Insulin-like Growth Factor Binding Protein-3 (IGFBP3) Induces Fetal Hemoglobin in Hematopoietic Stem and Progenitor Cells from Patients with Sickle Cell Anemia
Authors: Paikari Alireza , Mi Tian , Zhang Yankai , Rampersaud Evadnie , Kang Guolian , Wu Gang , Hankins Jane S. , Estepp Jeremie H. , Sumazin Pavel , Weiss Mitchell J. , Sheehan Vivien A 
Published: October 2019

Abstract
Background: Fetal hemoglobin (HbF, α2g2) induction is known to reduce the clinical complications of sickle cell anemia (SCA). Progress in identifying novel HbF inducing strategies has been slowed by an incomplete understanding of gamma-globin regulation. We have used natural genetic variation to identify novel genes and pathways associated with HbF levels in patients with SCA, beginning with whole exome sequencing (WES). This approach identified FOXO3, a transcription factor important for insulin signaling and erythroid maturation, among other functions, as a positive regulator of HbF. We then confirmed the role of FOXO3 in HbF regulation with functional studies in erythroid culture (Zhang, Blood 2018). To overcome the limitations of WES, namely the absence of regulatory and promotor sequencing data, we performed whole genome sequencing (WGS) on 658 pediatric SCA patients, and analyzed the data for common variants predictive of HbF levels.
Methods : WGS was performed on a cohort of 658 pediatric patients with HbSS and HbSβ0 with IRB approval from Texas Children's Hospital, and from St Jude Children's Research Hospital, as part of the Sickle Cell Clinical Research and Intervention Program (Hankins et al 2018). Baseline HbF levels, not on hydroxyurea therapy, was measured by HPLC. Subjects were between 6 months and 21 years of age from both institutions; 52% were male. We used mixed linear regression models to screen for variants associated with transformed HbF values, and performed ridge regression with 10-fold cross-validation to confirm associations after adjustment for HBG and BCL11A-associated variants, age, sex, and race (determined by principal components analysis).
Hematopoietic stem and progenitor cells (HSPCs) from 3 patients (all HbSS) were treated with recombinant human IGFBP3 (1µg/ml) beginning on day 7 of two-phase culture. Effects of IGFBP3 on gamma-globin expression were evaluated by RT-qPCR, and on HbF levels by HPLC, on days 14 and 21 of culture. The effects of IGFBP3 on known modifiers of HbF and the FOXO3 pathway were assessed by RT-qPCR and western blot on day 21 of culture. Erythroid maturation was assessed by flow cytometry using anti-CD71, GPA, and Band3 on day 21 of culture.
Results: Our whole genome sequencing data identified a strong association between all 11 variants 200 kb upstream of IGFBP3 and baseline HbF levels (p<1x10-9). The association of IGFBP3 variants with HbF remained after correcting for HBG and BCL11A variants in addition to patient age, sex, and race (p<0.001). Eleven SNPs were tested, and corrected for multiple testing. Mean HbF levels for patients heterozygous for an IGFBP3 variant predicted to alter IGFBP3 levels as determined by NESDA conditional eQTL catalog (Jenson et al, 2017) were 43% higher compared to patients without a variant (t-test p<0.0002).
In erythroblasts treated with IGFBP3, gamma globin expression doubled compared to untreated (p=0.01). %HbF in IGFBP3 treated cells at day 21 of culture was the same as on day 14, while the HbF of untreated cells declined (Figure 1). IGFBP3 did not alter expression of known gamma globin regulators BCL11A, KLF1, and MYB, nor did it alter erythroid maturation as measured by flow cytometry with anti-CD71, GPA, and Band3, and morphologic examination.
Conclusions: WGS analysis identified variants in the regulatory region of IGFBP3 as associated with higher levels of HbF. Addition of exogenous human recombinant IGFBP3 to HSPCs prevented the physiologic decline of HbF, resulting in higher %HbF levels in erythroblasts, functionally confirming the association. Exogenous IGFBP3 did not arrest maturation, supporting our hypothesis that IGFBP3 affects HbF production directly via gamma globin induction, rather than through manipulation of maturation. IGFBP3 did not alter expression of known regulators of HbF (BCL11A, KLF1, and MYB). These findings are compatible with our prior data identifying FOXO3 as a positive regulator of gamma globin, and our use of metformin as a HbF inducer in an ongoing clinical trial. Metformin increases IGFBP3 levels, and it has been shown that increases in IGFBP3 lead to activation of FOXO3 through the insulin signaling pathway. We therefore conclude that the insulin signaling pathway plays a significant role in gamma-globin regulation and is an important therapeutic target for HbF induction in patients with SCA.

Disclosures
Hankins: Global Blood Therapeutics: Research Funding; bluebird bio: Consultancy; Novartis: Research Funding; NCQA: Consultancy. Estepp:Global Blood Therapeutics: Consultancy, Research Funding; NHLBI: Research Funding; Daiichi Sankyo: Consultancy; ASH Scholar: Research Funding.