Finally, we checked-out the power of PHGDH inhibitors to your 4T1 tumors that have IDH2-high account
Because of one’s part out of PHGDH and you can PSAT1 from inside the mediating IDH2-founded metabolic restorations, we examined new proteomic aftereffects of these types of relationships. Protein employed in metabolic process, translation equipments, ribosome biogenesis, splicing, and mobile migration had been upregulated by the IDH2 and you will downregulated having PHGDH and PSAT1 knockouts (Secondary Fig. S8A and S8B; Secondary Table S6). Big metabolic protein integrated the cytochrome family members (CYCS, CYC1, CYB5R1), glutamine consumption and glutamate metabolic rate (SLC1A5 and GLUD1), solute provider transporters (SLC25A1 – CIC, citrate/malate transporter, SLC25A11 – OGC, alpha-ketoglutarate/malate transporter and you will SLC25A5 – Aurora escort ATP/ADP transporter), lipid metabolic rate (SOAT1, TSPO, ACAD9), and glycolytic protein (HK1 and PKM). We speculated one to a decrease in this new metabolic hobby through to PHGDH and you may PSAT1 knockout you’ll sign up for this new redox imbalance and you will sensitize the new cells to help you oxidative ruin. S8C). Therefore, PHGDH and you may PSAT1 gamble a significant part for the getting anabolic provide from nucleotides, lipids, and you will proteins in tissues with high IDH2, and you may help mobile worry opposition (Additional Fig. S8D).
In fact, losing PHGDH and PSAT1 induced susceptability so you’re able to oxidative damage as well as the cell survival was lower than the brand new handle tissue (Second 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.