HDAC inhibition effectively targets the C481S-mutant BTK clone in ibrutinib-resistant CLL. (A) Induction of miR-147b, miR-210, miR-425, miR-1253, miR-4269, and miR-4667-3p in response to abexinostat in paired CLL samples obtained at baseline or after acquisition of the C481S BTK clone while the patient was receiving ibrutinib therapy. Data represent mean ± SEM of 4 CLL samples (P < .01 for miR-210 before and after ibrutinib resistance had developed, and P < .01 for miR-4667-3p after ibrutinib resistance had developed; mixed effects model). (B) Effect of ibrutinib or abexinostat on BTK mRNA in paired CLL samples obtained at baseline or after acquisition of the C481S BTK clone while the patient received ibrutinib therapy. Data represent mean ± SEM of 4 CLL samples (P < .01; mixed effects model). (C) Effect of ibrutinib or abexinostat at 18 and 36 hours on BTK phosphorylation, protein, and signaling in paired CLL samples obtained at baseline or after acquisition of the C481S BTK clone while the patient received ibrutinib therapy. Levels of H3K4Ac3 were evaluated as a measure of HDAC inhibition, and H3 was measured as a control for H3K9Ac. (D) The decrease in BTK protein was quantitated; data represent mean ± SEM of 4 CLL samples (P < .001; mixed effects model). (E) Effect of abexinostat on CLL survival at 48 hours as measured by increase in percentage of annexin V–positive cells in paired CLL samples obtained at baseline or after acquisition of the C481S BTK clone while the patient received ibrutinib therapy. ***P ≤ .001.