F1 subunit-specific ATP synthase inhibition disrupts AML mitochondrial metabolism distinctly from other electron transport chain inhibitors Free

Targeting oxidative phosphorylation (OXPHOS) has long been pursued, but a suitable therapeutic window has been elusive in the development of new drugs. In the PRISM drug repurposing dataset, we observed an inverse relationship between AML cell line sensitivity to IACS-010759 (inhibitor of complex I) and oligomycin (OGM, inhibitor of complex V) (Pearson -0.74, P = 0.0011). This suggested that AML cells vary in their sensitivity to methods of OXPHOS perturbation. ATP synthase (complex V) has two main subunits, the F_O subunit which shuttles protons across the inner mitochondrial membrane, and the F₁ subunit which contains the catalytic ATP-generating unit. While F_O inhibitors have been studied, selective inhibition of the F₁ subunit has not been explored in AML. We report here the preclinical evaluation of EB2023, a novel F₁-selective inhibitor. Comparing metabolic consequences induced from a panel of OXPHOS inhibitors, we illustrate how selective F₁ inhibition offers a unique therapeutic opportunity in AML.

While both IACS and EB2023 treatment decreased proliferation and caused cell cycle arrest at the G₀ phase (Ki67/PI flow cytometry analysis), EB2023 induced significantly more cell death in 72h than IACS in 5 cell lines as well as involution of clonal HSPC in 5 ex vivo AML patient samples. To investigate the divergent activity profiles of OXPHOS inhibitors EB2023, OGM, and IACS in AML, we leveraged an array of mitochondrial and metabolomic assays to discern mechanistic differences. Flow cytometry showed gradual decreases in membrane potential in all cases over 24h (TMRM MFI). IACS treatment led to a rapid rise in mitochondrial ROS at 0.5h, whereas EB2023 and OGM led to decreases (14743 vs 2384 vs 2442 MitoSox MFI, respectively). Analysis of mitochondrial morphology of treated AML cells via Tunneling EM, revealed that, while all 3 inhibitors resulted in increased mitochondrial size, only EB2023 caused increased mitochondrial roundness with more significant loss of cristae architecture, signifying severe mitochondrial stress (Analysis with ImageJ software).

Seahorse XF analysis revealed that EB2023 and OGM, at nM concentrations, paradoxically increased oxygen consumption rate (OCR) over the first 2 hours of exposure in all cell lines tested, before ultimately halting OCR by 24h, whereas IACS halted OCR immediately. Rapid NAD+ depletion was observed with IACS treatment, but not with EB2023 or OGM. Accordingly, interruption of NAD+ dependent TCA cycle reactions was observed with IACS treatment at 24h by mass spectrometry (MS) analysis (malate and ketoglutarate accumulation, aspartate depletion) while the opposite pattern was seen with EB2023 and OGM. IACS treatment led to rapid extracellular acidification in MV-4-11 cells but not with EB2023 exposure. MS confirmed that cells treated with IACS, but not EB2023, accumulated intracellular lactate and increased incorporation of C13 atoms into lactate when fed C13-labeled glucose, suggesting compensatory glycolysis occured with IACS treatment but not with EB2023. This suggests that circumvention of metabolic adaptation may explain observed differences in cytotoxicity between F₁ ATP Synthase and Complex I OXPHOS inhibitors.

Finally, only EB2023-treated cells preserved their baseline ADP/ATP ratio after 4 hours of treatment (ADP/ATP ratio: OGM = 0.42, IACS = 0.39, EB2023 = 0.18, DMSO = 0.20 by luminescence assay). Nucleotide MS showed rapid interruption of nucleotide biosynthesis with all inhibitors by C13 incorporation but again recapitulated the relative preservation of ADP/ATP ratio with EB2023 treatment alone – which we suggest arises from EB2023 preventing the consumption of the existing ATP reservoir via compensatory ATP hydrolysis (reversal of ATP synthase).

In summary, complex I inhibition leads to immediate interruption of cellular respiration, Fo inhibition blocks protons from crossing the inner mitochondrial membrane and F₁ inhibition decouples ATP production from respiration while still allowing protons to cross the inner mitochondrial membrane. EB2023 inhibits both ATP synthase and the reverse reaction of ATP hydrolysis directly at the catalytic site, transiently maintaining a normal ADP/ATP ratio while OGM, like complex I inhibition, immediately depletes available ATP at nanomolar doses. Together, these findings offer compelling evidence that the selective F₁-targeting EB2023 offers a novel mechanism of OXPHOS inhibition, and improved potency in AML.