A phase II trial of nifurtimox combined with topotecan and cyclophosphamide for refractory or relapsed neuroblastoma and medulloblastoma.

Children with relapsed/refractory (R/R) neuroblastoma (NB) and medulloblastoma (MB) have poor outcomes. We evaluated the efficacy of nifurtimox (Nfx) in a clinical trial for children with R/R NB and MB. Subjects were divided into three strata: first relapse NB, multiply R/R NB, and R/R MB. All patients received Nfx (30 mg/kg/day divided TID daily), Topotecan (0.75 mg/m2 /dose, days 1-5) and Cyclophosphamide (250 mg/m2 /dose, days 1-5) every 3 weeks. Response was assessed after every two courses using International Neuroblastoma Response Criteria and Response Evaluation Criteria in Solid Tumors (RECIST) criteria. One hundred and twelve eligible patients were enrolled with 110 evaluable for safety and 76 evaluable for response. In stratum 1, there was a 53.9% response rate (CR + PR), and a 69.3% total benefit rate (CR + PR + SD), with an average time on therapy of 165.2 days. In stratum 2, there was a 16.3% response rate, and a 72.1% total benefit rate, and an average time on study of 158.4 days. In stratum 3, there was a 20% response rate and a 65% total benefit rate, an average time on therapy of 105.0 days. The most common side effects included bone marrow suppression and reversible neurologic complications. The combination of Nfx, topotecan and cyclophosphamide was tolerated, and the objective response rate plus SD of 69.8% in these heavily pretreated populations suggests that this combination is an effective option for patients with R/R NB and MB. Although few objective responses were observed, the high percentage of stabilization of disease and prolonged response rate in patients with multiply relapsed disease shows this combination therapy warrants further testing.

A subset analysis of a phase II trial evaluating the use of DFMO as maintenance therapy for high-risk neuroblastoma.

Neuroblastoma is a sympathetic nervous system tumor, primarily presenting in children under 6 years of age. The long-term prognosis for patients with high-risk neuroblastoma (HRNB) remains poor despite aggressive multimodal therapy. This report provides an update to a phase II trial evaluating DFMO as maintenance therapy in HRNB. Event-free survival (EFS) and overall survival (OS) of 81 subjects with HRNB treated with standard COG induction, consolidation and immunotherapy followed by 2 years of DFMO on the NMTRC003/003b Phase II trial were compared to a historical cohort of 76 HRNB patients treated at Beat Childhood Cancer Research Consortium (BCC) hospitals who were disease-free after completion of standard upfront therapy and did not receive DFMO. The 2- and 5-year EFS were 86.4% [95% confidence interval (CI) 79.3%-94.2%] and 85.2% [77.8%-93.3%] for the NMTRC003/003b subset vs 78.3% [69.5%-88.3%] and 65.6% [55.5%-77.5%] for the historical control group. The 2- and 5-year OS were 98.8% [96.4-100%] and 95.1% [90.5%-99.9%] vs 94.4% [89.3%-99.9%] and 81.6% [73.0%-91.2%], respectively. DFMO maintenance for HRNB after completion of standard of care therapy was associated with improved EFS and OS relative to historical controls treated at the same institutions. These results support additional investigations into the potential role of DFMO in preventing relapse in HRNB.

A Phase 1 Trial of TPI 287 as a Single Agent and in Combination With Temozolomide in Patients with Refractory or Recurrent Neuroblastoma or Medulloblastoma.

BACKGROUND: The primary aim of this Phase I study was to determine the maximum tolerated dose (MTD) of TPI 287 and the safety and tolerability of TPI 287 alone and in combination with temozolomide (TMZ) in pediatric patients with refractory or recurrent neuroblastoma or medulloblastoma. The secondary aims were to evaluate the pharmacokinetics of TPI 287 and the treatment responses. PROCEDURE: Eighteen patients were enrolled to a phase I dose escalation trial of weekly intravenous infusion of TPI 287 for two 28-day cycles with toxicity monitoring to determine the MTD, followed by two cycles of TPI 287 in combination with TMZ. Samples were collected to determine the pharmacokinetic parameters C(max), AUC(0-24), t(1/2), CL, and Vd on day 1 of cycles 1 (TPI 287 alone) and 3 (TPI 287 + TMZ) following TPI 287 infusion. Treatment response was evaluated by radiographic (CT or MRI) and radionuclide (MIBG) imaging for neuroblastoma. RESULTS: We determined the MTD of TPI 287 alone and in combination with temozolomide to be 125 mg/m(2). The non-dose-limiting toxicities at this dose were mainly anorexia and pain. The dose-limiting toxicities (DLTs) of two patients at 135 mg/m(2) were grade 3 hemorrhagic cystitis and grade 3 sensory neuropathy. CONCLUSIONS: Overall, TPI 287 was well tolerated by pediatric patients with refractory and relapsed neuroblastoma and medulloblastoma at a dose of 125 mg/m(2) IV on days 1, 8, and 15 of a 28 day cycle.

Phase I study of vinblastine and sirolimus in pediatric patients with recurrent or refractory solid tumors.

BACKGROUND: The combination of vinblastine and mammalian target of rapamycin (mTOR) inhibitor sirolimus inhibits the growth of neuroblastoma xenografts through pro-apoptotic and anti-angiogenic mechanisms. This phase I study aimed to explore the safety and toxicity of this combination in pediatric patients with advanced solid tumors. PROCEDURE: Patients ≤21 years of age with recurrent/refractory solid tumors (including CNS) were eligible. Sirolimus was administered daily by mouth or nasogastric (NG) tube, with doses adjusted to achieve a target trough concentration of 10-15 ng/ml, with weekly intravenous vinblastine (dose escalated 4-6 mg/m(2)/dose according to 3 + 3 phase I design). RESULTS: Fourteen patients were enrolled (median age 8.7 years; range 2.3-19) of whom 12 were evaluable for toxicity and 11 for response. One patient experienced a dose-limiting toxicity (grade 3 mucositis) at the highest vinblastine dose level. Myelosuppression was the most common toxicity. Dose-adjusted sirolimus trough concentrations were significantly lower in patients receiving drug via NG tube (1.50 ± 0.75 ng/ml/mg vs. 2.25 ± 1.07 ng/ml/mg for oral administration). Correlative biomarker analysis demonstrated a significant reduction in serum concentration of soluble vascular endothelial growth factor receptor (sVEGFR2) at 28 days compared to baseline consistent with inhibition of angiogenesis. One patient had a partial response and three had stable disease for more than 3 months. CONCLUSIONS: The combination of mTOR inhibitor and vinblastine given over an extended continuous schedule is safe, associated with a reduction in circulating angiogenic factor (CAF) VEGFR2 and resulted in clinical responses. Future studies using the intravenously administered mTOR inhibitor temsirolimus are planned.

DFMO inhibition of neuroblastoma tumorigenesis.

BACKGROUND: Most high-risk neuroblastoma patients who relapse succumb to disease despite the existing therapy. We recently reported increased event-free and overall survival in neuroblastoma patients receiving difluoromethylornithine (DFMO) during maintenance therapy. The effect of DFMO on cellular processes associated with neuroblastoma tumorigenesis needs further elucidation. Previous studies have shown cytotoxicity with IC50 values >5-15 mM, these doses are physiologically unattainable in patients, prompting further mechanistic studies at therapeutic doses. METHODS: We characterized the effect of DFMO on cell viability, cell cycle, apoptosis, neurosphere formation, and protein expression in vitro using five established neuroblastoma cell lines (BE2C, CHLA-90, SHSY5Y, SMS-KCNR, and NGP) at clinically relevant doses of 0, 50, 100, 500, 1000, and 2500 µM. Limiting Dilution studies of tumor formation in murine models were performed. Statistical analysis was done using GraphPad and the level of significance set at p = 0.05. RESULTS: There was not a significant loss of cell viability or gain of apoptotic activity in the in vitro assays (p > 0.05). DFMO treatment initiated G1 to S phase cell cycle arrest. There was a dose-dependent decrease in frequency and size of neurospheres and a dose-dependent increase in beta-galactosidase activity in all cell lines. Tumor formation was decreased in xenografts both with DFMO-pretreated cells and in mice treated with DFMO. CONCLUSION: DFMO treatment is cytostatic at physiologically relevant doses and inhibits tumor initiation and progression in mice. This study suggests that DFMO, inhibits neuroblastoma by targeting cellular processes integral to neuroblastoma tumorigenesis at clinically relevant doses.

Eflornithine as Postimmunotherapy Maintenance in High-Risk Neuroblastoma: Externally Controlled, Propensity Score-Matched Survival Outcome Comparisons.

PURPOSE: Long-term survival in high-risk neuroblastoma (HRNB) is approximately 50%, with mortality primarily driven by relapse. Eflornithine (DFMO) to reduce risk of relapse after completion of immunotherapy was investigated previously in a single-arm, phase II study (NMTRC003B; ClinicalTrials.gov identifier: NCT02395666) that suggested improved event-free survival (EFS) and overall survival (OS) compared with historical rates in a phase III trial (Children Oncology Group ANBL0032; ClinicalTrials.gov identifier: NCT00026312). Using patient-level data from ANBL0032 as an external control, we present new analyses to further evaluate DFMO as HRNB postimmunotherapy maintenance. PATIENTS AND METHODS: NMTRC003B (2012-2016) enrolled patients with HRNB (N = 141) after standard up-front or refractory/relapse treatment who received up to 2 years of continuous treatment with oral DFMO (750 ± 250 mg/m2 twice a day). ANBL0032 (2001-2015) enrolled patients with HRNB postconsolidation, 1,328 of whom were assigned to dinutuximab (ch.14.18) treatment. Selection rules identified 92 NMTRC003B patients who participated in (n = 87) or received up-front treatment consistent with (n = 5) ANBL0032 (the DFMO/treated group) and 852 patients from ANBL0032 who could have been eligible for NMTRC003B after immunotherapy, but did not enroll (the NO-DFMO/control group). The median follow-up time for DFMO/treated patients was 6.1 years (IQR, 5.2-7.2) versus 5.0 years (IQR, 3.5-7.0) for NO-DFMO/control patients. Kaplan-Meier and Cox regression compared EFS and OS for overall groups, 3:1 (NO-DFMO:DFMO) propensity score-matched cohorts balanced on 11 baseline demographic and disease characteristics with exact matching on MYCN, and additional sensitivity analyses. RESULTS: DFMO after completion of immunotherapy was associated with improved EFS (hazard ratio [HR], 0.50 [95% CI, 0.29 to 0.84]; P = .008) and OS (HR, 0.38 [95% CI, 0.19 to 0.76]; P = .007). The results were confirmed with propensity score-matched cohorts and sensitivity analyses. CONCLUSION: The externally controlled analyses presented show a relapse risk reduction in patients with HRNB treated with postimmunotherapy DFMO.

Molecular-guided therapy for the treatment of patients with relapsed and refractory childhood cancers: a Beat Childhood Cancer Research Consortium trial.

BACKGROUND: Children with relapsed central nervous system (CNS tumors), neuroblastoma, sarcomas, and other rare solid tumors face poor outcomes. This prospective clinical trial examined the feasibility of combining genomic and transcriptomic profiling of tumor samples with a molecular tumor board (MTB) approach to make real­time treatment decisions for children with relapsed/refractory solid tumors. METHODS: Subjects were divided into three strata: stratum 1-relapsed/refractory neuroblastoma; stratum 2-relapsed/refractory CNS tumors; and stratum 3-relapsed/refractory rare solid tumors. Tumor samples were sent for tumor/normal whole-exome (WES) and tumor whole-transcriptome (WTS) sequencing, and the genomic data were used in a multi-institutional MTB to make real­time treatment decisions. The MTB recommended plan allowed for a combination of up to 4 agents. Feasibility was measured by time to completion of genomic sequencing, MTB review and initiation of treatment. Response was assessed after every two cycles using Response Evaluation Criteria in Solid Tumors (RECIST). Patient clinical benefit was calculated by the sum of the CR, PR, SD, and NED subjects divided by the sum of complete response (CR), partial response (PR), stable disease (SD), no evidence of disease (NED), and progressive disease (PD) subjects. Grade 3 and higher related and unexpected adverse events (AEs) were tabulated for safety evaluation. RESULTS: A total of 186 eligible patients were enrolled with 144 evaluable for safety and 124 evaluable for response. The average number of days from biopsy to initiation of the MTB-recommended combination therapy was 38 days. Patient benefit was exhibited in 65% of all subjects, 67% of neuroblastoma subjects, 73% of CNS tumor subjects, and 60% of rare tumor subjects. There was little associated toxicity above that expected for the MGT drugs used during this trial, suggestive of the safety of utilizing this method of selecting combination targeted therapy. CONCLUSIONS: This trial demonstrated the feasibility, safety, and efficacy of a comprehensive sequencing model to guide personalized therapy for patients with any relapsed/refractory solid malignancy. Personalized therapy was well tolerated, and the clinical benefit rate of 65% in these heavily pretreated populations suggests that this treatment strategy could be an effective option for relapsed and refractory pediatric cancers. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02162732. Prospectively registered on June 11, 2014.

A pilot study of genomic-guided induction therapy followed by immunotherapy with difluoromethylornithine maintenance for high-risk neuroblastoma.

BACKGROUND: Survival for patients with high-risk neuroblastoma (HRNB) remains poor despite aggressive multimodal therapies. AIMS: To study the feasibility and safety of incorporating a genomic-based targeted agent to induction therapy for HRNB as well as the feasibility and safety of adding difluoromethylornithine (DFMO) to anti-GD2 immunotherapy. METHODS: Twenty newly diagnosed HRNB patients were treated on this multicenter pilot trial. Molecular tumor boards selected one of six targeted agents based on tumor-normal whole exome sequencing and tumor RNA-sequencing results. Treatment followed standard upfront HRNB chemotherapy with the addition of the selected targeted agent to cycles 3-6 of induction. Following consolidation, DFMO (750 mg/m2 twice daily) was added to maintenance with dinutuximab and isotretinoin, followed by continuation of DFMO alone for 2 years. DNA methylation analysis was performed retrospectively and compared to RNA expression. RESULTS: Of the 20 subjects enrolled, 19 started targeted therapy during cycle 3 and 1 started during cycle 5. Eighty-five percent of subjects met feasibility criteria (receiving 75% of targeted agent doses). Addition of targeted agents did not result in toxicities requiring dose reduction of chemotherapy or permanent discontinuation of targeted agent. Following standard consolidation, 15 subjects continued onto immunotherapy with DFMO. This combination was well-tolerated and resulted in no unexpected adverse events related to DFMO. CONCLUSION: This study demonstrates the safety and feasibility of adding targeted agents to standard induction therapy and adding DFMO to immunotherapy for HRNB. This treatment regimen has been expanded to a Phase II trial to evaluate efficacy.

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 (I-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.

Genomic and Transcriptomic Analysis of Relapsed and Refractory Childhood Solid Tumors Reveals a Diverse Molecular Landscape and Mechanisms of Immune Evasion.

Children with treatment-refractory or relapsed (R/R) tumors face poor prognoses. As the genomic underpinnings driving R/R disease are not well defined, we describe here the genomic and transcriptomic landscapes of R/R solid tumors from 202 patients enrolled in Beat Childhood Cancer Consortium clinical trials. Tumor mutational burden (TMB) was elevated relative to untreated tumors at diagnosis, with one-third of tumors classified as having a pediatric high TMB. Prior chemotherapy exposure influenced the mutational landscape of these R/R tumors, with more than 40% of tumors demonstrating mutational signatures associated with platinum or temozolomide chemotherapy and two tumors showing treatment-associated hypermutation. Immunogenomic profiling found a heterogenous pattern of neoantigen and MHC class I expression and a general absence of immune infiltration. Transcriptional analysis and functional gene set enrichment analysis identified cross-pathology clusters associated with development, immune signaling, and cellular signaling pathways. While the landscapes of these R/R tumors reflected those of their corresponding untreated tumors at diagnosis, important exceptions were observed, suggestive of tumor evolution, treatment resistance mechanisms, and mutagenic etiologies of treatment. SIGNIFICANCE: Tumor heterogeneity, chemotherapy exposure, and tumor evolution contribute to the molecular profiles and increased mutational burden that occur in treatment-refractory and relapsed childhood solid tumors.

Nifurtimox induces apoptosis of neuroblastoma cells in vitro and in vivo.

Neuroblastoma is the most common extracranial solid tumor in children and, when disseminated, carries a poor prognosis. Even with aggressive combinations of chemotherapy, surgery, autologous bone marrow transplant, and radiation, long-term survival remains at 30% and new therapies are needed. Recently, a patient with neuroblastoma who acquired Chagas disease was treated with nifurtimox with subsequent reduction in tumor size. The effect of nifurtimox on the neuroblastoma cell lines CHLA-90, LA1-55n, LA-N2, SMS-KCNR, and SY5Y was examined. Nifurtimox decreased cell viability in a concentration-dependent manner. Cell morphology, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay, and caspase-3 activation indicate that cell death was primarily due to apoptosis. Nifurtimox also suppressed basal and TrkB-mediated Akt phosphorylation, and the cytotoxicity of nifurtimox was attenuated by a tyrosine hydroxylase inhibitor (alpha-methyl-tyrosine). Nifurtimox killed catecholaminergic, but not cholinergic, autonomic neurons in culture. In vivo xenograft models showed inhibition of tumor growth with a histologic decrease in proliferation and increase in apoptosis. These results suggest that nifurtimox induces cell death in neuroblastoma. Therefore, further studies are warranted to develop nifurtimox as a promising new treatment for neuroblastoma.

Maintenance DFMO Increases Survival in High Risk Neuroblastoma.

High risk neuroblastoma (HRNB) accounts for 15% of all pediatric cancer deaths. Despite aggressive therapy approximately half of patients will relapse, typically with only transient responses to second-line therapy. This study evaluated the ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) as maintenance therapy to prevent relapse following completion of standard therapy (Stratum 1) or after salvage therapy for relapsed/refractory disease (Stratum 2). This Phase II single agent, single arm multicenter study enrolled from June 2012 to February 2016. Subjects received 2 years of oral DFMO (750 ± 250 mg/m2 twice daily). Event free survival (EFS) and overall survival (OS) were determined on an intention-to-treat (ITT) basis. 101 subjects enrolled on Stratum 1 and 100 were eligible for ITT analysis; two-year EFS was 84% (±4%) and OS 97% (±2%). 39 subjects enrolled on Stratum 2, with a two-year EFS of 54% (±8%) and OS 84% (±6%). DFMO was well tolerated. The median survival time is not yet defined for either stratum. DFMO maintenance therapy for HRNB in remission is safe and associated with high EFS and OS. Targeting ODC represents a novel therapeutic mechanism that may provide a new strategy for preventing relapse in children with HRNB.

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

BACKGROUND: Nerve 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. RESULTS: As 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. CONCLUSION: Developing 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.

Feasibility of implementing molecular-guided therapy for the treatment of patients with relapsed or refractory neuroblastoma.

The primary objective of the study was to evaluate the feasibility and safety of a process which would utilize genome-wide expression data from tumor biopsies to support individualized treatment decisions. Current treatment options for recurrent neuroblastoma are limited and ineffective, with a survival rate of <10%. Molecular profiling may provide data which will enable the practitioner to select the most appropriate therapeutic option for individual patients, thus improving outcomes. Sixteen patients with neuroblastoma were enrolled of which fourteen were eligible for this study. Feasibility was defined as completion of tumor biopsy, pathological evaluation, RNA quality control, gene expression profiling, bioinformatics analysis, generation of a drug prediction report, molecular tumor board yielding a treatment plan, independent medical monitor review, and treatment initiation within a 21 day period. All eligible biopsies passed histopathology and RNA quality control. Expression profiling by microarray and RNA sequencing were mutually validated. The average time from biopsy to report generation was 5.9 days and from biopsy to initiation of treatment was 12.4 days. No serious adverse events were observed and all adverse events were expected. Clinical benefit was seen in 64% of patients as stabilization of disease for at least one cycle of therapy or partial response. The overall response rate was 7% and the progression free survival was 59 days. This study demonstrates the feasibility and safety of performing real-time genomic profiling to guide treatment decision making for pediatric neuroblastoma patients.

A Phase I Trial of DFMO Targeting Polyamine Addiction in Patients with Relapsed/Refractory Neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is the most common cancer in infancy and most frequent cause of death from extracranial solid tumors in children. Ornithine decarboxylase (ODC) expression is an independent indicator of poor prognosis in NB patients. This study investigated safety, response, pharmacokinetics, genetic and metabolic factors associated with ODC in a clinical trial of the ODC inhibitor difluoromethylornithine (DFMO) ± etoposide for patients with relapsed or refractory NB. METHODS AND FINDINGS: Twenty-one patients participated in a phase I study of daily oral DFMO alone for three weeks, followed by additional three-week cycles of DFMO plus daily oral etoposide. No dose limiting toxicities (DLTs) were identified in patients taking doses of DFMO between 500-1500 mg/m2 orally twice a day. DFMO pharmacokinetics, single nucleotide polymorphisms (SNPs) in the ODC gene and urinary levels of substrates for the tissue polyamine exporter were measured. Urinary polyamine levels varied among patients at baseline. Patients with the minor T-allele at rs2302616 of the ODC gene had higher baseline levels (p=0.02) of, and larger decreases in, total urinary polyamines during the first cycle of DFMO therapy (p=0.003) and had median progression free survival (PFS) that was over three times longer, compared to patients with the major G allele at this locus although this last result was not statistically significant (p=0.07). Six of 18 evaluable patients were progression free during the trial period with three patients continuing progression free at 663, 1559 and 1573 days after initiating treatment. Median progression-free survival was less among patients having increased urinary polyamines, especially diacetylspermine, although this result was not statistically significant (p=0.056). CONCLUSIONS: DFMO doses of 500-1500 mg/m2/day are safe and well tolerated in children with relapsed NB. Children with the minor T allele at rs2302616 of the ODC gene with relapsed or refractory NB had higher levels of urinary polyamine markers and responded better to therapy containing DFMO, compared to those with the major G allele at this locus. These findings suggest that this patient subset may display dependence on polyamines and be uniquely susceptible to therapies targeting this pathway. TRIAL REGISTRATION: Clinicaltrials.gov NCT#01059071.

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.

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 (IC(50) = 6-25 µM) against six representative NB cell lines compared to its parent analog Nifurtimox (currently in phase 2). Pre-formulated RKS262 (150 mg/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 G(2)/M-phase arrest and suppressed cdc-2, cyclin B1, p21, and cyclin D1/D4 expression. N-acetyl-cysteine (NAC; 10 mM) pre-treatment rescued cell viability of RKS262 (23 µM)-treated SMSKCNR cells, and pre-treatment with ascorbic acid (100 µM) and a MAPK inhibitor SB203580 (20 µM) reversed SAPK/JNK, caspase-3 activation, PARP-1 cleavage, and suppression of IGF-1R, PI3K, and PKC phosphorylation. Further, treatment with exogenous BDNF (50 nM) did not suppress SAPK/JNK or ROS activation due to RKS262. Rather, BDNF (50 nM), EGF (100 nM) and IGF-1 (100 nM) co-treatment with RKS262 induced a remarkable S-phase arrest rather than a G(2)/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.

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.

Induction of cytotoxicity, apoptosis and cell cycle arrest by 1-t-butyl carbamoyl, 7-methyl-indole-3-ethyl isothiocyanate (NB7M) in nervous system cancer cells.

Our group has recently developed 1-(t)butyl carbamoyl, 7-methyl-indole-3-ethyl isothiocyanate (NB7M), a novel indole ethyl isothiocyanate analog. We now describe its selective cytotoxicity in both central nervous system (CNS) and neuroblastoma (NB) cancer cells. In an effort to understand its mechanism of action we examined the effects of NB7M on apoptosis, cell cycle arrest, and pro-survival/mitogen-activated protein kinase (MAPK) signaling in neuroblastoma cells. NB7M proved highly cytotoxic to NB cell lines (SMS-KCNR, SK-N- SH, SH-SY5Y, IMR-32) with IC(50) values ranging from 1.0-2.0 microM, whereas lung fibroblasts were less affected (IC(50) > or =10 microM). In the NCI 60 cell screen 1-dose assay, NB7M (10 microM) reduced the growth (-89 to -27 % growth) of CNS cancer cell lines SF-268, SF-295, SNB-75 (glioblastoma), SF-539 (gliosarcoma), and U251 (astroglioma) while SNB-19 glioblastoma cells were relatively resistant (19% growth). Hoechst staining of SMS-KCNR cells treated with NB7M (3 microM) for 24 hrs exhibited significant chromatin condensation and DNA fragmentation, whereas Annexin-v/7AAD staining revealed that the majority of cells accumulated in the early-apoptotic and late-apoptotic/necrotic stages. NB7M treatment of SMS-KCNR and SH-SY5Y cells also led to the cleavage of procaspases-3, and PARP-1 while causing activation of pro-apoptotic MAPKs and down-regulation of pro-survival factors AKT and PI-3K. Furthermore, NB7M treatment caused S-phase arrest in SMSKCNR and G1-phase arrest in SH-SY5Y cells. NB7M is active against CNS cancers and NB.

Effect of indole ethyl isothiocyanates on proliferation, apoptosis, and MAPK signaling in neuroblastoma cell lines.

Several indole ethyl isothiocyanate (IEITC) analogs were designed, synthesized, and screened to evaluate their cytotoxicity against neuroblastoma (NB) cells in-vitro. In NB, predominantly a tumor of early childhood, survival remains low despite aggressive treatments. Therefore, novel treatment strategies are greatly needed. The objective of the present study was to study the therapeutic potential of IEITC by analyzing the cytotoxic, anti-proliferative, and apoptotic effects on NB cell lines. 7-Methyl-indole-3-ethyl isothiocyanate (7Me-IEITC) proved to be cytotoxic to various NB cell lines (SMS-KCNR, SK-N-SH, SH-SY5Y, and IMR-32) with an IC(50) at 2.5-5.0 microM, while primary control cells (lung fibroblasts) were not affected. 7Me-IEITC led to the activation of apoptotic markers caspase-3, -8, and -9, caused activation of pro-apoptotic p38 MAPK and SAP/JNK, and down-regulated pro-survival factor AKT in SMS-KCNR cells. Moreover, 7Me-IEITC displayed anti-proliferative effects (IC(50) at 600 nM) and caused an arrest in cell cycle progression. This wide effect of 7Me-IEITC on NB cell signaling and survival suggests that it could be developed as a therapeutic agent against neuroblastoma.