Overcoming Ven/Aza Resistance through Imetelstat-Mediated Lipophagy in Acute Myeloid Leukemia

Imetelstat is a first-in-class telomerase inhibitor with recent FDA approval for the treatment of myelodysplastic syndromes (Platzbecker et al. Lancet 2024). We have previously shown that imetelstat is effective in randomized, preclinical trials in Acute Myeloid Leukemia (AML), inducing AML cell death via polyunsaturated fatty acid (PUFA) phospholipid synthesis (Bruedigam et al. Nature Cancer 2023). Detailed analysis of lipidomic datasets revealed two predominant, acute effects of imetelstat on AML cell lipid metabolism in vitro: desaturation of triglycerides, and chain length reduction of cholesteryl esters. Triglycerides and cholesteryl esters are the main constituents of lipid droplets. Here, we investigated whether short-term imetelstat treatment induces AML cell death through the autophagy of lipid droplets (lipophagy; n = 8 AML cell lines). The autophagy inhibitors chloroquine (CQ), bafilomycin A1, or 3-methyladenine prevented imetelstat-induced cell death, lipid peroxidation, lipid ROS production, and colocalization of lipid droplets with the late endosomal marker LAMP-1. These findings correlated with reduced lysosomal acidification in AML cells treated with these drug combinations. Moreover, the autophagy inhibitors restored neutral lipid content that was significantly reduced by imetelstat treatment alone. These data demonstrate that imetelstat is an acute inducer of lipophagy in AML cells in vitro. To validate the proposed mechanism in vivo, we performed targeted lipidomics on 500 lipid species on sorted AML patient-derived cells from PDX that had been treated continuously with imetelstat or vehicle control (n = 12 individual AMLs with n = 3 PDX per treatment group; n = 72 PDX in total). Overall, imetelstat treatment significantly altered the relative abundance of 43 lipid species (p < 0.05). Phosphatidylcholine (PC) species containing PUFAs were depleted in AML cells from imetelstat-treated PDXs, while the levels of lysophosphatidylcholine (LPC) species were increased. These findings suggest that PC-PUFAs are oxidized by imetelstat in AML cells, followed by their degradation or phospholipase-mediated cleavage, increasing the levels of LPCs. Lipidomic responses to imetelstat differed between individual AML patient samples based on their treatment response category. Specifically in samples that responded to imetelstat, the relative abundance of PUFA triglycerides was increased by imetelstat, whereas this effect was reversed in the intermediate and poor imetelstat responding samples. In summary, these findings demonstrate imetelstat-mediated, ferroptosis-associated lipidomic alterations in AML cells that correlate with imetelstat treatment responses in vivo. AML leukemic stem cells (LSC) are particularly dependent on lipophagy as they uniquely rely on oxidative phosphorylation to fulfill their energy demands through fatty acid oxidation (beta-oxidation) and amino acid metabolism. Importantly, increased fatty acid metabolism in pre-treatment AML LSCs is associated with resistance to venetoclax and azacitidine (ven/aza) combination therapy. Our proposed mechanism of action of imetelstat-induced lipophagy led to the hypothesis that imetelstat may target ven/aza resistant AML subclones. As proof-of-concept in vivo, we sequenced ven/aza therapy prior to administration of imetelstat in AML PDX (n = 6 /group; n = 24 NRGS recipients in total). Combination therapy reduced peripheral blood AML donor chimerism when compared to imetelstat monotherapy (mean 1.02% vs. 64.96%; p < 0.0001), ven/aza alone (1.02% vs. 26.35%; p < 0.0001), or vehicle control (1.02% vs. 96.55%; p < 0.0001). Moreover, thrombocytopenia was reversed in the combination therapy group only. Additional analyses in multiple independent PDX cohorts including survival are currently ongoing. In summary, we have identified imetelstat-mediated, ferroptosis-associated lipidomic alterations in AML cells that correlate with imetelstat treatment responses in vivo. Imetelstat is an acute inducer of lipophagy, promoting the formation of PUFA-containing phospholipids, causing excessive levels of lipid peroxidation and oxidative stress resulting in ferroptosis in AML. These mechanistic insights may be leveraged to develop an optimized therapeutic strategy using imetelstat to target ven/aza resistant AML subclones, providing significantly improved disease control for AML.