Molecular Interactions between Mitochondrial Ferroptosis and Apoptosis Pathways in Acute Myeloid Leukemia

Background: Acquired resistance to apoptosis-targeted therapies is a major mechanism underlying relapsed and refractory acute myeloid leukemia (AML). Exploring non-apoptotic regulated cell death (RCD) could circumvent apoptosis-related resistance, therefore, has the potential of improving outcomes in AML. Ferroptosis is a form of RCD driven by iron-dependent lipid peroxidation (LP), which is negatively regulated by GPX4. We recently reported that ferroptosis in AML uniquely relies on the LP of mitochondrial membranes, which we term “mitochondrial ferroptosis” (Leukemia;38:729-740, 2024). This result prompted us to investigate the potential interrelationship between mitochondrial ferroptosis and apoptosis pathways in AML. Here, we evaluated the combinatorial anti-AML efficacy of dual induction of RCD, by inducing apoptosis and ferroptosis, and investigated the molecular interactions.

Results: We evaluated the anti-AML efficacy of dual induction of apoptosis and ferroptosis utilizing venetoclax (Ven) and ML210, a specific GPX4 inhibitor, as apoptosis and ferroptosis inducers, respectively. Treatment with ML210 in combination with Ven induced synergistic cell death in various AML cell lines: the combination index was 0.71, 0.53, and 0.57 in OCI-AML3, MOLM13, and MV4;11 cells, respectively. This synergism was also recapitulated in doxycycline-inducible GPX4 knockdown cells, supporting the on-target effects of pharmacologic GPX4 inhibition. Interestingly, Ven-resistant (Ven-R) cell lines exhibited more prominent synergism compared to the parental Ven-sensitive cells. Furthermore, the synergism was also observed in AML stem/progenitor cells (CD34⁺CD38^-) obtained from Ven-R as well as Ven-naïve AML patients.

As starting point for our investigation of interactions between mitochondrial ferroptosis and apoptosis, we observed that ferroptosis of AML cells induces cytochrome c (CytC) release from mitochondria, which is a hallmark of apoptosis. Importantly, the ferroptotic CytC release was mitochondrial lipid peroxidation-dependent but BAX/BAK-independent, suggesting a mechanistic divergence from apoptotic BAX/BAK-dependent CytC release by Ven. Since metabolic rewiring to fatty acid metabolism underlies Ven-R in AML, we hypothesized that Ven affects lipid metabolism and may further sensitize AML cells to ferroptosis. Indeed, while Ven alone only slightly induced LP in AML cells, the combination of ML210 and Ven significantly enhanced LP compared to ML210 alone. The synergistic effect was attenuated by ferrostatin-1, a selective ferroptosis inhibitor. Notably, a mitochondria-targeted antioxidant, MitoTEMPO, completely blocked LP and cell death induction by the combinatorial treatment. Consistently, mass-spectrometry-based mitochondrial proteome analysis suggested that Ven reduced glutathione reductase, which is critical for the function of GPX4 by catalyzing the conversion of oxidized glutathione (GSSG) to its reduced form GSH. These results suggest that mitochondrial ROS accumulation is an essential trigger for the synergistic anti-AML effects. Furthermore, pretreatment with Ven followed by ML210 significantly increased LP and cell death compared to concomitant treatment or pretreatment with ML210 followed by Ven. This indicates a non-apoptotic role of Ven in priming AML cells into a pro-ferroptotic state.

Conclusion: Mitochondrial ferroptosis exhibits molecular interactions with apoptosis pathways in AML: ferroptosis induces CytC release in a BAX/BAK-independent manner, while apoptosis sensitizes AML cells to LP possibly by affecting mitochondrial lipid and oxidative metabolism. Targeting the ferroptosis-apoptosis interactions could evolved into a novel therapeutic strategy for AML.

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