Abstract
Aim:Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers that play a fundamental role in transcription regulation. Preclinical and early clinical evidence sustain BET targeting as an anti-cancer approach. BET degraders are chimeric compounds comprising of a BET inhibitor, which allows the binding to BET bromodomains, linked to a small molecule, binder for an E3 ubiquitin ligase complex, triggering BET proteins degradation via the proteasome. These degraders, called proteolysis-targeting chimeras (PROTACs), can exhibit greater target specificity compared to BET inhibitors and overcome some of their limitations, such as the upregulation of the BET proteins themselves. Here are presented data on the anti-tumor activity and the mechanism of action of the BET degrader MZ1 in diffuse large B cell lymphoma (DLBCL) of the activated B-cell like (ABC, ABC DLBCL), using a BET inhibitor as a comparison.Methods:Established lymphoma cell lines were exposed for 72 h to increasing doses of the compounds. Cell proliferation was evaluated by using an 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide (MTT) assay. Fluorescent-Activated Cell Sorter (FACS) analysis was performed to measure apoptotic activation and RNA sequencing (RNA-Seq) to study the transcriptional changes induced by the compounds.Results:MZ1, and not its negative control epimer cisMZ1, was very active with a median half maximal inhibitory concentration (IC50) of 49 nmol/L. MZ1 was more in vitro active than the BET inhibitor birabresib (OTX015). Importantly, MZ1 induced cell death in all the ABC DLBCL cell lines, while the BET inhibitor was cytotoxic only in a fraction of them. BET degrader and inhibitor shared partially similar changes at transcriptome level but the MZ1 effect was stronger and overlapped with that caused cyclin-dependent kinase 9 (CDK9) inhibition.Conclusions:The BET degrader MZ1 had strong cytotoxic activity in all the ABC DLBCL cell lines that were tested, and, at least in vitro, it elicited more profound effects than BET inhibitors, and encourages further investigations.
Highlights
Diffuse large B cell lymphoma (DLBCL) comprises two biologically distinct entities that are referred to as germinal center B-cell (GCB) and activated B-cell (ABC) subtype [1,2,3]
Bromodomain and extra-terminal domain (BET) inhibitors are active in all ABC diffuse large B cell lymphoma (DLBCL) cell lines, their activity is largely cytostatic with a strong induction of apoptosis limited to cell lines bearing somatic mutations in essential genes such as myeloid differentiation factor 88 (MYD88) and cluster of differentiation 79B (CD79B) [9, 10], representative of the genetically defined subclusters MCD/cluster 5 (C5) [4,5,6]
Seven cell lines derived from ABC DLBCL were exposed for 72 h to the BET degrader MZ1, its negative control epimer cisMZ1 and, as a comparison, the pan-BET inhibitor birabresib which has shown early clinical activity [12, 27]
Summary
Diffuse large B cell lymphoma (DLBCL) comprises two biologically distinct entities that are referred to as germinal center B-cell (GCB) and activated B-cell (ABC) subtype [1,2,3]. BET proteins are epigenetic readers that play a fundamental role in transcription regulation [13,14,15] They include the ubiquitously expressed bromodomain-containing protein 2 (BRD2), BRD3, and BRD4 and the testis-restricted bromodomain testis-specific protein (BRDT). BET inhibitors are active in all ABC DLBCL cell lines, their activity is largely cytostatic with a strong induction of apoptosis limited to cell lines bearing somatic mutations in essential genes such as myeloid differentiation factor 88 (MYD88) and cluster of differentiation 79B (CD79B) [9, 10], representative of the genetically defined subclusters MCD/C5 [4,5,6]
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