Overcoming Temozolomide Resistance using MGMT-targeting Spherical Nucleic Acids

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DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM) is the most prevalent primary central nervous system malignancy. Due to the aggressive nature of these tumors and our inability to adequately treat them, only 3-5% of patients survive longer than 3 years post-diagnosis. The standard of care for newly diagnosed GBM is surgical resection followed by adjuvant radiotherapy and temozolomide (TMZ) chemotherapy. TMZ cytotoxicity is mediated primarily through methylation of the O6-position of guanine. In the majority of patients, this methyl group is rapidly removed by the enzyme O6-methylguanine-DNA methyltransferase (MGMT), conferring resistance to the chemotherapy. However, in a subset of GBM patients, sometime during the course of their tumor development the promoter region for MGMT is methylated. This epigenetic silencing of MGMT activity allows TMZ to induce apoptosis in glioblastoma cells and drastically increases survival in GBM patients. Patients with a methylated promoter region have a much higher two-year survival rate (49%) than patients without a methylated MGMT promoter region (15%). Furthermore, in the rare long-term survivors of GBM, 74% of patients were found to have a methylated MGMT promoter region. This project seeks to knockdown MGMT expression in GBM cells and subsequently administer TMZ to recapitulate the improved survival phenotype observed in patients with a methylated MGMT promoter region. We will utilize small interfering RNA (siRNA) duplexes densely conjugated to the surface of gold nanoparticles (MGMTi-Spherical Nucleic Acids (SNAs)). These particles possess unique characteristics that confer advantages over other gene transfection reagents, including (1) simultaneous transfection and gene regulation independent of auxiliary transfection agents or lipoplexes, (2) rapid internalization by all cell types including neurons, (3) superior stability i physiological environments including resistance to nuclease degradation, (4) minimal activation of the innate immune response and no acute toxicity at high doses in animal models, and (5) capacity to cross the blood-brain barrier (BBB) and blood-tumor barrier (BTB), penetrate xenografted, intracranial tumors, silence GBM oncogenes, and increase survival in mice. We will use patient-derived tumor neurospheres (TNS) for in vitro and in vivo experiments; for in vivo experiments, we will generate intracranial xenografts from TNS cultures. Prior to running in vivo experiments, we will establish a relationship between MGMT, TMZ, and cell death in our TNS cultures. MGMT knockdown by MGMTi-SNAs will be assessed with RT-qPCR, western blot and an MGMT repair assay. Quantification of apoptotic markers will include: annexin V positivity, mitochondrial membrane integrity, caspase activity, and assessment of proteolytic cleavage. In vivo studies with orthotopic GBM mouse models will assess the combination treatment of MGMTi-SNAs and TMZ; we intend to (1) enhance intratumoral apoptosis, (2) reduce glioma formation, and (3) increase survival time.