Therapeutic impact of BET inhibitor BI 894999 treatment: backtranslation from the clinic

BET inhibitors have been tested in several clinical trials, and despite encouraging results from preclinical cancer models, substantial clinical benefit in monotherapy has been limited. An important aspect is the proper translation of preclinical observations into clinical evaluation. The clinical BET inhibitor BI 894999 was tested in mechanistic studies, assessing pathway modulation and rational drug combinations at clinically achievable concentrations. We used NUT carcinoma (NC) cell lines with bona fide oncogenic drivers fusions including BRD4-NUT and BRD3-NUT. Proliferation assays, pathway modulation studies, and in vivo xenografts were performed with BI 894999, in monotherapy, and in combination with the clinical p300/CBP inhibitor CCS1477. Detachment of the BET complex from chromatin in vitro was assessed by LSM immunofluorescence, and by immunohistochemistry in tumour sections. Clinical findings on pathway modulation marker such as HEXIM1 and HIST2H2BF as well as human PK data were back translated to the preclinic setting. The clinical phase I scheduling regimen of 1 week-on (with a loading dose on day 1) and one week-off, derived from modelling of preclinical and clinical data, achieved plasma levels of about 20 nM. This concentration led to the full removal of BRD-NUT from chromatin and resulted in sustained and prolonged PD modulation of HEXIM1 and HIST2H2BF, accompanied with better patient tolerability. Rational combination with the p300/CBP inhibitor CCS1477 led to tumour regression in all three NC xenograft models tested. Preclinical mechanistic studies with the BET inhibitor BI 894999, back translation of clinical PK/PD data, and modelling provided guidance for an optimal clinical schedule. BI 894999 holds significant potential as a combination drug and preclinical data identified p300/CBP inhibitors as highly relevant combination partner for NUT carcinoma and beyond, pending clinical trials. NUT midline carcinoma cell lines were grown in media and treated with either DMSO (control) or compound(s). BI 894999 (WO2014076237) is the clinical compound synthesized by Boehringer Ingelheim. CCS1477 was purchased from Chemie Tek, Indianapolis, USA. The experiment was performed in biological triplicates per condition/cell lines, two samples however had to be excluded from the final analysis as these samples did not meet the quality criteria of sequencing coverage. Information about sampling time points, compounds, compound concentrations, and replicates are detailed out in the respective sample descriptions. All samples were subjected to RNA isolation, followed by library preparation (QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina, Lexogen), and messenger RNA sequencing on an Illumina NextSeq 500 system. Update 2022-08-05: In contrast to the sample titles, the compound BI00894999 was dosed with 10nM in all respective samples, in contrast to the title claiming a dose of 1000nM.