STAT3-VDAC1 Axis Modulates Mitochondrial Function and Plays a Critical Role in the Survival of Leukemic Stem Cells

Signal transducer and activator of transcription 3 (STAT3) is critical to the survival of acute myeloid leukemia (AML) cells. It has previously been shown to regulate mitochondrial functions such as oxidative phosphorylation (OXPHOS) via a MYC-SLC1A5-mediated pathway (Amaya et al. 2022) and regulate the transcription of important mitochondrial genes such as MCL-1 (Shastri et al. 2018). While STAT3 has also been shown to translocate to the mitochondria of AML cells, its role in mitochondria is less well defined in this cell population. In this study, we sought to understand the role of mitochondrial STAT3 in AML and its leukemia stem cell (LSC) population. We demonstrate that primary human AML cells have constitutive STAT3 activation via phosphorylation of S727 (pSTAT3 S727), which has been shown to signal mitochondrial localization. Further, we show that STAT3 is localized in the mitochondria of the AML cell-line MOLM-13, as well as in primary AML patient samples. To understand the role of STAT3 in mitochondria, both transcriptomic (siRNA) and pharmacologic (Stattic) inhibition of STAT3 were used in AML cells. STAT3 inhibition results in decreased pSTAT3 S727, with subsequent decreased translocation of STAT3 to the mitochondria. Additionally, inhibition of STAT3 results in decreased oxygen consumption rate (OCR) and increased reactive oxygen species (ROS), suggesting an important role of STAT3 in mitochondrial function. To determine whether STAT3 interacts with other mitochondrial proteins, we performed immunoprecipitation assays with STAT3 antibody on mitochondrial fraction isolates followed by mass spectrometry. This revealed an interaction between STAT3 and voltage-dependent anion channel 1 (VDAC1), an outer mitochondrial membrane protein involved in calcium and metabolite transport as well as in apoptosis. Interestingly, STAT3 inhibition led to decreased VDAC1 in mitochondrial fractions, as well as decreased mitochondrial calcium, decreased TCA cycle metabolites, decreased intracellular ATP, and increased ROS, all functions which are associated with VDAC1. In addition, direct VDAC1 inhibition resulted in similar effects on mitochondrial OCR, calcium, and ROS suggesting STAT3 and VDAC1 share a common pathway. To confirm that STAT3's regulatory effects in the mitochondria are achieved through its interaction with VDAC1, we used a transient VDAC1 overexpression system in MOLM-13 cells. Inhibition of STAT3 in the context of VDAC1 overexpression was able to partially restore mitochondrial calcium and decrease mitochondrial ROS. To determine whether STAT3 is important for survival of AML cells, we first performed in vitro viability assays showing that inhibition of STAT3 led to significantly decreased viability in MOLM-13 and primary AML cells. In addition, similar effects were seen upon VDAC1 inhibition. Finally, by utilizing a patient-derived xenograft (PDX), we tested whether STAT3 inhibition affects leukemia stem cell function. Treatment of 3 AML patient samples with a STAT3 inhibitor (Stattic) vs vehicle control resulted in significantly impaired engraftment of LSCs. Of note, AML samples resistant to the standard treatment venetoclax were sensitive to STAT3 inhibition, suggesting this could be a promising therapeutic target in the context of venetoclax-resistance. To conclude, in this study we show that STAT3 plays a key role in mitochondrial function of AML cells. STAT3 inhibition leads to decreased mitochondrial calcium and decreased OCR, increased ROS, and eventually cell death. Additionally, we discovered a novel role of STAT3 through its interaction with VDAC1, which modulates several mitochondrial functions including calcium and ROS balance. We further show that the STAT3-VDAC1 axis is critical to the survival of LSCs and could serve as a novel target in AML. Given the promising therapeutic implications of targeting STAT3, inhibitors of this protein are currently being investigated in AML (NCT05986240) as well as other cancers (NCT03195699), with thus far no reports of drug-limiting toxicities.