Fig. 4.

SRPK1 inhibition affects BRD4 chromatin recruitment at the BCL2 enhancer locus and synergizes with iBET-151 to inhibit growth of AML cells in vivo. a Overlap between loci with reduced BRD4 binding after treatment with iBET in MOLM14 from Pelish et al.²¹ and after BRD4S-to-BRD4L isoform switching by BRD4-ex12-SA_A gRNA in THP-1 cells. Both MOLM-14 and THP-1 harbor the MLL-AF9 oncogene (Fisher’s exact test). b BRD4 ChIP-seq of THP-1 cells targeted with empty gRNA or gRNA BRD4-ex12-SA_A, 5 days post-transduction, showing eviction of BRD4 from the 3’ BCL2 enhancer (left). c, d ChIP-qPCR experiments in THP-1 cells showing reduced binding of BRD4 after exposure to 1.5 μM SPHINX31 for 72 h or BRD4-ex12-SA_A gRNA (6 days post-transduction). (mean ± s.d., n = 3). e Box plot showing correlation of BRD4 eviction from promoters or linked distal intergenic regions with reduced expression of the affected genes, when compared to unselected genes genome-wide (All) (*p < 0.001; Wilcoxon test). Red, dashed line corresponds to no change in the gene expression. f Location of gRNAs targeting the 3’ BCL2 enhancer. g Competitive co-culture showing the requirement for the BCL2 3’ enhancer for MOLM-13 and THP-1 cell growth and proliferation. Results were normalized to day 4 (mean ± s.d., n = 3). h Reduction of BCL2 protein in THP-1 cells by gRNA targeting of the 3’ BCL2 enhancer (i) and by BRD4-ex12-SA_A gRNA. j Quantification of luminescence for mice transplanted with luciferase-labeled THP-1 cells and treated with low dose of iBET-151 (10 mg/kg) or SPHINX31 (0.8 mg/kg) or both, showing a synergistic effect between the two drugs. **P < 0.01. ***P < 0.001. k Survival of mice transplanted with THP-1 cells treated as described in j (n = 8–9 animals per group). **P < 0.01. compared to vehicle (black). ***P < 0.001. compared to iBET-151(green) or SPHINX31 (blue). ***P < 0.001. Log-rank (Mantel–Cox) test was performed for the survival assays in k