Combined BRD4 and CDK9 inhibition as a new therapeutic approach in malignant rhabdoid tumors

Abstract

Rhabdoid tumors are caused by the deletion of SMARCB1, whose protein encodes the SMARCB1 subunit of the chromatin remodeling complex SWI/SNF that is involved in global chromatin organization and gene expression control. Simultaneously inhibiting the main players involved in the deregulated transcription machinery is a promising option for preventing exaggerated tumor cell proliferation and survival as it may bypass compensatory mechanisms. In support of this hypothesis, we report efficient impairment of cellular proliferation and strong induction of cell death elicited by inhibition of bromodomain protein BRD4 and transcription kinase CDK9 using small molecular compounds. Combination of both compounds efficiently represses antiapoptotic genes and the oncogene MYC. Our results provide a novel approach for the treatment of RT.

Keywords: BRD4; CDK9; SMARCB1; rhabdoid tumors; synergistic.

Figure 1. Simultaneous inhibition of BRD4 and CDK9 synergistically induces apoptosis in rhabdoid tumor cells in vitro and inhibits cell proliferation in vivo

The indicated RT cell lines were treated with BRD4 and CDK9 inhibitors as single compounds or as a combination of both at different concentrations. Evaluation of apoptosis by flow cytometry shows a strong synergism on apoptosis induction after combined treatments with JQ1/LDC067 (A) or iBET/DRB (B). Rhabdoid tumor xenograft mice were treated with Vehicle, JQ1, LDC067 or simultaneously with JQ1 plus LDC067 during 3 weeks, followed by an observation period of three additional weeks. Tumor size was measured twice a week by digital caliper (C). Box-plots show the average tumor volume at the end of the experiment (D). *p<0.05, **p<0.01, ***p<0.001, ****p < 0.0001 (ANOVA One-way test).

Figure 2. Down-regulation of anti-apoptotic genes and MYC expression in response to combined CDK9 and BRD4 inhibition

Rhabdoid tumor cell lines were treated for 120 min with increasing concentrations of BRD4 or CDK9 inhibitors alone or in combination. Expression of anti-apoptotic genes was analyzed by RT-qPCR. A significant down-regulation of anti-apoptotic genes is evident after exposure to JQ1/ LDC067 (A) or iBET/DRB (B). RT-qPCR analysis of de novo transcription was performed to assess if simultaneous treatment of RT cell lines can affect newly synthesized MYC mRNA using exon-exon primers (MYCe2e3) or intron-intron primers (MYCi1i1) after treatment with JQ1/ LDC067 (C) or iBET/DRB (D). Despite repressed MYC mRNA production, protein levels remain stable 24h after treatment of G401 cells with iBET or with DRB at low doses. Combination of both compounds synergistically reduces MYC protein levels (E). *p<0.05, **p < 0.01, ***p < 0.001 (ANOVA One-way Test).

Figure 3. Combined BRD4 and CDK9 targeting acts synergistically on inhibition of general transcription

To detect effects of combined treatment strategy on general transcription, the indicated RT cell lines were incubated for 120 min with different concentrations of JQ1/ LDC067 (A, B) or iBET/DRB (C, D) alone or in combination of both inhibitors. Gene expression was analyzed by RT-qPCR using different exon-intron and exon-exon primer pairs. Newly synthesized mRNA of the housekeeping genes RPL3 (A, C) and GAPDH (B, D) is shown. Simultaneous application of the inhibitors significantly reduces the expression of these genes indicating a general blockade of the transcription process. *p<0.05, **p < 0.01, ***p < 0.001 (ANOVA One-way Test).