In vitro activity: PLX51107 is a novel, potent and selective BET (Bromodomain and Extra-Terminal motif) inhibitor, also called BRD4 (bromodomain and extra terminal domain) inhibitor with Kd values of 1.6, 2.1, 1.7, and 5 nM for BD1 and 5.9, 6.2, 6.1, and 120 nM for BD2 of BRD2, BRD3, BRD4, and BRDT, respectively; BRD4 is overexpressed in CLL and is enriched proximal to genes upregulated or de novo expressed in CLL with known functions in disease pathogenesis and progression. PLX51107 also interacts with the bromodomains of CBP and EP300 with Kd in the 100 nM range. PLX51107 (0.156-10 μM) suppresses the CpG-induced proliferation of primary chronic lymphocytic leukemia (CLL) cells. PLX51107 also causes accumulation of p21 and IκBα, reduces c-MYC level, and modulates proapoptotic and antiapoptotic proteins. PLX51107 selectively modulates CLL driver gene.

Kinase Assay: DAVID functional annotation tool was used to identify enriched gene ontology terms in a given gene list. IPA (Qiagen Bioinformatics) software was used for functional annotation of differentially expressed genes regulated by BRD4 inhibition. Fold change information from microarray gene expression data was also provided as an input to IPA. Significantly overrepresented canonical pathways were reported. Upstream transcriptional regulators that could explain the observed changes in gene expression were identified by IPA upstream regulator analysis with a z-score cutoff of +2 for activation and –2 for inhibition. Overrepresented diseases and biological functions with a predicted activation state were identified by IPA diseases/biofunctions analysis with a z-score cutoff of 2 for increase and –2 for decrease.

Cell Assay: Primary CLL cells (1 × 107 cells per condition) were treated with vehicle (DMSO), or 1 μmol/L PLX51107 with or without CpG oligonucleotides (3.2 μmol/L) for 4 hours. Cells were fixed with 1% formaldehyde for 15 minutes and quenched with 0.125 mol/L glycine. Chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300 to 500 bp. See Supplementary Information for detailed cross-link procedure. Genomic DNA regions of interest were isolated using 4 μg antibody against BRD4, H3K27ac, and RNA Pol II. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Cross-links were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol–chloroform extraction and ethanol precipitation.