Abstract 3850: The potent catalytic p300HAT inhibitor A-485 induces rapid decay and reduction in breast cancer ERBB2 mRNA expression
Authors: Scott Gary K. , Rose Jacob , Wei Lei , Schilling Birgit , Benz Christopher 
Published: August 2019

Abstract
We have recently demonstrated that siRNA knockdown of p300 promotes the selective loss of oncogenic breast cancer transcripts such as ERBB2 while housekeeping transcripts like GAPDH are largely unaffected. Utilizing the potent p300 HAT inhibitor A-485 to treat the ERBB2 overexpressing SKBR3 breast cancer cells, ERBB2 transcripts experienced accelerated decay relative to decay induced by actinomycin D with cells entering growth arrest by 24 h, a situation similar to the growth arrest observed following p300 siRNA knockdown. Interestingly, previous studies from our lab identified a 3’UTR dependent mechanism promoting accelerated decay of ERBB2 transcripts following inhibition of class-1 histone deacetylases (e.g. HDAC1/2) using the approved therapeutic Romidepsin (FK228). In this situation mass spectrometry (MS) determined that within 2 h of FK228 treatment p300 becomes hyperacetylated at multiple lysine sites (K970, K1542, K1546, K1549, K1550, K1551, K1554, K1555, K1558, K1560, K1674, K1760) which include those lysines thought to regulate the activity of its HAT domain. The rapid reduction in ERBB2 mRNA levels induced by either 2-4 h of FK228 or A-485 treatment raises the interesting question of how either gain or loss of p300HAT enzymatic function induces accelerated ERBB2 mRNA decay? In regard to FK228 treatment, p300 siRNA experiments identified a 28 kDa species, presumed to be monoubiquitinated H2B, dependent upon p300 for FK228 induced acetylation and where knockdown of RNF20, an E3 ligase promoting H2B monoubiquitination, blunted the FK228 induced decay of ERBB2 mRNA. By immunoprecipitating p300 from cells treated with either vehicle or FK228 (2 h) and then assaying immunoprecipitates for histone acetyl transferase activity, we observed that FK228 induced hyperacetylation appears to increase p300HAT enzymatic activity. Additionally, it was observed that pretreating cells for 1 h with A-485 (2 μM) followed by 5 h of FK228 treatment (0.5 uM) produced complete loss of FK228 induced p300 hyperacetylation together with greatly reduced ERBB2 mRNA levels. Altogether, these findings point to a novel 3’UTR-dependent transcript regulatory mechanism wherein active but not hyperacetylated or hypoacetylated p300 stabilizes both new and mature ERBB2 transcripts, whose stability is then selectively and rapidly lost when p300 is either hyperacetylated by FK228 or functionally crippled by either siRNA knockdown or A-485 catalytic inhibition. While this novel ERBB2 mRNA stability mechanism is still in need of further structural definition, it can be effectively targeted by mechanistically different approaches and small molecule anticancer agents including the clinically approved class-I HDAC inhibitor, Romidepsin (FK228), and the investigational catalytic p300HAT inhibitor, A-485.
Citation Format: Gary K. Scott, Jacob Rose, Lei Wei, Birgit Schilling, Christopher Benz. The potent catalytic p300HAT inhibitor A-485 induces rapid decay and reduction in breast cancer ERBB2 mRNA expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3850.