Abstract
Background: Fetal hemoglobin (HbF, α2g2) induction has long been an area of investigation, as it is known to reduce the clinical complications of sickle cell disease (SCD) and beta thalassemia. Progress in identifying novel HbF inducing strategies has been stymied by an incomplete understanding of gamma-globin regulation. We used natural genetic variation to identify novel genes and pathways associated with HbF levels in patients with SCD. Our whole exome sequencing analysis of 1290 samples from patients with SCD identified the insulin signaling pathway to be related to HbF regulation. Functional studies performed in hematopoietic stem and progenitor cells (HSPCs) from patients with SCD established that FOXO3 is a positive regulator of HbF, and that metformin, a FOXO3 and AMPK activator, can induce HbF (Zhang et al, Blood 2018). We hypothesized that other proteins in the insulin signaling pathway, particularly AMPK, a direct activator of FOXO3, may contribute to HbF regulation and be a potential target for pharmacologic induction of HbF.
Objectives: We now seek to determine the role of AMPK and AMPK activators such as piceatannol in HbF regulation through functional studies in HSPCs from patients with SCD.
Methods: HSPCs from 3 unique patients with SCD were transduced with AMPK shRNA on day 5 of two phase primary erythroid culture. AMPK, FOXO3, gamma and beta globin gene expression were measured by RT-qPCR and HbF by HPLC respectively on day 14 of culture. HSPCs from 3 unique patients with SCD were treated with AICAR, piceatannol at 12.5µM and metformin at 100 µM on day 7 of erythroid culture. Cell lysate was collected on day 14, and AMPK, FOXO3, gamma and beta globin gene expression and protein levels measured by RT-qPCR and western blot respectively. Levels of pAMPK, at Thr172, were quantified by western blot. 1 µM Compound C was added with piceatannol and with metformin in separate erythroid cultures on day 7, and the effect on gamma globin and phosphorylation of AMPK at Thr172 was measured on day 14 by RT-qPCR and western blot respectively.
Results: 70% knockdown of AMPK resulted in a 50% decrease in HbF (p<0.01) and a three-fold reduction in gamma-globin expression (p<0.001). HSPCs treated with metformin or piceatannol exhibited a 2-3 fold rise in AMPK, FOXO3 and gamma globin gene expression (p<0.001). HSPCs treated with piceatannol and metformin showed an increase in pAMPK at Thr172, the activated form of AMPK. In the presence of a specific AMPK inhibitor, Compound C, metformin and piceatannol, no induction of gamma globin was observed (Figure 1), and pAMPK was reduced to untreated levels.
Conclusions: Knockdown of AMPK in HSPCs reduces gamma globin expression and %HbF, supporting the role of AMPK in gamma globin regulation. Drugs known to activate AMPK, metformin and piceatannol, increase gamma globin in SCD patient derived HSPCs. Pharmacologic blockage of AMPK activity with Compound C results in reduction of HbF induction, and reduces the gamma globin induction of metformin and piceatannol to untreated levels. We therefore conclude that AMPK is a positive regulator of HbF, and that pharmacologic induction of HbF with metformin and piceatannol requires AMPK activity. Further work is needed to establish if FOXO3 and AMPK alone are instrumental in HbF regulation, or if other proteins in the insulin signaling pathway may play a role in HbF regulation.
No relevant conflicts of interest to declare.
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