Giselle L. Saulnier Sholler

A phase 1 study of nifurtimox in patients with relapsed/refractory neuroblastoma.

The primary aim of this phase 1 study was to determine the maximum tolerated dose (MTD) and evaluate the safety of nifurtimox alone and in combination with cyclophosphamide and topotecan in multiple relapsed/refractory neuroblastoma pediatric patients. The secondary aim was to evaluate the pharmacokinetics of nifurtimox and the treatment response. To these ends, we performed a phase 1 dose escalation trial of daily oral nifurtimox with toxicity monitoring to determine the MTD, followed by 3 cycles of nifurtimox in combination with cyclophosphamide and topotecan. Samples were collected to determine the pharmacokinetic parameters maximum concentration, time at which maximum concentration is reached, and area under the curve between 0 and 8 hours. Treatment response was evaluated by radiographic and radionuclide (123I-metaiodobenzylguanidine) imaging, measurement of urinary catecholamines, and clearance of bone marrow disease. We determined the MTD of nifurtimox to be 30 mg/kg/d. The non-dose-limiting toxicities were mainly nausea and neuropathy. The dose-limiting toxicities of 2 patients at 40 mg/kg/d were a grade 3 pulmonary hemorrhage and a grade 3 neuropathy (reversible). Overall, nifurtimox was well tolerated by pediatric patients at a dose of 30 mg/kg/d, and tumor responses were seen both as a single agent and in combination with chemotherapy. A Phase 2 study to determine the antitumor efficacy of nifurtimox is currently underway.

Anti‐proliferative and Pro‐apoptotic Properties of 3‐Bromoacetoxy Calcidiol in High‐risk Neuroblastoma

The cytotoxic, anti‐proliferative and apoptotic effects of 3‐Bromoacetoxy Calcidiol (B3CD), a derivative of vitamin D3 precursor calcidiol, on human neuroblastoma (NB) cells were examined. NB, predominantly a tumor of early childhood, is the most common extracranial solid tumor. Despite aggressive treatments, survival for advanced stages remains low and novel treatment strategies are needed. B3CD‐induced apoptosis in various neuroblastic cells via caspases‐3 and ‐9 activation. B3CD upregulated mitochondrial pro‐apoptotic Bax and anti‐apoptotic Bcl‐2 expression, caused cytochrome c release, downregulated N‐Myc expression and activated pro‐survival marker Akt. Accordingly, B3CD treatment dose dependently reduced the viability of NB cells with IC50 values between 1 and 3 μm. The cytotoxicity of B3CD was significantly higher than for the calcemic parent‐compound vitamin D3 (IC50 between 10 and 30 μm). Further studies revealed that B3CD treatment inhibits the proliferation of NB cells at low concentrations (IC50 between 30 and 100 nm). Cell cycle analysis showed a dramatic increase in the apoptotic sub‐diploidal population along with a cell cycle block. In summary, the present study shows that B3CD is toxic to NB cells via suppression of cell proliferation and cell viability by caspase activation and regulation of survival signals. These results suggest that B3CD could be developed as a treatment for NB.

Embryonic sympathoblasts transiently express TrkB in vivo and proliferate in response to brain-derived neurotrophic factor in vitro

BackgroundNerve growth factor and neurotrophin-3 are involved in the development of sympathetic neurons; however, whether brain derived neurotrophic factor also plays a role is not known. The purpose of this study was to determine whether BDNF and its receptor, TrkB, are expressed during the development of paravertebral sympathetic ganglia in vivo and to determine the effect of BDNF in vitro.ResultsAs neural crest cells coalesce to form sympathetic ganglia, TrkB-positive cells are seen in both chicken and mouse embryos. In chicken embryos, TrkB-expressing cells first appear at Hamburger-Hamilton Stage (St) 27 and they co-express HNK-1, confirming that they are migrating neural crest cells. The TrkB-positive cells lack neural markers at this stage; however, they migrate with other neurally differentiating cells that are TrkA and TrkC-positive. By St. 29/30, TrkB-positive cells begin to express the neural specific markers Hu C/D and Islet-1; eventually, all TrkB positive cells commence neural differentiation. By St. 34, TrkB and TrkC staining are lost. BDNF transcript expression parallels that of TrkB. In the mouse, TrkB-positive cells surround newly formed sympathetic ganglia and a small number of TrkB positive cells that co-express tyrosine hydroxylase are seen within ganglia between E13.5-15. In cell culture, many cells from St. 29–30 chicken lumbar sympathetic ganglia express neural markers and are dividing, indicating that they are sympathoblasts. Sympathoblasts and neurons require both nerve growth factor and neurotrophin-3 for survival. BDNF increases the number of cells expressing neural markers in culture by increasing number of cells that incorporate bromodeoxyuridine. In contrast, most TrkB-positive sympathetic cells in vivo are not actively proliferating between E6–E8.ConclusionDeveloping paravertebral sympathetic ganglia in avian and murine embryos contain a subpopulation of sympathoblasts that transiently express TrkB and ultimately commence neuronal differentiation. These TrkB expressing sympathoblasts are not actively dividing in vivo; yet, when placed in vitro, will divide in response to BDNF. This suggests that the availability of BDNF in vivo fails to reach a threshold necessary to induce proliferation. We suggest that excess TrkB stimulation of sympathoblasts in vivo may lead to the genesis of neuroblastoma.

Oral RKS262 reduces tumor burden in a neuroblastoma xenograft animal model and mediates cytotoxicity through SAPK/JNK and ROS activation in vitro.

Patients diagnosed with high-risk neuroblastoma (NB), an extracranial solid tumor in children, have metastases and low survival (30%) despite aggressive multi-modal therapy. Therefore new therapies are urgently needed. We show significant in vitro and in vivo antitumor efficacy of RKS262 in NB. RKS262 showed superior cytotoxicity (IC50 = 6-25μM) against six representative NB cell lines compared to its parent analog Nifurtimox (currently in phase 2). Pre-formulated RKS262 (150mg/kg/daily) pellets administered orally, suppressed tumor growth (60%, p = 0.021) in NB xenograft mice within 28 days. RKS262-treated SMSKCNR cells showed TUNEL-positive DNA nicks and activation of ROS, MAPKs (SAPK/JNK), caspase-3, and p53, along with suppression of the IGF-1R/PI3K/PKC pathway and the Bcl2 family of proteins. RKS262 caused G2/M-phase arrest and suppressed cdc-2, cyclin B1, p21, and cyclin D1/D4 expression. N-acetyl-cysteine (NAC; 10mM) pre-treatment rescued cell viability of RKS262 (23µM)-treated SMSKCNR cells, and pre-treatment with ascorbic acid (100μM) and a MAPK inhibitor SB203580 (20μM) reversed SAPK/JNK, caspase-3 activation, PARP-1 cleavage, and suppression of IGF-1R, PI3K, and PKC phosphorylation. Further, treatment with exogenous BDNF (50nM) did not suppress SAPK/JNK or ROS activation due to RKS262. Rather, BDNF (50nM), EGF (100nM) and IGF-1 (100nM) co-treatment with RKS262 induced a remarkable S-phase arrest rather than a G2/M phase arrest when RKS262 was used alone. In summary, RKS262 shows oral efficacy in NB xenograft animals, and induces apoptosis in vitro in SMSKCNR cells via cell cycle arrest, MAPK and ROS activation, and suppression of IGF-1R/PI3K/PKC and Bcl2 family proteins in a growth factor (BDNF/EGF/IGF-1)-independent fashion.

Antitumor activity of nifurtimox is enhanced with tetrathiomolybdate in medulloblastoma.

Medulloblastoma, a neuroectodermal tumor arising in the cerebellum, is the most common brain tumor found in children. We recently showed that nifurtimox induces production of reactive oxygen species (ROS) and subsequent apoptosis in neuroblastoma cells both in vitro and in vivo. Tetrathiomolybdate (TM) has been shown to decrease cell proliferation by inhibition of superoxide dismutase-1 (SOD1). Since both nifurtimox and TM increase ROS levels in cells, we investigated whether the combination of nifurtimox and TM would act synergistically in medulloblastoma cell lines (D283, DAOY). Genome-wide transcriptional analysis, by hybridizing RNA isolated from nifurtimox and TM alone or in combination treated and control cells (D283) on Affymetrix exon array gene chips was carried out to further confirm synergy. We show that nifurtimox and TM alone and in combination decreased cell viability and increased ROS levels synergistically. Examination of cell morphology following drug treatment (nifurtimox + TM) and detection of caspase-3 activation via Western blotting indicated that cell death was primarily due to apoptosis. Microarray data from cells treated with nifurtimox and TM validated the induction of oxidative stress, as many Nrf2 target genes (HMOX1, GCLM, SLC7A11 and SRXN1) (p<10(-5)) were upregulated. Other genes related to apoptosis, oxidative stress, DNA damage, protein folding and nucleosome formation were differentially involved in cells following treatment with nifurtimox + TM. Taken together, our results suggest nifurtimox and TM act synergistically in medulloblastoma cells in vitro, and that this combination warrants further studies as a new treatment for medulloblastoma.

Chemotherapeutic Effect of Calcidiol Derivative B3CD in a Neuroblastoma Xenograft Model

Bromoacetoxy‐calcidiol (B3CD), a pro‐apoptotic and cytotoxic agent in neuroblastoma (NB) cell lines, displayed therapeutic potential in vivo as an anticancer drug in a NB xenograft mouse model. Tumors of all animals treated intraperitoneally with B3CD went into regression within 10–30 days of treatment, while tumors in control animals grew aggressively. The response mechanisms of NB cells to B3CD in vitro were studied and included differential targeting of cell cycle key regulators p21 and cyclin D1 on the transcriptional and expression level leading to arrest in G0/G1 phase. In contrast to the effect in ovarian cancer cells, B3CD‐induced cell death in SMS‐KCNR NB cells was only marginally mediated by the p38 MAPK signaling pathway. Signaling induced by exogenous recombinant EGF leads to a partial restoration of the negative effects of B3CD on SMS‐KCNR cell proliferation and survival. Upon combinational treatment of SMS‐KCNR cells with B3CD and recombinant EGF, the EGF receptor (EGF‐R) was highly activated. We suggest future studies to include analysis of the effects of B3CD in combination therapy with pharmacological inhibitors of cell cycle regulators or with EGF‐R‐targeting inhibitors, ‐toxins or ‐antibodies in vitro and their translation into in vivo models of tumor development.

Single tumor imaging with multiple antibodies targeting different antigens

Antibodies are important drugs for treating cancer and there is strong rationale for using multiple antibodies to improve outcomes. We labeled two breast cancer binding antibodies, anti-ErbB2 and anti-EpCAM, with infrared fluorescence dyes of different wavelengths and determined their in vivo distribution in a breast cancer xenograft model using a near-infrared (NIR) fluorescence imaging system. Our data show that these two antibodies can be readily assessed simultaneously in mouse xenograft model. This will help guide design of dosing strategies for multiple antibodies and identify potential interaction that could affect pharmacokinetics and possible side effects.

Abstract 1621: Tolcapone, a catechol-O-methyltransferase inhibitor, alone and in combination with oxaliplatin induces cell death in neuroblastoma

Background: Neuroblastoma (NB) is an aggressive childhood cancer that arises from neural crest cells of the sympathetic nervous system. These cells contain cathecholamines which may be a therapeutic target. Catechol-O-methyltransferase (COMT) metabolizes catechol-containing compounds, including dopamine. COMT inhibitors, including tolcapone, are used as an adjunctive treatment for Parkinson9s disease, as they increase dopamine within cells. Increase in dopamine and other catecholamines within cells may result in cell toxicity. Methods: Cell viability was measured using Calcein AM fluorescent assay at tolcapone doses 1.5625 μM - 200 μM, both alone and in combination with oxaliplatin at doses 1.5 μM - 6 μM. Western blot analysis was used to measure cleaved poly ADP ribose polymerase (PARP) and cleaved and full caspase-3 levels. ATP level per cell was measured using CyQuant fluorescent DNA assay combined with the Cell Titer GLO luminescent cell viability assay. Expression of COMT was confirmed using microarray analysis and immunofluorescence. Reactive oxygen species (ROS) levels were measured using DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit. IncuCyte ZOOM machine was used in conjugation with SYTOX Green dye to measure cell death and CellPlayer™ Kinetic Caspase-3/7 Apoptosis Assay Reagent to measure apoptosis over 48 hours. Microarray analysis using U133+ RNA expression profiles were used to evaluate gene expression changes after cells were treated with tolcapone for 12 hours. Results: Tolcapone IC50 values ranged from 13.33 μM to 156 μM in seven different NB cell lines. Concentrations of 25 μM and 50 μM increased levels of cleaved PARP and cleaved caspase-3 over 48, 72, and 96 hours treated. Intracellular ATP decreased significantly with concentrations ranging from 12.5-200 μM treated over 48 hours and ROS levels increased significantly with concentrations ranging from 12.5-200 μM treated over 24 hours. IncuCyte Zoom analysis displayed dose dependent levels of cell death and presence of cleaved caspase-3 and 7 with increasing concentrations ranging from 1.5625 μM -200 μM. The combination of tolcapone at 25 μM and oxaliplatin at 3.5 μM show synergy in cell viability assays. Conclusion: There is no curative therapy for relapsed/refractory NB patients. The preclinical evidence suggests that patients with overexpression of COMT may respond to tolcapone via catecholamine induced ROS and cytotoxicity, especially when combined with oxaliplatin. Therefore, tolcapone and oxaliplatin may be a potential new therapy for children with NB with plans to be evaluated in a Phase I/II trial. Citation Format: David E. Hayes, Ping Zhao, Austin Voydanoff, Abhinav Nagulapally, Jeff Bond, Giselle Sholler. Tolcapone, a catechol-O-methyltransferase inhibitor, alone and in combination with oxaliplatin induces cell death in neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1621. doi:10.1158/1538-7445.AM2015-1621

Abstract 3942: A pilot trial testing the feasibility of using molecular-guided therapy in patients with refractory or recurrent neuroblastoma

Background: Neuroblastoma is the most common extracranial solid tumor in children. The prognosis for infants with neuroblastoma is good, while only 30% of children diagnosed after 12-15 months of age survive despite aggressive multimodal therapies. There are currently few treatment options from which pediatric oncologists can select with any degree of confidence to improve the management of multiply recurrent neuroblastoma patients. The identification of agents through genomic profiling that target specific molecular pathways associated with the development and/or progression of neoplastic diseases holds promise. Improved approaches that identify effective existing agents in a rational, data-driven fashion may result in a survival benefit in the clinical setting, while avoiding the toxicity associated with agents that are unlikely to be beneficial. Method: The primary objective of this pilot study was to evaluate the feasibility of using predictive modeling based on genome-wide mRNA expression profiles of neuroblastoma tumor biopsies to make real-time treatment decisions. Feasibility was defined as the completion of the following in a two week time period: tumor biopsy, quality RNA extraction, mRNA U133 2+ Affymetrix gene chip hybridization, analysis utilizing a series of predictive methodologies, report generation, tumor board review with formulated treatment plan, and medical monitor review. Results: There were 5 patients enrolled between April-June 2010 with multiply relapsed or refractory neuroblastoma. Patients had received between 2-13 previous relapsed therapies and were between 2-6.5 years post diagnosis. All patients had soft tissue disease which was able to be biopsied. All biopsies were adequate by pathology evaluation (>75% viable tumor) and RNA quality (>6.5 RIN). Gene chips were completed in 3-7 days, report generation was 1-5 days, tumor board was 1-3 days, medical monitor sign off was 1 day. The total time was 10-12 days for all patients. The tumor board was able to create individualized therapy regimens for all patients. Correlative biology specimens were obtained and grown in culture. Mice xenografts of 2/5 patients were established. Cultures and xenografts were used for validation studies of predicted drug sensitivity. Conclusion: It is feasible to obtain real-time genomic profiling for molecularly guided therapy for use in treatment decision making. This warrants further testing in a Phase I study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3942. doi:10.1158/1538-7445.AM2011-3942

Abstract 5260: Bortezomib and HDAC inhibitor PCI-24781 combination therapy for neuroblastoma

Background: Neuroblastoma is an aggressive solid tumor with poor prognosis. Novel therapies are needed to help increase the survival and quality of life for affected children. It has been previously shown that bortezomib results in apoptosis in neuroblastoma both in vitro and in vivo. PCI-24781 is a novel pan-HDAC inhibitor which has been shown to be synergistic with bortezomib in lymphoma models. We hypothesize the combination of bortezomib with PCI-24781 would increase cytotoxicity to neuroblastoma. Methods: Several established neuroblastoma cell lines as well as patient-derived primary neuroblastoma cultures were grown in 96-well plates and treated with bortezomib and PCI-24781, both separately and in combination for 48 hours. Cell viability was assessed by calcein AM assays and IC509s and drug synergy was determined at each cell line tested. mRNA was collected from drug treated cell lines and effects on cell signaling was evaluated using U133 mRNA expression arrays and Ingenuity analysis. Cell lysates prepared from drug treated cells were evaluated by western blot for caspase-3 and PARP cleavage. Nude mice were injected with 107 SMS-KCNR cells subcutaneously in the left flank. Mice were treated with daily doses of either 0.5 mg/kg bortezomib, 12.5 mg/kg PCI-24781, or the combination. Mice tumors were imaged using the IVIS lumina imager twice per week and caliper measurements were also obtained weekly until tumor max was reached. Results: All neuroblastoma cell lines and patient cells tested showed sensitivity to bortezomib and PCI-24781 treatment in calcien AM cell viability assays with IC509s for bortezomib less than 50nM and IC509s for PCI-24781 less than 500nM. The combination of bortezomib and PCI-24781 was more cytotoxic than either drug alone. Cells treated with combined treatment showed an increase in caspase-3 and PARP activity confirming apoptosis. Expression analysis showed differential expression in the Notch and Wnt pathways between drug treated and vehicle treated cells. The xenograft models showed a significant decrease in tumor volume in mice treated with both bortezomib and PCI-24781 when compared to the single agent treatment groups or to the control group. Conclusions: Bortezomib and PCI-24781 inhibit neuroblastoma growth both in vitro and in vivo to a greater extent than either drug alone. The mechanism of action is currently being investigated further but certainly involves caspase-3 mediated apoptosis. This combination therapy appears to be effective and well tolerated in the mouse model and would be a novel therapy for neuroblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5260.