Potent Induction of Human γ Globin Gene Expression by Largazole, a New Histone Deacetylase Inhibitor.

Abstract 2022

Poster Board I-1044

Previous studies have demonstrated that Histone Deacetylase (HDAC) inhibitors such as butyrate and several short chain fatty acids, can induce fetal hemoglobin in humans and animal models; however induction of Hb F is achieved in relatively high concentrations of these compounds. We have previously investigated the induction of human γ globin gene activity by the prototypical HDAC inhibitor, FK228. The results demonstrated that FK228 is a more potent γ globin gene inducer compared to other HDAC inhibitors we have tested before (Am J Hematol. 12:981). In this study, we investigated the induction of human γ globin gene function of largazole and it's thiol analogue in vitro in cultures of normal human adult BFUe and in vivo in the mice carrying a human γ globin transgene. Largazole is a HDAC inhibitor which was recently isolated from a marine vyanobacterium by Luesch and co-workers. Structural features of largazole, a macrocyclic depsopeptide, closely resemble those of FK228, FR901375 and spiruchostatin. We have reported that largazole and numerous synthetic analogues are highly potent Class I histone deacetylase inhibitors (J Am Chem Soc. 130:11219, J Am Chem Soc. 2009 Feb 4).

We used flow cytometry to measure the in vitro effect of largazole and it's derivatives on the frequency of HbF-positive erythroblasts in BFUe cultures from normal individuals; real-time quantitative PCR (RT-qPCR) and high performance liquid chromatography (HPLC) were used to measure the in vivo effects of largazole on human γ globin induction in γ transgenic mice carrying a human γ globin gene..

Our results show that largazole and it's thiol derivative are potent γ hemoglobin gene inducers. In the human BFUe cultures, largazole increased the levels of fetal hemoglobin positive cells from 21.9% (control level) to 62.8% at a concentration of 0.1μM; largazole thiol increased the levels of fetal hemoglobin positive cells to 62.0% at a concentration of 1μM. Transgenic mice carrying the human μLCR ^Aγ construct continue to express the human γ gene in the adult stage (Blood. 77:1326). Largazole was administered through IP injection at the dosages of 0.3mg/kg/day and 0.6mg/kg/day, 5 days per week, for 2 weeks to two cohorts of transgenic mice. Largazole at the dose of 0.3mg/kg/day increased the level of human γ mRNA at the end of injection by 160.7%; at a dose of 0.6mg/kg/day human γ mRNA increased by 174.7%. At the 0.6mg/kg/day dosage the level of fetal hemoglobin in the peripheral blood of the animals increased by 3.4 and 3.2 fold at day 21 and day 28, respectively.

These results provide strong in vitro and in vivo evidence that Largazole and it's thiol analogue are potent HbF inducers acting at low concentrations, and thus provide promising alternatives to compounds currently considered for induction of Hb F in patients with sickle cell disease and thalassemia.