CN470 is a BET/CBP/p300 multi-bromodomain inhibitor and has an anti-tumor activity against MLL-rearranged acute lymphoblastic leukemia

Abstract

Acute lymphoblastic leukemia with chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene (MLL-r ALL) remains an incurable disease. Thus, development of a safe and effective therapeutic agent to treat this disease is crucial to address this unmet medical need. BRD4, a member of the bromodomain and extra-terminal domain (BET) protein family, and cyclic AMP response element binding protein binding protein (CBP) and p300, two paralogous histone acetyltransferases, are all considered cancer drug targets and simultaneous targeting of these proteins may have therapeutic advantages. Here, we demonstrate that a BET/CBP/p300 multi-bromodomain inhibitor, CN470, has anti-tumor activity against MLL-r ALL in vitro and in vivo. CN470, potently inhibited ligand binding to the bromodomains of BRD4, CBP, and p300 and suppressed the growth of MLL-r ALL cell lines and patient-derived cells with MLL rearrangements. CN470 suppressed mRNA and protein expression of MYC and induced apoptosis in MLL-r ALL cells, following a cell cycle arrest in the G1 phase. Moreover, CN470 reduced BRD4 binding to acetylated histone H3. The in vivo effects of CN470 were investigated using SEM^Luc/GFP cells expressing luminescent markers in an orthotopic mouse model. Mice administered CN470 daily had prolonged survival compared to the vehicle group. Further, CN470 also showed anti-tumor effects against an MLL-r ALL patient-derived xenograft model. These findings suggest that inhibition of BET/CBP/p300 by the multi-bromodomain inhibitor, CN470, represents a promising therapeutic approach against MLL-r ALL.

Keywords: ALL; BET; Bromodomain; CBP/p300; MLL; PDX.

Fig. 1.

Effects of CN470 on MLL-r ALL cell proliferation and induction of apoptosis. (A) BROMOscan™ assay for CN470. For BRD4, a target containing both bromodomains, BRD4 (BD1-BD2), was used. (B, C, D) Growth inhibitory effects of CN470 on MLL-r ALL (B, C) and patient-derived (D) cells. (B) WST-8 assay data are represented as mean ± standard error (SE) of three independent experiments, each with four replicates experiments. (C) IC₅₀ values for cell proliferation analyses in this study. (D) Cell-Titer Glo^® 2.0 viability assay results are presented as mean ± standard deviation (SD) of four replicates for each concentration. (E) CN470 induced changes in SEM and KOPN-1 cell cycle status. (F) Induction of SEM and KOPN-1 cell apoptosis was assessed. Cell cycle and apoptosis assay data are presented as mean ± SD of three independent experiments.

Fig. 2.

Evaluation of mRNA transcript and protein expression levels in MLL-r ALL cells treated with CN470. (A) SEM and KOPN-1 cells were incubated with CN470 or OTX015 at the doses of 0.75 × and 1.5 × their respective IC₅₀ values for 24 h, and qRT-PCR then performed. Results are presented as mean ± SE of three independent experiments, each with three replicates. (B) SEM and KOPN-1 cells were incubated with CN470 or OTX015 at the doses of 0.75 × and 1.5 × their respective IC₅₀ values for 72 h, and western blotting was then performed. Data are representative of two independent experiments. (C) Co-IP assay using an anti-BRD4 antibody. The IC₅₀ values of CN470 and OTX015 against SEM were 71.2 and 141.1 nM, respectively, and those against KOPN-1 were 106.1 and 194.4 nM, respectively.

Fig. 3.

Effects of in vivo CN470 administration. (A) Schedule of the in vivo study. BRJ mice were transplanted intravenously with SEM^Luc/GFP cells. Treatment was initiated when bioluminescence was detected in mice using IVIS system. (B) Survival rates of SEM^Luc/GFP-xenografted mice. Solid and dashed lines represent the survival rates of the CN470-treated (n = 6 mice) and the vehicle-treated (n = 5 mice) groups, respectively (**p = 0.003). (C) Bioluminescent images generated using the IVIS system and quantification of ventral bioluminescent intensity of SEM^Luc/GFP-xenografted mice on 14 and 21 days after treatment. Data are shown as mean + SE ventral total flux for each group (day 14, *p = 0.015; day21, *p = 0.022).

Fig. 4.

Effect of CN470 on a PDX mouse model using K110 cells. (A) CN470 reduced chimerism in PB. Data are shown as mean + SE of chimerism in PB for each group (day 42, *p = 0.02). The dot plots shown are from representative experiments. (B) CN470 prolonged the survival of PDX model mice (**p = 0.002). Solid and dashed lines represent the survival rates of the CN470-treated (n = 5 mice) and the vehicle-treated (n = 4 mice) groups, respectively.