Novel Oxidative Phosphorylation Inhibitor IACS-010759 Induces AMPK-Dependent Apoptosis of AML Cells

Acute myeloid leukemia (AML) cells frequently adjust to increased energy/substrate demand under stress conditions in the bone marrow microenvironment. Recent studies indicate that AML cells including leukemia-initiating cells are highly dependent on oxidative phosphorylation (OxPhos) for survival, while normal hematopoietic stem cells predominantly utilize glycolysis for energy homeostasis.

The aim of this study is to assess the biomarkers of anti-leukemia efficacy of the first-in-class oxidative phosphorylation inhibitor (OxPhosi) IACS-010759. IACS-010759 at nanomolar concentrations blocks cellular respiration through inhibition of complex I of the electron transport chain (Molina et al., AACR 2016), is orally bioavailable and is currently in Phase 1 clinical trial in AML (NCT #02882321).We utilized primary AML samples from patients with newly diagnosed or relapsed/refractory AML (11 sensitive and 3 resistant to IACS-010759) as well as OxPhosi-sensitive OCI-AML3 and -resistant MOLM13 AML cell lines . To identify transcript abundances for the transcriptome profiling, Cap Analysis of Gene Expression (CAGE)-Seq analysis has been conducted. CAGE-Seq identifies and quantifies the 5' ends of capped mRNA transcripts, which enables the identification of transcription start sites (TSS) and allows investigating promoter structures necessary for gene expression. CAGE-Seq analysis identified recurrent baseline differences of TSS gene expression between IACS-010759-sensitive and -resistant primary AML samples; 4 genes were higher and 124 genes were lower in sensitive samples compared to resistant samples (false positive rate; FDR<0.05). In IACS-010759 sensitive OCI-AML3 cells the expression levels of 1,623 genes were higher and 1,821 were lower than in resistant MOLM13 cells (triplicate, FDR<0.05). Based on these transcriptome differences, the Ingenuity Pathway Analysis (IPA) bioinformatics platform highlighted consistent overexpression of 4 factors in IACS-010759-resistant AML primary samples and cell line compared to the sensitive AML. These include mammalian target of rapamycin (mTOR) kinase, a positive regulator of cell metabolism; Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2) that enhances kinase-mediated activation; erythropoietin (EPO), a positive regulator of Jak-Stat pathway; and IL3, a potent growth-promoting cytokine. These findings indicate that the basal metabolic energetic capacity might affect the sensitivity of AML cells to IACS-010759. AMPK is a crucial cellular energy sensor and regulates energy metabolism through mTOR inhibition. We therefore studied the role of energy metabolism regulator AMP-activated protein kinase (AMPK) and of mTOR in anti-leukemia efficacy. To validate the role of AMPK, we utilized AMPK knockdown (sh AMPK) OCI-AML3 cells, and examined the sensitivity against IACS-010759, comparing to parental shC OCI-AML3 cells. By immunoblotting, IACS-010759 induced significant increase of p-AMPK in parental OCI-AML3 cells along with decrease of mTOR downstream target phospho- (p-) 4EBP1. OCI-AML3 stably infected with sh AMPK predictably overexpressed p-4EBP1 which was not modulated by IACS-010759. sh AMPK OCI-AML3 cells were more than 10-fold less sensitive against IACS-010759 compared to shC OCI-AML3 (IC₅₀; 8.6±4.2 nM vs 0.5±0.1 nM, respectively), albeit remained in low nM concentrations. Interestingly, baseline level of p-AMPK expression in parental OCI-AML3 cells was markedly suppressed under physiologic hypoxic (1% oxygen) conditions, which caused activation of mTOR signaling (upregulation of p-4EBP1) and decreased sensitivity to IACS-010759 (IC₅₀; 14.5±7.1 nM).

Taken together, these results indicate that the responsiveness of AML to IACS-010759 depend at least in part on the AMPK-mTOR signaling, the gauge of cellular energy metabolic capacity. In sensitive AML cells IACS-010759 induces AMPK activation leading to mTOR suppression which results in cell growth inhibition in AML cells. In contrast, in cells with lower levels of AMPK, or in highly proliferating cells (such as stimulated by IL-3), mTOR is constitutively activated conferring resistance to Oxphosi, possibly through glycolytic adaptation. Altogether, these findings highlight AMPK and mTOR as putative biomarkers of anti-leukemia activity of the novel OxPhosi IACS-010759.