Metformin Induction of Fetal Hemoglobin

Background: Elevated fetal hemoglobin (HbF, α2γ2) ameliorates the pathophysiology of sickle cell disease (SCD) by diluting HbS and inhibiting its polymerization in patient red blood cells. We identified FOXO3, a potentially "druggable" transcription factor, as a candidate HbF inducer through analysis of rare variants from whole exome sequence data from SCD patients. We then treated hematopoietic stem and progenitor cells (HSPCs) derived from patients with SCD with metformin, an FDA-approved drug used for type 2 diabetes, which is known to activate FOXO3. Metformin significantly increased HbF alone, and additively with hydroxyurea.

Objective: Determine the mechanism by which metformin induces HbF in SCD patient derived HSPCs, and report preliminary clinical trial results of metformin in patients with SCD.

Methods: HSPCs from patients with SCD were obtained from discarded red blood cell exchange product. 100 µM Metformin was added on day 7 of two phase primary erythroid culture, and HPLC analysis of hemoglobins or RNA extraction and RT-qPCR or RNASeq was performed on day 14 of culture. BCL11A, pAKT and pAMPK were quantified by Western blot, performed in triplicate.

Patients with HbSS between the ages of 16 and 40 who produced less than 20% HbF at maximum tolerated dose of hydroxyurea or were not taking hydroxyurea were recruited to participate in an IRB approved pilot clinical trial of metformin in patients with hemoglobinopathies (Fetal Hemoglobin Induction with Metformin, NCT02981329). RNA was collected from CD45+ can CD71+ cells collected from their peripheral blood at study entry and after 3 months at 1500 mg metformin, and analyzed by RNASeq.

Results: Metformin significantly increased HbF production in 80% of the HSPCs derived from unique SCD patients, in contrast with hydroxyurea, which failed to increase HbF in 33% of patient derived HSPCs. When FOXO3 is knocked out in HSPCs, metformin no longer induces HbF. Expression of known gamma globin regulators BCL11A, KLF1, and MYB are not altered by metformin treatment of patient derived HSPCs, nor are they altered by metformin treatment in patient derived RNA from CD45+ or CD71+ cells. Metformin treatment of SCD patient derived HSPCs causes FOXO3 to accumulate in the nucleus, reduces the active, phosphorylated form of AKT, a FOXO3 inhibitor, and increases the active, phosphorylated form of AMPK, a FOXO3 activator. Our clinical trial is ongoing, has enrolled 10 patients to date; results will be presented at the American Society of Hematology meeting.

Conclusions: Metformin induction of HbF in SCD patient derived HSPCs requires FOXO3 expression, and affects the activity of known FOXO3 activator AMPK and FOXO3 inhibitor AKT. Metformin does not alter expression levels of known regulators of HbF either in treated HSPCs from patients with SCD or in samples from clinical trial subjects treated with metformin compared to their trial entry samples. We conclude that metformin induces HbF through the FOXO3-AMPK-AKT pathway, and may be a viable pharmacologic HbF inducer alone or in combination with hydroxyurea.