Radiosensitization of Glioblastoma Using Targeted Inhibition of N-Myristoylation

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Treating Glioblastoma remains a major challenge. Radiotherapy has a long-standing role in extending survival, but almost all tumors are expected to recur and be fatal. Historic dose-escalation studies do not yield additional benefit beyond the current 60 Gy dose. Further therapeutic improvement can be gained with radiosensitizers. An undiscovered potential radiosensitization approach may exist in inhibiting the N-myristoylation pathways upregulated in malignant cells. Inhibiting this pathway has been shown to have pleotropic effects including decreasing PARP-1 activity and other cancer-driving pathways that may synergize with radiotherapy. This is a first report in using a targeted N-myristoyltransferase (NMT) inhibitor as a radiosensitizer in glioblastoma. Immortalized glioma cell lines (U87 and U251) and patient-derived de novo (ED501) and post-chemoradiation recurrent (ED512) cell lines were used to a NMT1/2 inhibitor with radiotherapy. Cell surface expression levels for NMT1 and NMT2 were established using immunofluorescence microscopy. In vitro cell viability studies via mitochondrial reduction of resazurin dye and clonogenic assays were done with single fraction 2 & 4 Gy irradiation with 5 days of drug exposure (5-150 nM 3 days prior to irradiation and 2 days post) to account for drug pharmacodynamics. PARP-1 expression by western blot was tested with exposure to drug and/or radiation. Whole exome sequencing and methylation profiling were done to find predictive markers of radiosensitization. Preliminary results show significant radiosensitization effect with a dose enhancement factor ranging from 1.33 to 2.71x higher than radiation alone. The amount of radiosensitization varied per cell line, with U251 and ED512 being more sensitive. The cell line sensitivity was not reliably predicted by the NMT cell surface receptor expression. PARP-1 activity increased with radiotherapy but was inhibited with drug exposure. Exome sequencing showed the radiosensitizing effect correlated with mutations in DNA damage repair and myristoylation pathways. N-myristoylation inhibition appears to be a novel method of radiosensitization for glioblastoma. N-myristoylation affects multiple oncogenic pathways including PARP-1 downregulation, which impedes DNA damage repair and may be what leads to radiosensitization. Future studies are aimed at further predictive markers and in vivo efficacy.