Agonistic targeting of TLR1/TLR2 inhibits leukemia-initiating cells and provides antileukemic effects in vivo. (A-E) Murine c-Kit⁺MLL-AF9 AML cells were cultured for 3 days with or without (control) Pam3CSK4. (A) Percentage of Annexin V⁺ and (B) CD11b⁺ cells (n = 3). (C) May-Grünwald-Giemsa–stained cytospin slides from representative samples (40× magnification). (D) 30 000 or (E) 10 000 leukemia cells were seeded in wells and cultured with Pam3CSK4 and SCF (Pam3CSK4) or only SCF (control), and then transplanted into sublethally irradiated recipient mice (6 mice per group). For the “30 000 seeded cells,” on average, 9200 control and 115 000 Pam3CSK4-treated cells were injected per mouse. For the “10 000 seeded cells”: 1100 control and 27 000 Pam3CSK4-treated cells were injected per mouse. Kaplan-Meier curves showing overall survival of the mice. (F-G) 100 000 c-Kit⁺MLL-AF9 AML cells were transplanted into sublethally irradiated recipient mice (5 mice per group in 2 independent experiments) and treated with Pam3CSK4 (100 μg/dose) or PBS control by intraperitoneal injections 3 times per week starting 1 day posttransplantation. (F) Percentage of Annexin V⁺ cells in BM at day 10 posttransplantation. (G) Percentage of dsRed⁺ leukemia cells in BM at day 10 (left) or day 15 (right) posttransplantation. Mean values and standard deviations are presented. *P < .05; **P < .01; ***P < .001; ****P < .0001.