Complex I-independent adaptation of SDH-deficient cancer cells.

(A) Schematic illustrating the generation of clonal SDHB-knockout 143B cells (B) Representative western blot showing levels of SDHB and tubulin loading control in wild-type parental cells (WT) and SDHB knockout clones 1-5 (C) Absolute proliferation rates (mean +/-S.D.) of SDHB KO clones 1-3 with or without 20 mM aspartate supplementation. (n=3) (D) Schematic illustrating the effects of CI/SDH co-suppression 15 on alternative aspartate synthesis and cell fitness. (E) Relative whole-cell aspartate levels (mean +/-S.D.), measured using LCMS, in SDHB-KO clones 1-3 after 6 hours of treatment with vehicle control or 50 nM rotenone (ROT). Levels are normalized to the vehicle-treated condition in each respective clone. (n=3) (F) Absolute proliferation rates (mean +/-S.D.) of SDHB KO clones 1-3 with or without 50nM rotenone (ROT) supplementation. (n=3) (G) Average inter-passage proliferation rates (see methods) of five SDHB-knockout clones over 120 cumulative population doublings. See Figure S1 for plots highlighting individual clones. (H) Absolute proliferation rates (mean +/-S.D.) of early passage (EP) and late passage (LP) clones 1-3 with or without 20 mM aspartate supplementation. (n=3) (I) Representative western blot showing levels of NDUFA8 and tubulin loading control in wild-type parental cells (WT), a previously-adapted (PA) SDHB-KO clone that adapted by suppressing complex I 15, and late passage (LP) SDHB knockout clones 1-3. Normalized NDUFA8 band densities are shown below each respective lane. (J) Normalized oxygen consumption rate (OCR) traces (mean +/-S.D.) for wild-type parental cells (WT), parental cells treated with 5 µM Atpenin A5 (WT + AA5), PA, and early passage (EP) and late passage (LP) SDHB knockout clones 1-3. Injections of rotenone (ROT) and antimycin (ANTI) are shown with arrows. (n=7-12) (K) Basal mitochondrial oxygen consumption rates (OCR) (mean +/-S.D.) quantified from data in (J). (n=7-12). Statistical significance determined using an ordinary two-way ANOVA and uncorrected Fisher’s LSD with single pooled variance (C, E-F) or ordinary one-way (K) or two-way (H) ANOVA and Tukey’s multiple comparisons test with a single pooled variance.

CI-high and CI-low adapted SDH-deficient cells are metabolically distinct.

(A) Schematic illustrating the creation of NDUFA8-KO (A8KO) cells from EP SDHB KO clone 2 (2 EP), and the subsequent comparison between A8KO and LP SDHB KO clone 2 (2 LP) (which had adapted without suppressing CI). (B) Representative western blot showing levels of NDUFA8 and tubulin loading control in 2 EP and A8KO. (C) Relative whole-cell aspartate levels (mean +/-S.D.) in 2 EP and A8KO 24 hours after media change. Levels are normalized to 2 EP. (n=3) (D) Proliferation rates (mean +/-S.D.) of 2 EP and A8KO with or without 20 mM aspartate supplementation. (n=3) (E) Heatmap depicting log2-transformed relative abundances of the indicated metabolites in 2 LP and A8KO treated with vehicle control (veh.) or 50 nM rotenone (ROT) for 24 hours. Abundances are normalized to the vehicle-treated 2 LP condition. (n=3) (F) Schematic depicting U-13C glucose or U-13C glutamine tracing to determine routes of alternative aspartate synthesis in SDH-deficient cells. The experimental procedure for results in panels G-H is shown in the box. (G) Fractional isotopolog distributions for the indicated metabolites in 2 LP and A8KO following 24 hours of tracing in media containing U-13C glucose. P-values represent results from comparing the M+3 fractions. (n=3) (H) Fractional isotopolog distributions for the indicated metabolites in 2 LP and A8KO following 24 hours of tracing in media containing U-13C glutamine. P-values represent results from comparing the M+3 fractions. (n=3) (I) Schematic illustrating the existence of at least two distinct adaptive trajectories in SDHB-KO cells, which differ in complex I activity status and in the major metabolic source of aspartate [glucose (Glc) vs. glutamine (Gln)]. Statistical significance determined using an unpaired t-test (C), ordinary two-way ANOVA and uncorrected Fisher’s LSD with single pooled variance (D) or ordinary two-way ANOVA and Tukey’s multiple comparisons test with a single pooled variance (G-H).

Shared and distinct aspartate-related metabolic dependencies of CI-high and CI-low adapted SDH-deficient cells

(A) Schematic illustrating the role of the mitochondrial pyruvate carrier (MPC) in alternative aspartate synthesis and its knockout using CRISPR/Cas9 (B) Representative western blot showing levels of MPC1, V5-tagged MPC1, and tubulin loading control in parental, MPC1-knockout (KO) and MPC1-addback (AB) cells for late passage SDHB-KO clone 2 (2 LP) and 2 EP NDUFA8-KO (A8KO). (C-D) Proliferation rates (mean +/-S.D.) of MPC KO and addback (AB) 2 LP (C) or A8KO (D) with or without 20 mM aspartate supplementation. (n=3) (E) Relative aspartate levels (mean +/-S.D.) of MPC KO and addback (AB) 2 LP and A8KO 24 hours after media change. Levels are normalized to each corresponding AB. (n=3) (F) Schematic illustrating the role of pyruvate carboxylase (PC) in alternative aspartate synthesis and its knockout using CRISPR/Cas9 (G) Representative western blot showing levels of PC, V5-tagged PC, and tubulin loading control in parental, PC-knockout (KO) and PC-addback (AB) 2 LP and A8KO cells (H-I) Proliferation rates (mean +/-S.D.) of PC KO and addback (AB) 2 LP (H) or A8KO (I) with or without 20 mM aspartate supplementation. (n=3) (J) Relative aspartate levels (mean +/-S.D.) of PC KO and addback (AB) 2 LP and A8KO 24 hours after media change. Levels are normalized to each corresponding AB. (n=3) (K) Schematic illustrating compartmentalized aspartate synthesis by GOT1/2 (L) Representative western blot showing levels of GOT1, GOT2, and tubulin loading control in parental, GOT1-KO and GOT2-KO 2 LP and A8KO cells (M) Proliferation rates (mean +/-S.D.) of GOT1- and GOT2-KO 2 LP and A8KO cells in all combinations of 20 mM aspartate and 50 nM rotenone treatments. (n=3) (N-O) Relative aspartate levels (mean +/-S.D.) of parental, GOT1-, and GOT2-KO 2 LP (N) or A8KO (O) cells 24 hours after treatment with vehicle control or 50 nM rotenone. Levels are normalized to each corresponding parental cell vehicle treatment. (n=3) (P) Schematic depicting aspartate synthesis in wild-type and SDH-deficient 143B cells before and after adaptation along two distinct trajectories. Abbreviations: GOT1, glutamic-oxaloacetic aminotransferase 1; GOT2, glutamic-oxaloacetic aminotransferase 2; ASP, aspartate; OAA, oxaloacetate; cyto., cytosol; mito., mitochondria; GLC, glucose; GLN, glutamine; PYR, pyruvate; SDH, succinate dehydrogenase; SUC, succinate; PC, pyruvate carboxylase; CI, respiratory complex I; CIT, citrate. Statistical significance determined using an ordinary two-way ANOVA and uncorrected Fisher’s LSD with single pooled variance (C-E, H-J) or ordinary two-way ANOVA and Tukey’s multiple comparisons test with a single pooled variance (M-O).

PC expression can be rate-limiting for aspartate synthesis and proliferation in SDH-deficient cells.

(A) Schematic depicting U-13C glucose tracing to monitor aspartate synthesis via the MPC-PC-GOT2 pathway (B) Absolute isotopolog distributions (mean +/-S.D.) for aspartate in early passage (EP) or late passage (LP) SDHB-KO clone 2 following 24 hours of tracing in media containing U-13C glucose. (n=3) (C) Representative western blots showing levels of MPC1, PC, GOT2, and tubulin loading control in 2 EP/LP (D) Quantification of PC protein levels from 2 EP/LP using western blotting. Statistical significance determined using Welsh’s t-test. (n=3) (E) Representative western blots showing levels of PC, V5-tagged PC, and tubulin loading control in 2 EP or PC-overexpressing 2 EP (2 EP PC OE). (F) Absolute isotopolog distributions (mean +/-S.D.) for aspartate in 2 EP and 2 EP PC OE after 24 hours of tracing in media containing U-13C glucose. (n=3) (G) Proliferation rates (mean +/-S.D.) of 2 EP and 2 EP PC OE with or without 20 mM aspartate (ASP) supplementation. Statistical significance determined using an ordinary two-way ANOVA and uncorrected Fisher’s LSD with a single pooled variance. (n=3) (H) Representative western blots showing levels of PC and tubulin loading control in wild-type 143B cells or PC-overexpressing 143B cells (143B PC OE). (I) Proliferation rates (mean +/-S.D.) of 143B and 143B PC OE at the indicated doses of Atpenin A5 (AA5) with or without 20 mM aspartate (ASP) supplementation. Proliferation rates are normalized to the 0 µM 5 treatment condition for each respective trace. P-values shown correspond to statistical testing comparing the relative proliferation rate of 143B vs. 143B PC OE at the indicated dose of AA5. (n=3) (J) Schematic illustrating the approach in (K), described further in the main text. (K) Mean SDHA-D DepMap gene effect scores for the top and bottom 5% of expressors of the indicated metabolic genes. Statistical significance determined using an ordinary one-way ANOVA and Sidak’s multiple comparisons test with a single pooled variance. (n=57) Statistical significance determined using Welch’s t-test (D), an ordinary two-way ANOVA and uncorrected Fisher’s LSD with single pooled variance (G), ordinary two-way ANOVA and Tukey’s multiple comparisons test with a single pooled variance (I), or ordinary one-way ANOVA with Sidak’s multiple comparisons test with a single pooled variance (K).

SDH-deficient cells adapt to overcome pyrimidine deficiency and replication stress.

(A) Schematic depicting metabolic fates of aspartate in proliferating cells and the effect of SDH loss on pyrimidine biosynthesis 24 (B-D) Relative levels of carbamoyl-aspartate (carb-asp) (B), orotate (C), and uridine monophosphate (UMP) (D) (mean +/-S.D.) in early passage (EP), aspartate reared (AR), and late passage (LP) SDHB knockout clones 1-3. Levels are normalized to each corresponding EP clone. (n=3) (E) Schematic illustrating the cell cycle analysis experimental setup and normalized univariate histograms of propidium iodide (PI) staining in early passage (EP) and late passage (LP) knockout clone 2. Approximate regions corresponding to G1, S, and G2 cell cycle phases are labeled at the top of the chart. One replicate of each cell line is shown for clarity. (F) Cell cycle phase distributions of 2 EP/LP quantified from data in (E). P-value shown corresponds to statistical testing comparing the fraction of cells in S phase in each condition. (n=3) (G) Schematic illustrating the finding that SDH loss causes replication stress, the role of ATR kinase in the replication stress response, and the ATR inhibitor BAY-1895344 (BAY). (H-K) Proliferation rates (mean +/-S.D.) of EP/LP SDHB knockout clones 1-3 (H-J) or 2 EP, PC-overexpressing 2 EP (2 EP PC OE), 2 LP, and NDUFA8-KO 2 EP (A8KO) (K) in the indicated doses of BAY, with or without 500 µM uridine supplementation. Proliferate rates are normalized to the 0 nM BAY dose in each condition. P-values shown correspond to statistical testing comparing the relative proliferation rate of EP vs. LP at the indicated dose of BAY. Statistical significance determined using ordinary two-way ANOVAs and uncorrected Fisher’s LSD with single pooled variance.