Pharmacologic suppression out of PHGDH sensitizes structure with a high IDH2 and prevents tumefaction development in vivo

Ultimately, i tested the effectiveness of PHGDH inhibitors on the 4T1 tumors that have IDH2-high account

Because of the part out-of PHGDH and you will PSAT1 into the mediating IDH2-mainly based metabolic renovations, we investigated the brand new proteomic negative effects of these relationships. Necessary protein involved in k-calorie burning, translation gadgets, ribosome biogenesis, splicing, and cell migration were upregulated from the IDH2 and you can downregulated which have PHGDH and you may PSAT1 knockouts (Second Fig. S8A and you may S8B; Second Dining table S6). Big metabolic protein integrated the newest cytochrome members of the family (CYCS, CYC1, CYB5R1), glutamine use and you may glutamate k-calorie burning (SLC1A5 and you will GLUD1), solute provider transporters (SLC25A1 – CIC, citrate/malate transporter, SLC25A11 – OGC, alpha-ketoglutarate/malate transporter and you may SLC25A5 – ATP/ADP transporter), lipid metabolism (SOAT1, TSPO, ACAD9), and you may glycolytic protein (HK1 and PKM). We speculated you to definitely a reduction in the brand new metabolic interest up on PHGDH and you will PSAT1 knockout you’ll sign up for the fresh redox imbalance and you may sensitize the latest tissues to oxidative ruin. S8C). Thus, PHGDH and PSAT1 play an essential character during the providing anabolic source of nucleotides, lipids, and amino acids for the structure with high IDH2, and you can service cellular fret resistance (Supplementary Fig. S8D).

Indeed, the increased loss of PHGDH and you will PSAT1 created vulnerability so you can oxidative damage together with telephone survival is lower than the brand new handle tissue (Secondary Fig

Aiming to translate the SDL interaction to cancer therapy, we examined the sensitivity of IDH2-high cells to PHGDH inhibitors, in vitro and in vivo. Cells with stable IDH2 overexpression and IDH2 knockout were treated with PHGDH inhibitor (NCT-fifty2) for 48 hours in RPMI medium without serine and glycine. Initial metabolic analysis showed that PHGDH inhibition reduced serine (m3) and glycine (m2) labeling from 13 C₆-glucose (Supplementary Fig. S8E-S8H). The dose range of NCT-502 was calibrated for each cell line (HCC38 and HCC1143), due to basal differences in cell line sensitivities. In agreement with the SDL prediction, HCC38 cells with IDH2 overexpression were more sensitive to NCT-502 treatment (IC₅₀: 0.05 ?mol/L) compared with the control cells with low IDH2 expression (IC₅₀: 0.18 ?mol/L; Fig. 7A). Control knockout HCC1143 cells with high basal IDH2 were more sensitive to NCT-502 (IC₅₀: 0.5 ?mol/L) compared with the cells with IDH2 knockout (IC₅₀: 2.2 ?mol/L; Fig. 7B). Next, we examined the efficacy of the PHGDH inhibitor in an in vivo murine model, 4T1 TN breast cancer cells, with high basal IDH2 and PHGDH expression. We knocked down IDH2 using stable shRNA constructs and the knockdown was confirmed by Western blotting (Supplementary Fig. S8I). 4T1 cells exhibited reduced cell proliferation and colony formation upon IDH2 knockdown (Fig. 7C and D). In addition, DMKG supplement to the murine 4T1 cells with IDH2 knockdown rescued the reduced cell proliferation and colony formation (Fig. 7C and D). 4T1 cells with high and low IDH2 expression were injected orthotopically to mammary glands of female mice and treated with the PHGDH inhibitor NCT-503 (Supplementary Fig. S8J), which is reported to have increased solubility in vivo (42). Analysis of tumor growth revealed that 4T1 tumors with high IDH2 showed enhanced tumor growth with larger tumor size and weight compared with the tumors with low IDH2 (Fig. 7E–G; Supplementary Fig. S8K). In addition, only the IDH2-high tumors treated with NCT-503 showed reduced tumor size and weight compared with the IDH2-high tumors treated with vehicle (Fig. 7E–G). IDH2-low tumors treated with either NCT-503 or vehicle were not affected by the treatment. Altogether, pharmacologic inhibition of serine biosynthesis using PHGDH inhibitor affects only the growth of IDH2-high cells. This in vivo validation demonstrated the SDL interaction between PHGDH and IDH2 and strengthened the metabolic alterations and the in vitro protumorigenic phenotypes. Our study emphasizes PHGDH inhibition as a promising therapeutic approach for TN breast tumors with high IDH2.