Extended Data Fig. 5: α-KG is the key SSP product for glutamine-independent growth.

a, Western blot validation of CRISPR/Cas9-mediated knockout of PHGDH or PSAT1 using independent sgRNAs. b, Sensitivity of breast cancer cell lines to 6 days BI-4916 treatment. Data are presented as mean values ± SD, n = 3 biological replicates. c, Western blot validation of knockdown of PHGDH or PSAT1 by independent shRNA constructs. d, Western blot validation of knockdown of PSPH by shRNA. e, Sensitivity of adapted Hs 578T cells to 6 days CB-839 treatment. Data are presented as mean values ± SD, n = 3 biological replicates. f, Growth of GlnIND BT-549 cells treated with 12 µM NCT-503 in glutamine-free medium supplemented with dimethyl α-KG (500 µM) or serine at the indicated concentrations over 6 days. Viable untreated cell count in complete medium was set as 100%. Data are presented as mean values ± SD, n = 3 biological replicates. Two-tailed unpaired t-test. ** P ≤ 0.01; NS, not significant. g, Serine supplementation fails to rescue the growth of CB839RS Hs 578T cells treated with 12 µM NCT-503 or 2 µM BI-4916, in medium also containing 500 nM CB-839, over 6 days. Viable untreated cell count in complete medium was set as 100%. Data are presented as mean values ± SD, n = 3 biological replicates. Two-tailed unpaired t-test. *** P ≤ 0.001; NS, not significant. h, Dimethyl α-KG supplementation rescues the growth of CB839RS Hs 578T cells treated with 12 µM NCT-503 or 2 µM BI-4916, in medium also containing 500 nM CB-839, over 6 days. Viable untreated cell count in complete medium was set as 100%. Data are presented as mean values ± SD, n = 3 biological replicates. i, Growth of adapted BT-549 cells in glutamine-free medium over 6 days. Dimethyl α-KG was used at 500 µM. Viable control cell count in the absence of dimethyl α-KG was set as 100%. Data are presented as mean values ± SD, n = 3 biological replicates. Two-tailed unpaired t-test. ** P ≤ 0.01; *** P ≤ 0.001; NS, not significant. j, Glutamate supplementation fails to rescue the growth of CB839RS Hs 578T cells treated with 12 µM NCT-503 or 2 µM BI-4916, in medium also containing 500 nM CB-839, over 6 days. Viable untreated cell count in complete medium was set as 100%. Data are presented as mean values ± SD, n = 3 biological replicates. Two-tailed unpaired t-test. *** P ≤ 0.001; NS, not significant. k, Supplementation with 500 µM dimethyl α-KG, 4 mM serine, or 4 mM glutamate leads to increased intracellular abundance of these metabolites in GlnIND MDA-MB-231 cells. Data are presented as mean values ± SD, n = 3 biological replicates. Two-tailed unpaired t-test. *** P ≤ 0.001. Parental MDA-MB-231 BI-4916 dose curve is shared between Fig. 3a, b and Extended Data Fig. 5b, and parental BT-549 BI-4916 dose curve is shared between Extended Data Fig. 4d and Extended Data Fig. 5b, for different comparisons. Control and NCT-503 only (no supplementation) data are shared between Extended Data Fig. 5g, h because the data were obtained from the same experiment. Control and BI-4916 only data are shared between Extended Data Fig. 5g, h, j because the data were obtained from the same experiment. CB839RS, CB-839-resistant; GlnIND, glutamine-independent; SSP, serine synthesis pathway; DMα-KG, dimethyl α-ketoglutarate; PHGDH, D-3-phosphoglycerate dehydrogenase; PSPH, phosphoserine phosphatase.

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