Fig. 4: A metabolic corollary identifies NAD biosynthesis as a targetable vulnerability to SDH loss.

A Overview of NAD biosynthesis pathways. NA nicotinic acid, NAM nicotinamide, NR nicotinamide riboside, NMN nicotinamide mononucleotide, NAAD nicotinic acid adenine dinucleotide, MeNAM N-methyl-nicotinamide, NAMN nicotinic acid mononucleotide, ADPR adenosine diphosphate ribose, Qa quinolinic acid, TRP tryptophan, ACMS alpha-amino-beta-carboxymuconate-epsilon-semialdehyde, SAM S-adenosyl-methionine, SAH S-adenosyl-homocysteine. In blu, the following enzymes: NNMT nicotinamide N-methyltransferase, NAMPT nicotinamide phosphoribosyltransferase, NMNAT1-3 nicotinamide nucleotide adenylyltransferase, NAPRT nicotinate phosphoribosyltransferase, NADSYN1 NAD synthetase 1, QPRT quinolinate phosphoribosyltransferase, IDO/TDO indoleamine-2,3-dioxygenase/tryptophan-2,3-dioxygenase, NMRK1-2 nicotinamide riboside kinase 1-2. PARPs poly [ADP-ribose] polymerases, SIRTs sirtuins. B NAD⁺ levels in both Sdhb^fl/fl and Sdhb^Δ/Δ cells cultured for 24 h in the presence/absence of 6 nM FK866 in the medium. Data were presented as mean ± s.e.m. of n = 4 replicates. **P < 0.01; ***P < 0.001; # P < 0.005 (compared with FK866-untreated Sdhb^fl/fl cells); §§§ P < 0.001 (compared with FK866-treated Sdhb^fl/fl cells) (two-tailed Student’s t-test). C Heatmap depicting the intracellular levels of the indicated metabolites in Sdhb^fl/fl and Sdhb^Δ/Δ cells cultured for 24 h in the presence or absence of 6 nM FK866 in a medium containing U-¹³C₆-glucose. Data were presented as row normalized Z-scores of peak area/ug proteins of n = 4 replicates. D ATP levels in both Sdhb^fl/fl and Sdhb^Δ/Δ cells cultured for 48 h in the presence or absence of 6 nM FK866 in a medium containing U-¹³C₆-glucose. The sum of all ATP isotopologues is reported for clarity. Data were presented as mean ± s.e.m. of n = 4 replicates. **P < 0.01 (two-tailed Student’s t-test). E Number of Sdhb^fl/fl and Sdhb^Δ/Δ cells measured after 72 h of culture in the presence^/absence of 6–12 nM FK866 in the medium. Data were presented as mean ± s.e.m. of at least n = 7 replicates. ***P < 0.001 (one-way ANOVA), n.s. not significant. F Number of Gpt2-silenced Sdhb^Δ/Δ cells measured after 5 days of culture in the presence/absence of 3 nM FK866 in the medium. Data are presented as mean ± s.e.m. of at least n = 5 replicates. *P < 0.05 (two-tailed Student’s t-test). G Effect of pharmacological inhibition of SDH activity on the response of RENCA cells to FK866 treatment. Top, percent of RENCA cell numbers measured after 6 days of culture in the presence/absence of 20 mM malonate (MAL), 3 mM dimethyl malonate (DMM), 5 µM d-α-tocopherol succinate (α-TOS), or 12 nM FK866 in the medium compared to vehicle-treated cells. Data were presented as mean ± s.e.m. of n = 6 replicates. ***P < 0.001 (one-way ANOVA followed by Dunnett’s multiple comparisons test, compared with FK866-treated cells). Bottom, representative images of crystal violet⁺ colonies from cells treated as in the top panel. H Volumes of tumors originating from the subcutaneous transplant of RENCA cells in BALB/c mice treated daily with FK866 (20 mg/Kg), α-TOS (50 mg/Kg), or the combination of both compounds via intraperitoneal injections for 11 days. ***P < 0.001 (two-way ANOVA). I Weight of tumors isolated from mice treated as described in (H). *P < 0.05 (one-way ANOVA).