survival rate of 35%. EOC cells often spread to the greater omentum where they take-up cholesterol. Excessive amounts of cholesterol can be cytocidal, suggesting that cholesterol efflux through transporters may be important to maintain homeostasis, and this may explain the observation that high expression of the ATP-binding cassette A1 (ABCA1) cholesterol transporter has been associated with poor outcome in EOC patients. METHODS: ABCA1 expression was silenced in EOC cells to investigate the effect of inhibiting cholesterol efflux on EOC biology through growth and migration assays, three-dimensional spheroid culture and cholesterol quantification. RESULTS: ABCA1 suppression significantly reduced the growth, motility and colony formation of EOC cell lines as well as the size of EOC spheroids, whilst stimulating expression of ABCA1 reversed these effects. In serous EOC cells, ABCA1 suppression induced accumulation of cholesterol. Lowering cholesterol levels using methyl-B-cyclodextrin rescued the effect of ABCA1 suppression, restoring EOC growth. Furthermore, we identified FDA-approved agents that induced cholesterol accumulation and elicited cytocidal effects in EOC cells. CONCLUSIONS: Our data demonstrate the importance of ABCA1 in maintaining cholesterol balance and malignant properties in EOC cells, highlighting its potential as a therapeutic target for this disease.

miR-99b-5p, miR-380-3p, and miR-485-3p are novel chemosensitizing miRNAs in high-risk neuroblastoma.

Neuroblastoma is a deadly childhood cancer arising in the developing sympathetic nervous system. High-risk patients are currently treated with intensive chemotherapy, which is curative in only 50% of children and leaves some surviving patients with life-long side effects. microRNAs (miRNAs) are critical regulators of neural crest development and are deregulated during neuroblastoma tumorigenesis, making miRNA-based drugs an attractive therapeutic avenue. A functional screen of >1,200 miRNA mimics was conducted in neuroblastoma cell lines to discover miRNAs that sensitized cells to low doses (30% inhibitory concentration [IC30]) of doxorubicin and vincristine chemotherapy used in the treatment of the disease. Three miRNAs, miR-99b-5p, miR-380-3p, and miR-485-3p, had potent chemosensitizing activity with doxorubicin in multiple models of high-risk neuroblastoma. These miRNAs underwent genomic loss in a subset of neuroblastoma patients, and low expression predicted poor survival outcome. In vitro functional assays revealed each of these miRNAs enhanced the anti-proliferative and pro-apoptotic effects of doxorubicin. We used RNA sequencing (RNA-seq) to show that miR-99b-5p represses neuroblastoma dependency genes LIN28B and PHOX2B both in vitro and in patient-derived xenograft (PDX) tumors. Luciferase reporter assays demonstrate that PHOX2B is a direct target of miR-99b-5p. We anticipate that restoring the function of the tumor-suppressive miRNAs discovered here may be a valuable therapeutic strategy for the treatment of neuroblastoma patients.

Whole-genome sequencing facilitates patient-specific quantitative PCR-based minimal residual disease monitoring in acute lymphoblastic leukaemia, neuroblastoma and Ewing sarcoma.

BACKGROUND: Minimal residual disease (MRD) measurement is a cornerstone of contemporary acute lymphoblastic leukaemia (ALL) treatment. The presence of immunoglobulin (Ig) and T cell receptor (TCR) gene recombinations in leukaemic clones allows widespread use of patient-specific, DNA-based MRD assays. In contrast, paediatric solid tumour MRD remains experimental and has focussed on generic assays targeting tumour-specific messenger RNA, methylated DNA or microRNA. METHODS: We examined the feasibility of using whole-genome sequencing (WGS) data to design tumour-specific polymerase chain reaction (PCR)-based MRD tests (WGS-MRD) in 18 children with high-risk relapsed cancer, including ALL, high-risk neuroblastoma (HR-NB) and Ewing sarcoma (EWS) (n = 6 each). RESULTS: Sensitive WGS-MRD assays were generated for each patient and allowed quantitation of 1 tumour cell per 10-4 (0.01%)-10-5 (0.001%) mononuclear cells. In ALL, WGS-MRD and Ig/TCR-MRD were highly concordant. WGS-MRD assays also showed good concordance between quantitative PCR and droplet digital PCR formats. In serial clinical samples, WGS-MRD correlated with disease course. In solid tumours, WGS-MRD assays were more sensitive than RNA-MRD assays. CONCLUSIONS: WGS facilitated the development of patient-specific MRD tests in ALL, HR-NB and EWS with potential clinical utility in monitoring treatment response. WGS data could be used to design patient-specific MRD assays in a broad range of tumours.

In vitro and in vivo drug screens of tumor cells identify novel therapies for high-risk child cancer.

Biomarkers which better match anticancer drugs with cancer driver genes hold the promise of improved clinical responses and cure rates. We developed a precision medicine platform of rapid high-throughput drug screening (HTS) and patient-derived xenografting (PDX) of primary tumor tissue, and evaluated its potential for treatment identification among 56 consecutively enrolled high-risk pediatric cancer patients, compared with conventional molecular genomics and transcriptomics. Drug hits were seen in the majority of HTS and PDX screens, which identified therapeutic options for 10 patients for whom no targetable molecular lesions could be found. Screens also provided orthogonal proof of drug efficacy suggested by molecular analyses and negative results for some molecular findings. We identified treatment options across the whole testing platform for 70% of patients. Only molecular therapeutic recommendations were provided to treating oncologists and led to a change in therapy in 53% of patients, of whom 29% had clinical benefit. These data indicate that in vitro and in vivo drug screening of tumor cells could increase therapeutic options and improve clinical outcomes for high-risk pediatric cancer patients.

The important role of routine cytopathology in pediatric precision oncology.

BACKGROUND: The development of high-throughput drug screening (HTS) using primary cultures provides a promising, clinically translatable approach to tailoring treatment strategies for patients with cancer. However, this has been challenging for solid tumors because of often limited amounts of tissue available. In most cases, in vitro expansion is required before HTS, which may lead to overgrowth and contamination by non-neoplastic cells. METHODS: In this study, hematoxylin and eosin staining and immunohistochemical staining were performed on 129 cytopathology cases from 95 patients. These cytopathology cases comprised cell block preparations derived from primary tumor specimens or patient-derived xenografts as part of a pediatric precision oncology trial. Cytopathology cases were compared with the morphology and immunohistochemical staining profile of the original tumor. Cases were reported as tumor cells present, equivocal, or tumor cells absent. The HTS results from cytopathologically validated cultures were incorporated into a multidisciplinary tumor board report issued to the treating clinician to guide clinical decision making. RESULTS: On cytopathologic examination, tumor cells were present in 77 of 129 cases (60%) and were absent in 38 of 129 cases (29%), whereas 14 of 129 cases (11%) were equivocal. Cultures that contained tumor cells resembled the tumors from which they were derived. CONCLUSIONS: Cytopathologic examination of tumor cell block preparations is feasible and provides detailed morphologic characterization. Cytopathologic examination is essential for ensuring that samples submitted for HTS contain representative tumor cells and that in vitro drug sensitivity data are clinically translatable.

Dual Targeting of Chromatin Stability By The Curaxin CBL0137 and Histone Deacetylase Inhibitor Panobinostat Shows Significant Preclinical Efficacy in Neuroblastoma.

PURPOSE: We investigated whether targeting chromatin stability through a combination of the curaxin CBL0137 with the histone deacetylase (HDAC) inhibitor, panobinostat, constitutes an effective multimodal treatment for high-risk neuroblastoma. EXPERIMENTAL DESIGN: The effects of the drug combination on cancer growth were examined in vitro and in animal models of MYCN-amplified neuroblastoma. The molecular mechanisms of action were analyzed by multiple techniques including whole transcriptome profiling, immune deconvolution analysis, immunofluorescence, flow cytometry, pulsed-field gel electrophoresis, assays to assess cell growth and apoptosis, and a range of cell-based reporter systems to examine histone eviction, heterochromatin transcription, and chromatin compaction. RESULTS: The combination of CBL0137 and panobinostat enhanced nucleosome destabilization, induced an IFN response, inhibited DNA damage repair, and synergistically suppressed cancer cell growth. Similar synergistic effects were observed when combining CBL0137 with other HDAC inhibitors. The CBL0137/panobinostat combination significantly delayed cancer progression in xenograft models of poor outcome high-risk neuroblastoma. Complete tumor regression was achieved in the transgenic Th-MYCN neuroblastoma model which was accompanied by induction of a type I IFN and immune response. Tumor transplantation experiments further confirmed that the presence of a competent adaptive immune system component allowed the exploitation of the full potential of the drug combination. CONCLUSIONS: The combination of CBL0137 and panobinostat is effective and well-tolerated in preclinical models of aggressive high-risk neuroblastoma, warranting further preclinical and clinical investigation in other pediatric cancers. On the basis of its potential to boost IFN and immune responses in cancer models, the drug combination holds promising potential for addition to immunotherapies.

Preclinical small molecule WEHI-7326 overcomes drug resistance and elicits response in patient-derived xenograft models of human treatment-refractory tumors.

Targeting cell division by chemotherapy is a highly effective strategy to treat a wide range of cancers. However, there are limitations of many standard-of-care chemotherapies: undesirable drug toxicity, side-effects, resistance and high cost. New small molecules which kill a wide range of cancer subtypes, with good therapeutic window in vivo, have the potential to complement the current arsenal of anti-cancer agents and deliver improved safety profiles for cancer patients. We describe results with a new anti-cancer small molecule, WEHI-7326, which causes cell cycle arrest in G2/M, cell death in vitro, and displays efficacious anti-tumor activity in vivo. WEHI-7326 induces cell death in a broad range of cancer cell lines, including taxane-resistant cells, and inhibits growth of human colon, brain, lung, prostate and breast tumors in mice xenografts. Importantly, the compound elicits tumor responses as a single agent in patient-derived xenografts of clinically aggressive, treatment-refractory neuroblastoma, breast, lung and ovarian cancer. In combination with standard-of-care, WEHI-7326 induces a remarkable complete response in a mouse model of high-risk neuroblastoma. WEHI-7326 is mechanistically distinct from known microtubule-targeting agents and blocks cells early in mitosis to inhibit cell division, ultimately leading to apoptotic cell death. The compound is simple to produce and possesses favorable pharmacokinetic and toxicity profiles in rodents. It represents a novel class of anti-cancer therapeutics with excellent potential for further development due to the ease of synthesis, simple formulation, moderate side effects and potent in vivo activity. WEHI-7326 has the potential to complement current frontline anti-cancer drugs and to overcome drug resistance in a wide range of cancers.

Targeted Therapy of TERT-Rearranged Neuroblastoma with BET Bromodomain Inhibitor and Proteasome Inhibitor Combination Therapy.

PURPOSE: TERT gene rearrangement with transcriptional superenhancers leads to TERT overexpression and neuroblastoma. No targeted therapy is available for clinical trials in patients with TERT-rearranged neuroblastoma. EXPERIMENTAL DESIGN: Anticancer agents exerting the best synergistic anticancer effects with BET bromodomain inhibitors were identified by screening an FDA-approved oncology drug library. The synergistic effects of the BET bromodomain inhibitor OTX015 and the proteasome inhibitor carfilzomib were examined by immunoblot and flow cytometry analysis. The anticancer efficacy of OTX015 and carfilzomib combination therapy was investigated in mice xenografted with TERT-rearranged neuroblastoma cell lines or patient-derived xenograft (PDX) tumor cells, and the role of TERT reduction in the anticancer efficacy was examined through rescue experiments in mice. RESULTS: The BET bromodomain protein BRD4 promoted TERT-rearranged neuroblastoma cell proliferation through upregulating TERT expression. Screening of an approved oncology drug library identified the proteasome inhibitor carfilzomib as the agent exerting the best synergistic anticancer effects with BET bromodomain inhibitors including OTX015. OTX015 and carfilzomib synergistically reduced TERT protein expression, induced endoplasmic reticulum stress, and induced TERT-rearranged neuroblastoma cell apoptosis which was blocked by TERT overexpression and endoplasmic reticulum stress antagonists. In mice xenografted with TERT-rearranged neuroblastoma cell lines or PDX tumor cells, OTX015 and carfilzomib synergistically blocked TERT expression, induced tumor cell apoptosis, suppressed tumor progression, and improved mouse survival, which was largely reversed by forced TERT overexpression. CONCLUSIONS: OTX015 and carfilzomib combination therapy is likely to be translated into the first clinical trial of a targeted therapy in patients with TERT-rearranged neuroblastoma.

Suppression of ABCE1-Mediated mRNA Translation Limits N-MYC-Driven Cancer Progression.

The ability of the N-MYC transcription factor to drive cancer progression is well demonstrated in neuroblastoma, the most common extracranial pediatric solid tumor, where MYCN amplification heralds a poor prognosis, with only 11% of high-risk patients surviving past 5 years. However, decades of attempts of direct inhibition of N-MYC or its paralogues has led to the conclusion that this protein is "undruggable." Therefore, targeting pathways upregulated by N-MYC signaling presents an alternative therapeutic approach. Here, we show that MYCN-amplified neuroblastomas are characterized by elevated rates of protein synthesis and that high expression of ABCE1, a translation factor directly upregulated by N-MYC, is itself a strong predictor of poor clinical outcome. Despite the potent ability of N-MYC in heightening protein synthesis and malignant characteristics in cancer cells, suppression of ABCE1 alone selectively negated this effect, returning the rate of translation to baseline levels and significantly reducing the growth, motility, and invasiveness of MYCN-amplified neuroblastoma cells and patient-derived xenograft tumors in vivo. The growth of nonmalignant cells or MYCN-nonamplified neuroblastoma cells remained unaffected by reduced ABCE1, supporting a therapeutic window associated with targeting ABCE1. Neuroblastoma cells with c-MYC overexpression also required ABCE1 to maintain cell proliferation and translation. Taken together, ABCE1-mediated translation constitutes a critical process in the progression of N-MYC-driven and c-MYC-driven cancers that warrants investigations into methods of its therapeutic inhibition. SIGNIFICANCE: These findings demonstrate that N-MYC-driven cancers are reliant on elevated rates of protein synthesis driven by heightened expression of ABCE1, a vulnerability that can be exploited through suppression of ABCE1.

Interplay between MycN and c-Myc regulates radioresistance and cancer stem cell phenotype in neuroblastoma upon glutamine deprivation.

Targeting glutamine metabolism has emerged as a potential therapeutic strategy for Myc overexpressing cancer cells. Myc proteins contribute to an aggressive neuroblastoma phenotype. Radiotherapy is one of the treatment modalities for high-risk neuroblastoma patients. Herein, we investigated the effect of glutamine deprivation in combination with irradiation in neuroblastoma cells representative of high-risk disease and studied the role of Myc member interplay in regulating neuroblastoma cell radioresistance. Methods: Cell proliferation and viability assays were used to establish the effect of glutamine deprivation in neuroblastoma cells expressing c-Myc or MycN. Gene silencing and overexpression were used to modulate the expression of Myc genes to determine their role in neuroblastoma radioresistance. qPCR and western blot investigated interplay between expression of Myc members. The impact of glutamine deprivation on cell response following irradiation was explored using a radiobiological 3D colony assay. DNA repair gene pathways as well as CSC-related genes were studied by qPCR array. Reactive Oxygen Species (ROS) and glutathione (GSH) levels were detected by fluorescence and luminescence probes respectively. Cancer-stem cell (CSC) properties were investigated by sphere-forming assay and flow cytometry to quantify CSC markers. Expression of DNA repair genes and CSC-related genes was analysed by mining publicly available patient datasets. Results: Our results showed that glutamine deprivation decreased neuroblastoma cell proliferation and viability and modulated Myc member expression. We then demonstrated for the first time that combined glutamine deprivation with irradiation led to a selective radioresistance of MYCN-amplified neuroblastoma cells. By exploring the underlying mechanism of neuroblastoma radioresistance properties, our results highlight interplay between c-Myc and MycN expression suggesting compensatory mechanisms in Myc proteins leading to radioresistance in MYCN-amplified cells. This result was associated with the ability of MYCN-amplified cells to dysregulate the DNA repair gene pathway, maintain GSH and ROS levels and to increase the CSC-like population and properties. Conversely, glutamine deprivation led to radiosensitization in non-MYCN amplified cell lines through a disruption of the cell redox balance and a trend to decrease in the CSC-like populations. Mining publicly available gene expression dataset obtained from pediatric neuroblastoma patients, we identified a correlation pattern between Myc members and CSC-related genes as well as a specific group of DNA repair gene pathways. Conclusions: This study demonstrated that MycN and c-Myc tightly cooperate in regulation of the neuroblastoma CSC phenotypes and radioresistance upon glutamine deprivation. Pharmacologically, strategies targeting glutamine metabolism may prove beneficial in Myc-driven tumors. Consideration of MycN/c-Myc status in selecting neuroblastoma patients for glutamine metabolism treatment will be important to avoid potential radioresistance.

Combination therapy with the CDK7 inhibitor and the tyrosine kinase inhibitor exerts synergistic anticancer effects against MYCN-amplified neuroblastoma.

Patients with neuroblastoma due to MYCN oncogene amplification and consequent N-Myc oncoprotein overexpression have very poor prognosis. The cyclin-dependent kinase 7 (CDK7)/super-enhancer inhibitor THZ1 suppresses MYCN gene transcription, reduces neuroblastoma cell proliferation, but does not cause significant cell death. The protein kinase phosphatase 1 nuclear targeting subunit (PNUTS) has recently been shown to interact with c-Myc protein and suppresses c-Myc protein degradation. Here we screened the U.S. Food and Drug Administration-Approved Oncology Drugs Set V from the National Cancer Institute, and identified tyrosine kinase inhibitors (TKIs), including ponatinib and lapatinib, as the Approved Oncology Drugs exerting the best synergistic anticancer effects with THZ1 in MYCN-amplified neuroblastoma cells. Combination therapy with THZ1 and ponatinib or lapatinib synergistically induced neuroblastoma cell apoptosis, while having little effects in normal nonmalignant cells. Differential gene expression analysis identified PNUTS as one of the genes most synergistically reduced by the combination therapy. Reverse transcription polymerase chain reaction and immunoblot analyses confirmed that THZ1 and the TKIs synergistically downregulated PNUTS mRNA and protein expression and reduced N-Myc protein but not N-Myc mRNA expression. In addition, PNUTS knockdown resulted in decreased N-Myc protein but not mRNA expression and decreased MYCN-amplified neuroblastoma cell proliferation and survival. As CDK7 inhibitors are currently under clinical evaluation in patients, our data suggest the addition of the TKI ponatinib or lapatinib in CDK7 inhibitor clinical trials in patients.

Mouse models of high-risk neuroblastoma.

Informative and realistic mouse models of high-risk neuroblastoma are central to understanding mechanisms of tumour initiation, progression, and metastasis. They also play vital roles in validating tumour drivers and drug targets, as platforms for assessment of new therapies and in the generation of drug sensitivity data that can inform treatment decisions for individual patients. This review will describe genetically engineered mouse models of specific subsets of high-risk neuroblastoma, the development of patient-derived xenograft models that more broadly represent the diversity and heterogeneity of the disease, and models of primary and metastatic disease. We discuss the research applications, advantages, and limitations of each model type, the importance of model repositories and data standards for supporting reproducible, high-quality research, and potential future directions for neuroblastoma mouse models.

Accelerating development of high-risk neuroblastoma patient-derived xenograft models for preclinical testing and personalised therapy.

BACKGROUND: Predictive preclinical models play an important role in the assessment of new treatment strategies and as avatar models for personalised medicine; however, reliable and timely model generation is challenging. We investigated the feasibility of establishing patient-derived xenograft (PDX) models of high-risk neuroblastoma from a range of tumour-bearing patient materials and assessed approaches to improve engraftment efficiency. METHODS: PDX model development was attempted in NSG mice by using tumour materials from 12 patients, including primary and metastatic solid tumour samples, bone marrow, pleural fluid and residual cells from cytogenetic analysis. Subcutaneous, intramuscular and orthotopic engraftment were directly compared for three patients. RESULTS: PDX models were established for 44% (4/9) of patients at diagnosis and 100% (5/5) at relapse. In one case, attempted engraftment from pleural fluid resulted in an EBV-associated atypical lymphoid proliferation. Xenogeneic graft versus host disease was observed with attempted engraftment from lymph node and bone marrow tumour samples but could be prevented by T-cell depletion. Orthotopic engraftment was more efficient than subcutaneous or intramuscular engraftment. CONCLUSIONS: High-risk neuroblastoma PDX models can be reliably established from diverse sample types. Orthotopic implantation allows more rapid model development, increasing the likelihood of developing an avatar model within a clinically useful timeframe.

CCI52 sensitizes tumors to 6-mercaptopurine and inhibits MYCN-amplified tumor growth.

The antimetabolite 6-mercaptopurine (6-MP) is an important component in the treatment of specific cancer subtypes, however, the development of drug resistance and dose-limiting toxicities can limit its effectiveness. The therapeutic activity of 6-MP requires cellular uptake, enzymatic conversion to thio-GMP and incorporation of thio-GTP into RNA and DNA, as well as inhibition of de novo purine synthesis by methyl-thio-IMP. Mechanisms that prevent 6-MP entry into the cell, prevent 6-MP metabolism or deplete thiopurine intermediates, can all lead to 6-MP resistance. We previously conducted a high-throughput screen for inhibitors of the multidrug transporter MRP4 using 6-MP sensitivity as the readout. In addition to MRP4-specific inhibitors, we identified a compound, CCI52, that sensitized cell lines to 6-MP independent of this transporter. CCI52 and its more stable analogue CCI52-14 also function as effective chemosensitizers in vivo, substantially extending survival in a transgenic mouse cancer model treated with 6-MP. Chemosensitization was associated with an increase in thio-IMP, suggesting that CCI52 functions directly on 6-MP uptake or metabolism. In addition to its chemosensitizing effects, CCI52 and CCI52-14 inhibited the growth of MYCN-amplified high-risk neuroblastoma cell lines and delayed tumor progression in a MYCN-driven, transgenic mouse model of neuroblastoma. These multifunctional inhibitors may be useful for the further development of anticancer agents and as tools to better understand 6-MP metabolism.

Inhibition of polyamine synthesis and uptake reduces tumor progression and prolongs survival in mouse models of neuroblastoma.

Amplification of the MYCN oncogene is associated with an aggressive phenotype and poor outcome in childhood neuroblastoma. Polyamines are highly regulated essential cations that are frequently elevated in cancer cells, and the rate-limiting enzyme in polyamine synthesis, ornithine decarboxylase 1 (ODC1), is a direct transcriptional target of MYCN. Treatment of neuroblastoma cells with the ODC1 inhibitor difluoromethylornithine (DFMO), although a promising therapeutic strategy, is only partially effective at impeding neuroblastoma cell growth due to activation of compensatory mechanisms resulting in increased polyamine uptake from the surrounding microenvironment. In this study, we identified solute carrier family 3 member 2 (SLC3A2) as the key transporter involved in polyamine uptake in neuroblastoma. Knockdown of SLC3A2 in neuroblastoma cells reduced the uptake of the radiolabeled polyamine spermidine, and DFMO treatment increased SLC3A2 protein. In addition, MYCN directly increased polyamine synthesis and promoted neuroblastoma cell proliferation by regulating SLC3A2 and other regulatory components of the polyamine pathway. Inhibiting polyamine uptake with the small-molecule drug AMXT 1501, in combination with DFMO, prevented or delayed tumor development in neuroblastoma-prone mice and extended survival in rodent models of established tumors. Our findings suggest that combining AMXT 1501 and DFMO with standard chemotherapy might be an effective strategy for treating neuroblastoma.

TPD52 expression increases neutral lipid storage within cultured cells.

Tumor protein D52 (TPD52) is amplified and/or overexpressed in cancers of diverse cellular origins. Altered cellular metabolism (including lipogenesis) is a hallmark of cancer development, and protein-protein associations between TPD52 and known regulators of lipid storage, and differential TPD52 expression in obese versus non-obese adipose tissue, suggest that TPD52 might regulate cellular lipid metabolism. We found increased lipid droplet numbers in BALB/c 3T3 cell lines stably expressing TPD52, compared with control and TPD52L1-expressing cell lines. TPD52-expressing 3T3 cells showed increased fatty acid storage in triglyceride (from both de novo synthesis and uptake) and formed greater numbers of lipid droplets upon oleic acid supplementation than control cells. TPD52 colocalised with Golgi, but not endoplasmic reticulum (ER), markers and also showed partial colocalisation with lipid droplets coated with ADRP (also known as PLIN2), with a proportion of TPD52 being detected in the lipid droplet fraction. Direct interactions between ADRP and TPD52, but not TPD52L1, were demonstrated using the yeast two-hybrid system, with ADRP-TPD52 interactions confirmed using GST pulldown assays. Our findings uncover a new isoform-specific role for TPD52 in promoting intracellular lipid storage, which might be relevant to TPD52 overexpression in cancer.