Sirtuin 3 Inhibition Targets AML Stem Cells through Perturbation of Fatty Acid Oxidation

Acute myeloid leukemia (AML) in adults has a 5-year survival of approximately 30% and a high rate of disease recurrence in part due to our inability to eliminate the disease-initiating leukemic stem cells (LSCs) (Shlush et al. Nature, 2017). Previous studies have shown that LSCs uniquely rely on oxidative phosphorylation (OXPHOS) for survival (Lagadinou et al. Cell Stem Cell, 2013). Thus, novel therapies that are designed to target LSC metabolism have the potential to improve patient outcomes.

Work from our group and others has demonstrated that a critical metabolite for OXPHOS regulation in LSCs is the coenzyme NAD ⁺ (Jones et al. Cell Stem Cell, 2020; Mitchell et al. Blood Advances 2019). One family of NAD ⁺ dependent proteins important in cancer biology, and AML specifically (Yan et al. Blood Cancer Discovery, 2021), are sirtuins. To determine if any sirtuins are important in LSC function we knocked down each sirtuin family member (sirtuin 1-7) with siRNA in four primary AML specimens and measured viability and colony forming ability. Knockdown of sirtuin 3 (SIRT3) decreased viability and colony forming potential of all AML specimens tested. SIRT3 is a mitochondrial de-acetylase with a multi-faceted role in metabolic regulation and oncogenesis (Finley, et al. Trends in Molecular Medicine, 2016). SIRT3 interacts with pathways upstream of OXPHOS including the tricarboxylic acid (TCA) and fatty acid oxidation (FAO). Importantly, a SIRT3 inhibitor (YC8-02) has been developed and has been shown to be effective pre-clinically for the treatment of B-cell lymphoma (Li et al. Cancer Cell, 2019).

To further understand the significance of SIRT3 in LSCs, we assessed viability and colony forming potential upon YC8-02 treatment. LSCs were enriched from primary specimens based upon relative reactive oxygen species (ROS) level as previously described (Lagadinou et al. Cell Stem Cell, 2013). LSCs and blasts enriched from ten primary AML, and four AML cell lines (MOLM13, TEX, OCI-AML2, OCI-AML3) were cultured for 48 hours with or without YC8-02 before assessing viability and colony forming ability. YC8-02 treatment resulted in a significant decrease in colony forming potential of AML cells compared to control (data not shown). Similarly, LSCs, blasts, and cell lines showed a significant decrease in viability upon YC8-02 treatment (Fig 1A and data not shown). Cord blood and mobilized peripheral blood samples conversely did not show a change in colony forming potential following SIRT3 knockdown or YC8-02 treatment, respectively (data not shown). To assess YC8-02's effect on LSC function, three AML samples were treated with 10µM of drug for 24 hours and transplanted into NSG-S mice. YC8-02 treatment resulted in a significant decrease in AML engraftment, indicating a decrease in LSC function (Fig 1B).

To determine the mechanism by which SIRT3 inhibition causes cell death, LSCs enriched from three primary specimens were treated with YC8-02; metabolite and lipid levels were determined by mass spectrometry. This analysis revealed a significant accumulation of fatty acids post YC8-02 treatment. To further characterize these changes, MOLM13 cells were treated with ¹³C ₁₆-palmitic acid following 4 hours of incubation with 10µM YC8-02. Cells were collected 4 and 16 hours after introduction of palmitic acid and metabolic tracing was assessed by mass spectrometry. We found an accumulation of long and very long chain fatty acids and a decrease in TCA cycle intermediates (Fig 1C). FAO normally supplies TCA with intermediate acetyl-CoA; thus, these data indicate a decrease in FAO upon YC8-02 treatment. Accordingly, we measured changes in OXPHOS in response to treatment with YC8-02, in primary LSCs (Fig 1D) and AML cell lines (data not shown) and found a significant decrease in basal oxygen consumption. Further, ATP levels were significantly decreased upon YC8-02 treatment in LSCs (Fig 1E).

In conclusion, we show that SIRT3 plays a pivotal role in FAO and LSC function. When SIRT3 is inhibited, FAO activity decreases resulting in the accumulation of long and very long chain fatty acids. This change in FAO activity reduces the availability of products for the TCA cycle, limiting necessary intermediates for OXPHOS, decreasing ATP production, and ultimately causing cell death. Therefore, our data suggests that SIRT3 is a potential therapeutic target for LSCs and should be considered in future pre-clinical and clinical investigations.

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