Person:

Mazitschek, Ralph

The worldwide burden of sickle cell disease is enormous, with over 200,000 infants born with the disease each year in Africa alone. Induction of fetal hemoglobin is a validated strategy to improve symptoms and complications of this disease. The development of targeted therapies has been limited by the absence of discrete druggable targets. We developed a unique bead-based strategy for the identification of inducers of fetal hemoglobin transcripts in primary human erythroid cells. A small-molecule screen of bioactive compounds identified remarkable class-associated activity among histone deacetylase (HDAC) inhibitors. Using a chemical genetic strategy combining focused libraries of biased chemical probes and reverse genetics by RNA interference, we have identified HDAC1 and HDAC2 as molecular targets mediating fetal hemoglobin induction. Our findings suggest the potential of isoform-selective inhibitors of HDAC1 and HDAC2 for the treatment of sickle cell disease.

We examined the effects of isoform-specific histone deacetylase (HDAC) inhibitors on β-catenin posttranslational modifications in neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs). β-catenin is a multifunctional protein with important roles in the developing and adult central nervous system. Activation of the Wnt pathway results in stabilization and nuclear translocation of β-catenin, resulting in activation of multiple target genes. In addition, β-catenin forms a complex with cadherins at the plasma membrane as part of the adherens junctions. The N-terminus of β-catenin has phosphorylation, ubiquitination, and acetylation sites that regulate its stability and signaling. In the absence of a Wnt signal, Ser33, Ser37, and Thr41 are constitutively phosphorylated by glycogen synthase kinase 3β (GSK3β). β-Catenin phosphorylated at these sites is recognized by β-transducin repeat-containing protein (βTrCP), which results in ubiquitination and degradation by the ubiquitin-proteasome pathway. The N-terminal regulatory domain of β-catenin also includes Ser45, a phosphorylation site for Casein Kinase 1α (CK1α) and Lys49, which is acetylated by the acetyltransferase p300/CBP-associated factor (PCAF). The relevance of Lys49 acetylation and Ser45 phosphorylation to the function of β-catenin is an active area of investigation. We find that HDAC6 inhibitors increase Lys49 acetylation and Ser45 phosphorylation but do not affect Ser33, Ser37, and Thr41 phosphorylation. Lys49 acetylation results in decreased ubiquitination of β-catenin in the presence of proteasome inhibition. While increased Lys49 acetylation does not affect total levels of β-catenin, it results in increased membrane localization of β-catenin.