EB2023 primes mitochondria for BCL2 dependence and induces pyroptotic cell death via AMPK signaling and the unfolded protein response

Abstract The F1 catalytic domain of ATP synthase performs the final and pivotal step of oxidative phosphorylation (OXPHOS). EB2023 is a selective F1 inhibitor derived from a rare actinobacterium with anti-proliferative properties in AML. Here we show that EB2023 has broad anti-cancer activity and leads to improved OS in an AML mouse model, while identifying distinct mechanisms of cell death for the monotherapy and combination therapy with BCL2 inhibitors. EB2023 exhibited potent anti-proliferative effects across multiple cancer cell lines including AML, ALL, multiple myeloma, prostate cancer, breast cancer, and melanoma, with IC50 values ranging 5-91 nM, confirmed by an ATP-independent (CellTiter-Fluor) viability assay. EB2023 dose-dependently decreased oxygen consumption rate (OCR) in MV-4-11 cells with 24 hours of treatment by Seahorse XF analysis and induced phosphorylation of AMPK and ACC by western blot analysis, consistent with on target effects of OXPHOS inhibition and ATP depletion. EB2023 was broadly active in an ex vivo screen of AML patient samples and this activity was found to correlate with baseline OCR, suggesting OXPHOS reliant cells are most susceptible to EB2023. Finally, daily M-F i.p. administration of EB2023 significantly extended survival in the MLL-AF9 murine AML model (median 49 vs 33 days, p<0.05). To survey cellular consequences of F1 inhibition, we performed bulk RNA-sequencing analysis of MV-4-11 and THP-1 cells treated with EB2023 for 24 hours. Gene set enrichment analysis showed significant suppression of OXPHOS and cell cycling alongside increases in unfolded protein response (UPR), oxidative stress, and pyroptosis pathways in both cell lines. Notably, there was evidence of severe and prolonged activation of the PERK-mediated UPR pathway, evidenced by increased expression of ATF4, DDIT3 and PPP1R15A, known effectors of cell death in response to prolonged UPR activity. This was further supported by visualization of endoplasmic reticulum dilation in EB2023-treated MV-4-11 cells [tunneling electron microscopy (TEM) image analysis using ImageJ, p = 0.02]. Interestingly, TEM images also showed morphologic evidence of non-apoptotic cell death changes, including cell membrane perforation and lack of cell volume decrease or chromatin condensation. The absence of EB2023-induced caspase 3/7 activity and cleavage of gasdermin-E suggests pyroptotic cell death may predominate. It has previously been reported that signaling through phosphorylated-AMPK, via PERK and ATF4, primes AML cells to become BCL2 dependent (Grenier, Cell Rep 2022) and so we postulated that EB2023 would be synergistic with the BCL2 inhibitor, venetoclax (ven). We observed potent synergy when EB2023 was combined with ven to treat a panel of AML cell lines (Average ZIP synergy score = 21.0, CTG assay), including in the relatively ven-resistant, NRAS-mutated THP-1 cell line (ZIP synergy score = 48.4). Unlike with EB2023 monotherapy, the combination of ven and EB2023 induced increases in Caspase 3/7 activity in primary human AML samples. Seahorse XF analysis showed that the compensatory increase in glycolytic capacity seen in response to EB2023 as a monotherapy in Molm-13 cells was completely abolished in still viable cells when combined with ven, indicating this combination can circumvent transient metabolic adaptation. BH3 profiling analysis of THP-1 cells revealed significantly increased apoptotic priming and BCL2-dependence with EB2023 treatment. Finally, daily EB2023 administration combined with a low dose azacitidine/ven regimen significantly decreased AML bone marrow chimerism in patient-derived xenograft NSGS mouse models (P=0.002). Together, these results support the conclusion that F1 inhibition induces non-apoptotic cell death in AML cells, likely via pyroptosis, and simultaneously increases cell dependency on BCL-2 for survival, priming them for venetoclax-induced apoptosis. This insight, and the favorable pharmacology and considerable therapeutic window observed in mouse models make EB2023 a promising candidate for development with and without combination with ven-based regimens in AML.