Mary K. Danks

Efficacy of topoisomerase I inhibitors, topotecan and irinotecan, administered at low dose levels in protracted schedules to mice bearing xenografts of human tumors

The efficacy of protracted schedules of therapy of the topoisomerase I inhibitors 9-dimethylaminomethyl-10-hydroxycamptothecin (topotecan) and 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin (irinotecan; CPT-11) were evaluated against a panel of 21 human tumor xenografts derived from adult and pediatric malignancies. Tumors included eight colon adenocarcinomas, representing an intrinsically chemorefractory malignancy, six lines derived from childhood rhabdomyosarcoma (three embryonal, three alveolar) representing a chemoresponsive histiotype, sublines of rhabdomyosarcomas selected in vivo for resistance to vincristine and melphalan, and three pediatric brain tmors. All tumors were grown at the subcutaneous site. Topotecan was administered by oral gavage 5 days per week for 12 consecutive weeks. The maximum tolerated dose (MTD) was 1.5 mg/kg per dose. Irinotecan was given by i.v. administration daily for 5 days each week for 2 weeks [(d×5)2] (one cycle of therapy), repeated every 21 days. The MTD for three cycles was 10 mg/kg per dose. Treatment was started against advanced tumors. Topotecan caused a high frequency of objective regressions in one of eight colon tumor lines, whereas irinotecan caused complete regressions (CR) of all tumors in three colon lines and a high frequency of CRs in three additional lines. Both drugs demonstrated similar activity against rhabdomyosarcoma xenografts. Topotecan caused CR of all tumors in four of six lines, and irinotecan in five of six lines evaluated. Both agents retained full activity against tumors selected for primary resistance to vincristine, but only irinotecan retained activity against a tumor selected for primary resistance to melphalan. Both agents demonstrated good activity against brain tumor xenografts with irinotecan causing CR in two of three lines and topotecan inducing CR in one of three lines. Results indicate that low-dose protracted schedules of daily administration of these topoisomerase I inhibitors is either equi-effective or more efficacious than more intense shorter schedules of administration reported previously.

Stem and progenitor cell-mediated tumor selective gene therapy

The poor prognosis for patients with aggressive or metastatic tumors and the toxic side effects of currently available treatments necessitate the development of more effective tumor-selective therapies. Stem/progenitor cells display inherent tumor-tropic properties that can be exploited for targeted delivery of anticancer genes to invasive and metastatic tumors. Therapeutic genes that have been inserted into stem cells and delivered to tumors with high selectivity include prodrug-activating enzymes (cytosine deaminase, carboxylesterase, thymidine kinase), interleukins (IL-2, IL-4, IL-12, IL-23), interferon-β, apoptosis-promoting genes (tumor necrosis factor-related apoptosis-inducing ligand) and metalloproteinases (PEX). We and others have demonstrated that neural and mesenchymal stem cells can deliver therapeutic genes to elicit a significant antitumor response in animal models of intracranial glioma, medulloblastoma, melanoma brain metastasis, disseminated neuroblastoma and breast cancer lung metastasis. Most studies reported reduction in tumor volume (up to 90%) and increased survival of tumor-bearing animals. Complete cures have also been achieved (90% disease-free survival for >1 year of mice bearing disseminated neuroblastoma tumors). As we learn more about the biology of stem cells and the molecular mechanisms that mediate their tumor-tropism and we identify efficacious gene products for specific tumor types, the clinical utility of cell-based delivery strategies becomes increasingly evident.

Crystal Structure of Human Carboxylesterase 1 Complexed with the Alzheimer's Drug Tacrine: From Binding Promiscuity to Selective Inhibition

Human carboxylesterase 1 (hCE1) is a broad-spectrum bioscavenger that plays important roles in narcotic metabolism, clinical prodrug activation, and the processing of fatty acid and cholesterol derivatives. We determined the 2.4 A crystal structure of hCE1 in complex with tacrine, the first drug approved for treating Alzheimers disease, and compare this structure to the Torpedo californica acetylcholinesterase (AcChE)-tacrine complex. Tacrine binds in multiple orientations within the catalytic gorge of hCE1, while it stacks in the smaller AcChE active site between aromatic side chains. Our results show that hCE1s promiscuous action on distinct substrates is enhanced by its ability to interact with ligands in multiple orientations at once. Further, we use our structure to identify tacrine derivatives that act as low-micromolar inhibitors of hCE1 and may provide new avenues for treating narcotic abuse and cholesterol-related diseases.

Tumor-Targeted Enzyme/Prodrug Therapy Mediates Long-term Disease-Free Survival of Mice Bearing Disseminated Neuroblastoma

Neural stem cells and progenitor cells migrate selectively to tumor loci in vivo. We exploited the tumor-tropic properties of HB1.F3.C1 cells, an immortalized cell line derived from human fetal telencephalon, to deliver the cDNA encoding a secreted form of rabbit carboxylesterase (rCE) to disseminated neuroblastoma tumors in mice. This enzyme activates the prodrug CPT-11 more efficiently than do human enzymes. Mice bearing multiple tumors were treated with rCE-expressing HB1.F3.C1 cells and schedules of administration of CPT-11 that produced levels of active drug (SN-38) tolerated by patients. Both HB1.F3.C1 cells and CPT-11 were given i.v. None of the untreated mice and 30% of mice that received only CPT-11 survived long term. In contrast, 90% of mice treated with rCE-expressing HB1.F3.C1 cells and 15 mg/kg CPT-11 survived for 1 year without detectable tumors. Plasma carboxylesterase activity and SN-38 levels in mice receiving both rCE-expressing HB1.F3.C1 cells (HB1.F3.C1/AdCMVrCE) and CPT-11 were comparable with those in mice receiving CPT-11 only. These data support the hypothesis that the antitumor effect of the described neural stem/progenitor cell-directed enzyme prodrug therapy (NDEPT) is mediated by production of high concentrations of active drug selectively at tumor sites, thereby maximizing the antitumor effect of CPT-11. NDEPT approaches merit further investigation as effective, targeted therapy for metastatic tumors. We propose that the described approach may have greatest use for eradicating minimum residual disease.

Development of a Tumor-Selective Approach to Treat Metastatic Cancer

Background Patients diagnosed with metastatic cancer have almost uniformly poor prognoses. The treatments available for patients with disseminated disease are usually not curative and have side effects that limit the therapy that can be given. A treatment that is selectively toxic to tumors would maximize the beneficial effects of therapy and minimize side effects, potentially enabling effective treatment to be administered. Methods and Findings We postulated that the tumor-tropic property of stem cells or progenitor cells could be exploited to selectively deliver a therapeutic gene to metastatic solid tumors, and that expression of an appropriate transgene at tumor loci might mediate cures of metastatic disease. To test this hypothesis, we injected HB1.F3.C1 cells transduced to express an enzyme that efficiently activates the anti-cancer prodrug CPT-11 intravenously into mice bearing disseminated neuroblastoma tumors. The HB1.F3.C1 cells migrated selectively to tumor sites regardless of the size or anatomical location of the tumors. Mice were then treated systemically with CPT-11, and the efficacy of treatment was monitored. Mice treated with the combination of HB1.F3.C1 cells expressing the CPT-11-activating enzyme and this prodrug produced tumor-free survival of 100% of the mice for >6 months (P<0.001 compared to control groups). Conclusions The novel and significant finding of this study is that it may be possible to exploit the tumor-tropic property of stem or progenitor cells to mediate effective, tumor-selective therapy for metastatic tumors, for which no tolerated curative treatments are currently available.

Brain Tumor Oncolysis with Replication-Conditional Herpes Simplex Virus Type 1 Expressing the Prodrug-Activating Genes, CYP2B1 and Secreted Human Intestinal Carboxylesterase, in Combination with Cyclophosphamide and Irinotecan

The treatment of malignant glioma is currently ineffective. Oncolytic viruses are being explored as a means to selectively lyse tumor cells in the brain. We have engineered a mutant herpes simplex virus type 1 with deletions in the viral UL39 and gamma(1)34.5 genes and an insertion of the two prodrug activating genes, CYP2B1 and secreted human intestinal carboxylesterase. Each of these can convert the inactive prodrugs, cyclophosphamide and irinotecan (CPT-11), into their active metabolites, respectively. This new oncolytic virus (MGH2) displays increased antitumor efficacy against human glioma cells both in vitro and in vivo when combined with cyclophosphamide and CPT-11. Importantly, cyclophosphamide, CPT-11, or the combination of cyclophosphamide and CPT-11 does not significantly affect oncolytic virus replication. Therefore, MGH2 provides effective multimodal therapy for gliomas in preclinical models when combined with these chemotherapy agents.

p53 mutation and MDM2 amplification frequency in pediatric rhabdomyosarcoma tumors and cell lines

BACKGROUND The p53 tumor suppressor gene is the most commonly mutated gene in human cancer, and mutations arise in a wide variety of tumor types. Wild-type p53 functions as a regulator of apoptosis, so mutations in the p53 gene are generally associated with aggressive tumors and a poor prognosis. PROCEDURE We have investigated the p53 mutation and MDM2 amplification frequencies in biopsies from pediatric rhabdomyosarcoma (RMS) tumors and cell lines by SSCP and Southern analyses. RESULTS A mutation was detected in only 1 of 20 tumor specimens (5%), whereas the frequency in established RMS cell lines was significantly higher (6/10, 60%). p53 Mutations were more common in cell lines derived from tumors previously exposed to chemotherapy compared to those derived from tumors at di-agnosis, and it is likely that these mutations enhanced the probability of successful long-term culture. The frequency of MDM2 gene amplification in patient biopsies was also low (2/20, 10%). Interestingly, complete responses to treatment were obtained in the two patients with tumors that demonstrated amplification of MDM2. The response to treatment of patients with tumors wild-type for p53 and without MDM2 amplification was quite varied, indicating that expression of a wild-type p53 gene at diagnosis cannot always facilitate a favorable outcome. CONCLUSIONS p53 mutation and MDM2 gene amplification frequencies are extremely low in RMS tumors, but a wild-type p53 genotype is not always associated with a favorable prognosis.

New therapeutic targets for the treatment of high-risk neuroblastoma

High‐risk neuroblastoma remains a major problem in pediatric oncology, accounting for 15% of childhood cancer deaths. Although incremental improvements in outcome have been achieved with the intensification of conventional chemotherapy agents and the addition of 13‐cis‐retinoic acid, only one‐third of children with high‐risk disease are expected to be long‐term survivors when treated with current regimens. In addition, the cost of cure can be quite high, as surviving children remain at risk for additional health problems related to long‐term toxicities of treatment. Further advances in therapy will require the targeting of tumor cells in a more selective and efficient way so that survival can be improved without substantially increasing toxicity. In this review we summarize ongoing clinical trials and highlight new developments in our understanding of the molecular biology of neuroblastoma, emphasizing potential targets or pathways that may be exploitable therapeutically. J. Cell. Biochem. 107: 46–57, 2009.

Drug resistance associated with altered DNA topoisomerase II

Abstract OLX-209 has readily measurable activity, is safe in experimental animals, and is efficacious in model systems. These results support the concept of OLX-209 and provide groundwork for further development of this oncoprotein targeted agent.

Decreased drug accumulation in a mitoxantrone-resistant gastric carcinoma cell line in the absence of P-glycoprotein

An established gastric‐carcinoma cell line, EPG85‐257P, is extremely sensitive to mitoxantrone (IC50, 0.12 ng/ml). Stepwise selection with mitoxantrone for 3 years resulted in a cell line (EPG85‐257RN) that is 7,056‐fold resistant to mitoxantrone (IC50, 846 ng/ml) and displays cross‐resistance to the topoisomerase(topo)‐II poisons ametantrone (411×), etoposide (112×) and teniposide (60×) as well as the topo‐I poisons 7‐ethyl‐10‐hydroxycamptothecin (331×) and topotecan (58×). We now show that this resistance is multifactorial. Western blotting revealed a 5‐fold decrease in topo‐IIα polypeptide in the mitoxantrone‐resistant cells. Immunohistochemistry and Western blotting failed to demonstrate P‐glycoprotein over‐expression. Formation of trapped topo‐II–DNA complexes in the resistant cells required higher mitoxantrone concentrations than in parental cells, even though nuclei isolated from the EPG85‐257RN cells formed cleavage complexes normally. In agreement with these observations, which suggest the possibility of a defect in mitoxantrone accumulation, examination of mitoxantrone accumulation in both cell lines by confocal laser microscopy revealed that the EPG85‐257RN cells accumulate less mitoxantrone at steady state. From these results, we propose that mitoxantrone accumulation, along with alterations in topo‐IIα expression, contribute to the resistance to mitoxantrone observed in these cells.Int. J. Cancer 71:817‐824, 1997.