Inhibition of USP7 sensitized CLL cells to DNA cross-linking agents. (A) HBX19818 potentiated the induction of γH2AX in Mec1 cells treated with either MMC or 4HC for 72 hours. β-actin was the loading control. HBX19818 (8 µM) significantly (P < .05) increased the sensitivity of Mec1 cells (n = 3) (B,E) and proliferating p53-defective primary CLL cells (n = 3) (C,F) to either 4HC (B-C) or MMC (E-F). Inserts indicate the activity of HBX19818 alone. The accompanying combination index plots indicate synergism of HBX19818 with 4HC (D) and MMC (G) in both Mec1 and p53-defective primary CLL cells. (H) Tumor load was reduced by all treatment regimens, both single agents (5 and 10 mg/kg HBX19818, 20 mg/kg cyclophosphamide [CycloP], and 0.5 mg/kg rituximab [RTX]) and combinations with HBX1918 vs control (DMSO) treatment. Tumor load reduction induced by CycloP (20 mg/kg; n = 4) alone was significantly enhanced when coadministered with the higher dose of HBX19818 (10 mg/kg; n = 4). This demonstrated that HBX19818 increased the in vivo efficacy of CycloP in the Mec1 murine xenograft model. Data were compared using 2-way analysis of variance (B-C,E-F,) or 2-tailed Student t test (H), and statistical significance denoted by: *P ≤ .05, ***P ≤ .001.