Pharmacological Inhibition of CBP/p300 Blocks Estrogen Receptor Alpha (ERα) Function through Suppressing Enhancer H3K27 Acetylation in Luminal Breast Cancer

Abstract

Estrogen receptor alpha (ER) is the oncogenic driver for ER+ breast cancer (BC). ER antagonists are the standard-of-care treatment for ER+ BC; however, primary and acquired resistance to these agents is common. CBP and p300 are critical ER co-activators and their acetyltransferase (KAT) domain and acetyl-lysine binding bromodomain (BD) represent tractable drug targets, but whether CBP/p300 inhibitors can effectively suppress ER signaling remains unclear. We report that the CBP/p300 KAT inhibitor A-485 and the BD inhibitor GNE-049 downregulate ER, attenuate estrogen-induced c-Myc and Cyclin D1 expression, and inhibit growth of ER+ BC cells through inducing senescence. Microarray and RNA-seq analysis demonstrates that A-485 or EP300 (encoding p300) knockdown globally inhibits expression of estrogen-regulated genes, confirming that ER inhibition is an on-target effect of A-485. Using ChIP-seq, we report that A-485 suppresses H3K27 acetylation in the enhancers of ER target genes (including MYC and CCND1) and this correlates with their decreased expression, providing a mechanism underlying how CBP/p300 inhibition downregulates ER gene network. Together, our results provide a preclinical proof-of-concept that CBP/p300 represent promising therapeutic targets in ER+ BC for inhibiting ER signaling.

CREBBP/EP300 acetyltransferase inhibition disrupts FOXA1-bound enhancers to inhibit the proliferation of ER+ breast cancer cells

“…A key observation from our epigenomics experiments is the difference between the broad changes in H3K27ac response and the comparatively more subtle changes observed in chromatin accessibility upon CPI-1612 treatment. Similar to published findings, we observed a global reduction in H3K27 acetylation, with downregulated peaks enriched in enhancers relative to transcriptional start sites [ 8 , 33 , 34 ]. However, significant changes in chromatin accessibility were much less abundant, but were notably enriched with ER target genes.…”

“…Activity of CREBBP/EP300 HAT inhibition in TNBC cell lines has been noted previously [ 15 ], but the biomarkers that predict activity have not been elucidated. However, dependence on the acetyltransferase activity of CREBBP/EP300 in ER+ breast cancer is consistent with the known physical and functional links between CREBBP/EP300 and ER and has been corroborated in another recently published study [ 33 ]. However, while Waddell et al established a link between H3K27 acetylation at enhancers and ER target gene expression, key points were not addressed: comparison of CREBBP/EP300 HAT inhibition with direct ER targeting, the involvement of chromatin accessibility dynamics in transcriptional regulation by CREBBP/EP300, and the features that define the recruitment of CREBBP/EP300 activity to specific loci.…”

Theoretical and practical refinements of sans spike-in quantitative ChIP-seq with application to p300/CBP inhibition

“…Indeed, this conclusion is reported in several papers. [16–19] In one study, the increase in H3K18ac signal at TssA was described as an ’increase in the average level of H3K18ac and H3K27ac.’[28] In another study, using spike-ins, it was ’observed that the H3K27ac mark was reduced at [TssA] whereas H3K18ac was slightly increased.’[17] Yet another recent report observed that ’surprisingly, only 226 [of 807] downregulated genes exhibited hypoacetylation of H3K27 under the condition of CBP/p300 HAT inhibition.’[19] Consistently, treating unscaled ChIP-seq data as though it were quantitative leads to the conclusion that the targeted acetylation may be unaffected or even increased in some genomic regions — counter to expectations and contrary to the indication of global loss in the isotherms (IP mass capture, Figure 3), which mirror what is seen by other global measures like western blots (Figure 3a of Reference 28 and SI-Fig. 4).…”

Single-Cell Image-Based Analysis Reveals Chromatin Changes during the Acquisition of Tamoxifen Drug Resistance

“…The expression of estrogen receptor (ER) is H3K27ac dependent [ 51 ]. The ER signaling pathway is inhibited by H3K27 acetyltransferase inhibitors [ 52 ], indicating that our observed H3K27ac decrease may have contributed to the downregulation of ER signaling and subsequently conferred Tam resistance. Overall, we observed a global increase in the gene silencing marker in TamR cells, including 5mC, H3K9me3, and H3K27me3, and a decrease in gene activation marker, H3K27ac.…”