Don W. Coulter

Clofarabine salvage therapy in refractory multifocal histiocytic disorders, including Langerhans cell histiocytosis, juvenile xanthogranuloma and Rosai-Dorfman disease

Existing therapies for recurrent or refractory histiocytoses, including Langerhans cell histiocytosis (LCH), juvenile xanthogranuloma (JXG), and Rosai–Dorfman disease (RDD), have limited effectiveness. We report our experience with using clofarabine as therapy in children with recurrent or refractory histiocytic disorders, including LCH (11 patients), systemic JXG (4 patients), and RDD (3 patients).

Refractory autoimmune hemolytic anemia after intestinal transplant responding to conversion from a calcineurin to mTOR inhibitor

AIHA is a rare and serious complication of solid organ transplantation. Herein, we report four cases of warm or mixed AIHA in pediatric patients following combined liver, small bowel and pancreas transplant. The hemolysis was refractory to multiple treatment modalities including steroids, rituximab, IVIG, plasmapheresis, cytoxan, discontinuation of prophylactic penicillin, and a change in immunosuppression from tacrolimus to cyclosporine. All patients had resolution or marked improvement of hemolysis after discontinuation of maintenance of CNI and initiation of sirolimus immunosuppression. One patient developed nephrotic syndrome but responded to a change in immunosuppression to everolimus. Three of the four patients continue on immunosuppression with sirolimus or everolimus without further hemolysis, evidence of rejection or medication side effects. Based on our experience and review of similar cases in the literature, we have proposed a treatment algorithm for AIHA in the pediatric intestinal transplant patient population that recommends an early change in immunosuppressive regimen from CNIs to sirolimus therapy.

Valproic acid reduces the tolerability of temsirolimus in children and adolescents with solid tumors

A pediatric study has established a maximum tolerated dose (MTD) for temsirolimus (Tem) of more than 150 mg/m2 intravenously/week. A phase I trial was conducted to establish the MTD for Tem in combination with valproic acid (VPA) in children and adolescents with refractory solid tumors. The secondary aims included expression of mammalian target of rapamycin (mTOR) markers on archival tumor tissue; Tem pharmacokinetics; assessment of histone acetylation (HA); and tumor response. Patients were treated with VPA (5 mg/kg orally three times daily) with a target serum level of 75–100 mcg/ml. Tem was started at an initial dose of 60 mg/m2/week. Pharmacokinetics and HA measurements were performed during weeks 1 and 5. Two of the first three patients experienced dose-limiting toxicity (grade 3 mucositis). Tem at 35 mg/m2/week was found to be tolerable. Peak Tem concentrations were higher in all patients compared with those in previously published reports of single agent Tem. Increases in HA are correlated with VPA levels. All tumor samples expressed mTORC1 and mTORC2. An objective response was observed in one patient (melanoma), whereas transient stable disease was observed in four other patients (spinal cord ependymoma, alveolar soft part sarcoma, medullary thyroid carcinoma, and hepatocellular carcinoma). The MTD of Tem when administered with VPA is considerably lower than when used as a single agent, with mucositis the major dose-limiting toxicity. The combination merits further study and may have activity in melanoma. Attention to drug–drug interactions will be important in future multiagent trials including Tem.

Butyrylcholinesterase as a Blood Biomarker in Neuroblastoma.

Blood-based biomarkers are important in the detection of the disease and in the assessment of responses to therapy. In this study, butyrylcholinesterase was evaluated as a potential biomarker in newly diagnosed neuroblastoma (NB) patients at diagnosis and longitudinally during treatment. Plasma butyrylcholinesterase activities in age-matched and sex-matched children were used as controls. Pretreatment butyrylcholinesterase levels in NB subjects are on an average 2 times lower than butyrylcholinesterase levels in healthy subjects. Significantly, butyrylcholinesterase activities are ∼40% lower in MYCN-amplified as compared with nonamplified disease. As the course of chemotherapy progresses, butyrylcholinesterase activities recover and normalize to control values. The evident response to treatment indicates that plasma butyrylcholinesterase is a good biomarker of tumor response to therapy. Depressed butyrylcholinesterase levels in NB subjects are not caused by hepatic deficits suggesting a specific role for butyrylcholinesterase in NB. Further examination of the mechanism of altered butyrylcholinesterase production require an animal model that best approximates human condition. Studies in mice show that murine NB allografts significantly reduce butyrylcholinesterase activity in plasma. This finding correlates with changes observed in NB patients. In contrast, human NB xenografts produce the opposite effect, that is, butyrylcholinesterase plasma levels rise as the xenograft size increases. In the absence of any liver damage, dissimilarities between butyrylcholinesterase production in murine and human NB models suggest species-specific signaling pathways. This disparity also suggests that human NB xenograft mouse models do not approximate the human disease.

Abstract 4362: Screening of investigational antimalarials for anticancer activity in high risk N-MYC amplified neuroblastoma (NB)

Introduction: NB is a pediatric tumor of neural crest origin. Patients who have N-MYC amplified metastatic tumors have poor outcomes. The importance of autophagy in protecting NB cells from chemotherapy has been postulated. In the following study we screened bisquinoline (BQ) and ozonide (OZ) antimalarials (potential autophagy inhibitors) in a highly N-MYC amplified NB cell line (BE-2) and a moderately amplified N-MYC NB cell line (IMR-32). A combination of monolayer cell culture and neurospheres were used to screen activity. Methods: Using a 96 well MMT based assay and 16 hour treatment with compounds dissolved in DMSO, cytotoxicity was calculated as a percent decline in absorbance compared to DMSO controls. 32 BQ and 13 OZ antimalarials were tested. All compounds were tested at a concentration of 1 mcg/ml; an achievable concentration in patients. Selected compounds that were cytotoxic in monolayer culture will be tested in neurosphere culture and then in a mouse xenograft model alone and combined with chemotherapy. Results: The bisquinolone antimalarials were consistently active in both BE-2 and IMR cell culture. Among the bisquinolones, Q1-9, Q2-5, Q2-61, and Q3-75 had the greatest cytotoxicity. There was no clear structure-activity-relationship for this small bisquinoline library. Among the ozonides, OZ465, OZ513, and OZ521 were the most active. From these data, we could infer that the presence of a weak base functional group was required, but was insufficient for cytotoxicity. Initial studies in BE-2 and IMR neurosphere culture using an LDH assay for cytotoxicity suggests similar activity. Monolayer results are shown in the Table below: *Percent decline in absorbance compared to DMSO control Conclusion: The above data indicates that two classes of antimalarials had moderate cytotoxicity in both 96-well monolayer assays as well as in a neurosphere assay (data not shown). The results suggest cytotoxic activity in high and moderately amplified N-MYC NB in both monolayers and neurospheres. Note: This abstract was not presented at the meeting. Citation Format: Don W. Coulter, Jonathan Vennerstrom, John G. Sharp, Yuxiang Dong, Xiaofang Wang, Erin McIntyre, Tim McGuire. Screening of investigational antimalarials for anticancer activity in high risk N-MYC amplified neuroblastoma (NB). [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 4362. doi:10.1158/1538-7445.AM2015-4362

Suppression of STAT3 NH2-terminal domain chemosensitizes medulloblastoma cells by activation of protein inhibitor of activated STAT3 via de-repression by microRNA-21

Medulloblastoma (MB) is a malignant pediatric brain tumor with poor prognosis. Signal transducers and activators of transcription‐3 (STAT3) is constitutively activated in MB where it functions as an oncoprotein, mediating cancer progression and metastasis. Here, we have delineated the functional role of activated STAT3 in MB, by using a cell permeable STAT3‐NH2 terminal domain inhibitor (S3‐NTDi) that specifically perturbs the structure/function of STAT3. We have implemented several biochemical experiments using human MB tumor microarray (TMA) and pediatric MB cell lines, derived from high‐risk SHH‐TP53‐mutated and MYC‐amplified Non‐WNT/SHH tumors. Treatment of MB cells with S3‐NTDi leads to growth inhibition, cell cycle arrest, and apoptosis. S3‐NTDi downregulated expression of STAT3 target genes, delayed migration of MB cells, attenuated epithelial‐mesenchymal transition (EMT) marker expressions and reduced cancer stem‐cell associated protein expressions in MB‐spheres. To elucidate mechanisms, we showed that S3‐NTDi induce expression of pro‐apoptotic gene, C/EBP‐homologous protein (CHOP), and decrease association of STAT3 to the proximal promoter of CCND1 and BCL2. Of note, S3‐NTDi downregulated microRNA‐21, which in turn, de‐repressed Protein Inhibitor of Activated STAT3 (PIAS3), a negative regulator of STAT3 signaling pathway. Furthermore, combination therapy with S3‐NTDi and cisplatin significantly decreased highly aggressive MYC‐amplified MB cell growth and induced apoptosis by downregulating STAT3 regulated proliferation and anti‐apoptotic gene expression. Together, our results revealed an important role of STAT3 in regulating MB pathogenesis. Disruption of this pathway with S3‐NTDi, therefore, may serves as a promising candidate for targeted MB therapy by enhancing chemosensitivity of MB cells and potentially improving outcomes in high‐risk patients.

Complexation of Chol-DsiRNA in place of Chol-siRNA greatly increases the duration of mRNA suppression by polyplexes of PLL(30)-PEG(5K) in primary murine syngeneic breast tumors after i.v. administration

&NA; RNA interference has tremendous potential for cancer therapy but is limited by the insufficient potency of RNAi molecules after i.v. administration. We previously found that complexation with PLL(30)‐PEG(5K) greatly increases the potency of 3′‐cholesterol‐modified siRNA [Chol‐siRNA] in primary murine syngeneic 4T1 breast tumors after i.v. administration but mRNA suppression decreases 24 h after the final dose. We hypothesized that complexation of cholesterol‐modified Dicer‐substrate siRNA (Chol‐DsiRNA) in place of Chol‐siRNA can increase the potency and duration of suppression by polyplexes of PLL(30)‐PEG(5K) in solid tumors. We found that replacing Chol‐siRNA with Chol‐DsiRNA increased polyplex loading and nuclease protection, suppressed stably expressed luciferase to the same extent in primary murine 4T1‐Luc breast tumors under the current dosage regimen, but maintained suppression ˜72 h after the final dose. The kinetics of suppression in 4T1‐Luc over 72 h, however, were similar between DsiLuc and siLuc after electroporation and between polyplexes of Chol‐DsiLuc and Chol‐siLuc after transfection, suggesting that Chol‐DsiRNA polyplexes increase the duration of mRNA suppression through differences in polyplex activities in vivo. Thus, replacing Chol‐siRNA with Chol‐DsiRNA may significantly increase the duration of mRNA suppression by polyplexes of PLL(30)‐PEG(5K) and possibly other PEGylated polycationic polymers in primary tumors and metastases after i.v. administration. Graphical abstract Figure. No caption available.

PD-L1, inflammation, non-coding RNAs, and neuroblastoma: Immuno-oncology perspective

Neuroblastoma is the most common pediatric solid tumor of neural crest origin. The current treatment options for neuroblastoma produce severe side effects. Programmed death-ligand 1 (PD-L1), chronic inflammation, and non-coding RNAs are known to play a significant role in the pathogenesis of neuroblastoma. Cancer cells and the surrounding cells in the tumor microenvironment express PD-L1. Programmed death-1 (PD-1) is a co-receptor expressed predominantly by T cells. The binding of PD-1 to its ligands, PD-L1 or PD-L2, is vital for the physiologic regulation of the immune system. Chronic inflammation is involved in the recruitment of leukocytes, production of cytokines and chemokines that in turn, lead to survival, metastasis, and angiogenesis in neuroblastoma tumors. The miRNAs and long non-coding (lnc) RNAs have emerged as a novel class of non-coding RNAs that can regulate neuroblastoma associated cell-signaling pathways. The dysregulation of PD-1/PD-L1, inflammatory pathways, lncRNAs, and miRNAs have been reported in clinical and experimental samples of neuroblastoma. These signaling molecules are currently being evaluated for their potential as the biomarker and therapeutic targets in the management of neuroblastoma. A monoclonal antibody called dinutuximab (Unituxin) that attaches to a carbohydrate molecule GD2, on the surface of many neuroblastoma cells, is being used as an immunotherapy drug for neuroblastoma treatment. Atezolizumab (Tecentriq), an engineered monoclonal antibody against PD-L1, are currently in clinical trial for neuroblastoma patients. The lncRNA/miRNA-based therapeutics is being developed to deliver tumor suppressor lncRNAs/miRNAs or silencing of oncogenic lncRNAs/miRNAs. The focus of this review is to discuss the current knowledge on the immune checkpoint molecules, PD-1/PD-L1 signaling, inflammation, and non-coding RNAs in neuroblastoma.

Abstract B07: Treatment of a chemoresistant human neuroblastoma cell-line (BE-2c) with experimental ozonide antimalarials

Objective: To evaluate the anti-tumor activity of a class of experimental antimalarial drugs in an MYCN amplified chemoresistant neuroblastoma cell line (BE-2c) intended to model poor prognostic neuroblastoma. Methods: Etoposide cytotoxicity was studied at concentrations (250 ng/ml,500 ng/ml, 1 mcg/ml, 2 mcg/ml, and 3 mcg/ml) to confirm chemoresistance to a commonly used cytotoxic agent in the treatment of neuroblastoma. Concentrations chosen for the ozonide antimalarials were those that have been achieved in-vivo when studied in a malarial model in rodents. Ozonide antimalarial drugs were tested in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay which measures cell viability colorimetrically. A series of 13 ozonide compounds were evaluated for activity using a concentration response scheme (0, 250 ng/ml, 500 ng/ml, 1 mcg/ml, 5 mcg/ml, and 10 mcg/ml) that allowed the generation of IC50 for each agent based on mean MTT absorption values from 10 replicates at each concentration. The most active ozonide (OZ513) identified was then studied in a cell cycle flow cytometry analysis using propidium idodide with measurement of the A0 peak which estimates apoptosis. A neurosphere assay was also used which is considered enriched for “cancer stem cells” and is able to model movement of drug into a small avascular tumor was used to further evaluate the activity of OZ513. Spheres were counted and characterized after treatment and compared to non-treatment controls. Because it has been postulated that the anti-cancer mechanism of action of the ozonide antimalarials is the modulation of cancer cell metabolism (autophagy, disruption of oxidative metabolism) the effect of OZ513 on mitochondrial oxidative metabolism and glycolysis was evaluated in BE-2c cells using Seahorse metabolic analysis. Results: Etoposide had no cytotoxic activity at any of the concentrations studied. OZ513 antimalarials were consistently active in BE-2c cell culture with the MTT assay generating IC50s on two separate experiments of 0.03 and 0.05 mcg/ml. The MTT results were confirmed using trypan blue staining to directly measure cell death. The Ao peak on PI flow cytometry increased after treatment with OZ513 indicating an increase in apoptosis. In addition, 5 mcg/ml of OZ513 increased G1, S, and G2 aneuploid fraction on propidium iodide cell cycle analysis. OZ513 disrupted BE-2c neurospheres confirming their activity in these multicellular tumorspheres. Metabolic studies demonstrated little effect on mitochondria based oxidative metabolism or glycolysis. Structure activity relationship suggests that the alcohol of the ozonide structure is required for optimal activity. Conclusion: The antimalarial agent OZ513 has promising activity against the chemoresistant neuroblastoma cell-line BE-2c by increasing apoptosis by mechanisms that remain unclear, but increased cell cycle aneuploidy with treatment of cells with 5 mcg/ml of OZ513 indicates defects in mitosis and cytokinesis. Citation Format: Don W. Coulter, Timothy R. McGuire, John G. Sharp, Yuxiang Dong, Xiaofang Wang, Erin McIntyre, Xiaoyu Chen, Jon Vennerstrom. Treatment of a chemoresistant human neuroblastoma cell-line (BE-2c) with experimental ozonide antimalarials. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B07.

Abstract B05: Improved therapy for neuroblastoma using small molecule inhibitors in combination with chemotherapy

Neuroblastoma is the most common pediatric malignancy with poor response to current therapy. The NF-kB/mTOR, hedgehog and PLK1 pathways/molecules are aberrantly expressed and activated in high-risk neuroblastoma, thereby targeting these pathways/molecules is an attractive therapeutic strategy. Therefore, we investigated the efficacy and associated molecular mechanism(s) of NF-kB/mTOR dual inhibitor 13-197, hedgehog inhibitor Vismodegib and PLK1 inhibitor BI2536 alone or in combination with Topotecan against high-risk neuroblastoma in vitro and in vivo. Using three neuroblastoma cell lines, the in vitro efficacy of the inhibitors as single agents or in combination with Topotecan on cell growth and apoptosis along with associated molecular mechanism(s) were investigated. In addition, the combined efficacy of Vismodegib and Topotecan was determined in vivo using a xenograft mouse model. Results showed that 13-197, BI2536 and Vismodegib significantly decreased neuroblastoma cell growth and induced apoptosis by targeting associated pathways and molecules in vitro. The 13-197 and BI2536 as single agents showed similar efficacies and were most potent in inhibiting cell growth and survival of neuroblastoma cells. In combination with Topotecan, BI2536 or Vismodegib further significantly decreased neuroblastoma cell growth/survival and neurospheres formation. Corresponding changes in the expression of targeted molecules following therapy were observed. Together, in vitro data demonstrated that hedgehog pathway inhibitor Vismodegib was most efficacious in potentiating Topotecan-induced antineuroblastoma effects. Therefore, as a next logical step, we tested the combined efficacy of Vismodegib and Topotecan against neuroblastoma in vivo using NOD-SCID Gamma deleted (NSG) mice. Our in vivo results showed that Vismodegib combined with Topotecan significantly (p Citation Format: Nagendra K. Chaturvedi, McGuire R. Timothy, Don W. Coulter, Ashima Shukla, Erin M. McIntyre, J. Graham Sharp, Shantaram S. Joshi. Improved therapy for neuroblastoma using small molecule inhibitors in combination with chemotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B05.