Cori Bradley

Targeting Oxidative Stress in Embryonal Rhabdomyosarcoma

Rhabdomyosarcoma is a soft-tissue sarcoma with molecular and cellular features of developing skeletal muscle. Rhabdomyosarcoma has two major histologic subtypes, embryonal and alveolar, each with distinct clinical, molecular, and genetic features. Genomic analysis shows that embryonal tumors have more structural and copy number variations than alveolar tumors. Mutations in the RAS/NF1 pathway are significantly associated with intermediate- and high-risk embryonal rhabdomyosarcomas (ERMS). In contrast, alveolar rhabdomyosarcomas (ARMS) have fewer genetic lesions overall and no known recurrently mutated cancer consensus genes. To identify therapeutics for ERMS, we developed and characterized orthotopic xenografts of tumors that were sequenced in our study. High-throughput screening of primary cultures derived from those xenografts identified oxidative stress as a pathway of therapeutic relevance for ERMS.

Targeting the p53 Pathway in Retinoblastoma with Subconjunctival Nutlin-3a

Retinoblastoma is a rare childhood cancer of the retina that begins in utero and is diagnosed in the first years of life. The goals of retinoblastoma treatment are ocular salvage, vision preservation, and reduction of short- and long-term side effects without risking mortality because of tumor dissemination. To identify better chemotherapeutic combinations for the treatment of retinoblastoma, several groups have developed genetic mouse models and orthotopic xenograft models of human retinoblastoma for preclinical testing. Previous studies have implicated the MDMX protein in the suppression of the p53 pathway in retinoblastoma and shown that the MDM2/MDMX antagonist, Nutlin-3a, can efficiently induce p53-mediated cell death in retinoblastoma cell lines. However, Nutlin-3a cannot be administered systemically to treat retinoblastoma, because it has poor penetration across the blood-ocular barrier. Therefore, we developed an ocular formulation of Nutlin-3a, Nutlin-3a(OC), and tested the pharmacokinetics and efficacy of this new formulation in genetic and human retinoblastoma orthotopic xenograft models of retinoblastoma. Here, we show that Nutlin-3a(OC) specifically and efficiently targets the p53 pathway and that the combination of Nutlin-3a(OC) with systemic topotecan is a significantly better treatment for retinoblastoma than currently used chemotherapy in human orthotopic xenografts. Our studies provide a new standardized approach to evaluate and prioritize novel agents for incorporation into future clinical trials for retinoblastoma.

Targeting the DNA Repair Pathway in Ewing Sarcoma

Ewing sarcoma (EWS) is a tumor of the bone and soft tissue that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive, but patients with metastatic or recurrent disease have a poor outcome. We found that EWS cell lines are defective in DNA break repair and are sensitive to PARP inhibitors (PARPis). PARPi-induced cytotoxicity in EWS cells was 10- to 1,000-fold higher after administration of the DNA-damaging agents irinotecan or temozolomide. We developed an orthotopic EWS mouse model and performed pharmacokinetic and pharmacodynamic studies using three different PARPis that are in clinical development for pediatric cancer. Irinotecan administered on a low-dose, protracted schedule previously optimized for pediatric patients was an effective DNA-damaging agent when combined with PARPis; it was also better tolerated than combinations with temozolomide. Combining PARPis with irinotecan and temozolomide gave complete and durable responses in more than 80% of the mice.

Preclinical models for neuroblastoma: establishing a baseline for treatment.

Background Preclinical models of pediatric cancers are essential for testing new chemotherapeutic combinations for clinical trials. The most widely used genetic model for preclinical testing of neuroblastoma is the TH-MYCN mouse. This neuroblastoma-prone mouse recapitulates many of the features of human neuroblastoma. Limitations of this model include the low frequency of bone marrow metastasis, the lack of information on whether the gene expression patterns in this system parallels human neuroblastomas, the relatively slow rate of tumor formation and variability in tumor penetrance on different genetic backgrounds. As an alternative, preclinical studies are frequently performed using human cell lines xenografted into immunocompromised mice, either as flank implant or orthtotopically. Drawbacks of this system include the use of cell lines that have been in culture for years, the inappropriate microenvironment of the flank or difficult, time consuming surgery for orthotopic transplants and the absence of an intact immune system. Principal Findings Here we characterize and optimize both systems to increase their utility for preclinical studies. We show that TH-MYCN mice develop tumors in the paraspinal ganglia, but not in the adrenal, with cellular and gene expression patterns similar to human NB. In addition, we present a new ultrasound guided, minimally invasive orthotopic xenograft method. This injection technique is rapid, provides accurate targeting of the injected cells and leads to efficient engraftment. We also demonstrate that tumors can be detected, monitored and quantified prior to visualization using ultrasound, MRI and bioluminescence. Finally we develop and test a “standard of care” chemotherapy regimen. This protocol, which is based on current treatments for neuroblastoma, provides a baseline for comparison of new therapeutic agents. Significance The studies suggest that use of both the TH-NMYC model of neuroblastoma and the orthotopic xenograft model provide the optimal combination for testing new chemotherapies for this devastating childhood cancer.

Orthotopic patient-derived xenografts of paediatric solid tumours

Paediatric solid tumours arise from endodermal, ectodermal, or mesodermal lineages. Although the overall survival of children with solid tumours is 75%, that of children with recurrent disease is below 30%. To capture the complexity and diversity of paediatric solid tumours and establish new models of recurrent disease, here we develop a protocol to produce orthotopic patient-derived xenografts at diagnosis, recurrence, and autopsy. Tumour specimens were received from 168 patients, and 67 orthotopic patient-derived xenografts were established for 12 types of cancer. The origins of the patient-derived xenograft tumours were reflected in their gene-expression profiles and epigenomes. Genomic profiling of the tumours, including detailed clonal analysis, was performed to determine whether the clonal population in the xenograft recapitulated the patient’s tumour. We identified several drug vulnerabilities and showed that the combination of a WEE1 inhibitor (AZD1775), irinotecan, and vincristine can lead to complete response in multiple rhabdomyosarcoma orthotopic patient-derived xenografts tumours in vivo.

Pharmacokinetics and efficacy of the spleen tyrosine kinase inhibitor r406 after ocular delivery for retinoblastoma.

PurposeRetinoblastoma is a childhood cancer of the retina. Clinical trials have shown that local delivery of broad spectrum chemotherapeutic agents is efficacious. Recent studies characterizing the genomic and epigenomic landscape of retinoblastoma identified spleen tyrosine kinase (SYK) as a promising candidate for targeted therapy. The purpose of this study was to conduct preclinical testing of the SYK antagonist R406 to evaluate it as a candidate for retinoblastoma treatment.MethodsThe efficacy of the SYK antagonist R406 delivered locally in a human orthotopic xenograft mouse model of retinoblastoma was tested. Intraocular exposure of R406 was determined for various routes and formulations.ResultsThere was no evidence of efficacy for subconjunctival. R406. Maximal vitreal concentration was 10-fold lower than the minimal concentration required to kill retinoblastoma cells in vitro. Dosage of R406 subconjunctivally from emulsion or suspension formulations, direct intravitreal injection of the soluble prodrug of R406 (R788), and repeated topical administration of R406 all increased vitreal exposure, but failed to reach the exposure required for retinoblastoma cell death in culture.ConclusionTaken together, these data suggest that R406 is not a viable clinical candidate for the treatment of retinoblastoma. This study highlights the importance of pharmacokinetic testing of molecular targeted retinoblastoma therapeutics.

Development and characterization of a human orthotopic neuroblastoma xenograft

Neuroblastoma is a pediatric cancer of the developing sympathoadrenal lineage. The tumors are known to develop from the adrenal gland or paraspinal ganglia and have molecular and cellular features of sympathetic neurons such as dense core vesicles and catecholamine production. Here we present the detailed molecular, cellular, genetic and epigenetic characterization of an orthotopic xenograft derived from a high-risk stage 4 neuroblastoma patient. Overall, the xenografted tumor retained the high risk features of the primary tumor and showed aggressive growth and metastasis in the mouse. Also, the genome was preserved with no additional copy number variations, structural variations or aneuploidy. There were 13 missense mutations identified in the xenograft that were not present in the patient’s primary tumor and there were no new nonsense mutations. None of the missense mutations acquired in the xenograft were in known cancer genes. We also demonstrate the feasibility of using the orthotopic neuroblastoma xenograft to test standard of care chemotherapy and molecular targeted therapeutics. Finally, we optimized a new approach to produce primary cultures of the neuroblastoma xenografts for high-throughput drug screening which can be used to test new combinations of therapeutic agents for neuroblastoma.

Targeting ALK in pediatric RMS does not induce antitumor activity in vivo

PurposeThe anaplastic lymphoma kinase (ALK) has been demonstrated to be a valid clinical target in diseases such as anaplastic large cell lymphoma and non-small cell lung cancer. Recent studies have indicated that ALK is overexpressed in pediatric rhabdomyosarcoma (RMS) and hence we hypothesized that this kinase may be a suitable candidate for therapeutic intervention in this tumor.MethodsWe evaluated the expression of ALK in a panel of pediatric RMS cell lines and patient-derived xenografts (PDX), and sensitivity to ALK inhibitors was assessed both in vitro and in vivo.ResultsEssentially, all RMS lines were sensitive to crizotinib, NVP-TAE684 or LDK-378 in vitro, and molecular analyses demonstrated inhibition of RMS cell proliferation following siRNA-mediated reduction of ALK expression. However, in vivo PDX studies using ALK kinase inhibitors demonstrated no antitumor activity when used as single agents or when combined with standard of care therapy (vincristine, actinomycin D and cyclophosphamide). More alarmingly, however, crizotinib actually accelerated the growth of these tumors in vivo.ConclusionsWhile ALK appears to be a relevant target in RMS in vitro, targeting this kinase in vivo yields no therapeutic efficacy, warranting extreme caution when considering the use of these agents in pediatric RMS patients.

Abstract A09: Preclinical trial using the combination of panobinostat with bortezomib in human orthotopic xenograft mouse models of rhabdomyosarcoma

Background: Rhabdomyosarcoma (RMS) is an aggressive malignancy of childhood which has a poor prognosis when it has metastasized. Although there has been improvement in overall survival for low and intermediate risk patients, there has been no significant improvement in outcome in the past 25 years for patients with high risk disease. Orthotopic patient derived mouse xenografts (O-PDXs) have been created from patients with high risk disease to facilitate preclinical trials and provide useful information regarding efficacy and toxicity of novel therapeutic agents. Histone deacetylases (HDAC) have emerged as a relevant clinical target in RMS and other pediatric solid tumors, and the synergistic combination of HDAC inhibitors with proteasome inhibitors has led to increased cellular stress and apoptosis in other malignancies. The HDAC inhibitor panobinostat combined with bortezomib, a proteasome inhibitor, is a combination currently in consideration for clinical trial development for this patient population. Methods: Orthotopic xenografts were created by injecting patient derived RMS cells into the hind-leg muscle of CD-1 nude mice. Detailed molecular and histopathologic characterization of the tumors was performed to ensure they recapitulate human disease. Primary cultures of dissociated O-PDXs were used for high-throughput drug screening in dose response. Pharmacokinetic studies on RMS tumor-bearing mice were performed to determine matched human AUC-guided dosing and schedule of chemotherapy agents. Preclinical phase I tolerability studies were performed for all drug combinations. The following preclinical phase II study was performed using 2 different O-PDXs (SJRBH000026_X1, SJRHB012_Y) both derived from patients with metastatic recurrent embryonal RMS: (SJRBH000026_X1, SJRHB012_Y) Group 1 (n = 5, n = 4) Panobinostat/Bortezomib Group 2 (n = 3, n = 3) Vincristine/Irinotecan (relapse standard of care) Group 3 (n = 3, n = 3) Control (no chemotherapy) Enrollment into the 3 treatment groups was randomized for each of the O-PDXs tested. Two courses of therapy were given on a clinically relevant schedule with each course consisting of 21 days. The study endpoint was reached and mice were classified as progressive disease when tumor approached 20% of body weight. Results: Both O-PDXs tested in vitro were found to be highly sensitive to panobinostat and bortezomib with EC50 values in the nanomolar range for both drugs. Drug combinations were found to be well tolerated in preclinical phase I studies at the doses tested with only temporary mild dehydration observed in the panobinostat/bortezomib group. In the preclinical phase II study, mice in group 1 were found to have an average survival of 16.5 days (avg. of 11 days, 22 days respectively). Mice receiving the standard of care therapy (group 2) survived an average of 40.7 days (avg. of 49 days, 32.3 days respectively). Mice in group 3 were determined to have progressive disease on average at 17 days (avg. of 12.3 days, 21.7 days respectively). Conclusions: On the basis of preclinical in vitro data, combining HDAC and proteasome inhibitors is an attractive strategy for RMS and was found to be tolerable in preclinical phase I studies. However, the use of this combination in O-PDXs failed to show meaningful efficacy. The lack of response in relevant RMS models raises concerns about the sensitivity observed in in vitro studies and additional biologic and pharmacokinetic studies are indicated to identify factors leading to this discrepancy prior to further clinical development of these agents for this patient population. Citation Format: Elizabeth Stewart, Cori Bradley, Anang Shelat, Alberto Pappo, Michael A. Dyer. Preclinical trial using the combination of panobinostat with bortezomib in human orthotopic xenograft mouse models of rhabdomyosarcoma. [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 A09.