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PDF file - 102K, EWSR1-FLI1 expression upon Enzastaurin treatment

PDF file - 114K, Rescuing shRNAs Inhibition of PRKCB

PDF file - 94K, Stable A673-shPRKCB463_B5M3 cell line

PDF file - 75K, H3K4 trimethylation upon PRKCA inhibition

XLS file - 3.4MB, Expression profiles analyses and primer table

PDF file - 66K, Combined effect of TNFa with Enzastaurin on Ewing cell growth

PDF file - 122K, PRKC isoforms expression

PDF file - 196K

PDF file - 94K, EWSR1-FLI1 transcriptional influence on PRKCA and PRKCB

Pediatric patients with recurrent and refractory cancers are in most need for new treatments. This study developed patient-derived-xenograft (PDX) models within the European MAPPYACTS cancer precision medicine trial (NCT02613962). To date, 131 PDX models were established following heterotopical and/or orthotopical implantation in immunocompromised mice: 76 sarcomas, 25 other solid tumors, 12 central nervous system tumors, 15 acute leukemias, and 3 lymphomas. PDX establishment rate was 43%. Histology, whole exome and RNA sequencing revealed a high concordance with the primary patient’s tumor profile, human leukocyte-antigen characteristics and specific metabolic pathway signatures. A detailed patient molecular characterization, including specific mutations prioritized in the clinical molecular tumor boards are provided. Ninety models were shared with the IMI2 ITCC Paediatric Preclinical Proof-of-concept Platform (IMI2 ITCC-P4) for further exploitation. This new PDX biobank of unique recurrent childhood cancers provides an essential support for basic and translational research and new treatments development in advanced pediatric malignancies.

The ALK gene is a major oncogene of neuroblastoma cases exhibiting ALK activating mutations. Here, we characterized two neuroblastoma cell lines established from a stage 4 patient at diagnosis either from the primary tumor (PT) or from the bone marrow (BM). Both cell lines exhibited similar genomic profiles. All cells in the BM-derived cell line exhibited an ALK F1174L mutation, whereas this mutation was present in only 5% of the cells in the earliest passages of the PT-derived cell line. The BM-derived cell line presented with a higher proliferation rate in vitro and injections in Nude mice resulted in tumor formation only for the BM-derived cell line. Next, we observed that the F1174L mutation frequency in the PT-derived cell line increased with successive passages. Further Whole Exome Sequencing revealed a second ALK mutation, L1196M, in this cell line. Digital droplet PCR documented that the allele fractions of both mutations changed upon passages, and that the F1174L mutation reached 50% in late passages, indicating clonal evolution. In vitro treatment of the PT-derived cell line exhibiting the F1174L and L1196M mutations with the alectinib inhibitor resulted in an enrichment of the L1196M mutation. Using xenografts, we documented a better efficacy of alectinib compared to crizotinib on tumor growth and an enrichment of the L1196M mutation at the end of both treatments. Finally, single-cell RNA-seq analysis was consistent with both mutations resulting in ALK activation. Altogether, this study provides novel insights into ALK mutation dynamics in a neuroblastoma model harbouring two ALK mutations.

MET is expressed on neuroblastoma cells and may trigger tumor growth, neoangiogenesis and metastasis. MET upregulation further represents an escape mechanism to various anticancer treatments including VEGF signaling inhibitors. We developed in vitro a resistance model to pan-VEGFR inhibition and explored the simultaneous inhibition of VEGFR and MET in neuroblastoma models in vitro and in vivo using cabozantinib, an inhibitor of the tyrosine kinases including VEGFR2, MET, AXL and RET. Resistance in IGR-N91-Luc neuroblastoma cells under continuous in vitro exposure pressure to VEGFR1-3 inhibition using axitinib was associated with HGF and p-ERK overexpression. Cabozantinib exhibited anti-proliferative effects in neuroblastoma cells and reduced cell migration in vitro as measured by phase-contrast with IncuCyte system. In vivo, an enhanced number of animals with IGR-N91-Luc metastases was noted following axitinib treatment as compared to control animals. Orally administered cabozantinib per gavage at 30 and 60 mg/kg/day significantly inhibited tumor growth of orthotopic adrenal IGR-N91-Luc and metastatic IMR-32-Luc xenografts. Antitumor activity was associated with decreased vascularization, inhibition of p-SRC and induction of apoptotic cell death. Activation of the HGF-mediated MET pathway is involved in escape to selective VEGFR inhibition in neuroblastoma suggesting combined inhibition of MET and VEGFR signaling to reduce secondary resistance and enhanced invasiveness.

Osteosarcoma, the most common bone malignancy with a peak incidence at adolescence, had no survival improvement since decades. Persistent problems are chemo-resistance and metastatic spread. We developed in-vitro osteosarcoma models resistant to chemotherapy and in-vivo bioluminescent orthotopic cell-derived-xenografts (CDX). Continuous increasing drug concentration cultures in-vitro resulted in five methotrexate (MTX)-resistant and one doxorubicin (DOXO)-resistant cell lines. Resistance persisted after drug removal except for MG-63. Different resistance mechanisms were identified, affecting drug transport and action mechanisms specific to methotrexate (RFC/SCL19A1 decrease, DHFR up-regulation) for MTX-resistant lines, or a multi-drug phenomenon (PgP up-regulation) for HOS-R/DOXO. Differential analysis of copy number abnormalities (aCGH) and gene expression (RNAseq) revealed changes of several chromosomic regions translated at transcriptomic level depending on drug and cell line, as well as different pathways implicated in invasive and metastatic potential (e.g., Fas, Metalloproteinases) and immunity (enrichment in HLA cluster genes in 6p21.3) in HOS-R/DOXO. Resistant-CDX models (HOS-R/MTX, HOS-R/DOXO and Saos-2-B-R/MTX) injected intratibially into NSG mice behaved as their parental counterpart at primary tumor site, however, they exhibited a slower growth rate and lower metastatic spread, although they retained resistance and CGH main characteristics without drug pressure. These models represent valuable tools to explore resistance mechanisms and new therapies in osteosarcoma.

Busulfan-melphalan high-dose chemotherapy followed by autologous stem cell transplantation is an essential consolidation treatment of high-risk neuroblastoma in children. Main treatment limitation is hepatic veno-occlusive disease, the most severe and frequent extra-hematological toxicity. This life threatening toxicity has been related to a drug interaction between busulfan and melphalan which might be increased by prior disturbance of iron homeostasis, i.e. an increased plasma ferritin level.We performed an experimental study of busulfan and melphalan pharmacodynamic and pharmacokinetics in iron overloaded mice.Iron excess dramatically increased the toxicity of melphalan or busulfan melphalan combination in mice but it did not modify the clearance of either busulfan or melphalan. We show that prior busulfan treatment impairs the clearance of melphalan. This clearance alteration was exacerbated in iron overloaded mice demonstrating a pharmacokinetic interaction. Additionally, iron overload increased melphalan toxicity without altering its pharmacokinetics, suggesting a pharmacodynamic interaction between iron and melphalan. Based on iron homeostasis disturbance, we postulated that prior induction of ferritin, through Nrf2 activation after oxidative stress, may be associated with the alteration of melphalan metabolism.Iron overload increases melphalan and busulfan-melphalan toxicity through a pharmacodynamic interaction and reveals a pharmacokinetic drug interaction between busulfan and melphalan.

The antitumor drug, ifosfamide (IFO), requires activation by cytochrome P450 (CYP) to form the active metabolite, 4-hydroxyisfosfamide (4-OHIFO), leading to toxic by-products at high dose. In order to overcome these drawbacks, preactivated ifosfamide derivatives (RXIFO) were designed to release 4-OHIFO without CYP involvement. A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the simultaneous quantification of 4-OHIFO, IFO and four derivatives RXIFO in mouse plasma using multiple reaction monitoring. Because of its instability in plasma, 4-OHIFO was immediately converted to the semi-carbazone derivative, 4-OHIFO-SCZ. For the six analytes, the calibration curves were linear from 20 to 5000ng/mL in 50μL plasma and the lower limit of quantitation was determined at 20ng/mL with accuracies within ±10% of nominal and precisions less than 12%. Their recoveries ranged from 62 to 96% by using liquid-liquid extraction. With an improved assay sensitivity compared to analogues, the derivative 4-OHIFO-SCZ was stable in plasma at 4°C for 24h and at -20°C for three months. For all compounds, the assay was validated with accuracies within ±13% and precisions less than 15%. This method was applied to a comparative pharmacokinetic study of 4-OHIFO from IFO and three derivatives RXIFO in mice. This active metabolite was produced by some of the novel conjugates with good pharmacokinetic properties.

Osteosarcoma is one of the most common primary bone tumors in childhood and adolescence. Metastases occurrence at diagnosis or during disease evolution is the main therapeutic challenge. New drug evaluation to improve patient survival requires the development of various preclinical models mimicking at best the complexity of the disease and its metastatic potential. We describe here the development and characteristics of two orthotopic bioluminescent (Luc/mKate2) cell‐derived xenograft (CDX) models, Saos‐2‐B‐Luc/mKate2‐CDX and HOS‐Luc/mKate2‐CDX, in different immune (nude and NSG mouse strains) and bone (intratibial and paratibial with periosteum activation) contexts. IVIS SpectrumCT system allowed both longitudinal computed tomography (CT) and bioluminescence real‐time follow‐up of primary tumor growth and metastatic spread, which was confirmed by histology. The murine immune context influenced tumor engraftment, primary tumor growth, and metastatic spread to lungs, bone, and spleen (an unusual localization in humans). Engraftment in NSG mice was found superior to that found in nude mice and intratibial bone environment more favorable to engraftment compared to paratibial injection. The genetic background of the two CDX models also led to distinct primary tumor behavior observed on CT scan. Saos‐2‐B‐Luc/mKate2‐CDX showed osteocondensed, HOS‐Luc/mKate2‐CDX osteolytic morphology. Bioluminescence defined a faster growth of the primary tumor and metastases in Saos‐2‐B‐Luc/mKate2‐CDX than in HOS‐Luc/mKate2‐CDX. The early detection of primary tumor growth and metastatic spread by bioluminescence allows an improved exploration of osteosarcoma disease at tumor progression, and metastatic spread, as well as the evaluations of anticancer treatments. Our orthotopic models with metastatic spread bring complementary information to other types of existing osteosarcoma models.

International audience; Neuroblastoma is a tumor of the peripheral sympathetic nervous system(1), derived from multipotent neural crest cells (NCCs). To define core regulatory circuitries (CRCs) controlling the gene expression program of neuroblastoma, we established and analyzed the neuroblastoma super-enhancer landscape. We discovered three types of identity in neuroblastoma cell lines: a sympathetic noradrenergic identity, defined by a CRC module including the PHOX2B, HAND2 and GATA3 transcription factors (TFs); an NCC-like identity, driven by a CRC module containing AP-1 TFs; and a mixed type, further deconvoluted at the single-cell level. Treatment of the mixed type with chemotherapeutic agents resulted in enrichment of NCC-like cells. The noradrenergic module was validated by ChIP-seq. Functional studies demonstrated dependency of neuroblastoma with noradrenergic identity on PHOX2B, evocative of lineage addiction. Most neuroblastoma primary tumors express TFs from the noradrenergic and NCC-like modules. Our data demonstrate a previously unknown aspect of tumor heterogeneity relevant for neuroblastoma treatment strategies.

Polo-like kinase 1 (PLK1) controls the main cell-cycle checkpoints, suggesting utility of its inhibition for cancer treatment, including of highly proliferative pediatric cancer. This preclinical study explored the selective PLK1 inhibitor volasertib (BI 6727) alone and combined with chemotherapy in pediatric malignancies.Inhibition of proliferation was explored in vitro using dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium (MTS) assay. Mice bearing human xenografts were treated with weekly intravenous injections of volasertib.Volasertib inhibited proliferation in all 40 cell lines tested, with a mean half-maximal growth inhibitory concentration of 313 nmol/l (range: 4-5000 nmol/l). Volasertib was highly active against RMS-1 alveolar rhabdomyosarcoma xenografts, resulting in 100% tumor regression. Activity was associated with complete and prolonged G2/M arrest and subsequent apoptotic cell death. Volasertib showed synergistic activity with vincristine but antagonistic effects with etoposide.These findings support the further exploration of volasertib for pediatric malignancies, particularly alveolar rhabdomyosarcoma, and its combination with mitotic spindle poison.

International audience; INTRODUCTION:Papillary Glioneuronal Tumor (PGNT) is a grade I tumor which was classified as a separate entity in the World Health Organization Classification of the Central Nervous System 2007 in the group of mixed glioneuronal tumors. This tumor is rare and subclassifying PGNT represents a challenge. Recently, a fusion between SLC44A1 and PRKCA which encodes a protein kinase C involved in MAPK signaling pathway has been described in two studies (five cases). The current study aimed at raising the cytogenetic, histological and molecular profiles of PGNT and to determine if SLC44A1-PRKCA fusion represented a specific diagnostic marker to distinguish it from other glioneuronal tumors.RESULTS:We report on four pediatric cases of PGNT, along with clinico-radiologic and immunohistological features for which SLC44A1-PRKCA fusion assessment by fluorescence in situ hybridization, BRAF V600E and FGFR1 mutation by immunohistochemistry and direct DNA sequencing and KIAA1549-BRAF fusion by RT-PCR were performed. MAPK signaling pathway activation was investigated using phospho-ERK immunohistochemistry and western blot. We analyzed fifteen cases of tumors with challenging histological or clinical differential diagnoses showing respectively a papillary architecture or periventricular location (PGNT mimics). fluorescence in situ hybridization analysis revealed a constant SLC44A1-PRKCA fusion signal in all PGNTs. None of PGNT mimics showed the SLC44A1-PRKCA fusion signal pattern. All PGNTs were negative for BRAF V600E and FGFR1 mutation, and KIAA1549-BRAF fusion. Phospho-ERK analysis provides arguments for the activation of the MAPK signaling pathway in these tumors.CONCLUSIONS:Here we confirmed and extended the molecular data on PGNT. These results suggest that PGNT belong to low grade glioma with MAPK signaling pathway deregulation. SLC44A1-PRKCA fusion seems to be a specific characteristic of PGNT with a high diagnostic value and detectable by FISH.

Insulin-like growth factor 1 receptor (IGF-1R) is overexpressed in many tumours and contributes to tumourigenicity, cell proliferation, metastasis and resistance, thus representing a promising therapeutic target. The human IGF-1R antagonistic monoclonal antibody EM164 (murine AVE1642) has shown activity in adult cancers and is being evaluated in patients with advanced malignancies. We investigated the EM164 for its therapeutic potential against childhood neuroblastoma. EM164 at 0.07, 0.7 and 7 μg/mL exhibited anti-proliferative activity against all nine cell lines tested in (3)H-thymidine incorporation assay in vitro. Cell proliferation after EM164 exposure ranged between 24% and 80% compared to controls. Sensitivity was independent from culture serum conditions, intensity of IGF-1R expression and IGF-II secretion, although associated with inhibition of AKT activation. In vivo, EM164 administered intravenously at 40 mg/kg twice weekly for 4 weeks yielded significant tumour growth delays (TGD) of 13.4d in advanced stage IGR-N91 and 12.9 d in SK-N-AS tumours compared to controls (p = 0.02 and p = 0.0059, respectively). Simultaneous treatment of EM164 0.7 μg/mL and temozolomide resulted in enhanced activity in vitro. In vivo, treatment with temozolomide at the maximum tolerated dose (100mg/kg/d for 5 consecutive days) and EM164 yielded a significantly greater TGD of 29.1d (p

Notch signaling is altered in many cancers. Our previous findings in primary pediatric ependymoma support a role for NOTCH in glial oncogenesis. The present study evaluates the γ-secretase inhibitor RO4929097 in glial tumor models. The expression of Notch pathway genes was evaluated using real-time RT-PCR in 21 ependymoma and glioma models. NOTCH1 mutations were analyzed by DNA sequencing. RO4929097 activity was evaluated in vitro and in vivo, as a single agent and in combination, in glioma and ependymoma models. Notch pathway genes are overexpressed in ependymomas and gliomas along with FBXW7 downregulation. NOTCH1 mutations in the TAD domain were observed in 20% (2/10) of ependymoma primary cultures. Blocking the Notch pathway with the γ-secretase inhibitor RO4929097 reduced cell density and viability in ependymoma short-term cultures. When combined with chemotherapeutic agents, RO4929097 enhanced temozolomide effects in ependymoma short-term cultures and potentiated the cytotoxicity of etoposide, cisplatinum, and temozolomide in glioma cells. RO4929097, in combined treatment with mTOR inhibition, potentiated cytotoxicity in vitro, but did not enhance antitumor effects in vivo. In contrast, RO4929097 enhanced irradiation effects in glioma and ependymoma xenografts and showed tumor growth inhibition in advanced-stage IGRG121 glioblastoma xenografts. RO4929097-mediated effects were independent of NOTCH1 mutation status or expression levels, but associated with low IL-6 levels. In established glial tumor models, NOTCH inhibition had limited effects as a single agent, but enhanced efficacy when combined with DNA-interfering agents. These preclinical data need to be considered for further clinical development of NOTCH inhibitors in glial tumors.

Treatment of metastatic neuroblastoma remains a challenge in pediatric oncology. Relevant preclinical models may improve exploration of oncogenesis and new therapies. We developed new orthotopic and metastatic models derived from stage 4 neuroblastoma.Orthotopic and systemic models were established in BalbC Rag2(-/-)gammaC(-/-) mice following adrenal and intravenous injection of luciferase-transfected IMR-32 and IGR-N91 cells, respectively.All four models exhibited 100% tumor take rate. Metastatic spread of orthotopic IMR-32-Luc cells was observed mainly to the lung, liver and bone; that of IGR-N91-Luc cells to liver, spleen and adrenals. Interestingly, systemic IMR-32-Luc cells metastasized rather to the lung, liver and bone, and IGR-N91-Luc to liver, lung, spleen and adrenals. Feasibility of non-invasive, real-time antitumor response evaluation was validated in the systemic models.These neuroblastoma models with distinct patterns of metastatic spread represent relevant tools for exploring local and metastatic tumor cell tropism, mechanisms of spread and evaluating new cancer therapeutics.

International audience; Identification of druggable targets is a prerequisite for developing targeted therapies againstEwing sarcoma. We report the identification of Protein Kinase C Beta (PRKCB) as a proteinspecifically and highly expressed in Ewing sarcoma as compared to other pediatric cancers.Its transcriptional activation is directly regulated by the EWSR1-FLI1 oncogene. Gettinginsights in PRKCB activity we show that, together with PRKCA, it is responsible for thephosphorylation of histone H3T6, allowing global maintenance of H3K4 trimethylation on avariety of gene promoters. In the long term, PRKCB RNA interference induces apoptosis invitro. More importantly, in xenograft mice models, complete impairment of tumor engraftmentand even tumor regression were observed upon PRKCB inhibition, highlighting PRKCB as amost valuable therapeutic target. Deciphering PRKCB roles in Ewing sarcoma usingexpression profiling, we found a strong overlap with genes modulated by EWSR1-FLI1 andan involvement of RPKCB in regulating crucial signaling pathways. Altogether, we show thatPRKCB may have two important independent functions and should be considered as highlyvaluable for understanding Ewing sarcoma biology and as a promising target for newtherapeutic approaches in Ewing sarcoma.

The multikinase inhibitor regorafenib (BAY 73–4506) exerts both anti-angiogenic and anti-tumorigenic activity in adult solid malignancies mainly advanced colorectal cancer and gastrointestinal stromal tumors. We intended to explore preclinically the potential of regorafenib against solid pediatric malignancies alone and in combination with anticancer agents to guide the pediatric development plan. In vitro effects on cell proliferation were screened against 33 solid tumor cell lines of the Innovative Therapies for Children with Cancer (ITCC) panel covering five pediatric solid malignancies. Regorafenib inhibited cell proliferation with a mean half maximal growth inhibition of 12.5 μmol/L (range 0.7 μmol/L to 28 μmol/L). In vivo, regorafenib was evaluated alone at 10 or 30 mg/kg/d or in combination with radiation, irinotecan or the mitogen-activated protein kinase kinase (MEK) inhibitor refametinib against various tumor types, including patient-derived brain tumor models with an amplified platelet-derived growth factor receptor A (PDGFRA) gene. Regorafenib alone significantly inhibited tumor growth in all xenografts derived from nervous system and connective tissue tumors. Enhanced effects were observed when regorafenib was combined with irradiation and irinotecan against PDGFRA amplified IGRG93 glioma and IGRM57 medulloblastoma respectively, resulting in 100% tumor regressions. Antitumor activity was associated with decreased tumor vascularization, inhibition of PDGFR signaling, and induction of apoptotic cell death. Our work demonstrates that regorafenib exhibits significant antitumor activity in a wide spectrum of preclinical pediatric models through inhibition of angiogenesis and induction of apoptosis. Furthermore, radio- and chemosensitizing effects were observed with DNA damaging agents in PDGFR amplified tumors.

Ifosfamide (IFO) is an alkylating agent used in routine clinical practices for treatment of cancer for many years. As a prodrug it requires cytochrome P450 bio-activation (Giraud et al. Expert Opin.Drug Metab. Toxicol. 2010, 6, 919-938) oxidising the position C-4 leading to the formation of the 4-HO-IFO (10%) which releases of the active compound (isophosphoramide mustard) responsible for the DNA cross links. However, during high dose protocols, this bio-activation leads to the release of two toxic metabolites: i) acrolein, an urotoxic compound, concomitantly generated with the active isophosphoramide mustard and, ii) chloroacetaldehyde, a neurotoxic and nephrotoxic compound, arising from the oxidation of the side chains (Kerbusch et al. Clin Pharmacokinet. 2001, 40, 41-62) responsible for limiting side-effects. To circumvent these toxic side-effects, we have designed several pre-activated compounds by covalent binding of several O- and S-alkyl moieties on the carbon C-4 to avoid cytochrome bio-activation favoring the release of the 4-HO-IFO, control this release by the physico-chemical properties of the engrafted moiety and limit the chloroacetaldehyde release. Among these designed compounds, some showed a self assembling property leading to nano-assemblies formulation. Moreover, the in vitro evaluation of these compounds in absence of cytochrome has revealed that one of them: the geranyloxy-ifosfamide showed a greater activity under nano-assemblies (600nM) compared to the bulk form (15μM) on rhabdomyosarcoma. This interesting finding has led us to deepen our investigation of this compound to understand and explain the meaning of this increased activity. We present here the latest results obtained for this compound from additional in vitro evaluation on a wide panel of cancer cell line, internalization studies within the cell. Moreover, transmission electron microscopy imaging of this formulation allows the determination of the shape of these nano-assemblies. Previous studies regarding the release kinetic profile of 4-HO-IFO of this compound in vitro in mice plasma at 37°C showed an interesting release profile which was increased when tested as nano-assemblies compared to the bulk form. To validate this finding, a pharmacokinetic study has also been investigated compared to IFO, showing a higher release of 4-HO-IFO compound suggesting that this compound might have an increased therapeutic index. Also, in vivo studies are being carried out at different dose to evaluate the efficacy of this compound (as bulk and nano-assemblies) on rhabdomyosarcoma xenografted mice compared to IFO. These results increase acknowledge of this compound which could become a promising candidate for both oral and IV administration in a wide panel of tumors. Citation Format: Charles Skarbek, Didier Desmaele, Alain Deroussent, Lea Lesueur, Estelle Daudigeos-Dubus, Ludivine Le Dret, Michael Rivard, Thierry Martens, Gilles Vassal, Patrick Couvreur, Angelo PACI. Geranyloxy-ifosfamide: A pre-activated ifosfamide analogue showing an increased therapeutic index. [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 4537. doi:10.1158/1538-7445.AM2015-4537

10049 Background: Angiogenesis plays a pivotal role in tumor growth and metastatic spread in children. High VEGF expression is correlated with invasion, metastases and risk for recurrence. In sever...

Oxazaphosphorines are alkylating antineoplasic agents used in the treatment of cancer and have demonstrated activity against various tumor types, such as soft tissue sarcomas or lymphomas. Oxazaphosphorines are non-toxic prodrugs that require cytochrome P450 (CYP) bioactivation. In the case of Ifosfamide (IFO), the bioactivation produces the 4-OH-IFO, which after ring opening liberates the active nitrogen mustard displaying DNA cross-links. Associated toxicities of IFO are due to 1) acrolein, an urotoxic metabolite concomitantly generated with the nitrogen mustard and 2) chloroacetaldehyde, a neuro and nephrotoxic metabolite obtained by oxidation of the side chains (Kerbusch et al., 2001). To circumvent these toxicities, we have designed pre-activated oxazaphosphorines through electrochemical oxidation and binding of various moieties such as O-alkyl groups and S-alkyl groups. IFO is activated by oxidation of position 4 with labile moieties leading to pre-activated cytotoxic entities. We observed physical-chemical characteristics leading to nano-assemblies depending on the length of the linked moieties, which could bring tissue specificity for drug delivery purposes. Some of these pre-activated prodrugs may be constitutive of drug delivery systems, such as nanoparticules, aiming to address alkylating moieties to their target. We present here the use of different length moieties, from C1 (methoxy) to C30 (Squalenoyl, SQ) with intermediate length of C5 (Pentoxy) and C10 (Geranioxy and DimethylOctanoxy). These compounds are pre-activated formulation of IFO with C4-oxidation allowing to by-pass the CYP activation with direct release of 4-OH-IFO and then the alkylating mustard. Some of them, SQ-(O)-IFO, SQ-(S)-IFO and Geranioxy-IFO are able to self-assembly leading to nanoparticules. The length and nature of the engrafted moieties allow the study of the parameters that lead the kinetics of liberation of the activated compound. The nanoparticules have been fully characterized with a mean diameter of 182 nm for SQ-(O)-IFO and SQ-(S)-IFO and 130 nm for Geranioxy-IFO. The cytotoxic activity of SQ-(O)-IFO and SQ-(S)-IFO has been studied in vitro on a panel of 15 human and murine cancer lines (RMS-1, RD, TC71, A673, SK-N-MC, UW 479, Lan-5, SAOS-2, IGR-OV1, MCF-7(MDR), A549, M109, KB 3.1, B16F10, MiaPaCa-2). The cytotoxic activity of Pentoxy-IFO, Geranioxy-IFO and DimethylOctanoxy-IFO has been studied in vitro on a panel of 2 human cancer lines (A673 and RMS-1). All IFO derivatives present high in vitro activity while IFO has no activity on these cells. This proves the pre-activated property of these new compounds and the lack of CYP activation requirements. Their therapeutic activity is currently studied in vivo on human Ewing sarcoma TC71 and human Lung cancer A549 xenografts in mice after i.v. administration. These compounds are now patented. Citation Format: Charles Skarbek, Hubert Chapuis, Léa Lesueur, Michael Rivard, Thierry Martens, Didier Desmaele, Estelle Daudigeos-Dubus, Jean-Rémi Bertrand, Alain Deroussent, Gilles Vassal, Patrick Couvreur, Angelo Paci. Enhanced activity of pre-activated oxazaphosphorine prodrugs designed for drug delivery strategy: influence of the length of the engrafted group. [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 5671. doi:10.1158/1538-7445.AM2013-5671

Background: The treatment of metastatic neuroblastoma remains a challenge in pediatric oncology. Multiple angiogenesis inhibitors targeting VEGF signaling pathway have been therapeutically validated in preclinical cancer models and several are explored in clinical trials. While antitumor effect and survival benefit are described, relapse or progressive disease typically ensues, reflecting multiple mechanisms of adaption to angiogenic therapies. Material and Methods: Rag-gammaC mice bearing orthotopic IGR-N91Luc human neuroblastoma following injection of 10 cells into the left adrenal gland were treated with the specific VEGFR-1−3 tyrosine kinase inhibitor at 30mg/kg/day during 28 days. Tumor growth was followed by ultrasound and bioluminescence; tumors and organs were harvested 24 hours after end of treatment to evaluate the presence of tumor cells. Comparative genomic analysis was performed on treated versus control tumors, metastases versus their respective primary tumors. Results: Significant tumor inhibition was detected at Day 25 (p = 0.0255) with a heterogeneous distribution of tumor volumes (median = 18mm, range 3−136) in treated mice as compared to controls (median= 55mm, range 9−330), associated with a decrease of vasculature density. While VEGFR1 and AKT remain activated in control and non responding tumors, both pathways were found inhibited in tumors that were responding to the specific VEGFR-1−3 tyrosine kinase inhibitor whereas SRC was activated. At autopsy, the pan-VEGFR inhibitor treated mice presented more frequently with macrometastases in liver and spleen as compared to controls. Metastases were confirmed by bioluminescence imaging. Through histology analyses, we detected metastases independent of primary tumor size and presence of infiltrating micrometastases. Gene expression analysis is currently ongoing. Conclusion: Inhibition of VEGFR pathways with the pan-VEGFR inhibitor exhibited moderate antitumor activity in the orthotopic IGR-N91 neuroblastoma model associated with inhibition of neovascularization. Enhanced detection of macrometastases and infiltrating micrometastases suggest activation of different signaling pathways associated with proliferation or migration involved in the escape mechanism to antiangiogenic treatment.

9555 Background: Notch pathway signaling is deregulated in many human cancers, notably leukemia. Our recent findings in primary ependymoma suggest the NOTCH1 pathway is also implicated in glial onc...