Targeting the CoREST complex with dual histone deacetylase and demethylase inhibitors

Abstract

Here we report corin, a synthetic hybrid agent derived from the class I HDAC inhibitor (entinostat) and an LSD1 inhibitor (tranylcypromine analog). Enzymologic analysis reveals that corin potently targets the CoREST complex and shows more sustained inhibition of CoREST complex HDAC activity compared with entinostat. Cell-based experiments demonstrate that corin exhibits a superior anti-proliferative profile against several melanoma lines and cutaneous squamous cell carcinoma lines compared to its parent monofunctional inhibitors but is less toxic to melanocytes and keratinocytes. CoREST knockdown, gene expression, and ChIP studies suggest that corin's favorable pharmacologic effects may rely on an intact CoREST complex. Corin was also effective in slowing tumor growth in a melanoma mouse xenograft model. These studies highlight the promise of a new class of two-pronged hybrid agents that may show preferential targeting of particular epigenetic regulatory complexes and offer unique therapeutic opportunities.

Fig. 1

Strategy for combining the pharmacophores of clinically used HDAC inhibitors and a preclinical LSD1 inhibitor to generate dual action HDAC-LSD1 inhibitors. Blue—incorporated LSD1 inhibitor features, Green—incorporated HDAC inhibitor features, Orange—shared structural features, Black—features not incorporated into dual inhibitors

Fig. 2

Dual inhibitors exhibit unique activity against the CoREST complex. a Coomassie stained gel depicting the three components of the CoREST ternary complex after purification by size exclusion chromatography. b Dose response produced by inhibition of LSD1 as part of the CoREST complex by corin and structurally matched compound 7. c Inhibition curves generated for corin and MS-275 against HDAC1 as part of the CoREST complex. d Corin inhibited the deacetylation of reconstituted nucleosomes by the CoREST complex as determined by Western blot (CoREST complex = 100 nM, nucleosome = 100 nM). Data (mean ± SEM) are representative of at least two independent experiments

Fig. 3

Corin exhibits sustained inhibition of CoREST complex HDAC activity. a Relative CoREST complex HDAC activity. b Relative MiDAC complex HDAC activity. Protein complexes at 500 nM and all compounds at 5 µM preincubated for 30 min prior to dialysis and dilution to measure HDAC activity at 2 nM (CoREST complex) or 8 nM (MiDAC complex). 50 µM acetylated P53_379–382 tetrapeptide (RHKK(Ac)) was used as substrate. c Corin exhibits near irreversible inhibition of CoREST complex HDAC activity relative to unifunctional LSD1 and HDAC inhibitors after jump dilution (CoREST complex = 0.5 µM, inhibitor = 5 µM, substrate = 200 µM acetylated P53_379–382 tetrapeptide RHKK(Ac)). d Model depicting dual engagement mechanism of corin leading to sustained inhibition of CoREST complex HDAC activity. Data (mean ± SEM) are representative of at least two independent experiments

Fig. 4

Pharmacologic effects of corin and related compounds on melanoma cells and the role of CoREST. a Western blot depicting increases in histone H3K4 methylation and H3K9 acetylation in WM983B melanoma cells induced by 10 µM inhibitor treatment. b Corin (EC₅₀ = 0.095 ± 0.017 µM) was more potent than MS-275 (EC₅₀ = 0.42 ± 0.07 µM) and more efficacious at inducing histone H3K9 acetylation in WM983B melanoma cells as determined by ELISA after 24 h treatment (n = 3). c Treatment with corin (1 µM) for 72 h potently inhibited cell growth across a panel of ten melanoma cell lines without significantly affecting primary melanocytes whereas MS-275 (1 µM) was similarly potent toward transformed and non-transformed cells (n = 3). d Knockdown of CoREST1 inhibits WM983B melanoma cell proliferation (n = 3). e, f Knockdown of CoREST1 enhances the potency of MS-275 but does not affect the potency of corin as determined after 72 h treatment (n = 3). g, h Knockdown of SIN3A inhibits WM983B melanoma cell proliferation but does not sensitize cells to the antiproliferative effects of MS-275 after 72 h treatment (n = 3). Note that cell proliferation was determined using the PicoGreen^® cell proliferation assay and data were normalized to zero inhibitor concentration for individual cell lines (scrambled shRNA and CoREST shRNA1). Data (mean ± SEM) are representative of at least two independent experiments (unpaired t test, *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 5

Analysis of gene knockout effects in HCT116 cells and gene expression changes in melanoma associated with corin treatment. a Knockout of LSD1 in HCT116 cells decreased the rate of cancer cell proliferation by ~40% (n = 4). b, c Knockout of LSD1 enhanced the potency of MS-275 but did not affect the potency of corin in HCT116 cells as determined by [³H]thymidine incorporation after 48 h treatment (n = 4). d, e Venn diagrams depicting increases in global and tumor suppressor gene transcription (fold change ≥ 2σ, p < 0.05), respectively, upon 2.5 µM MS-275 or corin treatment (n = 3). f, g qRT-PCR validation of gene expression changes identified by microarray. WM983B melanoma cells were treated with inhibitor (2.5 µM) for 24 h prior to RNA isolation and analysis (n = 3). h ChIP-PCR localized CoREST complex components HDAC1 and LSD1 to the promoter region of CHOP (n = 3). i ChIP-PCR localized HDAC1, but not LSD1, to the promoter region of SYN1 (n = 3). Data (mean ± SEM) are representative of at least three independent experiments (unpaired t test, *p < 0.05, ***p < 0.001)

Fig. 6

In vivo analysis of corin in a melanoma xenograft. a Daily IP administration of corin (30 mg kg⁻¹) potently inhibited tumor growth in an SK-MEL-5 melanoma cell mouse xenograft model over the course of a 28-day treatment regimen (n = 10 mice per condition). b Western blot depicting increases in histone H3K4 methylation and H3K9 acetylation in tumor tissue obtained from the SK-MEL-5 melanoma xenograft. c Gene expression changes induced by corin in mouse xenograft tumor tissue as determined by qRT-PCR. d Corin treatment decreased the expression of Ki67, a biomarker for cell proliferation, in mouse xenograft tumor tissue (scale = 100 µm). Data (mean ± SEM) are representative of at least two independent experiments (unpaired t test, **p < 0.01, ***p < 0.001)