Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Leukemia stem cells (LSCs) drive the initiation and perpetuation of AML, are quantifiably associated with worse clinical outcomes, and often persist after conventional chemotherapy resulting in relapse 1-5. In this report, we show that treatment of older AML patients with the B-cell lymphoma 2 (BCL-2) inhibitor venetoclax in combination with azacitidine results in deep and durable remissions and is superior to conventional treatments. We hypothesized that these promising clinical results were due to targeting LSCs. Analysis of LSCs from patients undergoing treatment with venetoclax + azacitidine showed disruption of the TCA cycle manifested by decreased alpha-ketoglutarate and increased succinate levels, suggesting inhibition of electron transport chain complex II. In vitro modeling confirmed inhibition of complex II via reduced glutathionylation of succinate dehydrogenase. These metabolic perturbations suppress oxidative phosphorylation (OXPHOS), which efficiently and selectively targets LSCs. Our findings show for the first time that a therapeutic intervention can eradicate LSCs in AML patients by disrupting the metabolic machinery driving energy #

Summary: In this study we interrogated the metabolome of human acute myeloid leukemia (AML) stem cells to elucidate properties relevant to therapeutic intervention. We demonstrate that amino acid uptake, steady-state levels, and catabolism are all elevated in the leukemia stem cell (LSC) population. Furthermore, LSCs isolated from de novo AML patients are uniquely reliant on amino acid metabolism for oxidative phosphorylation and survival. Pharmacological inhibition of amino acid metabolism reduces oxidative phosphorylation and induces cell death. In contrast, LSCs obtained from relapsed AML patients are not reliant on amino acid metabolism due to their ability to compensate through increased fatty acid metabolism. These findings indicate that clinically relevant eradication of LSCs can be achieved with drugs that target LSC metabolic vulnerabilities.

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SUMMARY There are still gaps in our understanding of the complex processes by which p53 suppresses tumorigenesis. Here we describe a novel role for p53 in suppressing the mevalonate pathway, which is responsible for biosynthesis of cholesterol and nonsterol isoprenoids. p53 blocks activation of SREBP-2, the master transcriptional regulator of this pathway, by transcriptionally inducing the ABCA1 cholesterol transporter gene. A mouse model of liver cancer reveals that downregulation of mevalonate pathway gene expression by p53 occurs in premalignant hepatocytes, when p53 is needed to actively suppress tumorigenesis. Furthermore, pharmacological or RNAi inhibition of the mevalonate pathway restricts the development of murine hepatocellular carcinomas driven by p53 loss. Like p53 loss, ablation of ABCA1 promotes murine liver tumorigenesis and is associated with increased SREBP-2 maturation. Our findings demonstrate that repression of the mevalonate pathway is a crucial component of p53-mediated liver tumor suppression and outline the mechanism by which this occurs.

To study mechanisms underlying resistance to the BCL2 inhibitor venetoclax in acute myeloid leukemia (AML), we used a genome-wide CRISPR/Cas9 screen to identify gene knockouts resulting in drug resistance. We validated TP53 , BAX , and PMAIP1 as genes whose inactivation results in venetoclax resistance in AML cell lines. Resistance to venetoclax resulted from an inability to execute apoptosis driven by BAX loss, decreased expression of BCL2, and/or reliance on alternative BCL2 family members such as BCL2L1. The resistance was accompanied by changes in mitochondrial homeostasis and cellular metabolism. Evaluation of TP53 knockout cells for sensitivities to a panel of small-molecule inhibitors revealed a gain of sensitivity to TRK inhibitors. We relate these observations to patient drug responses and gene expression in the Beat AML dataset. Our results implicate TP53 , the apoptotic network, and mitochondrial functionality as drivers of venetoclax response in AML and suggest strategies to overcome resistance. SIGNIFICANCE: AML is challenging to treat due to its heterogeneity, and single-agent therapies have universally failed, prompting a need for innovative drug combinations. We used a genetic approach to identify genes whose inactivation contributes to drug resistance as a means of forming preferred drug combinations to improve AML treatment.

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Red blood cell storage in the blood bank promotes the progressive accumulation of metabolic alterations that may ultimately impact the erythrocyte capacity to cope with oxidant stressors. However, the metabolic underpinnings of the capacity of RBCs to resist oxidant stress and the potential impact of donor biology on this phenotype are not known. Within the framework of the REDS-III RBC-Omics study, RBCs from 8,502 healthy blood donors were stored for 42 days and tested for their propensity to hemolyze following oxidant stress. A subset of extreme hemolyzers donated a second unit of blood, which was stored for 10, 23, and 42 days and profiled again for oxidative hemolysis and metabolomics (599 samples). Alterations of RBC energy and redox homeostasis were noted in donors with high oxidative hemolysis. RBCs from females, donors over 60 years old, donors of Asian/South Asian race-ethnicity, and RBCs stored in additive solution-3 were each independently characterized by improved antioxidant metabolism compared to, respectively, males, donors under 30 years old, Hispanic and African American race ethnicity donors, and RBCs stored in additive solution-1. Merging metabolomics data with results from an independent GWAS study on the same cohort, we identified metabolic markers of hemolysis and G6PD-deficiency, which were associated with extremes in oxidative hemolysis and dysregulation in NADPH and glutathione-dependent detoxification pathways of oxidized lipids. Donor sex, age, ethnicity, additive solution and G6PD status impact the metabolism of the stored erythrocyte and its susceptibility to hemolysis following oxidative insults.

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