Figure 7.

Blocking TLR9 reduces CLL cell migration and is synergistic with ibrutinib. (A) PBMCs from 6 different patients were split into 4 fractions. One fraction was stimulated with ODN2006 alone, 1 fraction stimulated in the presence of ibrutinib, one fraction stimulated in the presence of the TLR9 inhibitor ODN INH-18, and 1 fraction stimulated in the presence of both (2:1 fixed molar ratio of ODN INH-18 to ibrutinib). After overnight incubation, cells were harvested and then transferred into transwell migration chambers and incubated under the conditions described above. The migrated and nonmigrated cells were collected, stained with CD5 and CD19 for CLL cell identification, and then quantitated volumetrically. The percent change compared with the normalized stimulated fraction was then assessed. The combination of ODN INH-18 and ibrutinib gave maximum CLL cell migration inhibition. (B) Combination index (CI) analysis showed that the 2 drugs were synergistic in all 6 samples using 1 μM ibrutinib + 2 μM ODN INH-18 (ie, CI <1). The mean CI at the half maximal effective dose (ED50) for the combination of the 2 drugs was 0.2, indicating a strong synergistic effect. (C,E) CLL cells from 11 or 12 different patients were split and treated as above, but stimulated for 4 hours. After activation, cells were harvested and stained for CD5, CD19, and intracellular p-STAT3, p-p65 NF-κB, or an isotype matched control and then assessed by using flow cytometry and the MFI recorded. The combination of ODN INH-18 and ibrutinib gave the maximum inhibition of both p-STAT3 and p-p65 NF-κB. CI analysis showed that the 2 drugs were synergistic at reducing p-STAT3 in 10 of 11 patients (D) and p-p65 NF-κB in all 12 patients (F) using 1 μM ibrutinib + 2 μM ODN INH-18 (ie, CI <1). The mean CI at the ED50 for the combination of the 2 drugs was 0.5 for p-STAT3 and 0.4 for p-p65 NF-κB, indicating a strong synergistic effect.