Abstract 2017: Dual targeting of glutathione-GPX4 nexus and mitochondrial protease CLPP causes synergistic anti-leukemia effects in acute myeloid leukemia (AML)
Authors: Akiyama Hiroki , Zhao Ran , Ostermann Lauren B. , Ishizawa Jo , Andreeff Michael 
Published: July 2021

Abstract
Cancer cells depend on aberrant antioxidant mechanisms to survive increased oxidative stress, which is particularly critical for cancer stem cell maintenance and its therapy resistance phenotype. Mitochondria is the primary site of oxygen consumption and reactive oxygen species (ROS) production, thus is essential for regulating cellular redox homeostasis. We have recently reported that hyperactivation of mitochondrial (mito-) protease CLPP by imipridones (ONC201/212) exerts cancer selective lethality through degradation of mito-proteins including respiratory chain complexes and induction of oxidative stress (Ishizawa et al., Cancer Cell 2019). However, the cell death mechanisms remain to be fully determined. We hypothesized that deregulation of mito-redox homeostasis induces a distinct mode of cell death in CLPP-hyperactivated cells and that concomitant inhibition of both mito- and cytosolic antioxidant mechanisms could enhance the anti-leukemia effects. We first tested if inhibition of glutathione (GSH) synthesis enhances the anti-leukemia effects by CLPP hyperactivation by using buthionine sulfoximine (BSO), a glutamate-cysteine ligase inhibitor. As hypothesized, BSO highly sensitized AML cells to genetic or pharmacologic hyperactivation of CLPP by overexpressing CLPP-Y118A (an activating mutation) or ONC201 treatment. We next investigated if a downstream target of GSH depletion is causing the synergy, and re-analysis of recently published CRISPR-knockout screens using leukemic cells treated with imipridones identified GPX4 as one of the 15 top-hit genes whose knockout sensitized cells to CLPP hyperactivation. GPX4 is a selenoprotein that is known for its GSH-dependent reduction of lipid hydroperoxides to protect cells from oxidative cell death including ferroptosis. We found that CLPP hyperactivation increased GPX4 protein levels in AML cells, prominently in mitochondria. Consistently, the GPX4 inhibitors ML210 and JKE1674 sensitized cells to ONC201 in multiple AML cell lines. Overexpression of the activating CLPP-Y118A was also synthetically lethal to GPX4 inhibition, confirming the on-target effect of ONC201. Mechanistically, GPX4 inhibition induced iron-dependent lipid peroxidation and AML cell killing, thus defining ferroptosis as the operational mechanism of cell death. Dual targeting of CLPP and GPX4 induced enhanced lipid peroxidation and mito-ROS but ferroptosis inhibitors only partially blocked cell death, indicating that other modes of oxidative cell death are also involved in the combinatorial synergism. Collectively, our data indicate therapeutic potential of targeting GPX4 and CLPP in AML and potential identification of a novel mode of cancer cell death. Investigations are ongoing to elucidate the underlying molecular mechanisms and to assess in vivo efficacy of the combinatorial treatment.
Citation Format: Hiroki Akiyama, Ran Zhao, Lauren B. Ostermann, Jo Ishizawa, Michael Andreeff. Dual targeting of glutathione-GPX4 nexus and mitochondrial protease CLPP causes synergistic anti-leukemia effects in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2017.