Dual Targeting of Mitochondrial Unfolded Protein Response and BCL2 in Acute Myeloid Leukemia

Cellular stress response has dual aspects; cell-protective or lethal. Mitochondria have their unique organellar response termed "mitochondrial unfolded protein response (UPR^mt)" induced by damaged mitochondrial (mt) matrix proteins. While recent discoveries have successfully targeted BCL2, a regulator of mt integrity in acute myeloid leukemia (AML), the significance of UPR^mt is unknown. We hypothesized that priming UPR^mt towards cell death would be a novel therapeutic strategy for AML.

UPR^mt is generally induced by dysregulation of mt protein pools. Therefore, to test if UPR^mt signaling is also operational in AML cells, we selected classical or putative UPR^mt inducers; the mt translation inhibitors tetracycline and tigecycline, the mt protein transport inhibitor MitoBlock6, and the mtDNA damaging agent ethidium bromide. In OCI-AML3 and HL60 cells, these agents indeed induced the transcription factor ATF5, which was reported as a central inducer of UPR^mt, and its targets (e.g., LonP, HSPA9), triggering apoptosis in AML cells. In addition, we here report imipridones (ONC201 and ONC212), the activators of mt protein degradation, as novel UPR^mt inducers. We recently reported that imipridones non-covalently bind the mt protease ClpP and allosterically activate it. They induced prominent apoptosis in primary AML progenitor and leukemia initiating cells (LICs) in vitro and in vivo, but not in normal bone marrow cells, following "mitochondrial proteolysis" with reduction of selective mt matrix proteins (e.g., SDHB, NDUFA12) and resultant inhibition of oxidative phosphorylation (Oxphos) (Ishizawa, Zarabi et al, Cancer Cell 2019). We then postulated that dysregulation of mt protein pools by mitochondrial proteolysis can also induce UPR^mt. Indeed, our gene expression profiles of ONC201-treated Z138 and Jeko-1 cells were highly enriched for previously published UPR^mt gene signatures, and UPR^mt effectors were induced also in AML cells.

Of potentially high clinical significance is the finding of synergistic anti-leukemia effects of imipridones when combined with the selective BCL2 inhibitor venetoclax, in vitro and in vivo (Ishizawa et al. Science Signaling 2016, and Nii et al. Blood 2019). However, its underlying molecular mechanism is unclear. Since BCL2 is reported to be induced by UPR^mt, we hypothesized that BCL2 is critical for the ClpP-mediated UPR^mt to have the cell protective effects, contrary to lethal effects, as dual aspects of stress response. We utilized the tetracycline-inducible system of an activated mutant (Y118A) form of ClpP in OCI-AML3 cells, and demonstrated that venetoclax treatment sensitizes OCI-AML3 cells to genetic activation of ClpP towards apoptosis. Furthermore, other UPR^mt inducers (tetracycline, tigecycline, and MitoBlock6) in combination with venetoclax also synergistically induced apoptosis in AML cells, suggesting that BCL2 inhibition and UPR^mt induction generally exerts synergistic anti-leukemia effects.

We next focused on the enhanced effect observed for the combination of imipridones with venetoclax as compared to other UPR^mt inducers, searching for other targets that could further enhance the synergy. We then hypothesized that the synergism between ClpP activation and BCL2 inhibition involves SDHB, a respiratory chain complex II subunit degraded by activated ClpP but not targeted by any of other UPR^mt inducers. Consistently, SDHB knockdown sensitized OCI-AML3 cells to venetoclax-induced apoptosis, indicating that SDHB reduction and UPR^mt by ClpP activation concomitantly enhance the cell lethality by BCL2 inhibition.

Collectively, UPR^mt is a new potential therapeutic target for AML, which significantly enhances the cell death effects of BCL2 inhibition on AML cells. In particular, ClpP activation induces UPR^mt and, concomitantly, downregulates SDHB, thus targeting the respiratory chain complex II, which results in improved synergistic leukemia cell apoptosis when combined with BCL2 inhibition. Oxphos is also a hallmark of drug resistant AML stem cells, which supports the notion that Oxphos inhibition by this combination targets LICs. Based on promising preclinical anti-tumor efficacy, ONC201 as a single agent is being evaluated in early phase clinical trials, showing clinical responses in AML and midline gliomas. A clinical trial testing the combinatorial strategy of targeting ClpP and Bcl-2 is under development.