A Novel Integrated Approach For Deciphering The Mitochondrial Mutation Enigma In Glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most common adult primary brain tumor. It is highly malignant with 2-year survival of just 27.2% following diagnosis. Treatment is hampered by its unique underlying biology, including differential inter- and intra-tumor responses to therapy. Impaired mitochondrial function is a common feature of cancer cells in comparison to healthy cells. However, the precise contribution of single mitochondrial DNA (mtDNA) mutations to this impairment, and consequently to the development, progression and chemosensitivity of GBM remains poorly described. To address this we have developed a novel integrated approach where we have: (1) obtained complete mitochondrial genomes from multiple GBM samples, (2) employed 3D structural mapping and analysis to predict the function of non-synonymous mtDNA-encoded complex III and IV mutations; (3) measured oxidative phosphorylation (OXPHOS) enzyme complex activities of GBM cell lines. Finally, to circumvent the current inability to genetically transform human mtDNA, we have employed humanized yeast technology to decipher the specific impact of single key mtDNA mutations on cellular growth and OXPHOS complex activity. Over 200 mutations were identified in 42 GBM-mtDNAs using next generation sequencing. This included 9 functional candidates in complexes III and IV, with highly variable mutation loads, which were predicted to affect corresponding enzyme activity. Most were GBM-specific and not found in the general population, two were present in the germ-line and 43% of tumors carried at least one functional candidate. Preliminary spectrophotometric measurements indicate a striking diversity of OXPHOS enzyme complex activity profiles between the GBM cell lines. One cell line exhibited a combined increase in complex I, II, linked II/III and IV activity compared to control. In 5 GBM cell lines, all complex I activities were altered (either elevated or reduced) and all complex IV activities were altered (mostly decreased), while the remaining cell line exhibited decreases in both complex I and IV activity combined with an increase in linked complex II/III activity. Analysis of yeast cells that were genetically transformed to contain a single mutation that mimics the most prevalent functional candidate identified in GBM to date: F18L, revealed an alteration of complex III activity (quinol-cytochrome c reduction) probably caused by a modification of the substrate quinol binding. Using our approach, we show for the first time that a single GBM-associated mtDNA mutation can have a direct effect on mitochondrial function, raising the possibility that small mitogenomic elements could contribute to the heterogeneous biology of GBM. This study paves the way for the systematic study of other functional candidates using humanized yeast technology, as well as other studies that explore their contribution to GBM cell behavior and drug sensitivity. Citation Format: Rhiannon E. Lloyd, Kathleen Keatley, Anais Laleve, Samantha C. Higgins, Stavros Polyzoidis, Keyoumars Ashkan, Helen L. Fillmore, Simon J. Heales, John E. McGeehan, Iain Hargreaves, Brigitte Meunier, Geoffrey J. Pilkington. A novel integrated approach for deciphering the mitochondrial mutation enigma in glioblastoma. [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 3042. doi:10.1158/1538-7445.AM2015-3042