Role of Glycolytic Activity in Hematopoietic Stem and Progenitor Cells in Fetal Hemoglobin Induction By Hydroxyurea
Authors: McGaskey Nyeomi , Elliot Emani C. , Sanchez Ventura Luisanna M , Flanagan Jonathan M. 
Published: December 2024

Background: High levels of fetal hemoglobin (HbF) have well-established benefits on clinical outcomes for patients with sickle cell disease. Without the protection of robust HbF levels, patients suffer tremendous morbidity and early mortality. The main drug used to induce higher levels of HbF is hydroxyurea. Individual hematological responses to hydroxyurea treatment is highly variable, with induced levels ranging from 10 to greater than 30 %HbF even for compliant patients. The cytotoxic and cytostatic effects of hydroxyurea are thought to cause periods of temporary arrest in hematopoiesis in the bone marrow and stimulate the recruitment of early erythroid progenitors able to produce more HbF. We hypothesized that screening hematopoietic stem and progenitor cells (HSPCs) isolated from whole blood could be used to predict HbF response to hydroxyurea.

Objectives: The aim of the study is to determine whether glycotic activity in HSPCs can determine hematopoietic potential and HbF induction in patients with sickle cell disease.

Methods: We will obtain clinical and laboratory data from 10 pediatric patients from ages 0-21 with severe SCD genotype (homozygous HbS and sickle-beta0 thalassemia) seen at Texas Children's Hospital (TCH). Patients were classified according to their individual response to hydroxyurea into a moderate HbF group (&lt;15% HbF) and a high HbF group (&gt;15% HbF). We examined HSPCs isolated from peripheral blood. We used Lin and CD34 antibodies, as well as propidium iodide and a fluorescent derivative of glucose 2-NBDG to detect live HSPCs that were capable of glucose uptake. We incubated a minimum of 500,000 PBMCs with the antibodies and 1.75 μg/ml 2-NBDG for 1 hour. We will use flow cytometry to compare HSPC profiles from the two groups of patients to determine if there were: a) differences in HSPC progenitor numbers; and b) increased glucose uptake in the moderate HbF group that would indicate increased glycolysis associated with diminished hematopoietic potential and reduced ability to express HbF.

Results: There was a significant difference in age and HbF levels between the moderate and high HbF groups (15.7 versus 6.4 years of age, p=0.028; 10.4 %HbF versus 23.2 %HbF, p=0.0003). There were no major differences in treatment length, hydroxyurea dose, or myelosuppression based on ANC and WBC levels between the groups. From flow cytometry analysis of HSPCs detected PBMC samples from each patient, we saw no difference in the number of Lin- CD34+ HSPC between the two groups (8.4% versus 8.7% Lin- CD34+ cells). By measuring cellular uptake of 2-NBDG, approximately 90% of CD34+ HSPCs from all patients were capable of glucose uptake. We did not see any significant difference in glucose uptake between the moderate and high HbF groups.

Future directions: Moving forward, we will work to further optimize our assay to see if specific HSPC subsets have altered glycolytic activity that impacts HbF expression. Additionally, we will examine glycolytic activity in HSPCs from individuals without sickle cell disease.