Figure 4. Pharmacologic inhibition of H6PD and effects on enzalutamide resistance.

(A) A steady-state affinity model for rucaparib and H6PD was obtained by surface plasmon resonance (SPR). Purified H6PD protein was covalently immobilized, rucaparib was injected, and the data were fit into a steady-state affinity model. (B) Effects of rucaparib andolaparib on H6PD production by NADPH in vitro. Rucaparib or olaparib was added to a reaction cocktail with H6PD protein, D-glucosamine 6-phosphate and NADP, and after 1 hour incubation, NADPH production was measured using Glo Detection Reagent. (C) Effects of rucaparib or olaparib on H6PD activity in enz-resistant LAPC4 cells. Cells were treated with rucaparib for 24 hours, then microsomal NADPH was measured. (D) Cortisol metabolism in enz-resistant LAPC4 cells treated with rucaparib or olaparib. Cells were pretreated with rucaparib or olaparib for 1 hour, then [³H]-cortisol (100 nM) was added; the steroids were extracted, separated and quantitated by HPLC after the indicated incubation times. (E) Viability of enz-resistant LAPC4 cells treated with rucaparib. Cells were treated with 100 nM cortisol with 10 μM enz combined with the indicated drugs for 5 days and assayed using CellTiter-Glo. Viability is normalized to Ctrl. (F and H) Rucaparib and the inhibitory effect of enz on xenograft growth in the F, VCaP and H, LAPC4 mouse xenograft models. (G and I) Effects of rucaparib on progression-free survival in enz-treated xenograft models. G, VCaP or I, LAPC4 xenografts were grown in orchiectomized mice supplemented with DHEA and arbitrarily divided among 3 groups each for control or enz diet: control, olaparib or rucaparib. Tumor volume was compared between CTRL/Enz and RUCA/Enz or between RUCA/Ctrl and RUCA/Enz with an unpaired two-tailed t-test on day 21 (LAPC4) or 25 (VCaP). To compare progression-free survival, the difference between CTRL/Enz and RUCA/Enz or between RUCA/Ctrl and RUCA/Enz was calculated with a log-rank test. The number of mice in the LAPC4 CTRL/Ctrl, OLA/Ctrl, RUCA/Ctrl, CTRL/Enz, OLA/Enz and RUCA/Enz groups were 9, 9, 9, 8, 9 and 8, respectively. The number of mice in the VCaP CTRL/Ctrl, OLA/Ctrl, RUCA/Ctrl, CTRL/Enz, OLA/Enz and RUCA/Enz groups were 7, 7, 7, 8, 6 and 9, respectively. (J) Rucaparib treatment and the absolute concentration of corticosterone in LAPC4 xenograft tumors and (K) in sera. For all panels unless otherwise noted, error bars represent the SEM; p values were calculated using unpaired 2-tailed t-tests.