The inhibition of IRAK1 confers an exaggerated leukemic defect to IRAK4-deficient AML. (A) Growth curves of WT and IRAK1^KO MDSL and THP1 cells treated with CA-4948 (10 μM) or vehicle (2 independent experiments). (B) Colony formation of WT and IRAK1^KO MDSL and THP1 cells treated with CA-4948 (30 μM) or vehicle (3 independent experiments). (C) Colony formation of WT and IRAK1^KO MDSL and THP1 cells treated with IRAK4 degrader-1 (MDSL, 5 μM; THP1, 10 μM) or vehicle. (D) Immunoblots for IRAK1 and IRAK4 in WT and IRAK4^KO cell lines transduced with nontargeting control short hairpin RNA (shRNA; shControl) or shIRAK1. (E) Colony formation of WT and IRAK4^KO AML cell lines transduced with nontargeting control shRNA (shControl) or shIRAK1. (F) Representative colony images of WT and IRAK4^KO AML (1294) cells transduced with nontarget control shRNA (shControl) or shIRAK1 (original magnification ×40). (G) Immunoblots for IRAK1 and IRAK4 in WT, IRAK4^KO, IRAK1^KO, and IRAK1/4^dKO THP1 cells. (H) Colony formation of isogenic THP1 cells. (I) Kaplan-Meier survival analysis of NSGS mice (n = 7 mice per group) that received engraftment with WT, IRAK4^KO, IRAK1^KO, and IRAK1/4^dKO THP1 cells (Data represent 1 of 2 independent experiments with similar trends). (J) Bone marrow engraftment of WT (n = 4), IRAK4^KO (n = 5), IRAK1^KO (n = 5), and IRAK1/4^dKO (n = 5) THP1 cells in NSGS mice that underwent xenograftment at the time of death. Leukemic engraftment was determined as the percentage of huCD45⁺huCD33⁺ cells. (K) Liver engraftment of WT (n = 4), IRAK4^KO (n = 5), IRAK1^KO (n = 5), and IRAK1/4^dKO (n = 5) THP1 cells in NSGS mice that underwent xenograftment at the time of death. Leukemic engraftment was determined as the percentage of huCD45⁺huCD33⁺ cells normalized to the number of days. (L) Representative images of livers collected from NSGS mice that underwent xenograftment with WT, IRAK4^KO, IRAK1^KO, and IRAK1/4^dKO THP1 cells. Arrows indicate examples of AML cell infiltration. Significance was determined with a Student t test (∗P < .05). Error bars represent the standard deviation.