Fasting-mimicking diet and hormone therapy induce breast cancer regression.

Approximately 75% of all breast cancers express the oestrogen and/or progesterone receptors. Endocrine therapy is usually effective in these hormone-receptor-positive tumours, but primary and acquired resistance limits its long-term benefit1,2. Here we show that in mouse models of hormone-receptor-positive breast cancer, periodic fasting or a fasting-mimicking diet3-5 enhances the activity of the endocrine therapeutics tamoxifen and fulvestrant by lowering circulating IGF1, insulin and leptin and by inhibiting AKT-mTOR signalling via upregulation of EGR1 and PTEN. When fulvestrant is combined with palbociclib (a cyclin-dependent kinase 4/6 inhibitor), adding periodic cycles of a fasting-mimicking diet promotes long-lasting tumour regression and reverts acquired resistance to drug treatment. Moreover, both fasting and a fasting-mimicking diet prevent tamoxifen-induced endometrial hyperplasia. In patients with hormone-receptor-positive breast cancer receiving oestrogen therapy, cycles of a fasting-mimicking diet cause metabolic changes analogous to those observed in mice, including reduced levels of insulin, leptin and IGF1, with the last two remaining low for extended periods. In mice, these long-lasting effects are associated with long-term anti-cancer activity. These results support further clinical studies of a fasting-mimicking diet as an adjuvant to oestrogen therapy in hormone-receptor-positive breast cancer.

Apoptosis reprogramming triggered by splicing inhibitors sensitizes multiple myeloma cells to Venetoclax treatment.

Identification of novel vulnerabilities in the context of therapeutic resistance is emerging as a key challenge for cancer treatment. Recent studies have detected pervasive aberrant splicing in cancer cells, supporting its targeting for novel therapeutic strategies. Here, we evaluated the expression of several spliceosome machinery components in multiple myeloma (MM) cells and the impact of splicing modulation on tumor cell growth and viability. A comprehensive gene expression analysis confirmed the reported deregulation of spliceosome machinery components in MM cells, compared to normal plasma cells from healthy donors, with its pharmacological and genetic modulation resulting in impaired growth and survival of MM cell lines and patient-derived malignant plasma cells. Consistent with this, transcriptomic analysis revealed deregulation of BCL2 family members, including decrease of anti-apoptotic long form of myeloid cell leukemia-1 (MCL1) expression, as crucial for "priming" MM cells for Venetoclax activity in vitro and in vivo, irrespective of t(11;14) status. Overall, our data provide a rationale for supporting the clinical use of splicing modulators as a strategy to reprogram apoptotic dependencies and make all MM patients more vulnerable to BCL2 inhibitors.

Transcriptome analysis defines myocardium gene signatures in children with ToF and ASD and reveals disease-specific molecular reprogramming in response to surgery with cardiopulmonary bypass.

BACKGROUND: Tetralogy of Fallot (ToF) and Atrial Septal Defects (ASD) are the most common types of congenital heart diseases and a major cause of childhood morbidity and mortality. Cardiopulmonary bypass (CPB) is used during corrective cardiac surgery to support circulation and heart stabilization. However, this procedure triggers systemic inflammatory and stress response and consequent increased risk of postoperative complications. The aim of this study was to define the molecular bases of ToF and ASD pathogenesis and response to CPB and identify new potential biomarkers. METHODS: Comparative transcriptome analysis of right atrium specimens collected from 10 ToF and 10 ASD patients was conducted before (Pre-CPB) and after (Post-CPB) corrective surgery. Total RNA isolated from each sample was individually hybridized on Affymetrix HG-U133 Plus Array Strips containing 38,500 unique human genes. Differences in the gene expression profiles and functional enrichment/network analyses were assessed using bioinformatic tools. qRT-PCR analysis was used to validate gene modulation. RESULTS: Pre-CPB samples showed significant differential expression of a total of 72 genes, 28 of which were overexpressed in ToF and 44 in ASD. According to Gene Ontology annotation, the mostly enriched biological processes were represented by matrix organization and cell adhesion in ToF and by muscle development and contractility in ASD specimens. GSEA highlighted the specific enrichment of hypoxia gene sets in ToF samples, pointing to a role for hypoxia in disease pathogenesis. The post-CPB myocardium exhibited significant alterations in the expression profile of genes related to transcription regulation, growth/apoptosis, inflammation, adhesion/matrix organization, and oxidative stress. Among them, only 70 were common to the two disease groups, whereas 110 and 24 were unique in ToF and ASD, respectively. Multiple functional interactions among differentially expressed gene products were predicted by network analysis. Interestingly, gene expression changes in ASD samples followed a consensus hypoxia profile. CONCLUSION: Our results provide a comprehensive view of gene reprogramming in right atrium tissues of ToF and ASD patients before and after CPB, defining specific molecular pathways underlying disease pathophysiology and myocardium response to CPB. These findings have potential translational value because they identify new candidate prognostic markers and targets for tailored cardioprotective post-surgical therapies.

SIRT6 deacetylase activity regulates NAMPT activity and NAD(P)(H) pools in cancer cells.

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the NAD+ salvage pathway from nicotinamide. By controlling the biosynthesis of NAD+, NAMPT regulates the activity of NAD+-converting enzymes, such as CD38, poly-ADP-ribose polymerases, and sirtuins (SIRTs). SIRT6 is involved in the regulation of a wide number of metabolic processes. In this study, we investigated the ability of SIRT6 to regulate intracellular NAMPT activity and NAD(P)(H) levels. BxPC-3 cells and MCF-7 cells were engineered to overexpress a catalytically active or a catalytically inactive SIRT6 form or were engineered to silence endogenous SIRT6 expression. In SIRT6-overexpressing cells, NAD(H) levels were up-regulated, as a consequence of NAMPT activation. By immunopurification and incubation with recombinant SIRT6, NAMPT was found to be a direct substrate of SIRT6 deacetylation, with a mechanism that up-regulates NAMPT enzymatic activity. Extracellular NAMPT release was enhanced in SIRT6-silenced cells. Also glucose-6-phosphate dehydrogenase activity and NADPH levels were increased in SIRT6-overexpressing cells. Accordingly, increased SIRT6 levels reduced cancer cell susceptibility to H2O2-induced oxidative stress and to doxorubicin. Our data demonstrate that SIRT6 affects intracellular NAMPT activity, boosts NAD(P)(H) levels, and protects against oxidative stress. The use of SIRT6 inhibitors, together with agents inducing oxidative stress, may represent a promising treatment strategy in cancer.-Sociali, G., Grozio, A., Caffa, I., Schuster, S., Becherini, P., Damonte, P., Sturla, L., Fresia, C., Passalacqua, M., Mazzola, F., Raffaelli, N., Garten, A., Kiess, W., Cea, M., Nencioni, A., Bruzzone, S. SIRT6 deacetylase activity regulates NAMPT activity and NAD(P)(H) pools in cancer cells.

SIRT6 inhibitors with salicylate-like structure show immunosuppressive and chemosensitizing effects.

The NAD+-dependent deacetylase SIRT6 is an emerging cancer drug target, whose inhibition sensitizes cancer cells to chemo-radiotherapy and has pro-differentiating effects. Here we report on the identification of novel SIRT6 inhibitors with a salicylate-based structure. The new SIRT6 inhibitors show improved potency and specificity compared to the hit inhibitor identified in an in silico compound screen. As predicted based on SIRT6 biological roles, the new leads increase histone 3 lysine 9 acetylation and glucose uptake in cultured cells, while blocking TNF-α production and T lymphocyte proliferation. Notably, the new SIRT6 inhibitors effectively sensitize pancreatic cancer cells to gemcitabine. Finally, studies of compound fingerprinting and pharmacokinetics defined the drug-like properties of one of the new SIRT6 inhibitors, potentially allowing for subsequent in vivo proof-of-concept studies. In conclusion, new SIRT6 inhibitors with a salicylate-like structure were identified, which are active in cells and could potentially find applications in disease conditions, including cancer and immune-mediated disorders.

Use of Attribute Driven Incremental Discretization and Logic Learning Machine to build a prognostic classifier for neuroblastoma patients.

BACKGROUND: Cancer patient's outcome is written, in part, in the gene expression profile of the tumor. We previously identified a 62-probe sets signature (NB-hypo) to identify tissue hypoxia in neuroblastoma tumors and showed that NB-hypo stratified neuroblastoma patients in good and poor outcome 1. It was important to develop a prognostic classifier to cluster patients into risk groups benefiting of defined therapeutic approaches. Novel classification and data discretization approaches can be instrumental for the generation of accurate predictors and robust tools for clinical decision support. We explored the application to gene expression data of Rulex, a novel software suite including the Attribute Driven Incremental Discretization technique for transforming continuous variables into simplified discrete ones and the Logic Learning Machine model for intelligible rule generation. RESULTS: We applied Rulex components to the problem of predicting the outcome of neuroblastoma patients on the bases of 62 probe sets NB-hypo gene expression signature. The resulting classifier consisted in 9 rules utilizing mainly two conditions of the relative expression of 11 probe sets. These rules were very effective predictors, as shown in an independent validation set, demonstrating the validity of the LLM algorithm applied to microarray data and patients' classification. The LLM performed as efficiently as Prediction Analysis of Microarray and Support Vector Machine, and outperformed other learning algorithms such as C4.5. Rulex carried out a feature selection by selecting a new signature (NB-hypo-II) of 11 probe sets that turned out to be the most relevant in predicting outcome among the 62 of the NB-hypo signature. Rules are easily interpretable as they involve only few conditions. CONCLUSIONS: Our findings provided evidence that the application of Rulex to the expression values of NB-hypo signature created a set of accurate, high quality, consistent and interpretable rules for the prediction of neuroblastoma patients' outcome. We identified the Rulex weighted classification as a flexible tool that can support clinical decisions. For these reasons, we consider Rulex to be a useful tool for cancer classification from microarray gene expression data.

Cyclic fasting bolsters cholesterol biosynthesis inhibitors' anticancer activity.

Identifying oncological applications for drugs that are already approved for other medical indications is considered a possible solution for the increasing costs of cancer treatment. Under the hypothesis that nutritional stress through fasting might enhance the antitumour properties of at least some non-oncological agents, by screening drug libraries, we find that cholesterol biosynthesis inhibitors (CBIs), including simvastatin, have increased activity against cancers of different histology under fasting conditions. We show fasting's ability to increase CBIs' antitumour effects to depend on the reduction in circulating insulin, insulin-like growth factor-1 and leptin, which blunts the expression of enzymes from the cholesterol biosynthesis pathway and enhances cholesterol efflux from cancer cells. Ultimately, low cholesterol levels through combined fasting and CBIs reduce AKT and STAT3 activity, oxidative phosphorylation and energy stores in the tumour. Our results support further studies of CBIs in combination with fasting-based dietary regimens in cancer treatment and highlight the value of fasting for drug repurposing in oncology.

CD38-Induced Metabolic Dysfunction Primes Multiple Myeloma Cells for NAD+-Lowering Agents.

Cancer cells fuel growth and energy demands by increasing their NAD+ biosynthesis dependency, which therefore represents an exploitable vulnerability for anti-cancer strategies. CD38 is a NAD+-degrading enzyme that has become crucial for anti-MM therapies since anti-CD38 monoclonal antibodies represent the backbone for treatment of newly diagnosed and relapsed multiple myeloma patients. Nevertheless, further steps are needed to enable a full exploitation of these strategies, including deeper insights of the mechanisms by which CD38 promotes tumorigenesis and its metabolic additions that could be selectively targeted by therapeutic strategies. Here, we present evidence that CD38 upregulation produces a pervasive intracellular-NAD+ depletion, which impairs mitochondrial fitness and enhances oxidative stress; as result, genetic or pharmacologic approaches that aim to modify CD38 surface-level prime MM cells to NAD+-lowering agents. The molecular mechanism underlying this event is an alteration in mitochondrial dynamics, which decreases mitochondria efficiency and triggers energetic remodeling. Overall, we found that CD38 handling represents an innovative strategy to improve the outcomes of NAD+-lowering agents and provides the rationale for testing these very promising agents in clinical studies involving MM patients.

SIRT6 enhances oxidative phosphorylation in breast cancer and promotes mammary tumorigenesis in mice.

BACKGROUND: Sirtuin 6 (SIRT6) is a NAD+-dependent deacetylase with key roles in cell metabolism. High SIRT6 expression is associated with adverse prognosis in breast cancer (BC) patients. However, the mechanisms through which SIRT6 exerts its pro-oncogenic effects in BC remain unclear. Here, we sought to define the role of SIRT6 in BC cell metabolism and in mouse polyoma middle T antigen (PyMT)-driven mammary tumors. METHODS: We evaluated the effect of a heterozygous deletion of Sirt6 on tumor latency and survival of mouse mammary tumor virus (MMTV)-PyMT mice. The effect of SIRT6 silencing on human BC cell growth was assessed in MDA-MB-231 xenografts. We also analyzed the effect of Sirt6 heterozygous deletion, of SIRT6 silencing, and of the overexpression of either wild-type (WT) or catalytically inactive (H133Y) SIRT6 on BC cell pyruvate dehydrogenase (PDH) expression and activity and oxidative phosphorylation (OXPHOS), including respiratory complex activity, ATP/AMP ratio, AMPK activation, and intracellular calcium concentration. RESULTS: The heterozygous Sirt6 deletion extended tumor latency and mouse survival in the MMTV-PyMT mouse BC model, while SIRT6 silencing slowed the growth of MDA-MB-231 BC cell xenografts. WT, but not catalytically inactive, SIRT6 enhanced PDH expression and activity, OXPHOS, and ATP/AMP ratio in MDA-MB-231 and MCF7 BC cells. Opposite effects were obtained by SIRT6 silencing, which also blunted the expression of genes encoding for respiratory chain proteins, such as UQCRFS1, COX5B, NDUFB8, and UQCRC2, and increased AMPK activation in BC cells. In addition, SIRT6 overexpression increased, while SIRT6 silencing reduced, intracellular calcium concentration in MDA-MB-231 cells. Consistent with these findings, the heterozygous Sirt6 deletion reduced the expression of OXPHOS-related genes, the activity of respiratory complexes, and the ATP/AMP ratio in tumors isolated from MMTV-PyMT mice. CONCLUSIONS: Via its enzymatic activity, SIRT6 enhances PDH expression and activity, OXPHOS, ATP/AMP ratio, and intracellular calcium concentration, while reducing AMPK activation, in BC cells. Thus, overall, SIRT6 inhibition appears as a viable strategy for preventing or treating BC.

Safety and Feasibility of Fasting-Mimicking Diet and Effects on Nutritional Status and Circulating Metabolic and Inflammatory Factors in Cancer Patients Undergoing Active Treatment.

In preclinical studies, fasting was found to potentiate the effects of several anticancer treatments, and early clinical studies indicated that patients may benefit from regimes of modified fasting. However, concerns remain over possible negative impact on the patients' nutritional status. We assessed the feasibility and safety of a 5-day "Fasting-Mimicking Diet" (FMD) as well as its effects on body composition and circulating growth factors, adipokines and cyto/chemokines in cancer patients. In this single-arm, phase I/II clinical trial, patients with solid or hematologic malignancy, low nutritional risk and undergoing active medical treatment received periodic FMD cycles. The body weight, handgrip strength and body composition were monitored throughout the study. Growth factors, adipokines and cyto/chemokines were assessed by ELISA. Ninety patients were enrolled, and FMD was administered every three weeks/once a month with an average of 6.3 FMD cycles/patient. FMD was largely safe with only mild side effects. The patients' weight and handgrip remained stable, the phase angle and fat-free mass increased, while the fat mass decreased. FMD reduced the serum c-peptide, IGF1, IGFBP3 and leptin levels, while increasing IGFBP1, and these modifications persisted for weeks beyond the FMD period. Thus, periodic FMD cycles are feasible and can be safely combined with standard antineoplastic treatments in cancer patients at low nutritional risk. The FMD resulted in reduced fat mass, insulin production and circulating IGF1 and leptin. This trial was registered on Clinicaltrials.gov in July 2018 with the identifier NCT03595540.

Amino acid depletion triggered by ʟ-asparaginase sensitizes MM cells to carfilzomib by inducing mitochondria ROS-mediated cell death.

Metabolic reprogramming is emerging as a cancer vulnerability that could be therapeutically exploitable using different approaches, including amino acid depletion for those tumors that rely on exogenous amino acids for their maintenance. ʟ-Asparaginase (ASNase) has contributed to a significant improvement in acute lymphoblastic leukemia outcomes; however, toxicity and resistance limit its clinical use in other tumors. Here, we report that, in multiple myeloma (MM) cells, the DNA methylation status is significantly associated with reduced expression of ASNase-related gene signatures, thus suggesting ASNase sensitivity for this tumor. Therefore, we tested the effects of ASNase purified from Erwinia chrysanthemi (Erw-ASNase), combined with the next-generation proteasome inhibitor (PI) carfilzomib. We observed an impressive synergistic effect on MM cells, whereas normal peripheral blood mononuclear cells were not affected. Importantly, this effect was associated with increased reactive oxygen species (ROS) generation, compounded mitochondrial damage, and Nrf2 upregulation, regardless of the c-Myc oncogenic-specific program. Furthermore, the cotreatment resulted in genomic instability and DNA repair mechanism impairment via increased mitochondrial oxidative stress, which further enhanced its antitumor activity. Interestingly, carfilzomib-resistant cells were found to be highly dependent on amino acid starvation, as reflected by their higher sensitivity to Erw-ASNase treatment compared with isogenic cells. Overall, by affecting several cellular programs, Erw-ASNase makes MM cells more vulnerable to carfilzomib, providing proof of concept for clinical use of this combination as a novel strategy to enhance PI sensitivity in MM patients.

Mesenchymal Stem Cell-Derived Extracellular Vesicles as Mediators of Anti-Inflammatory Effects: Endorsement of Macrophage Polarization.

Mesenchymal Stem Cells (MSCs) are effective therapeutic agents enhancing the repair of injured tissues mostly through their paracrine activity. Increasing evidences show that besides the secretion of soluble molecules, the release of extracellular vesicles (EVs) represents an alternative mechanism adopted by MSCs. Since macrophages are essential contributors toward the resolution of inflammation, which has emerged as a finely orchestrated process, the aim of the present study was to carry out a detailed characterization of EVs released by human adipose derived-MSCs to investigate their involvement as modulators of MSC anti-inflammatory effects inducing macrophage polarization. The EV-isolation method was based on repeated ultracentrifugations of the medium conditioned by MSC exposed to normoxic or hypoxic conditions (EVNormo and EVHypo ). Both types of EVs were efficiently internalized by responding bone marrow-derived macrophages, eliciting their switch from a M1 to a M2 phenotype. In vivo, following cardiotoxin-induced skeletal muscle damage, EVNormo and EVHypo interacted with macrophages recruited during the initial inflammatory response. In injured and EV-treated muscles, a downregulation of IL6 and the early marker of innate and classical activation Nos2 were concurrent to a significant upregulation of Arg1 and Ym1, late markers of alternative activation, as well as an increased percentage of infiltrating CD206pos cells. These effects, accompanied by an accelerated expression of the myogenic markers Pax7, MyoD, and eMyhc, were even greater following EVHypo administration. Collectively, these data indicate that MSC-EVs possess effective anti-inflammatory properties, making them potential therapeutic agents more handy and safe than MSCs. Stem Cells Translational Medicine 2017 Stem Cells Translational Medicine 2017;6:1018-1028.

First Characterization of Human Amniotic Fluid Stem Cell Extracellular Vesicles as a Powerful Paracrine Tool Endowed with Regenerative Potential.

Human amniotic fluid stem cells (hAFS) have shown a distinct secretory profile and significant regenerative potential in several preclinical models of disease. Nevertheless, little is known about the detailed characterization of their secretome. Herein we show for the first time that hAFS actively release extracellular vesicles (EV) endowed with significant paracrine potential and regenerative effect. c-KIT+ hAFS were isolated from leftover samples of amniotic fluid from prenatal screening and stimulated to enhance EV release (24 hours 20% O2 versus 1% O2 preconditioning). The capacity of the c-KIT+ hAFS-derived EV (hAFS-EV) to induce proliferation, survival, immunomodulation, and angiogenesis were investigated in vitro and in vivo. The hAFS-EV regenerative potential was also assessed in a model of skeletal muscle atrophy (HSA-Cre, SmnF7/F7 mice), in which mouse AFS transplantation was previously shown to enhance muscle strength and survival. hAFS secreted EV ranged from 50 up to 1,000 nm in size. In vitro analysis defined their role as biological mediators of regenerative, paracrine effects while their modulatory role in decreasing skeletal muscle inflammation in vivo was shown for the first time. Hypoxic preconditioning significantly induced the enrichment of exosomes endowed with regenerative microRNAs within the hAFS-EV. In conclusion, this is the first study showing that c-KIT+ hAFS dynamically release EV endowed with remarkable paracrine potential, thus representing an appealing tool for future regenerative therapy. Stem Cells Translational Medicine 2017;6:1340-1355.

Nicotinic Acid Phosphoribosyltransferase Regulates Cancer Cell Metabolism, Susceptibility to NAMPT Inhibitors, and DNA Repair.

In the last decade, substantial efforts have been made to identify NAD+ biosynthesis inhibitors, specifically against nicotinamide phosphoribosyltransferase (NAMPT), as preclinical studies indicate their potential efficacy as cancer drugs. However, the clinical activity of NAMPT inhibitors has proven limited, suggesting that alternative NAD+ production routes exploited by tumors confer resistance. Here, we show the gene encoding nicotinic acid phosphoribosyltransferase (NAPRT), a second NAD+-producing enzyme, is amplified and overexpressed in a subset of common types of cancer, including ovarian cancer, where NAPRT expression correlates with a BRCAness gene expression signature. Both NAPRT and NAMPT increased intracellular NAD+ levels. NAPRT silencing reduced energy status, protein synthesis, and cell size in ovarian and pancreatic cancer cells. NAPRT silencing sensitized cells to NAMPT inhibitors both in vitro and in vivo; similar results were obtained with the NAPRT inhibitor 2-hydroxynicotinic acid. Reducing NAPRT levels in a BRCA2-deficient cancer cell line exacerbated DNA damage in response to chemotherapeutics. In conclusion, NAPRT-dependent NAD+ biosynthesis contributes to cell metabolism and to the DNA repair process in a subset of tumors. This knowledge could be used to increase the efficacy of NAMPT inhibitors and chemotherapy. Cancer Res; 77(14); 3857-69. ©2017 AACR.

Novel phage display-derived neuroblastoma-targeting peptides potentiate the effect of drug nanocarriers in preclinical settings.

Molecular targeting of drug delivery nanocarriers is expected to improve their therapeutic index while decreasing their toxicity. Here we report the identification and characterization of novel peptide ligands specific for cells present in high-risk neuroblastoma (NB), a childhood tumor mostly refractory to current therapies. To isolate such targeting moieties, we performed combined in vitro/ex-vivo phage display screenings on NB cell lines and on tumors derived from orthotopic mouse models of human NB. By designing proper subtractive protocols, we identified phage clones specific either for the primary tumor, its metastases, or for their respective stromal components. Globally, we isolated 121 phage-displayed NB-binding peptides: 26 bound the primary tumor, 15 the metastatic mass, 57 and 23 their respective microenvironments. Of these, five phage clones were further validated for their specific binding ex-vivo to biopsies from stage IV NB patients and to NB tumors derived from mice. All five clones also targeted tumor cells and vasculature in vivo when injected into NB-bearing mice. Coupling of the corresponding targeting peptides with doxorubicin-loaded liposomes led to a significant inhibition in tumor volume and enhanced survival in preclinical NB models, thereby paving the way to their clinical development.

The use of the orthotopic model to validate antivascular therapies for cancer.

The orthotopic model reproduces aspects of the tumour microenvironment and emulates a number of important biological features of cancer progression, angiogenesis, metastasis and resistance. Due to its parallels with human cancer, the model can be used to evaluate therapeutic responses to various therapies. This review outlines the importance of using the orthotopic implantation of tumour cells in mice models for evaluating the effectiveness of antivascular therapies.

Tumor regression and curability of preclinical neuroblastoma models by PEGylated SN38 (EZN-2208), a novel topoisomerase I inhibitor.

PURPOSE: Treatment of neuroblastoma is successful in less than half of patients with high-risk disease. The antitumor activity of a water soluble pegylated SN38 drug conjugate, EZN-2208, was compared with CPT-11 (a prodrug for SN38) in preclinical models of human neuroblastoma. EXPERIMENTAL DESIGN: The in vitro cytotoxicity of EZN-2208 was tested by counting trypan blue dye- and Annexin V-positive cells, whereas its therapeutic efficacy was evaluated, in terms of survival, and antitumor and antiangiogenic activities, in s.c. luciferase-transfected, pseudometastatic, and orthotopic neuroblastoma animal models. RESULTS: EZN-2208 was about 100-fold more potent than CPT-11 in vitro, by inducing apoptosis/necrosis and p53 expression and by reducing hypoxia-inducible factor (HIF)-1α/HIF-2α expression. EZN-2208 gave superior antitumor effects compared with CPT-11 in neuroblastoma xenografts. EZN-2208 treatment always resulted in lack of tumor detection at the end of trials whereas only small therapeutic effects were observed with CPT-11, as assessed by luciferase assay or tumor size, or even by staining histologic sections of tumors with antibodies recognizing neuroblastoma cells and cell proliferation. In a neuroblastoma model resistant to doxorubicin, cisplatin, vincristine, fenretinide, and topotecan, EZN-2208 induced 100% curability. It also blocked tumor relapse after topotecan-vincristine-doxorubicin combined treatment. Mechanistic experiments showed statistically significantly enhanced terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and Histone H2ax staining as well as decreased vascular endothelial growth factor, CD31, matrix metalloproteinase (MMP)-2, and MMP-9 expression in tumors removed from EZN-2208-treated mice and radiating vessels invading the tumor implanted onto the chorioallantoic membranes. CONCLUSIONS: EZN-2208 should be considered a most promising novel antineuroblastoma agent. An ongoing phase I study in pediatric patients should identify the optimal dose for a phase II study.