Targeting fatty acid metabolism in Glioblastoma
Introduction
High-grade astrocytomas are among the deadliest human cancers with a median survival of 15 to 19 months. The majority (>90%) of patients treated with current standard of care, concomitant temozolomide and radiotherapy, show tumor progression while on this treatment regimen. Consequently, recurrence is inevitable and the optimal management remains unclear. There is an urgent need to develop strategies for improving treatment options.
We characterized the dependence of patient-derived glioblastoma sphere-forming cells (GSCs) on mitochondrial metabolism of fatty acids, which we discovered using an in vivo functional genomics screening platform. Specifically, we focused on medium-chain acyl-CoA dehydrogenase (MCAD), the protein produced by acyl-CoA dehydrogenase medium chain (ACADM), based on its consistently high score across all of our screens, as well as its high expression level in GSCs derived from multiple patients and its upregulation in GBM compared to normal brain.
We demonstrated that depletion of MCAD in GSCs irreparably damages mitochondria and is acutely toxic, but there was no effect of MCAD depletion on the metabolism of NHAs or NSCs. However, tumor cells have a documented, remarkable adaptability to shunt substrates to different metabolism pathways to survive metabolic perturbations. It is thus essential to understand the molecular programs that support tumor cells escaping from MCAD inhibition especially in light of any future effort to target MCAD therapeutically
Methods
To obtain cells that escape MCAD dependency (‘escapers’), we passaged liquid suspension cultures of GSC 8.11 infected with control sgRNA (sgControl) or ACADM-targeting sgRNA (sgACADM) in the presence of puromycin to retain selective pressure for infected cells. We measured cell number over time for up to 48 days, which confirmed the expected consistent number of cells in sgControl cultures. In sgACADM cultures, we observed the anticipated rapid and robust cell killing followed by the appearance of surviving cell clones by approximately three weeks, that had likely escaped MCAD dependency. Indeed, we confirmed the absence of MCAD expression in the ‘escapers’ in 24- and 48-day cultures by immunoblotting and immunofluorescence.
With these escaper cells, we conducted in vitro characterization of lipid profiles in MCAD escapers. Mitochondrial activity of parental and escaper GSCs will be evaluated using Seahorse technology to measure oxygen consumption rate (OCR) as well as cell glycolytic metabolism measured as extracellular acidification rate (ECAR). We will also measure intracellular ATP content (bioluminescence assay). The content, morphology, and structural organization of mitochondria will be analyzed by confocal microscopy using mitotracker staining as well as by transmission electron microscopy (TEM). Next, we will continue lipidomic profiling in parental GSCs and escapers. To investigate cellular and mitochondrial lipids composition, we will conduct additional liquid chromatography/mass spectrometry (LC/MS)-based analysis of lipid classes on whole-cell extracts and on mitochondria isolated from parental GSCs and escaper cells.
Results
Preliminary results showing that these MCAD escapers cells have mitochondrial activity comparable to parental cells. TEM shows that in escaper cells, there are two population of cells with different morphology of mitochondria. Preliminary lipidomic show the enrichment of signaling lipids while storage lipid decrease. RNA seq results show that these escapers cells have epithelial-mesenchymal transition signature, indicating that escapers cells become more aggressive. This is also reflected in our transplatation of parental and escaper cells.
Conclusion
We established the characteristic of these escapers cells. It will be essential to figure out the escaper mechanism, especially in light of any future effort to target MCAD therapeutically.