Resveratrol Regulates Fetal Hemoglobin Production by Inhibiting KLF1 and BCL11A in KU812 Cells
Authors: Agyekum Davies G , Chen Zhong , Xiao Haiyan , Yu Megan , Patel Niren , Pace Betty S , Meiler Steffen E. 
Published: October 2019

Abstract
Abstract 1000

Objective:
The use of small molecules to reactive fetal hemoglobin (HbF) expression in patients with sickle cell disease (SCD) is a promising clinical strategy. Erythroid Kruppel-like factor (KLF1) and B cell lymphoma 11A (BCL11A) are key regulatory components of the developmental switch from fetal to adult hemoglobin. Current models suggest that KLF1 positively regulates the expression of the γ-globin gene repressor BCL11A by interacting with the BCL11A promoter. Knockdown studies of KLF1 in adult erythroid progenitors are consistent with this model and result in low BCL11A levels and derepression of γ-globin gene expression. Based on the critical role of KLF1 in regulating HbF production, we performed a limited screen of small molecules for their ability to suppress KLF1 expression. We identified resveratrol, a small polyphenolic compound, as the most potent suppressor of KLF1. The following studies were performed to confirm RSV's mechanism of action.

Methods:

Cell Culture.
KU812 leukemia cells express both fetal and adult hemglobin and erythroid markers similar to days 7–14 primary erythroid cells, which makes them a useful in vitro model for testing the activity of HbF-inducing compounds. KU812 cells (ATTC) were cultured at a density of 5×105 cells/mL for 72h in the presence of vehicle or RSV (50 μM).

Flow cytometry.
Fixed KU812 cells were labeled with monoclonal anti-human γ-globin (Invitrogen) and β-globin (Santa Cruz) antibodies to determine the γ- to β-globin mean fluorescent ratio.

Western Blot.
Whole cell lysates were prepared in RIPA buffer containing protease inhibitors and quantified by BCA protein assay. Protein samples (40 μg per lane) were resolved on a 4–15% SDS-PAGE gradient gel and transferred onto a PVDF membrane. Blots were incubated on at 4°C in primary antibody (KLF1, 1:200, Santa Cruz; BCL11A, 1:1000, Novus Biologicals) followed by 1h incubation with an HRP-conjugated secondary antibody at RT. Protein bands were visualized by chemiluminescence and analyzed by densitometry using Image J software.

Transient transfections.
KU812 cells were transfected with a constitutively active KLF1 expression construct (pCMVSPORT6-KLF1) or an empty vector control (pCMV-EV) using the Amaxa 4D Nucleofector kit (Lonza). 12 h post transfection, KU812 cells (5×105 cells/mL) were incubated with vehicle or RSV (50 μM) for 72 h.

Statistical analysis.
One-Way ANOVA followed by Student-Newman-Kuels (Sigma Stat). Data are reported as the mean ± SEM. A p-value of <0.05 was considered significant.

Results:
RSV (50μM) induced a significant increase in HbF expression compared to vehicle (γ-/β-globin ratio: Veh: 7.09±0.18; RSV: 11.02±0.84; p<0.001). Parallel measurements revealed a 65% decrease of KLF1 protein expression in response to RSV (KLF1/actin ratio: Veh: 1.04±0.06; RSV: 0.39±0.007; p<0.001). Concurrently, RSV reduced BCL11A protein levels by 77% (BCL11A/actin: Veh: 1.00±0.05; RSV: 0.23±0.02; p<0.001). Overexpression of KLF1 completely abrogated the induction of HbF by RSV (γ-/β-globin ratio: pCMV-EV+vehicle 7.97±0.67; pCMV-EV+RSV (50 μM) 14.06±0.61; pCMV-KLF1+RSV (50 μM) 8.83±0.89; p<0.001).

Summary & Conclusions:
Our results demonstrate that RSV, a natural polyphenol found in grapes and red wine, is a potent inducer of HbF in KU812 cells. The mechanism of HbF induction is mediated in part through suppression of KLF1 and BCL11A expression. This view is strengthened by the fact that overexpression of a constitutively active form of KLF1 completely abrogated RSV's HbF activity. These findings support further investigations of RSV's bioactivity in primary CD34+ erythroid progenitor cells and preclinical models of SCD.

Disclosures:
No relevant conflicts of interest to declare.