Loss of CD99 leads to an impairment in LSC function that can be rescued with rapamycin treatment. (A) OP-puro incorporation in MOLM13 cells after stable transduction with shRNAs against CD99 (59, 60, and 61) or NT control. (B) Correlation of CD99 transcript levels in 398 patients with AML from the ECOG1900 clinical trial with recurrent cytogenetic abnormalities and mutations (Somers’ D test). The top 15 CD99-associated genetic abnormalities (of 36 tested) are shown. (C) Schematic of transduction of c-kit⁺ HSPCs from 5-FU–treated CD99 Gt/Gt mice and WT littermate controls to coexpress AML1-ETO9a and GFP, followed by transplant into lethally irradiated primary recipients. Beginning 48 hours after transplant, recipients were treated with rapamycin or vehicle. (D) Overall survival of mice transplanted with transduced CD99 Gt/Gt or WT HSPCs and treated with vehicle or rapamycin (KO vehicle [n = 5], WT vehicle [n = 6], KO rapamycin [n = 6], and WT rapamycin [n = 6], respectively; n = 6 for each group except for n = 5 in the KO vehicle group). (E) Immunophenotype of GFP⁺ leukemia cells derived from WT and CD99 Gt/Gt mice treated with vehicle (left 2 panels) or rapamycin (right 2 panels). (F) Schematic of transplant of GFP⁺ leukemia cells that developed in primary recipients into secondary recipient mice at limiting dilution. The numbers of mice that received engraftment with leukemia of the total mice that received transplant with each cell number are indicated (table). Leukemia-initiating cell frequency is shown as calculated using Poisson statistics. (G) Overall survival of mice secondarily transplanted with KO vehicle, WT vehicle, KO rapamycin, and WT rapamycin leukemias at the 30 000, 7500, and 1000 cell doses (n = 15 for each group). Statistical significance was assessed using a 1-way analysis of variance test for panel A and log-rank (Mantel-Cox) tests for panels D,G (∗P < .05; ∗∗P < .01; ∗∗∗P < .005). Data are represented as mean ± standard deviation. 5-FU, 5-fluorouracil; CI, confidence interval; GFP, green fluorescent protein; IP, intraperitoneal injection; NT, nontargeting; shRNA, short-hairpin RNA.