Targeting SIRT5 By Succinylating Hadha Synergizes with Venetoclax in Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is genetically diverse, driven by multiple oncogenic factors. Metabolic dysregulation is a critical vulnerability in AML cells for targeted therapy. BCL2 has been previously reported to maintain mitochondrial oxidative phosphorylation (OXPHOS) in AML cells. One of the mechanisms of action of the combination of venetoclax and azacitidine is to inhibit amino acid metabolism, leading to cell death. Although venetoclax-based treatment regimens have shown promising clinical outcomes in AML patients, treatment resistance has become a newly emerged challenge. Leukemia stem cells isolated from venetoclax-resistant patients exhibit distinctive metabolic features, including elevated nicotinamide metabolism. SIRT5 is an NAD+-dependent deacylase enzyme, and its knockout leads to elevated succinylation of various enzymes involved in OXPHOS. Hence, we hypothesize that inhibiting SIRT5 may augment venetoclax's cytotoxic effect by dampening metabolism. To further elucidate this mechanism, we employed proteome analyses of succinylation to identify specific target molecules regulated by SIRT5.

Methods: To determine the effects of combination therapy on cell proliferation, apoptosis, and colony formation, AML cell lines were transduced with lentiviruses carrying either shNC or shSIRT5, or treated with NRD167 (a specific inhibitor of SIRT5), followed by venetoclax treatment. To evaluate the clearance of leukemia cells in vivo, NSG mice were transplanted with MOLM-13 cells (carrying shNC or shSIRT5) and then subjected to vehicle or venetoclax treatment (80 mg/kg/day) via oral gavage for 14 days. Weekly live animal in vivo imaging was conducted to monitor tumor engraftment and growth. The synergistic effect on the metabolic level of AML was assessed by measuring ATP, glutathione, mitochondrial superoxide, and oxygen consumption rates (OCR). Electron microscopy was employed to detect changes in cellular mitochondrial structure after SIRT5 knockdown and venetoclax utilization. Furthermore, lysine succinylation proteome analyses were performed to identify proteins and sites with altered succinylation on lysine before and after SIRT5 knockdown in MOLM-13 cells. Immunoprecipitation assays and Western blot were conducted to verify the interaction and altered succinylation levels of the relevant proteins.

Results: The combination of SIRT5 inhibition with venetoclax treatment resulted in further suppression of cell proliferation, reduced colony formation, and increased apoptosis in AML cells compared to venetoclax treatment alone. The combination led to alterations in the metabolic profile of leukemia cells, increased the generation of mitochondrial reactive oxygen species (ROS), reduced intracellular glutathione levels, inhibited ATP production, and suppressed OCR. Notably, combination therapy resulted in a significant reduction in the engraftment levels of human leukemia cells in NSG mice. Mechanistically, electron microscopy revealed that combination treatment with SIRT5 knockdown increased mitochondrial damage. Lysine succinylation proteome analyses demonstrated upregulation of succinylation modification on mitochondrial trifunctional enzyme subunit α (HADHA), a protein involved in the fatty acid oxidation (FAO) pathway. Co-immunoprecipitation of SIRT5 confirmed its interaction with HADHA, and the relative succinylation level of precipitated HADHA was upregulated after SIRT5 knockdown. Moreover, the combination of trimetazidine (a specific inhibitor of HADHA) with venetoclax demonstrated a preferential reduction in cell proliferation and induction of apoptosis in AML cells.

Conclusion: Targeting of SIRT5 in combination with venetoclax treatment results in synergistic cytotoxic effects in AML cells. We hypothesize that SIRT5 desuccinylase activity promotes FAO and reduces intracellular mitochondrial ROS levels by desuccinylating HADHA. Targeting SIRT5 inhibits fatty acid metabolism in AML cells, thereby enhancing the therapeutic effect of venetoclax. We will further investigate the site(s) of SIRT5 desuccinylation modification of HADHA through lysine site mutation and assess changes in HADHA enzyme activity to elucidate its regulatory mechanism.