Metformin induces FOXO3-dependent fetal hemoglobin production in human primary erythroid cells

Abstract

Induction of red blood cell (RBC) fetal hemoglobin (HbF; α2γ2) ameliorates the pathophysiology of sickle cell disease (SCD) by reducing the concentration of sickle hemoglobin (HbS; α2β^S2) to inhibit its polymerization. Hydroxyurea (HU), the only US Food and Drug Administration (FDA)-approved drug for SCD, acts in part by inducing HbF; however, it is not fully effective, reflecting the need for new therapies. Whole-exome sequence analysis of rare genetic variants in SCD patients identified FOXO3 as a candidate regulator of RBC HbF. We validated these genomic findings through loss- and gain-of-function studies in normal human CD34⁺ hematopoietic stem and progenitor cells induced to undergo erythroid differentiation. FOXO3 gene silencing reduced γ-globin RNA levels and HbF levels in erythroblasts, whereas overexpression of FOXO3 produced the opposite effect. Moreover, treatment of primary CD34⁺ cell-derived erythroid cultures with metformin, an FDA-approved drug known to enhance FOXO3 activity in nonerythroid cells, caused dose-related FOXO3-dependent increases in the percentage of HbF protein and the fraction of HbF-immunostaining cells (F cells). Combined HU and metformin treatment induced HbF additively and reversed the arrest in erythroid maturation caused by HU treatment alone. HbF induction by metformin in erythroid precursors was dependent on FOXO3 expression and did not alter expression of BCL11A, MYB, or KLF1. Collectively, our data implicate FOXO3 as a positive regulator of γ-globin expression and identify metformin as a potential therapeutic agent for SCD.

Graphical abstract

Figure 1.

Knockdown of FOXO3 reduces γ-globin expression and protein levels relative to β-globin, and overexpression of FOXO3 increases γ-globin expression and protein levels relative to β-globin. (A) An 80% knockdown of FOXO3 is achieved. (B) Effects of FOXO3 knockdown on γ-globin and β-globin, as measured by RT-qPCR (3 independent assays). (C) Western blot depicting FOXO3 knockdown and its effect on γ-globin and β-globin protein levels. (D) Western blot depicting effects of FOXO3 overexpression on γ-globin and β-globin. (E) Quantification of western blot (3 independent assays). Error bar represents SD. ***P < .0001, **P < .005, *P < .05. NS, not significant.

Figure 2.

Treatment of HSPCs with metformin (Met) increases FOXO3 expression, increases the active form of FOXO3, and causes FOXO3 to accumulate in the nucleus. (A) Effect of Met treatment on FOXO3 expression, as measured by RT-qPCR. HSPCs derived from patients with SCD were treated on day 7 of culture and analyzed on day 14. (B) Met treatment increases the active form of FOXO3 activator AMPK (Thr172 phosphorylated AMPK) and the active nuclear-localizing form of FOXO3 (Ser413 p-FOXO3) and decreases the active form of the FOXO3 inhibitor, AKT (Ser473 phosphorylated AKT), and the cytoplasmic form of FOXO3 (Ser253 p-FOXO3). HSPCs derived from patients with SCD were treated on day 13 of culture and analyzed 24 hours later on day 14. (C) Aggregate of 3 individual western blots from 3 experiments with unique CD34⁺ donors with SCD, quantified. (D) Met treatment causes FOXO3 to accumulate in the nucleus. HSPCs from normal donors were treated with various concentrations of Met for 24 hours, collected, separated into nuclear and cytoplasmic fractions, and analyzed by western blot. Error bar represents SD. ***P < .0001, **P < .005, *P < .01. N.S, not significant.

Figure 3.

Metformin (Met) increases the number of HbF-expressing cells in HSPCs and prevents sickling under hypoxic conditions. (A) CD34⁺ cells from normal individuals were cultured under conditions to promote erythroid maturation. The indicated drugs were added at day 7 and samples were collected on day 14. HbF-immunostained cells (F cells) were measured by flow cytometry at day 14. (B) Aggregate of 3 independent assays. (C) CD34⁺ cells from a patient with SCD, treated with the indicated drugs from days 7 to 28. On day 28, cells were placed in 2% O₂ for 4 hours, fixed, and stained with Wright/Giemsa; scale bars, 10 µm. Error bar represents SD from 3 experiments using CD34⁺ cells from 3 patients with SCD. ***P < .0001, **P < .005, *P < .01.

Figure 4.

Metformin (Met) and HU additively induce HbF in HSPCs from normal individuals and from patients with SCD. (A) HSPCs from a normal individual were treated with the indicated drugs on day 7 of culture and analyzed at day 14. HbA and HbF were measured by HPLC; representative graphs are shown. (B) Aggregate of results of drug treatment of HSPCs from normal individuals on HbF (%). (C) HSPCs from an individual with SCD were treated with the indicated drugs on day 7 of culture and analyzed at day 14. HbA and HbF were measured by HPLC; representative graphs are shown. (D) Aggregate of results of drug treatment of HSPCs from individuals with SCD on HbF (%), calculated as (HbF/HbF+HbA) * 100. Error bar represents SD. ***P < .0001, *P < .05.

Figure 5.

Treatment of primary erythroid culture with metformin (Met) does not arrest erythroid maturation. Normal CD34⁺ cells were cultured under conditions to promote erythroid maturation, and the indicated drugs were added at day 7. (A) Cells were collected and analyzed by FACS at day 14 for band 3 positivity. (B) Cells were collected and analyzed by FACS at day 14 for CD71/GPA positivity. (C) Aggregate of results from 3 unique SCD donors. (D) Cells were fixed and stained with Wright/Giemsa; scale bars, 10 µM. **P < .005, *P < .05. N.S, not significant.

Figure 6.

Metformin (Met) induces γ-globin protein and RNA in a FOXO3-dependent manner. (A) The effect of Met treatment on HbF in HSPCs with FOXO3 knocked out using CRISPR/Cas9, as determined by representative HPLC. (B) Three aggregate experiments. (C) Western blot showing the effect of CRISPR/Cas9 editing on FOXO3 protein. (D) Quantification of the effect of CRISPR/Cas9 editing on FOXO3 expression. Error bar represents SD. ***P < .0001, **P < .001. KO, knockout; N.S, not significant.

Figure 7.

Metformin (Met) does not act through known modifiers of HbF. (A) Treatment with Met does not alter expression levels of BCL11A, KLF1, or MYB. HSPCs from 3 patients with SCD were treated with Met (0 or 100 µM), and expression levels were analyzed by RT-qPCR. (B) Effect of Met treatment on BCL11A, as shown in a representative western blot. (C) Aggregate of 3 independent assays, with quantification of the BCL11A XL band. Error bar represents SD. ****P < .00001, *P < .01. N.S, not significant.