Lack of radiosensitization of M443 on normal brain cells.

The microtubule inhibitor vincristine is currently used to treat a variety of brain tumors, including low-grade glioma and anaplastic oligodendroglioma. Vincristine, however, does not penetrate well into brain tumor tissue, and moreover, it displays dose-limiting toxicities, including peripheral neuropathy. Mebendazole, a Food and Drug Administration-approved anthelmintic drug with a favorable safety profile, has recently been shown to display strong therapeutic efficacy in animal models of both glioma and medulloblastoma. Importantly, appropriate formulations of mebendazole yield therapeutically effective concentrations in the brain. Mebendazole has been shown to inhibit microtubule formation, but it is not known whether its potency against tumor cells is mediated by this inhibitory effect. To investigate this, we examined the effects of mebendazole on GL261 glioblastoma cell viability, microtubule polymerization and metaphase arrest, and found that the effective concentrations to inhibit these functions are very similar. In addition, using mebendazole as a seed for the National Cancer Institute (NCI) COMPARE program revealed that the top-scoring drugs were highly enriched in microtubule-targeting drugs. Taken together, these results indicate that the cell toxicity of mebendazole is indeed caused by inhibiting microtubule formation. We also compared the therapeutic efficacy of mebendazole and vincristine against GL261 orthotopic tumors. We found that mebendazole showed a significant increase in animal survival time, whereas vincristine, even at a dose close to its maximum tolerated dose, failed to show any efficacy. In conclusion, our results strongly support the clinical use of mebendazole as a replacement for vincristine for the treatment of brain tumors.

Abstract Medulloblastoma is a cerebellar tumor and the most common pediatric brain malignancy. Radiotherapy is part of the standard care for this tumor, but its effectiveness is accompanied by significant neurocognitive sequelae due to the deleterious effects of radiation on the developing brain. We have previously shown that the protein kinase MRK/ZAK protects tumor cells from radiation-induced cell death by regulating cell-cycle arrest after ionizing radiation. Here, we show that siRNA-mediated MRK depletion sensitizes medulloblastoma primary cells to radiation. We have, therefore, designed and tested a specific small molecule inhibitor of MRK, M443, which binds to MRK in an irreversible fashion and inhibits its activity. We found that M443 strongly radiosensitizes UW228 medulloblastoma cells as well as UI226 patient–derived primary cells, whereas it does not affect the response to radiation of normal brain cells. M443 also inhibits radiation-induced activation of both p38 and Chk2, two proteins that act downstream of MRK and are involved in DNA damage–induced cell-cycle arrest. Importantly, in an animal model of medulloblastoma that employs orthotopic implantation of primary patient–derived UI226 cells in nude mice, M443 in combination with radiation achieved a synergistic increase in survival. We hypothesize that combining radiotherapy with M443 will allow us to lower the radiation dose while maintaining therapeutic efficacy, thereby minimizing radiation-induced side effects. Mol Cancer Ther; 15(8); 1799–808. ©2016 AACR.

Medulloblastoma is the most common malignant pediatric brain tumor. Although significant progress has been made in the treatment of medulloblastoma patients over the past several decades, the five-year disease-free survival for high-risk patients, characterized by metastatic dissemination at presentation or significant post-operative residual tumor, remains relatively poor (25-40%). Group 3 medulloblastoma has by far the worst prognosis with a 5-year survival probability of approximately 30% regardless of stage. Current therapeutic modalities, in particular ionizing radiation (IR), have significant long-term side-effects within the pediatric population. Enhancement of current therapies through targeted radiosensitization may allow for improved response and lead to decreased late radiation-induced sequelae. A bioinformatics approach was employed to select a number of candidate radioresistance genes that are preferentially expressed in group 3 medulloblastoma tumors. Candidate genes were tested for radiosensitization using RNA interference. Through this approach, we have identified and validated the antiapoptotic gene Bcl-XL as an effective target for the radiosensitization of medulloblastoma group 3 tumors. The siRNA results were confirmed with ABT-263, a small molecule inhibitor of Bcl-2 family members. ABT-263 potently radiosensitized D425MED group 3 cells with a dose enhancement factor (DEF) at 40% cell death of 2.2 and a concentration of 200 nM. As ABT-263 is not blood-brain barrier permeable, a murine model utilizing a paramagnetic gadolinium-based nanoparticle delivery system is currently being developed to examine the radiosensitizing efficacy of ABT-263 in vivo. In summary, radiosensitization of group 3 tumors is expected to be useful in improving survival outcomes in patients with this type of medulloblastoma. In addition, the localized action of the radiosensitizing effect provided by the focal delivery of the drug-coated nanoparticles is expected to reduce potential ABT-263-associated side-effects.

Medulloblastoma is the most common malignant pediatric brain tumor. Although significant progress has been made in the treatment of medulloblastoma patients over the past several decades, the five-year disease-free survival for high-risk patients, characterized by metastatic dissemination at presentation or significant post-operative residual tumor, remains relatively poor (25-40%). Group C medulloblastoma has by far the worst prognosis with a 5-year survival probability of approximately 30% regardless of stage. Current therapeutic modalities, in particular ionizing radiation (IR), have significant long-term side-effects within the pediatric population. Enhancement of current therapies through targeted radiosensitization may allow for IR dose reduction and lead to decreased late radiation-induced sequelae. A bioinformatics approach was employed to select a number of candidate radioresistance genes that are preferentially expressed in group C medulloblastoma tumors. Candidate genes were screened using focused RNA interference to assay for sensitization to ionizing radiation using primary cell cultures derived from group C tumors. We have identified and validated the antiapoptotic gene Bcl-XL as an effective target for the radiosensitization of medulloblastoma group C tumors through this approach. Radiosensitization of both sonic hedgehog (SHH) and group C cell lines was achieved in vitro with increasing doses of the Bcl-2 family small molecule inhibitor, ABT-263 (MW = 974 Da). After a 24 hour drug pre-treatment followed by irradiation (up to 9 Gy), a dose enhancement factor (DEF) after 40% cell death of 1.7 for UW228 (SHH) and 2.2 for D425MED (group C) cell lines was obtained. Group C cells were significantly more sensitive to treatment with 50 fold less drug required (10μM ABT-263 for the SSH subgroup vs. 200nM ABT-263 for group C). Similarly, siRNA targeting Bcl-XL was able to achieve a DEF of 1.9 in UW228 cells. A murine model utilizing a paramagnetic gadolinium-based nanoparticle delivery system to bypass the BBB is currently being developed to test ABT-263 in vivo as an effective radiosensitizer of human group C medulloblastoma tumors. In summary, radiosensitization of group C tumors is expected to be useful in improving local control and survival outcomes in patients with more aggressive subtypes of medulloblastoma. In the future, pre-clinical data generated from this approach will provide a rationale for the design of an early-phase clinical trial in order to test the safety and efficacy of pharmacological Bcl-family inhibition with concurrent radiation in the treatment of patients with group C medulloblastoma.

Abstract Medulloblastoma is a cerebellar tumor and the most common pediatric brain malignancy. Radiation therapy is part of the standard care for this tumor, but its effectiveness is accompanied by significant neurocognitive sequelae due to the deleterious effects of radiation on the developing brain. We have previously shown that the protein kinase MRK/ZAK protects tumor cells from radiation-induced cell death by regulating cell cycle arrest after ionizing radiation. Here we show that siRNA-mediated MRK depletion sensitizes medulloblastoma primary cells to radiation. We have, therefore, designed and tested a small molecule inhibitor of MRK, M443, which binds to MRK in an irreversible fashion and inhibits its activity. We found that M443 strongly radio-sensitizes UW228 medulloblastoma cells as well as IMB226 patient-derived primary cells. M443 also inhibits radiation-induced activation of both p38 and Chk2, two proteins that act downstream of MRK and are involved in DNA damage-induced cell cycle arrest. We also tested the effect of M443 in an animal model of medulloblastoma that employs orthotopic implantation of IMB226 medulloblastoma cells in nude mice. Intra-tumoral delivery of M443 alone significantly extended animal survival by 5 days compared to vehicle treatment. Combination treatment of M443 with radiation at 2 × 3 Gy, that is not effective on its own (1 day of additional survival over control), achieved a synergistic increase in survival (15 days over the control survival time). Western blotting of tumor lysates demonstrated strong inhibition of MRK activity. In conclusion, we have developed a new small molecule inhibitor of MRK/ZAK that radio-sensitizes medulloblastoma cells. We hypothesize that combining radio-therapy with M443 will allow us to lower the radiation dose while maintaining therapeutic efficacy, thereby minimizing radiation-induced side effects. Citation Format: Rosamaria Ruggieri, Daniel Markowitz, Caitlin Powell, Nhan Tran, Magimairajanissai Vanan, Mingzu He, Yousef Al-Abed, Marc Symons. Pharmacological inhibition of MRK/ZAK kinase for the treatment of medulloblastoma. [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 3338. doi:10.1158/1538-7445.AM2015-3338