USP7 is overexpressed in CLL and can be targeted with HBX19818 without activating p53 phosphorylation. Immunoblotting (A) and densitometry (B) quantification of primary CLL and healthy donor PBMCs demonstrate overexpression of USP7 in CLL samples. USP7 expression was normalized to β-actin. (C) HBX19818 dose responses of quiescent primary CLL cells and healthy donor PBMCs and the resultant 50% effective concentration (EC₅₀) for quiescent (D) and proliferating (E) healthy donor and primary CLL cells after 72-hour treatment. HBX19818 treatment of a chemotherapy-resistant Mec1 CLL xenograft model led to reduction of splenic weight (F) and splenic tumor engraftment (G). (H) Xenotransplantation of confirmed USP7 knockdown Mec1 cells by siRNA transfection also led to reduced splenic engraftment. (I) In a panel of 5 wild-type (WT) CLLs, 6-hour HBX19818 treatment stabilized p53 but did not induce its phosphorylation. As a positive control for p53 phosphorylation, CLL9 and CLL57 were treated with 5-Gy ionising radiation (IR). Data were compared using a 2-tailed Student t test, and statistical significance denoted by: *P ≤ .05, ***P ≤ .001. siRNA, small interfering RNA.