Loss of CD99 leads to increased protein synthesis in AML-ETO–driven leukemias. (A-B) Ex vivo OP-puro incorporation in c-kit⁺ leukemia cells from primary or secondary recipients of CD99 Gt/Gt or WT HSPCs treated with vehicle or Rapa (KO vehicle, WT vehicle, KO Rapa, and WT Rapa, respectively). Western blots examining p-S6, S6 (C-F), ubiquitylated protein (G-H), p-eIF2α, and eIF2α (I-J) in 3 × 10⁴ bulk leukemia cells from each experimental group (derived from mice that received secondary or tertiary transplant, as indicated). Quantification of western blots performed on independent mice for p-S6:S6 ratio in panels D,F; ubiquitylated protein in panel H; and p-eIF2α in panel J, with measurements normalized to WT HSCs for panels D,H,J. (K) Heat map and (L) GSEA analysis depicting ribosomal protein transcripts differentially expressed in the 4 experimental groups (n = 3 biological replicates per group). (M) Model of differences between CD99 Gt/Gt and WT LSCs in function, protein synthesis rates, and response to rapamycin. Statistical significance was assessed using unpaired 2-tailed Student t tests (∗P < .05; ∗∗P < .01; ∗∗∗P < .005). P values for selected nonsignificant trends are also shown; data are represented as mean ± standard deviation. FDR, false discovery rate; NES, normalized enrichment score; Rapa, rapamycin.