Extended Data Fig. 4: Relative sensitivities of glioma cell lines to the pro-drug POMHEX versus the active Enolase inhibitor HEX.

Sensitivity of a panel of glioma cell lines with with varying ENO1 statuses to the Enolase inhibitors POMHEX and HEX. IC₅₀ values were calculated based on terminal cell density measured by crystal violet. D502 and U343 are ENO1-heterozygous deleted cell lines (~50% total Enolase^3,4). Consistent with our previous reports for pan-Enolase inhibitors^3,4, ENO1-homozygous deletion confers the greatest sensitivity to HEX, while ENO1-heterozygous deletion status shows intermediate sensitivity. While sensitivity to HEX is dependent on ENO1-deletion status, the sensitivity to POMHEX like depends not only on ENO1-deletion status but also expression of pro-drug bioactivation enzymes that transform POMHEX into active HEX enolase inhibitor (carboxylesterases, CES and phosphodiesterases, PDE). On average, the potency of POMHEX is ~75-fold greater than HEX though with substantial variation across cell lines. D502 is considerably more sensitive to POMHEX than U343 (IC₅₀ 82 vs 559 nM), yet U343 is more sensitive to HEX than D502 (IC₅₀ 19,723 nM vs 28,756 nM). This can be explained by higher levels of expression of pro-drug activating enzymes in the D502 glioma cell line result in greater sensitivity to POMHEX as compared to U343. Identification of the specific genes responsible, and their expression could be used for patient stratification, expanding the utility of Enolase inhibitors beyond those with ENO1-homozygous deletions.