P438: intracellular citrate levels modulate anti-apoptotic proteins to enhance venetoclax sensitivity in acute myeloid leukemia stem cells

Background: Acute Myeloid Leukemia (AML) is an aggressive hematologic malignancy with poor overall survival. While classic chemotherapy and novel agents, such as Venetoclax, lead to remission in majority of patients, relapses driven by therapy-resistant leukemic stem cells (LSCs) are commonly observed. Eradication of the residual LSC population is therefore imperative in improving patient survival. LSCs are characterized by their distinct gene expression programs and epigenetic signatures. Recent proteomics studies revealed increased expression of an array of metabolism-related proteins in LSCs. Among these, many mitochondrial proteins, including those participating in oxidative phosphorylation, such as Isocitrate Dehydrogenase 3 αsubunit (IDH3A), were shown to be upregulated in LSCs (Raffel et al., Blood, 2020). Aims: In this study, we characterized the role of IDH3A in AML to identify metabolic vulnerabilities that can be used to target chemotherapy-resistant LSCs. Methods: IDH3A knockdown (KD) in AML cell lines, primary patient samples and healthy hematopoietic stem and progenitor cells (HSPCs) was achieved by lentivirus-mediated, doxycycline-inducible or constitutive shRNA expression. Inducible overexpression (OE) of IDH3A was achieved by lentivirus-mediated cDNA expression. Metabolite levels of AML cell lines, patient samples and healthy controls were quantified using Mass Spectrometry. Extracellular acidification rates (ECAR) were evaluated using a Seahorse XF96 analyzer. Protein levels of BCL-2, MCL-1 and BCL-XL were quantified using flow cytometry. Results: IDH3A protein expression is upregulated in LSC populations compared to AML blasts and healthy HSPCs. Higher IDH3A gene expression is associated with poor overall survival in large AML patient cohorts. IDH3A KD in AML cells resulted in impaired proliferation and colony forming capacity whereas IDH3A OE cells showed higher proliferation rates and colony forming potential. In contrast, IDH3A depletion in healthy HSPCs did not affect proliferation and colony forming capacity. Transcriptome and proteome analyses of IDH3A depleted AML cells revealed downregulation in glucose metabolism related pathways. Consistently, IDH3A KD cells showed lower ECAR values in Seahorse experiments. Metabolome profiling revealed increased intracellular citrate levels upon IDH3A KD while IDH3A OE cells showed lower levels of citrate. Recapitulating the effects of IDH3A KD, citrate supplementation decreased viability of AML cells in a dose dependent manner. Furthermore, in line with its established role in suppressing glycolysis, supplementing AML cells with citrate led to decreased glycolytic activity. Untargeted metabolome analyses showed LSCs to contain lower levels of intracellular citrate compared to healthy HSPCs. Despite their smaller intracellular citrate pool, LSCs showed greater susceptibility to citrate supplementation in comparison to HSPCs. Both IDH3A KD and citrate supplementation were found to modulate the expression of pro-apoptotic proteins MCL-1, BCL-XL and BCL-2. In line with this observation, IDH3A depletion and citrate supplementation increased the sensitivity of AML cells to BCL-2 inhibition by Venetoclax. Summary/Conclusion: Our study identifies IDH3A as a critical regulator of central carbon metabolism in AML, mainly by controlling intracellular citrate levels. Particularly upregulated in LSCs, but not in HSPCs, IDH3A limits intracellular citrate. IDH3A KD or citrate supplementation modulates anti-apoptotic proteins in LSCs, leading to increased sensitivity to Venetoclax treatment. Keywords: Mitochondria, Acute myeloid leukemia, Venetoclax, Drug resistance