Figure 7. BCAT1-Driven Leukemia Are Sensitive to mTOR Inhibition.

A. Upregulation of mTOR signature genes in G12D/E2-KO relative to G12D LSK cells by GSEA.

B. Increased p-4EBP1 and p-S6K in G12D/E2-KO relative to WT or G12D LSK cells.

C. Protein synthesis rate was assessed by OP-puro incorporation in BM LSK and myeloid cells at 4, 8 and 12 weeks post-pIpC.

D. Schematic of experimental design to determine the effect of mTOR inhibition.

E. Inhibition of mTOR by rapamycin, PP242, or Torin1 abolished the colony-forming ability of G12D/E2-KO HSPCs. Representative photomicrographs for CFU-GM, BFU-E and CFU-GEMM colonies are shown. Scale bars, 100μm.

F. Representative pictures and quantification of spleen and liver of recipients transplanted with G12D/E2-KO HSPCs treated with vehicle or rapamycin. Scale bars, 0.5 cm (top) and 1 cm (bottom).

G. Survival curve of recipients transplanted with G12D/E2-KO HSPCs treated with vehicle or rapamycin (N = 5 and 8). P values were calculated using log-rank (Mantel-Cox) test.

H. BCAT1 inhibition by BCAT1i (40 μM) decreased mTOR activity (p-4EBP1 and p-S6K) in G12D/E2-KO HSPCs.

I. Overexpression of BCAT1 in WT or G12D HSPCs enhanced mTOR activity.

J. Inhibition of MCT1 by AZD-3965 or GLS by CB-839 impaired mTOR activity in G12D/E2-KO HSPCs.

K. Depletion of BCAT1 impaired mTOR activity in G12D/E2-KO HSPCs, which was restored by Leu supplementation (500 μM).

L. Leu supplementation promoted EZH2-deficient MPNs in vivo. Representative pictures are shown for spleens of recipients transplanted with BCAT1-depleted G12D/E2-KO HSPCs supplemented with vehicle or Leu. Scale bars, 0.5 cm.

M. Survival curve of recipients transplanted with BCAT1-depleted G12D/E2-KO HSPCs with vehicle or Leu. P values were calculated using log-rank (Mantel-Cox) test.

N. Model for the oncogenic cooperativity between EZH2 loss and RAS activation in myeloid neoplasms.

Results are mean ± SEM and analyzed by a repeated-measures one-way ANOVA with multiple comparisons, unless stated otherwise. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. not significant.