Role of HDACs in silencing the BTK-targeting miR in the TCL1 model of CLL and consequence of the combination of abexinostat and ibrutinib on BTK protein, signaling, and leukemia cell survival in vivo. (A) CLL cells were isolated from the spleen of TCL1 transgenic mice before (CD19⁺CD5⁺ cells <10%) and after developing CLL (CD19⁺CD5⁺ cells >90%). RNA was extracted and used to quantitate the expression of miR-210 and miR-425 (P < .05 for miR-425, two-sample Student t test). (B) CLL cells from TCL1 mice with leukemia (CD19⁺CD5⁺ cells >90%) were exposed to either panobinostat or abexinostat for 18 hours and then evaluated for the expression of miR-210 and miR-425 (P < .05 for both, paired Student t test). (C-D) Mice were randomly assigned to vehicle/vehicle (control), abexinostat/vehicle, ibrutinib/vehicle, or a combination of abexinostat and ibrutinib for 2 weeks. Spleen CLL cells were isolated and used to quantitate miR-210 and miR-425 (P < .05 for miR-210 with the combination; analysis of variance). (E) CLL cells from TCL1 mice with leukemia (CD19⁺CD5⁺ cells >90%) were left untreated or exposed to abexinostat, ibrutinib, or the combination before being assayed for p-BTK and BTK. GAPDH was used as a loading control, and H3K9/14 was assayed to measure HDAC inhibition. (F) BTK protein was quantified in CLL cells from (E) (P < .05 for the combination vs vehicle; paired Student t test). (G) Mice were randomly assigned to vehicle/vehicle (control), abexinostat/vehicle, ibrutinib/vehicle, or a combination of abexinostat and ibrutinib for 2 weeks. Spleen CLL cells were isolated from each group, and the number of CD19⁺CD5⁺ CLL cells was quantitated (P < .05 for the combination over treatment with each individual drug; mixed effects model). *P ≤ .05.