Inhibition of USP7 with HBX19818 induces accumulation of DNA damage. (A) DNA damage was significantly elevated both in primary CLL cells (n = 3) and healthy donor lymphocytes (n = 3) treated with 8-µM HBX19818 for 6 hours, as quantified from Comet assay–derived tail moments. HBX-induced DNA damage was increased 3.92-fold in CLL cells and 2.42-fold in healthy donor cells, relative to DMSO. Representative images of DMSO- and HBX19818-treated cells are depicted. (B) Levels of the DNA damage marker γH2AX increased after HBX19818 treatment of quiescent and proliferating primary CLL cells and proliferating healthy donor cells. Lamin B1 (LMNB1) was the loading control. Immunofluorescence labeling showed significant induction of 53BP1 foci, a DSB marker, in Mec1 cells after 6-hour treatment with HBX19818 in vitro (C) and in vivo (D) in the chemotherapy-resistant murine xenograft model. Representative images are shown (DAPI, blue; 53BP1, green) and the mean data from 3 independent experiments are presented. Original magnification ×60 (A,C-D). Data were compared using a 2-tailed Student t test, and statistical significance denoted by: *P ≤ .05.