Assays for Validating Histone Acetyltransferase Inhibitors

Waddell, Aaron

doi:10.3791/61289

Cited by 5 publications

(5 citation statements)

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“…Estrogen (E2, 1 nM) was added 6 h before the addition of Passive Lysis Buffer (PLB) for cell lysis (5X PLB: 125 mM Tris, pH 7.8, 10 mM 1,2-CDTA, 10 mM DTT, 5 mg/mL bovine serum albumin, 5% (vol/vol) Triton X-100 and 50% (vol/vol) glycerol in ddH 2 O). Immunoblotting procedures were performed as described previously [ 58 ]. Briefly, samples were run on a 4–20% gradient polyacrylamide gel (XP04205BOX, Invitrogen, ThermoFisher Scientific, Carlsbad, CA, USA) and transferred to a Immobilon ® -P polyvinylidene difluoride (PVDF) membrane (MilliporeSigma, Burlington, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

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

Waddell

Mahmud

Ding

et al. 2021

Cancers

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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.

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Section: Methodsmentioning

confidence: 99%

“…Once at the desired confluency, cells were incubated with A-485 at 3 µM for 24 h in complete media. ChIP was then performed as described previously [ 58 ]. Briefly, cells were fixed in 1% formaldehyde (final concentration) for 10 min; cells were lysed, and chromatin was sonicated to an average 200 bp.…”

Section: Methodsmentioning

confidence: 99%

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

Waddell

Mahmud

Ding

et al. 2021

Cancers

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“…On the 12 th day, cells were utilized for ChIP as described previously. 61,62 Briefly, cells were fixed in 1% formaldehyde (final concentration) in PBS and chromatin was extracted. The chromatin was then sheared to approximately 200 bp using the Diagenode Bioruptor Pico (Diagenode, Denville, NJ, USA) using 30 cycles (30 seconds on/30 seconds off) according to manufacturer instructions.…”

Section: Chip-qpcrmentioning

confidence: 99%

p300 KAT regulates SOX10 stability and function in human melanoma

Waddell,

Grbic,

Leibowitz

et al. 2024

Preprint

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SOX10 is a lineage-specific transcription factor critical for melanoma tumor growth, while SOX10 loss-of-function drives the emergence of therapy-resistant, invasive melanoma phenotypes. A major challenge has been developing therapeutic strategies targeting SOX10’s role in melanoma proliferation, while preventing a concomitant increase in tumor cell invasion. Here, we report that the lysine acetyltransferase (KAT)EP300andSOX10gene loci on Chromosome 22 are frequently co-amplified in melanomas, including UV-associated and acral tumors. We further show that p300 KAT activity mediates SOX10 protein stability and that the p300 inhibitor, A-485, downregulates SOX10 protein levels in melanoma cells via proteasome-mediated degradation. Additionally, A-485 potently inhibits proliferation of SOX10+ melanoma cells while decreasing invasion in AXLhigh/MITFlowmelanoma cells through downregulation of metastasis-related genes. We conclude that the SOX10/p300 axis is critical to melanoma growth and invasion, and that inhibition of p300 KAT activity through A-485 may be a worthwhile therapeutic approach for SOX10-reliant tumors.

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“…The panel of MDA-MB-231-derived cell lines (control, WT DAXX and DSM mutant OE) were cultured in complete DMEM. ChIP experiments were performed essentially as described 89 . Briefly, at about 90% confluency, the cells were crosslinked by adding 37% formaldehyde to the final concentration of 1% for 10 min at room temperature.…”

Section: Chip-seq Analysismentioning

confidence: 99%

DAXX drives de novo lipogenesis and contributes to tumorigenesis

et al. 2023

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Cancer cells exhibit elevated lipid synthesis. In breast and other cancer types, genes involved in lipid production are highly upregulated, but the mechanisms that control their expression remain poorly understood. Using integrated transcriptomic, lipidomic, and molecular studies, here we report that DAXX is a regulator of oncogenic lipogenesis. DAXX depletion attenuates, while its overexpression enhances, lipogenic gene expression, lipogenesis, and tumor growth. Mechanistically, DAXX interacts with SREBP1 and SREBP2 and activates SREBP-mediated transcription. DAXX associates with lipogenic gene promoters through SREBPs. Underscoring the critical roles for the DAXX-SREBP interaction for lipogenesis, SREBP2 knockdown attenuates tumor growth in cells with DAXX overexpression, and DAXX mutants unable to bind SREBP1/2 have weakened activity in promoting lipogenesis and tumor growth. Remarkably, a DAXX mutant deficient of SUMO-binding fails to activate SREBP1/2 and lipogenesis due to impaired SREBP binding and chromatin recruitment and is defective of stimulating tumorigenesis. Hence, DAXX’s SUMO-binding activity is critical to oncogenic lipogenesis. Notably, a peptide corresponding to DAXX’s C-terminal SUMO-interacting motif (SIM2) is cell-membrane permeable, disrupts the DAXX-SREBP1/2 interactions, and inhibits lipogenesis and tumor growth. These results establish DAXX as a regulator of lipogenesis and a potential therapeutic target for cancer therapy.

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Assays for Validating Histone Acetyltransferase Inhibitors

Cited by 5 publications

References 0 publications

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

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

p300 KAT regulates SOX10 stability and function in human melanoma

DAXX drives de novo lipogenesis and contributes to tumorigenesis

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