Extended Data Fig. 5. ENO1-deletion status predicts sensitivity to Enolase inhibitors in glioma sphere- forming cells (GSC).
GSCs and omics data were kindly shared by the Lang lab9,10. a. Copy number variation at the 1p36 locus identifies GSC296 (red) as ENO1-homozygous deleted and GSC231, 268, 275, 289 as heterozygous deleted (pink). Dark blue regions represent bi-allelic (homozygous) deletion, while light blue regions correspond to mono-allelic loss. b. Confirmed lack of ENO1 expression by western blot in GSC296 (red), with corresponding levels of ENO2 shown. The western blot analysis for GSC296 was performed at least 3 times with similar outcomes. c. RNA-seq mRNA expression of ENO1 (bottom) and ENO2 (top) in ENO1-homozygous deleted and heterozygous deleted GSCs, confirming lack of ENO1 expression. Each bar represents one biological replicate. d,e. Sensitivity of GSCs with varying ENO1-deletion status to POMHEX. Dissociated GSCs were seeded in 96-well round bottom-low attachment plates in duplicate and allowed to form spheroids. Spheroids were treated with a serial dilution of POMHEX for 1 week; media was changed every 3 days with fresh drug. Viability was visualized with 5 nM TMRE (red). TMRE was quantified by ImageJ and expressed as a function of vehicle controls. Each dot represents one biological replicate. GSC296 (ENO1−/−) was distinctly sensitive to POMHEX. ENO1-heterozygous deleted GSCs showed intermediate sensitivity. ENO1-WT GSCs with the lowest residual ENO2 expression showed the greatest sensitivity amongst ENO1-WT GSCs. Experiments with GSC296 were reproduced independently once.