Simvastatin and tBHQ Act Synergistically to Increase γ-Globin Gene Expression Through the Transcription Factors KLF2 and NRF2

Abstract 2149

While increased fetal hemoglobin (HbF) levels have proven therapeutic benefit for people with sickle cell disease and β-thalassemia, none of the current HbF inducing agents have the optimal combination of safety, efficacy and ease of use that would make them applicable to most hemoglobinopathy patients. In an effort to develop new strategies for HbF induction, we have recently shown that drugs that activate the NF-E2 related factor 2 (NRF2)/antioxidant response signaling pathway stimulate HbF production in primary human erythroid cells. This discovery prompted us to investigate ways to further enhance HbF levels achieved with NRF2 activators alone. Recent reports from the cardiovascular literature have uncovered a synergy between Kruppel-like factor 2 (KLF2) and NRF2. In vascular endothelial cells, shear stress induces a battery of genes that protect against atherosclerotic cardiovascular disease and this induction is mediated by the transcription factors KLF2 and NRF2 and includes synergistic activation of NRF2 target genes by the two factors. Interestingly, HMG-CoA reductase inhibitors (statins), are strong activators of the transcription factor, KLF2. These findings suggested to us that combining statins with drugs that activate NRF2 signaling might synergistically activate γ-globin gene expression and HbF production. An additional rationale for this approach is that several NRF2 activating drugs are either already approved or undergoing clinical testing and that statins are among the most widely used drugs.

To test this hypothesis, we first treated K562 cells with various concentrations of simvastatin and observed a dose dependent increase in KLF2 mRNA and protein expression, with 5μM statin resulting in more than 200-fold increase in steady state mRNA levels but no change in γ-globin mRNA. When combined with tBHQ, 5μM statin synergistically increased γ-globin levels compared to either drug alone at 24 and 48hrs. To investigate the specificity of this synergy, we created a stable K562 cell line that overexpressed murine klf2. Treating these cells with tBHQ enhanced γ-globin expression compared to tBHQ treated WT K562 cells, reproducing the effect we saw with statin and tBHQ combination treatment in WT K562 cells. This suggests that KLF2 is responsible for the synergistic effects of statin when combined with tBHQ. To further investigate the mechanism of statin action we performed KLF2 and NRF2 ChIP studies. Statin treatment strongly increased KLF2 binding to HS2 of the β-globin LCR (30-fold over IgG) while tBHQ induced NRF2 binding to the NF-E2 region of LCR HS2 30-fold and 10-fold over IgG in K562 and in primary human erythroid cells, respectively. Binding at HS1, HS3 or HS4 was not increased for either factor. In a single experiment performed so far, combined tBHQ and statin treatment of differentiating primary human erythroid cells increased KLF2 and NRF2 target gene NQO1 mRNA. γ-globin mRNA was induced to levels equivalent to those seen with 5-azacytidine. These data provide preliminary evidence suggesting that combining NRF2 activators with widely used statins may be a safe and effective way to achieve therapeutic HbF levels in β-thalassmia and sickle cell disease patients.