Enitociclib, a selective CDK9 inhibitor: in vitro and in vivo preclinical studies in multiple myeloma

Abstract

Multiple myeloma (MM) is a cancer of plasma cells that remains incurable despite advances in treatment options. In this study, a library of 216 clinically feasible small-molecule inhibitors was screened to identify agents that selectively inhibit MM cell proliferation. Enitociclib, a cyclin-dependent kinase 9-specific small-molecule inhibitor, was found to be highly effective in decreasing cell viability and inducing apoptosis in 4 MM cell lines. Enitociclib inhibited the phosphorylation of the carboxy-terminal domain (CTD) of RNA polymerase II at Ser2/Ser5 and repressed the protein expression of oncogenes c-Myc, myeloid cell leukemia-1 (Mcl-1), and proliferating cell nuclear antigen (PCNA) in MM cells. Additionally, enitociclib demonstrated synergistic effects with several anti-MM agents, including bortezomib, lenalidomide, pomalidomide, and venetoclax. These results suggest that enitociclib may represent a promising therapeutic option for the treatment of MM, either as a single agent or in combination with other anti-MM agents.

Graphical abstract

Figure 1.

Enitociclib decreases cell viability in MM cells. (A) Cell viability of small-molecule inhibitors from the pharmaceutical pipeline library treated at 1 μM for 96 hours in OPM-2 cells. A breakdown of molecular targets with a mean cell viability inhibition of >50% are shown. (B) Cell viability values of various CDK inhibitors from the library treated at 1 μM for 96 hours in OPM-2 cells are shown. (C) Western blotting of MM cell lines MM1.S, NCI-H929, OPM-2, and U266B1. Total cell lysates were prepared and analyzed by immunoblotting to detect the level of CDK9. β-actin was used as a loading control. Molecular masses are indicated in kilodaltons (kDa). (D) Dose response curves of MM cell lines treated with increasing concentrations (12.5-200 nM) of enitociclib for 96 hours. Cell viability was measured by Alamar Blue assay. Percent cell viability was normalized to corresponding treatment with dimethyl sulfoxide (DMSO; vehicle control). Mean percentages of cell viability were calculated from 3 technical replicates and standard deviations are shown. BET, Bromodomain and extraterminal; EZH1/2, Enhancer of zeste homolog 1/2; FLT3, Fms-like tyrosine kinase 3; HDAC, Histone deacetylase; HSP, Heat shock protein; IGF-1R, Insulin-like growth factor 1 receptor; KDM5A, Lysine (K)-specific demethylase 5A; mTOR, Mammalian target of rapamycin; NAE, NEDD8-activating enzyme; WNK, With no lysine (K); XPO1, Exportin-1.

Figure 2.

Enitociclib induces apoptosis by inhibition of RNA Pol II phosphorylation and oncogene expression in MM cells. Western blotting of NCI-H929 and OPM-2 MM cells treated with either DMSO (vehicle control; “–”) or 0.5 to 1 μM of enitociclib for up to 24 hours. Total cell lysates were prepared and analyzed by immunoblotting to detect levels of markers associated with apoptosis (total and cleaved PARP and caspase-3, Mcl-1, and Bim_EL), total and phosphorylated RNA Pol II CTD (S2/S5), and short half-life oncogene proteins c-Myc and PCNA. β-actin was used as a loading control. Molecular masses are indicated in kDa.

Figure 3.

Enitociclib is synergistic with anti-MM chemotherapies. Three-dimensional (3D) response surface plots for combinatory activity of enitociclib with bortezomib, lenalidomide, pomalidomide, or venetoclax treated in MM cell lines for 96 hours. Cell viability was measured by Alamar Blue assay. Percent cell viability was normalized to corresponding treatment with DMSO (vehicle control). Synergy score is calculated by SynergyFinder based on the ZIP interaction model. Red indicates synergism, and green indicates antagonism of the respective drug combinations. Maximal synergistic effects (MSEs) are indicated when the synergy score is >10, and the drug concentrations at which the MSE occurs are highlighted in yellow.

Figure 4.

Enitociclib enhances apoptosis and oncogene repression. Western blotting of OPM-2 cells treated with enitociclib in combination with bortezomib, lenalidomide, pomalidomide, or venetoclax for 6 hours. Total cell lysates were prepared and analyzed by immunoblotting to detect the levels of markers associated with apoptosis (total and cleaved PARP and caspase-3 and Mcl-1) and short half-life oncogene proteins (c-Myc and PCNA). β-actin was used as a loading control. Molecular masses are indicated in kDa.

Figure 5.

Enitociclib is effective against MM in vivo. (A-B) In vivo mechanism of action of enitociclib in mice bearing JJN-3 MM xenografts upon a single dose of 15 mg/kg enitociclib administered IV compared with 80% PEG400 vehicle. Messenger RNA transcript (A) and protein levels (B) were normalized to housekeeping genes in L32 and β-actin, respectively. (C) In vivo antitumor activity of enitociclib administered as a single agent. For JJN-3, NCI-H929, and OPM-2 MM xenografts, enitociclib was dosed 15 mg/kg IV once weekly compared with vehicle control. (D) To study combinations, enitociclib was dosed 15 mg/kg IV once weekly in combination with 50 mg/kg lenalidomide orally daily or 0.8 mg/kg bortezomib intraperitoneally twice weekly in OPM-2 MM xenografts. ∗P < .05.