The IRAK1/4 inhibitorR191/R221 inhibits TLR signaling and delays the growth of murine Eμ-TCL1 CLL cells in vivo. (A-B) Effect of R191 on proliferation of TLR-stimulated Eμ-TCL1 CLL cells. The percentage of proliferating cells was determined by analysis of 5-bromo-2'-deoxyuridine (BrdU) incorporation in viable Eμ-TCL1 leukemia cells that were cultured for 28 hours in the presence or absence of CpG-1668 (1 μM), LPS (5 μg/mL), and R191 (1 μM) and then for additional 20 hours in the presence of BrdU (10 μM). One representative experiment with TCL1-333 cells is shown in panel A, and a summary of 4 experiments with CLL cells derived from 4 different Eμ-TCL1 transgenic mice is shown in panel B. Statistical analysis was done using 1-way repeated measures analysis of variance (ANOVA) with Tukey test for multiple comparisons. (C) Analysis of leukemia cell counts (CD5⁺/CD19⁺) in peripheral blood of mice inoculated with TCL1-355 or TCL1-333 leukemia cells and treated with R221 or vehicle control (n = 7 per group). Statistical analysis was done using the t test or Mann-Whitney test, as appropriate. (D) Survival analysis of mice treated with R221 or vehicle control. Treatment was started 3 days after tumor transfer. X-axis indicates days from tumor transfer. Survival curves were estimated using the Kaplan-Meier method, and curve differences were assessed using the log-rank test.