Anti-Cancer Effects of CKD-581, a Potent Histone Deacetylase Inhibitor against Diffuse Large B-Cell Lymphoma

Abstract

Double-hit lymphoma (DHL) and double-expressor lymphoma (DEL) are aggressive forms of lymphoma that require better treatments to improve patient outcomes. CKD-581 is a new histone deacetylase (HDAC) inhibitor that exhibited a better safety profile in clinical trials compared to other HDAC inhibitors. Here, we demonstrate that CKD-581 inhibited the class I-II HDAC family via histone H3 and tubulin acetylation. CKD-581 treatment also up-regulated the phosphorylation of histone H2AX (γH2AX, DNA double-strand break marker), and reduced levels of MYC and anti-apoptotic proteins such as BCL-2, BCL-6, BCL-_XL, and MCL-1 in DH/DE-diffuse large B cell lymphoma (DLBCL) cell lines. Ultimately, CKD-581 also induced apoptosis via poly(ADP ribose) polymerase 1 (PARP1) cleavage. In a DLBCL SCID mouse xenograft model, CKD-581 exhibited anti-cancer effects comparable with those of rituximab (CD20 mAb). Our findings suggest that CKD-581 could be a good candidate for the treatment of DLBCL.

Keywords: CKD-581; MYC; diffuse large B cell lymphoma; histone deacetylase inhibitor.

Figure 1

CKD-581 is a potent HDAC inhibitor. (a) Acetylation of tubulin or histone H3. SU-DHL-2 cells were treated with vehicle control (C), CKD-581 (10–300 nM), or 300 nM SAHA (S) for 6 h, and total cell lysates were obtained. Acetylation of tubulin or histone H3 was determined by immunoblotting. (b) Comparison of inhibitory effects of CKD-581 and SAHA on cell viability of four DLBCL cell lines. SU-DHL-4, OCI-LY1, SU-DHL-2, and U2932 cells were incubated with CKD-581 and SAHA for 72 h, and cell viability was assessed by a CellTiter Bright-Glo assay. Data represent mean ± SEM (n = 3).

Figure 2

CKD-581 reduces the protein expression of prognostic markers for DLBCL. (a) SU-DHL-4, (b) OCI-LY1, (c) SU-DHL-2, and (d) U2932 cells were treated with vehicle control (C), various concentrations (10–300 nM) of CKD-581, or 300 nM SAHA (S) for 24 h, and total cell lysates were subjected to immunoblotting for MYC, BCL-2 and BCL-6. Data represent mean ± SEM of at least three independent experiments (n = 3, significant vs. control; * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

CKD-581 induces DNA damage and apoptosis. (a) SU-DHL-4, (b) OCI-LY1, (c) SU-DHL-2, and (d) U2932 cells were treated with vehicle control (C), CKD-581 (10–300 nM), or 300 nM SAHA (S) for 24 h, and total cell lysates were subjected to immunoblotting for γH2AX and PARP1. Data represent mean ± SEM (n = 3, significant vs. control; * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

CKD-581 decreases anti-apoptotic proteins in DLBCL. (a) SU-DHL-4, (b) OCI-LY1, (c) SU-DHL-2, and (d) U2932 cells were treated with vehicle control (C), CKD-581 (10‒300 nM), or 300 nM SAHA (S) for 24 h, and total cell lysates were subjected to immunoblotting for BCL-xL and MCL-1. Data represent mean ± SEM (n = 3, significant vs. control; * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5

CKD-581 suppressed DH-DLBCL tumor growth in the mouse xenograft models. (a) CD20 expression in DH-DLBCL cells. CD20 expression levels were compared in SU-DHL-4 and SU-DHL-2 cells by immunoblotting. (b,c) Xenograft tumor growth assays. NOD.CB17 SCID mice were implanted with (b) SU-DHL-4 (1 × 10⁶ cells/mouse) or (c) SU-DHL-2 (1 × 10⁶ cells/mouse) cells. When tumors grew to about 150 mm³, the mice were intraperitoneally injected with vehicle, CKD-581 (20 and 40 mg/kg), or rituximab (10 mg/kg) according to the treatment schedule. Data represent mean ± SEM ((n = 10, SU-DHL-4; n = 6, SU-DHL-2) significant vs. vehicle group; * p < 0.05, ** p < 0.01, *** p < 0.001).