Hydroxyurea responsiveness in β-thalassemic patients is determined by the stress response adaptation of erythroid progenitors and their differentiation propensity

Abstract

β-thalassemia is caused by mutations in the β-globin locus resulting in loss of, or reduced, hemoglobin A (adult hemoglobin, HbA, α2β2) production. Hydroxyurea treatment increases fetal γ-globin (fetal hemoglobin, HbF, α2γ2) expression in postnatal life substituting for the missing adult β-globin and is, therefore, an attractive therapeutic approach. Patients treated with hydroxyurea fall into three categories: i) 'responders' who increase hemoglobin to therapeutic levels; (ii) 'moderate-responders' who increase hemoglobin levels but still need transfusions at longer intervals; and (iii) 'non-responders' who do not reach adequate hemoglobin levels and remain transfusion-dependent. The mechanisms underlying these differential responses remain largely unclear. We generated RNA expression profiles from erythroblast progenitors of 8 responder and 8 non-responder β-thalassemia patients. These profiles revealed that hydroxyurea treatment induced differential expression of many genes in cells from non-responders while it had little impact on cells from responders. Part of the gene program up-regulated by hydroxyurea in non-responders was already highly expressed in responders before hydroxyurea treatment. Baseline HbF expression was low in non-responders, and hydroxyurea treatment induced significant cell death. We conclude that cells from responders have adapted well to constitutive stress conditions and display a propensity to proceed to the erythroid differentiation program.

Figure 1.

Erythroblasts derived from responders are less sensitive to HU treatment and express higher HbF at baseline. NR-HEPs (A) grow faster than R-HEPs (B). After HU treatment, growth curves of NR-HEPs (A) decline more than those of R-HEPs (B). (C) HU sensitivity curves for NR-HEPs and R-HEPs, normalized by taking the ratio of cell proliferation of HU-treated cells over non-treated cells. (D) Hemoglobin induction by 100 μM HU (5 days) in NR-HEPs and R-HEPs; as in (C). (A-D) P<0.05 are indicated. (E) Representative cytospins of NR-HEPs and R-HEPs treated with HU for three days. Cytospins were stained with histological dyes and neutral benzidine. Hemoglobinized cells are stained brown. Pyknotic cells are indicated by arrows.

Figure 2.

Responder HEPs constitutively express a stress-program that is induced by HU in non-responders HEPs. (A) The number of differentially expressed genes between NR-and R-HEPs before and after HU treatment. (B and C) Supervised clustering of differentially expressed genes without (B) and with (C) HU treatment. Responder: R; non-responder: NR; HU treated: +HU. (D) Functional annotation of differentially expressed genes in R- versus NR-HEPs before HU treatment. (E) Functional annotation of differentially expressed genes in R- versus NR-HEPs after HU treatment. Functional annotation for biological processes was processed by Ingenuity pathway analysis (P< 0.05).

Figure 3.

Expression dynamics of substantial numbers of differentially expressed genes in responder HEPs are towards terminal erythroid differentiation. (A) Overlap of differentially expressed genes in R-HEPs (R) versus NR-HEPs (NR), with genes involved in the erythroid differentiation program (28%; 91 of 327). (B) Clustering analysis of genes involved in the erythroid differentiation program that are also differentially expressed between R and NR-HEPs.