Nicotinamide Metabolism Mediates Resistance to Venetoclax in Relapsed Acute Myeloid Leukemia Stem Cells

Abstract

We previously demonstrated that leukemia stem cells (LSCs) in de novo acute myeloid leukemia (AML) patients are selectively reliant on amino acid metabolism and that treatment with the combination of venetoclax and azacitidine (ven/aza) inhibits amino acid metabolism, leading to cell death. In contrast, ven/aza fails to eradicate LSCs in relapsed/refractory (R/R) patients, suggesting altered metabolic properties. Detailed metabolomic analysis revealed elevated nicotinamide metabolism in relapsed LSCs, which activates both amino acid metabolism and fatty acid oxidation to drive OXPHOS, thereby providing a means for LSCs to circumvent the cytotoxic effects of ven/aza therapy. Genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide metabolism, demonstrated selective eradication of R/R LSCs while sparing normal hematopoietic stem/progenitor cells. Altogether, these findings demonstrate that elevated nicotinamide metabolism is both the mechanistic basis for ven/aza resistance and a metabolic vulnerability of R/R LSCs.

Keywords: NAD+; NAMPT; acute myeloid leukemia; leukemia stem cells; metabolism; nicotinamide; oxidative phosphorylation; relapse; therapy resistance; venetoclax.

Figure 1.. R/R LSCs Have Increased Nicotinamide Metabolism

(A) Principal-component analysis of de novo and R/R LSCs generated using MetaboAnalyst 4.0. AML specimens used in this analysis include AML1–AML9 and AML14–AML16. (B) Heatmap of amino acids levels in de novo and R/R LSCs. AML specimens used in this analysis include AML1–AML9 and AML14–AML16. (C) Amino acids levels in paired de novo and relapsed LSCs. AML specimens used in this analysis include AML17–AML20. (D) Nicotinamide levels in de novo and R/R LSCs. AML specimens used in this analysis include AML1–AML9 and AML14–AML20. The left graph represents nicotinamide levels in unpaired specimens, and the right graph represents nicotinamide levels in paired specimens. (E) NAD+ levels in paired de novo and relapsed LSCs and AML blasts (non-LSCs). AML specimens used in this analysis include AML17–AML21. (F) Levels of SIL nicotinamide in de novo and R/R LSCs 12 h after [¹³C₃,¹⁵N] nicotinamide incubation determined by mass spectrometry. AML2 and AML5 were used in this analysis. (G) Levels of SIL NAD+ from nicotinamide in de novo and R/R LSCs 12 h after [¹³C₃,¹⁵N] nicotinamide incubation determined by mass spectrometry. AML2 and AML5 were used in this analysis. (H) Ratio of SIL NAD+/nicotinamide in de novo and R/R LSCs 12 h after [¹³C₃,¹⁵N] nicotinamide incubation. AML2 and AML5 were used in this analysis. Statistical significance was determined using Student’s t test (B, D, F–H) or a paired Student’s t test (E). *p < 0.05, **p < 0.01, ***p < 0.005 ****p < 0.001.

Figure 2.. R/R LSCs Have Increased Energy Metabolism Compared with De Novo LSCs

(A) ATP levels from LSCs isolated from 3 de novo and 3 R/R AML patients determined by mass spectrometry. AML specimens used in this analysis include AML1– AML6. Statistical significance was determined using an unpaired Student’s t test. (B) Schematic of experimental design. LSCs were isolated from de novo and R/R AML patient specimens and then incubated with [¹³C₆] glucose, ¹³C¹⁵N amino acids, or[¹³C₁₆] palmitic acid for 1 or 8 h. Metabolites were then measured by mass spectrometry. (C) Heatmap of ¹³C¹⁵N amino acids in de novo and R/R LSCs after a 1-h incubation. Statistical significance was determined using a paired Student’s t test. AML2 and AML5 were used in this analysis. (D) TCA cycle intermediate citrate metabolized from ¹³C¹⁵N amino acids. (E) Palmitic acid uptake from [¹³C₁₆ palmitic acid. (F) TCA cycle intermediate citrate metabolized from [¹³C₁₆ palmitic acid. (G) Glucose uptake from [¹³C6] glucose. (G) lucose uptake from [¹³C6] glucose. (H) Pyruvae metabolized from [¹³C6] glucose. (I) TCA cycle intermediate, citrae metabolized from [¹³C6] glucose. (D–I) Statistical significance determined by two-way ANOVA, and AML2 and AML5 were used in the analysis. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001.

Figure 3.. Nicotinamide Metabolism Mediates Energy Metabolism and ven/aza Resistance

(A) NAD+/H levels upon a 1-h incubation with 128 μM nicotinamide. (B) Schematic of experimental design. LSCs were isolated from de novo AML patient specimens, incubated with 128 μM nicotinamide for 1 h, and then incubated with [¹³C₆] glucose, ¹³C,¹⁵N amino acids, or [¹³C₁₆] palmitic acid for 8 h. Metabolites were then measured by mass spectrometry. (C) Heatmap of amino acid upon in vehicle control and nicotinamide pretreatment LSCs. (D) TCA cycle intermediates metabolized from ¹³C¹⁵N amino acids. (E) Palmitate and TCA cycle intermediates metabolized from [¹³C₁₆] palmitic acid. (F) Glucose and pyruvate metabolized from [¹³C₆] glucose. (G) Viability of de novo LSCs pretreated with 128 μM nicotinamide for 1 h and then ven (500 nM)/aza (2.5 μM) for 24 h where indicated. Statistical significance was determined by a two-way ANOVA. AML specimens 1–3, 11, and 12 were used in this analysis. Statistical significance was determined using an unpaired Student’s t test (A and D–F) or a paired Student’s t test (C), and AML1–AML3 were used in the analysis. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001.

Figure 4.. Inhibition of Nicotinamide Metabolism Specifically Targets R/R LSCs

(A) Percentage of survival of AML patients stratified by the 25% highest and lowest NAMPT expression. Data mined from TCGA. (B) Viability of de novo and R/R LSCs after a 24-h treatment with 10 nM APO866. Each dot represents an individual patient specimen normalized to vehicle control. AML specimens used in this analysis are AML1–AML12. (C) Colony-forming ability of de novo and R/R LSCs after treatment with 10 nM APO866. Each dot represents an individual patient specimen normalized to vehicle control. AML specimens used in this analysis are AML1, AML2, AML4–AML6, and AML8–AML11. (D) Viability of de novo and R/R LSCs after a 24-h treatment with 100 nM KPT-9274. Each dot represents an individual patient specimen normalized to vehicle control. AML specimens used in this analysis include AML1–AML13. (E) Colony-forming ability of de novo and R/R LSCs after treatment with 100 nM KPT-9274. Each dot represents an individual patient specimen normalized to vehicle control. AML specimens used in this analysis include AML1, AML2, and AML4–AML10. (F) Frequency of normal HSPCs (CD34+/CD45+ cells) from normal mobilized peripheral blood after a 24-h treatment with 10 nM APO866 or 100 nm KPT-9274. Each dot represents an individual patient specimen. (G) Colony number from normal mobilized peripheral blood after a 24-h treatment with 10 nM APO866 or 100 nM KPT-9274. Each dot represents an individual patient specimen. (H) Viability of primary AML specimens 5, 8, and 9 after siRNA-mediated knockdown of NAMPT compared with scrambled control. (I) Colony-forming ability of AML specimens 5, 8, and 9 after siRNA-mediated knockdown of NAMPT compared with scrambled control. (J) Viability of LSCs isolated from paired de novo and relapsed specimens (AML specimens 17–20) after treatment with APO866, KPT-9274, ven/aza, or cytarabine compared with vehicle control. Statistical significance was determined using Student’s t test (H and I), an unpaired Student’s t test (B–F), or ANOVA (J). *p < 0.05, **p < 0.01, ****p < 0.001.

Figure 5.. Inhibition of Nicotinamide Metabolism Specifically Targets R/R LSCs In Vivo

(A) Paired specimens from the same patient, AML2 (de novo) and AML5 (relapse), were treated with ven/aza or APO866 for 24 h and then transplanted into immunodeficient mice. Engraftment was measured in the bone marrow. (B) Experimental schema for the in vivo treatment and secondary engraftment assays. (C) Leukemia burden in mouse bone marrow after 2 weeks of APO866 treatment and engraftment after in vivo APO866 treatment. Leukemia burden in mouse femur upon secondary engraftment. (D) Leukemia burden in mouse femur after 2 weeks of KPT-9274 treatment. (E) Levels of normal human cell engraftment (CD45+ cells) in mouse femur after 2 weeks of APO866 or KPT-9274 treatment. (F) Normal HSPC (CD45+/CD34+) in mouse femur after 2 weeks of APO866 or KPT-9274 treatment. (A and C–F) Statistical significance was determined using an unpaired Student’s t test. Each dot represents an individual mouse. **p < 0.01, ***p < 0.005, ****p < 0.001.

Figure 6.. Inhibition of Nicotinamide Metabolism Decreased OXPHOS in R/R LSCs

(A and B) Oxygen consumption (A) and spare oxygen consumption capacity (B) determined by Seahorse assay in de novo and R/R LSCs after a 4-h treatment with 10 nM APO866. Each dot represents an individual patient specimen normalized to vehicle control. AML specimens used in this analysis are AML1–AML6, AML8, AML10, and AML11. (C) Oxygen consumption determined by Seahorse assay in de novo and R/R LSCs after a 4-h treatment with 100 nM KPT-9274. Each dot represents an individual patient specimen normalized to vehicle control. AML specimens used in this analysis are AML1–AML6, AML8, and AML9. (D) NAD+/H levels in leukemic cells isolated from mice treated with APO866 or KPT-9274 for 24 h. (E) Oxygen consumption determined by Seahorse assay in leukemic cells isolated from mice treated with APO866 or KPT-9274 for 24 h. (F) Spare oxygen consumption capacity determined by Seahorse assay in leukemic cells isolated from mice treated with APO866 or KPT-9274 for 24 h. (G) Abundance of TCA cycle intermediates in de novo LSCs (red/pink) and R/R LSCs (blue/light blue) treated with vehicle control or APO866 for 4 h determined by mass spectrometry. Each dot represents an individual patient. AML specimens used in this analysis are AML1–AML6. Statistical significance was determined by a two-way ANOVA. Yellow stars indicate enzymes within the TCA cycle that are NAD+ dependent. (H) Activity of 2-oxoglutarate dehydrogenase, isocitrate dehydrogenase, malate dehydrogenase, or hexokinase in R/R LSCs upon treatment with vehicle control or 10 nM APO866 for 4 h. Each dot represents an individual patient specimen. AML specimens used in this analysis are AML4–AML6. (I) Enzyme activity was determined in de novo and R/R LSCs. AML1–AML3, AML5, AML6, and AML8 were used for this analysis. Each dot represents an individual AML specimen. Statistical significance was determined using a pair Student’s t test. Statistical significance was determined using an unpaired Student’s t test (A–C and H) or ANOVA (D–F). *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001.

Figure 7.. Nicotinamide Metabolism Inhibition Decreases Amino Acid and Fatty Acid Metabolism

(A) Schematic of experimental design. LSCs were isolated from R/R AML patient specimens, treated with vehicle control or 10 nM APO866 for 4 h, and then incubated with [¹³C₆] glucose, ¹³C¹⁵N amino acids, or [¹³C₁₆] palmitic acid for 8 h. Metabolites were then measured by mass spectrometry. (B) Heatmap of ¹³C¹⁵N amino acids levels upon control or 10 nM APO866 treatment. AML5 was used in this analysis. (C) Malate levels from ¹³C¹⁵N amino acids in control or 10 nM APO866-treated LSCs. AML5, AML8, and AML9 were used in this analysis. (D) [¹³C₁₆] palmitic acid levels in R/R LSCs treated with vehicle control or 10 nM APO866. AML5, AML8, and AML9 were used in this analysis. (E) TCA cycle intermediates metabolized from ¹³C₁₆ palmitic acid with vehicle or 10 nM APO866 treatment. AML5, AML8, and AML9 were used in this analysis. (F) Viability of primary AML specimens 5, 8, and 9 after siRNA-mediated knockdown of HADH compared with scrambled control upon ven/aza or vehicle treatment. (G) Colony-forming ability of primary AML specimens 5, 8, and 9 after siRNA-mediated knockdown of HADH compared with scrambled control upon ven/aza or vehicle treatment. (H) OXPHOS levels of primary AML specimens 5, 8, and 9 after siRNA-mediated knockdown of HADH compared with scrambled control upon ven/aza or vehicle treatment. (I) Diagram illustrating that LSCs isolated from R/R AML patients have increased nicotinamide metabolism and ven/aza resistance. Increased nicotinamide metabolism mediates overall energy metabolism in R/R LSCs, resulting in increased amino acid and fatty acid metabolism into the TCA cycle. (J) Summary diagram showing that increased NAD+ levels in R/R LSCs are necessary for sufficient function of TCA cycle enzymes and that inhibition of nicotinamide metabolism decreases TCA cycle enzyme function, resulting in decreased levels of OXPHOS and LSC death. Statistical significance was determined using Student’s t test (F–H), a paired Student’s t test (B), or an unpaired Student’s t test (C–E). *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001.