Mechanistic Insights Into Fetal Hemoglobin (HbF) Induction Through Chemical Inhibition Of Histone Deacetylase 1 and 2 (HDAC1/2)
Authors: Shearstone Jeffrey R , van Duzer John H , Jones Simon S , Jarpe Matthew 
Published: October 2019

Abstract
Induction of HbF is an established therapeutic strategy for the treatment of sickle cell disease (SCD), and could also be effective in treating beta-thalassemia (bT). Fetal beta-like globin gene (HbG) expression is silenced in adults partly through the nucleosome remodeling and histone deacetylase (NuRD) complex, which contains HDAC1/2 (Sankaran VG, Science, 2008). Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of HbG expression (Bradner JE, Proc Natl Acad Sci, 2010). Furthermore, we have previously shown that selective chemical inhibitors of HDAC1 and 2 elicit a dose and time dependent induction of HbG mRNA and HbF protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). However, the mechanism through which HDAC1/2 inhibition leads to activation of HbG remains largely unknown. In this work, we have utilized our proof of concept molecule, ACY-957, to investigate changes in gene expression and chromatin organization that result from inhibition of HDAC1/2.
Gene expression profiling was performed on cells treated with ACY-957 (n=3) or vehicle (n=3) using Affymetrix PrimeView GeneChips. Treatment of early erythroblasts (CD71+, GlyA-) resulted in the up and down regulation of 1294 and 681 transcript probe sets, respectively. In comparison, treatment of late erythroblasts (CD71+, GlyA+) resulted in a total of 255 transcript probe set changes. This finding is consistent with follow-up experiments demonstrating that ACY-957 is unable to induce HbG in cells positive for both CD71 and GlyA. Taken together, these results suggest that erythroblasts become less responsive to HDAC inhibition as they mature. Gene set enrichment analysis using public domain data revealed that genes up- or down-regulated by HDAC1/2 shRNA knockdown are significantly overrepresented in the list of genes induced or repressed by ACY-957, respectively; suggesting pharmacologic inhibition of HDAC1/2 recapitulates genetic ablation. We also identified significant enrichment in other gene sets involving targets linked to HbG regulation, including lysine-specific demethylase 1 (LSD1) (Shi L, Nature Medicine, 2012).
GeneChip and quantitative real-time PCR time course experiments show ACY-957 treatment leads to a decrease in Bcl11A (2-fold) and Sox6 (10-fold) mRNA, known repressors of fetal globin synthesis, and an increase in Klf2 (2-fold) and Gata2 (8-fold) mRNA, proposed fetal globin activators. This result is consistent with work by others that show Gata2 is suppressed, in part, by the NuRD complex (Hong W, EMBO Journal, 2005) and that Gata2 binding at the HbG promoter leads to increased levels of HbG expression (Zhu X, PLoS One, 2012). Interestingly, Gata2 induction preceded Sox6 suppression in ACY-957 treated cells and the Sox6 promoter contains 8 canonical WGATAR binding sites and one Gata2-specific binding motif, raising the possibility suppression of Sox6 by ACY-957 is mediated by Gata2 induction. To investigate these possibilities, we have performed chromatin immunoprecipitation coupled with next generation sequencing (ChIP-seq) for HDAC1, HDAC2, Gata2, and the HDAC2-specific histone modification H3K56 in ACY-957 and vehicle treated cells. These experiments will be discussed. ChIP-seq data, both by itself and in combination with gene expression data, will provide further insight into the mechanism through which HDAC1/2 regulates HbF synthesis.

Disclosures:
Shearstone: Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.