Electron transport chain inhibition increases cellular dependence on purine transport and salvage

Abstract

Mitochondria house many metabolic pathways required for homeostasis and growth. To explore how human cells respond to mitochondrial dysfunction, we performed metabolomics in fibroblasts from patients with various mitochondrial disorders and cancer cells with electron transport chain (ETC) blockade. These analyses revealed extensive perturbations in purine metabolism, and stable isotope tracing demonstrated that ETC defects suppress de novo purine synthesis while enhancing purine salvage. In human lung cancer, tumors with markers of low oxidative mitochondrial metabolism exhibit enhanced expression of the salvage enzyme hypoxanthine phosphoribosyl transferase 1 (HPRT1) and high levels of the HPRT1 product inosine monophosphate. Mechanistically, ETC blockade activates the pentose phosphate pathway, providing phosphoribosyl diphosphate to drive purine salvage supplied by uptake of extracellular bases. Blocking HPRT1 sensitizes cancer cells to ETC inhibition. These findings demonstrate how cells remodel purine metabolism upon ETC blockade and uncover a new metabolic vulnerability in tumors with low respiration.

Keywords: HPRT1; NAD(+):NADH ratio; electron transport chain; metabolomics; purine metabolism; stable isotopes.

Figure 1.. Metabolomic profiling of patients with mitochondrial disorders.

A. Illustration of the human mitochondrial defects analyzed in panels B-E. B. and C. Altered metabolite abundances in the TCA cycle (B) and purine metabolism (C) in fibroblasts from patients with the indicated mitochondrial defects. Each dot represents a fibroblast line from a patient with a disorder affecting the mitochondria. The mutated gene from each disorder is indicated. D. and E. Plasma metabolite levels from a patient with LIPT1 deficiency and healthy controls. n = 60 (healthy); n = 28 (LIPT1 deficiency; samples collected on different days). An unpaired, two-sided t test was used for the statistical analysis. BioRender was used to generate the illustration.

Figure 2.. Mitochondrial ETC deficiency causes accumulation of purine metabolites.

A. Oxygen consumption rates (OCR) of H460 cells pre-treated with DMSO or 25 nM IACS-010759. OA: oligomycin A; CCCP: Carbonyl cyanide m-chlorophenylhydrazone. Data represent one of three independent experiments. B. NAD⁺:NADH ratio in H460 cells treated with DMSO or 25 nM IACS-010759 for 24 hours (n=3). C. Volcano plot showing metabolomic changes in H460 cells treated with DMSO or 25 nM IACS-010759 for 24 hours. The pink circles are increased purine metabolites with FDR < 0.05. D. Schematic illustrating the mechanism of NDI1 rescue of ETC complex I blockade. E. OCR of control and NDI1-expressing H460 cells pre-treated with DMSO or 25 nM IACS-010759. Data represent one of three independent experiments. F. NAD⁺:NADH ratio in control and NDI1-expressing H460 cells treated with DMSO or 25 nM IACS-010759 for 24 hours (n=3). G. Growth rates of control and NDI1-expressing cells cultured in glucose or galactose medium and treated with DMSO or 25 nM IACS-010759 (n=6). Data are from one of three independent experiments. H. Principal component analysis of metabolomic profiles in control and NDI1-expressing H460 cells treated with DMSO or 25 nM IACS-010759 for 24 hours. I. Relative abundance of the indicated purine nucleotides in control and NDI1-expressing H460 cells treated with DMSO or 25 nM IACS-010759 for 24 hours (n=3). J. Metabolite set enrichment analysis comparing IACS-010759-treated control and NDI-expressing H460 cells. K. Western blot validating deletion of UQCRC2. Vinculin is the loading control. L. OCR in WT and UQCRC2^−/− H460 cells. AA; antimycin A. Data are from one of three independent experiments. M. Metabolic pathway analysis of differentially abundant metabolites in UQCRC2-depleted (UQCRC2^−/−) H460 cells compared to parental cells. N. Relative abundance of the indicated purine metabolites in WT and UQCRC2^−/− H460 cells (n=3). Unpaired, two-sided t tests were used for the statistical analyses. ****: P < 0.0001; ***: P < 0.001, **: P < 0.01, *: P < 0.05; n.s.: P > 0.05. Error bars denote SEM. BioRender was used to generate the illustration.

Figure 3.. Cytosolic NAD(H) imbalance impacts purine accumulation in ETC-deficient cells.

A. Schematic of LbNOX-catalyzed reaction. B. Western blot validating expression of Flag-tagged LbNOX in UQCRC2^−/− H460 cells. Vinculin is the loading control. C. Immunofluorescence showing the subcellular localization of the indicated Flag-tagged LbNOX proteins in UQCRC2^−/− H460 cells. HSP60 is a mitochondrial matrix marker. Scale bar represents 10 μm. D. NAD⁺:NADH ratio in UQCRC2^−/− cells expressing empty vector (EV), Mito-LbNOX or Cyto-LbNOX (n=3). E. Growth rates of UQCRC2^−/− H460 cells expressing empty vector (EV), Mito-LbNOX, or Cyto-LbNOX (n=10). Data are from one of three independent experiments. F. Relative abundance of aspartate in WT H460 cells and UQCRC2^−/− H460 cells expressing empty vector (EV), Mito-LbNOX or Cyto-LbNOX (n=3). G. OCR of WT and UQCRC2^−/− cells expressing empty vector (EV), Mito-LbNOX or Cyto-LbNOX. Data are from one of three independent experiments. H. Principal component analysis of metabolomic profiles in WT and UQCRC2^−/− cells expressing empty vector (EV), Mito-LbNOX, or Cyto-LbNOX. I. Metabolite set enrichment analysis comparing UQCRC2^−/− cells expressing empty vector (EV) or Cyto-LbNOX. J. Metabolite set enrichment analysis comparing UQCRC2^−/− cells expressing empty vector (EV) or Mito-LbNOX. K. Heatmap displaying metabolite abundance in WT H460 cells and UQCRC2^−/− H460 cells expressing empty vector (EV), Mito-LbNOX or Cyto-LbNOX. Unpaired, two-sided t tests were used for the statistical analyses. ****: P < 0.0001, ***: P < 0.001, **: P < 0.01, *: P < 0.05. Error bars denote SEM. BioRender was used to generate the illustration.

Figure 4.. ETC blockade suppresses de novo purine nucleotide synthesis.

A. Schematic illustrating ¹³C labeling of purines from [U-¹³C]glucose. B. ¹³C labeling in IMP after 6 hours of culture with [U-¹³C]glucose in control and NDI1-expressing H460 cells pre-treated with DMSO or 25 nM IACS-010759 for 24 hours (n=3). C. Schematic illustrating ¹⁵N labeling from [amide-¹⁵N]glutamine during de novo purine nucleotide synthesis. D. Time-dependent fractional enrichment of m+2 IMP and m+3 GMP in control and NDI1-expressing H460 cells pre-treated with DMSO or 25 nM IACS-010759 for 24 hours (n =3 at each time point). E. Schematic illustrating infusion of [amide-¹⁵N]glutamine into mice bearing H460 xenografts. F. Relative abundance of AICAR in H460 xenografts treated with vehicle or IACS-010759 for 5 days. Vehicle (n=10), IACS (n=8). G-H. Fractional enrichment of m+1 glutamine (G), m+2 IMP and m+2 AMP (H) in vehicle and IACS-010759-treated H460 xenografts after 4 hours of [amide-¹⁵N]glutamine infusion. Vehicle (n=10), IACS (n=8). I. Western blot validating overexpression of SLC1A3. Vinculin is the loading control. EV: empty vector; OE: overexpression. J. Fractional enrichment of m+2 IMP, m+2 AMP, and m+3 GMP after 6 hours of culture with [amide-¹⁵N]glutamine in empty vector-expressing control cells (EV) and SLC1A3-overexpressing (SLC1A3^OE) cells pretreated with or without DMSO, 25 nM IACS-010759, 150 μM aspartate (Asp), and 10 μM hypoxanthine (hypo) (n=3). Unpaired, two-sided t tests (F-H), two-way ANOVA (D), and one-way ANOVA (J) were used for the statistical analyses. ****: P < 0.0001; ***: P < 0.001, **: P < 0.01; *: P < 0.05; n.s.: P > 0.05. Error bars denote SEM. BioRender was used to generate the illustration.

Figure 5.. ETC blockade promotes purine salvage.

A. Schematic illustrating conversion of [¹⁵N₄]hypoxanthine to m+4 IMP during HPRT1-mediated salvage. B. Fractional enrichment of m+4 IMP from [¹⁵N₄]hypoxanthine during 6 hours of tracing in control and NDI1-expressing H460 cells pre-treated with DMSO or 25 nM IACS-010759 for 24 hours. C. Relative abundance of hypoxanthine in control (sgScr) or HPRT1-depleted (sgHPRT1) cells (n=3). D. Fractional enrichment of m+4 IMP, AMP, and GMP in control (sgScr) or HPRT1-depleted (sgHPRT1) cells after 24 hours of pre-treatment with 25 nM IACS-010759 followed by 6 hours of culture with [¹⁵N₄]hypoxanthine (n=3). E. Heatmap displaying purine nucleotide abundance in control (sgScr) or HPRT1-depleted (sgHPRT1) cells treated with DMSO or 25 nM IACS-010759 for 24 hours. F-G. Time-dependent fractional enrichment (F) and relative abundance (G) of m+5 PRPP in H460 cells pretreated with DMSO or 25 nM IACS-010759 for 24 hours (n=3 at each time point). H. Schematic illustrating ¹³C labeling of R5P from [1,2-¹³C]glucose. I-J. Relative abundance (I) and fractional enrichment (J) of m+1 and m+2 R5P after 6 hours of culture in [1,2-¹³C]glucose. Control and NDI1-expressing H460 cells were pre-treated with DMSO or IACS-010759 for 24 hours (n=3). K. Relative abundance of m+5 PRPP after 6 hours of culture in [U-¹³C]glucose. Control and NDI1-expressing H460 cells were pre-treated with DMSO or IACS-010759 for 24 hours (n=3). Unpaired, two-sided t tests (B, D and K), multiple t test (I and J), and two-way ANOVA (F and G) were used for the statistical analyses. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01, *: P < 0.05; n.s.: P > 0.05. Error bars denote SEM. BioRender was used to generate the illustration.

Figure 6.. HPRT1 supports NSCLC growth during ETC inhibition.

A. Cell growth rates of control and HPRT1-depleted H460 cells treated with DMSO or 25 nM IACS-010759 (n=6). Data are from one of three independent experiments. B. Subcutaneous growth of control and HPRT1-deficient H460 xenografts treated with vehicle or 5 mg/kg IACS-010759. The right panel shows individual tumor sizes on the day when the tumors were harvested. n=10 for sgScr Vehicle, sgHPRT1 #1 Vehicle, and sgHPRT1 #1 IACS-010759. n=9 for sgScr IACS-010759, sgHPRT1 #2 Vehicle, and sgHPRT1 #2 IACS-010759. C. HPRT1 mRNA levels in human LUAD and LUSC tumors (T) or nonmalignant lung tissue (N). Data and statistics were generated using TIMER2.0^,. D. Patient-matched HPRT1 expression in human NSCLC tumors (T) and adjacent, nonmalignant lung (NL) (n=20). E. Kaplan-Meier plot showing overall survival of LUAD patients with high (top 25%, n=120) and low (bottom 25%, n=120) HPRT1 expression. Hazard ratio (high) = 2.1. p(HR)=0.00097. The plot and statistics were generated using GEPIA 2. F. Schematic illustrating intra-operative [U-¹³C]glucose infusion in patients with NSCLC followed by tumor resection and multi-omics analyses. G. Fractional enrichment of m+2 malate and relative IMP abundance in tumors displaying low or high malate labeling. The analysis was performed on the top and bottom 25% of tumors for malate m+2 labeling (n=7 tumors each with both isotope tracing and metabolomics analysis). H. Fractional enrichment of m+2 malate and HPRT1 mRNA levels in tumors displaying low or high malate labeling. The analysis was performed on the top and bottom 25% of tumors for malate m+2 labeling (n=6 tumors each with both isotope tracing and RNA-Seq analysis). Unpaired, two-sided t tests (A, B, G, and H), and a paired t test (D) were used for the statistical analyses. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01, n.s.: P > 0.05. Error bars denote SEM. BioRender was used to generate the illustration.

Figure 7.. Purine uptake is required to supply salvage upon ETC blockade.

A. Relative abundance of extracellular [¹⁵N₄]hypoxanthine during 8 hours of culture of H460 cells treated with DMSO, 25 nM IACS-010759, or both 25 nM IACS-010759 and 50 μM NBMPR. B. Fractional enrichment of m+4 IMP, GMP, and AMP after 6 hours of culture with [¹⁵N₄]hypoxanthine, following 24-hours of treatment with DMSO or 25 nM IACS-010759, with or without 50 μM NBMPR (n=3). C. Relative abundance of the indicated purine nucleotides in H460 cells after 24 hours of treatment with DMSO or 25 nM IACS-010759, with or without 50 μM NBMPR (n=6). D. Growth rates of H460 cells treated with 50 μM NBMPR, 25 nM IACS-010759, or both (n=8). Data are from one of three independent experiments. E. Western blot validating overexpression of SLC29A1. Vinculin is the loading control. EV: empty vector; OE: overexpression. F. Relative abundance of extracellular [¹⁵N₄]hypoxanthine during 8 hours of culture of empty vector-expressing control cells (EV) and SLC29A1-overexpressing (SLC29A1-OE) H460 cells. G. Growth rates of control (EV) and SLC29A1-overexpressing (SLC29A1-OE) cells treated with DMSO or 25 nM IACS-010759 (n=8). Data are from one of three independent experiments. H. Tumor growth rates of control (EV) and SLC29A1-overexpression (SLC29A1-OE) H460 xenografts. n=14 for each group. I. SLC29A1 and SLC29A2 RNA levels in human lung adenocarcinoma (LUAD). N: nonmalignant lung; T: tumors. Data and statistics were generated using TIMER 2.0^,. J. Reprogramming of purine synthesis pathways upon ETC suppression. Unpaired, two-sided t tests (B-D), and a two-way ANOVA test (F and H) were used for the statistical analyses. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01, *: P < 0.05, n.s.: P > 0.05. Error bars denote SEM. BioRender was used to generate the illustration.