Figure 4 – Hypoxia maintains histone lysine demethylase activity by increasing αKG abundance via glutamine catabolism in PFA ependymoma.
A) Snapshot of relative metabolite abundance of Krebs cycle intermediates in PFA (n=15) versus ST-EPN (n=4) surgical biopsies. Significantly differentially abundant metabolites characterized by t-test with FDR correction (q-value < 0.05).
B) PFA cells growing in 1% O2, exhibit significantly increased ratio of αKG/Succinate in PFA cells. Statistical comparison was performed using ratio paired t-test.
C) Histone lysine 27 demethylase gene expression is not differentially regulated by oxygen tension in PFA cells. Statistical comparison was performed using Wald test with Benjamini-Hochberg adjustment.
D) Cartoon depicting glutamine tracing in PFA cells growing in 1% O2. Labelled glutamine was used as the carbon source (13C5 light blue; unlabeled carbon dark blue). PFA cells preferentially undergo glutaminolysis and reductive carboxylation.
E) Labelled glutamine tracing of PFA cells reveals an elevated αKG:succinate ratio in 1% compared to 21% O2. Statistical analysis performed using two-tailed t-test with HolmSidak correction.
F) Cartoon depicting the roles of αKG and the KDMs in histone demethylation. Drugs which inhibit lysine 27 demethylase activity and their targets are in red, while exogenous αKG, which increases activity is in green.
G) Dose response of GSK-J4 inhibition (7 days) of KDM6A and KDM6B perturbs PFA cell survival in 1% O2.
H) Dose response of CB-839 inhibition (7 days) of GLS perturbs PFA cell survival in 1% O2.
I) Supplementing exogenous cell permeable dimethyl αKG rescues the growth defect of PFA cells cultured in 21% O2.
J) Supplementing exogenous cell permeable dimethyl αKG rescues PFA cell survival while under CB-839 inhibition in 1% O2.
*p < 0.05, **p < 0.01 and ****p < 0.0001. Data are displayed as the mean ± SEM. Curves show logistic regression line of fit for G-H.