Targeting Lipid Droplet Biogenesis to Enhance Ferroptosis in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a highly aggressive hematologic malignancy with a high relapse rate primarily due to acquired therapy resistance. Accumulating evidence has shown that ferroptosis, an iron-dependent non-apoptotic form of cell death characterized by lipid peroxidation within cellular membranes, is a potential therapeutic strategy in AML that could overcome the therapy resistance.

Polyunsaturated fatty acid-phospholipids (PUFA-PLs) are crucial components of cellular membranes, while particularly susceptible to peroxidation, thus could prime cancer cells to ferroptosis. Cellular PUFAs can be conjugated with Coenzyme A (CoA) for PUFA-PL synthesis, converted to acyl-CoA for energy production through β-oxidation or stored in lipid droplets (LDs) as PUFA-triglycerides (TAGs). LDs serve as the dynamic organelles for storing neutral lipids, composed primarily of TAGs and cholesteryl esters, and regulating lipid flux to meet the needs of metabolism and lipid homeostasis. Therefore, LD biogenesis conditionally buffers PUFA in TAGs, and reduces lipid peroxidation and subsequent ferroptosis. Diacylglycerol acyltransferases (DGATs), DGAT1 and DGAT2, mediate the final and rate-limiting step in TAG synthesis that drives LD biogenesis, playing critical roles in cellular lipid metabolism and homeostasis.

We first confirmed that the exogenous supplement of PUFA (Linoleic acid) significantly enhanced ferroptosis in AML cells treated with the GPX4 inhibitor ML210, while monounsaturated fatty acid (MUFA, Oleic acid) suppressed GPX4 inhibition-induced ferroptosis, as expected. However, interestingly, the lipidomics analysis revealed increased levels of PUFA-TAG species in AML cells when GPX4 is inhibited. In addition, GPX4 inhibition increased cellular LD mass. Given that TAG species are the major components in LDs, we hypothesized that PUFAs are sequestered in LDs as an adaptive mechanism to mitigate oxidative lipid stress upon ferroptosis. Further analysis of lipidomics showed increased levels not only in oxidized PUFA-PL species but also in oxidized PUFA-TAG species. This implies that TAG in LDs may also scavenge the oxidized PUFA, not only unoxidized PUFA, to mitigate ferroptosis. To test the hypothesis, this study explores the potential of targeting DGATs to disrupt LD formation and enhance ferroptosis in AML cells. Supporting this notion, we demonstrated that DGAT1 inhibition by A922500 attenuated the LD formation, in contrast to negligible effects by DGAT2 inhibition. Notably, DGAT1 inhibition significantly enhanced the GPX4 inhibition-induced ferroptosis. The combination of DGAT1 inhibition with Docosahexaenoic acid (DHA), a widely used food supplement belonging to the PUFA family, further improved the ferroptotic cell death. To further validate the roles of DGAT-mediated LD formation in sensitizing AML cells to ferroptosis, we generated DGAT1 and DGAT2 stable knockout (KO) lines in OCI-AML3 and MOLM13 cells by CRISPR-Cas9. Fluorescence microscopy confirmed the obvious reduction of LDs in DGAT1-deficient AML cells. In line with the pharmacological inhibition results, DGAT1 KO increased ferroptotic cell death, while not significantly affected by DGAT2 KO, perhaps reflecting their differential functions in LD biogenesis.

In summary, our research demonstrates that inhibition of LD biogenesis by selective DGAT1 inhibition and supplemental dietary PUFA synergistically enhances ferroptosis in AML cells. These findings suggest a novel therapeutic strategy for AML by targeting lipid metabolism to potentiate ferroptosis. Further investigation into the molecular underpinnings of the synergy, in vivo validations, and the development of specific DGAT inhibitors will be critical for translating these findings into clinical applications.

Andreeff:Syndax: Honoraria, Research Funding; Aptose: Honoraria; Eterna: Current holder of stock options in a privately-held company, Honoraria, Research Funding; Kintor Pharmaceutical: Research Funding; Ona: Honoraria; Glycomimetics: Honoraria; Daiichi-Sankyo: Research Funding; Oxford Biomedical: Research Funding; SentiBio: Current holder of stock options in a privately-held company, Honoraria, Research Funding; Chimerix: Current holder of stock options in a privately-held company; Oncolyze: Current holder of stock options in a privately-held company; Ellipses: Research Funding; Roivant: Honoraria; Paraza: Honoraria; Sellas: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria.