Increased Expression of α-Hemoglobin Stabilizing Protein (AHSP) mRNA in Erythroid Precursor Cells Isolated from β-Thalassemia Patients Treated with Sirolimus (Rapamycin)

Abstract

Background/Objectives: in β-thalassemia, important clinical complications are caused by the presence of free α-globin chains in the erythroid cells of β-thalassemia patients. These free α-globin chains are present in excess as a result of the lack of β-globin chains to bind with; they tend to aggregate and precipitate, causing deleterious effects and overall cytotoxicity, maturation arrest of the erythroid cells and, ultimately, ineffective erythropoiesis. The chaperone protein α-hemoglobin-stabilizing protein (AHSP) reversibly binds with free α-globin; the resulting AHSP-αHb complex prevents aggregation and precipitation. Sirolimus (rapamycin) has been previously demonstrated to induce expression of fetal hemoglobin and decrease the excess of free α-globin chain in the erythroid cells of β-thalassemia patients. The objective of this study was to verify whether sirolimus is also able to upregulate AHSP expression in erythroid precursor cells (ErPCs) isolated from β-thalassemia patients. Methods: the expression of AHSP genes was analyzed by measuring the AHSP mRNA content by real-time quantitative PCR (RT-qPCR) and the AHSP protein production by Western blotting. Results: AHSP gene expression was found to be higher in ErPCs of β-thalassemia patients in comparison to ErPCs isolated from healthy subjects. In addition, AHSP expression was further induced by treatment of β-thalassemia ErPCs with sirolimus. Finally, AHSP mRNA was expressed at an increased level in ErPCs of sirolimus-treated β-thalassemia patients participating in the NCT03877809 Sirthalaclin clinical trial. Conclusions: this exploratory study suggests that AHSP expression should be considered as an endpoint in clinical trials based on sirolimus.

Keywords: alpha-hemoglobin stabilizing protein; erythroid precursor cells (ErPCs); rapamycin; sirolimus; β-thalassemia.

Figure 1

Expression of AHSP in Erythroid Precursor Cells (ErPCs) isolated from healthy subjects and β-thalassemia patients. (A). RT-qPCR analysis showing the relative content of AHSP mRNA (GAPDH was chosen as internal control). (B,C). Western blotting analysis. Autoradiograms are shown in panel (B) (the uncut version of the gels is shown in Supplementary Figure S2), the densitometric quantification of obtained bands is shown in panel (C). * = p < 0.05 (statistically significant); ** = p < 0.01 (statistically highly significant); unpaired t-test was employed.

Figure 2

(A). Effect of sirolimus treatment on AHSP mRNA accumulation in ErPCs isolated from three β-thalassemia patients. (B). GAPDH gene expression normalized on β-actin, showing no difference following sirolimus treatment. The quantification of the AHSP mRNA was performed using β-actin as internal reference sequence, as elsewhere described. The genotypes of the patients were β⁺IVSI-6/β⁺IVSI-6, β⁰ 39/β⁰ 39, β⁺IVSI-110/β⁺IVSI-110. ** = p < 0.01 (statistically highly significant). Data and relative analyses were generated using the Prism Software v9.02 and paired t-test.

Figure 3

Scheme of this study, based on the Sirthalaclin NCT03877809 clinical trial. Modified from Zuccato et al. [43].

Figure 4

AHSP RNA content in ErPCs isolated from sirolimus-treated β-thalassemia patients participating to the Sirthalaclin (NCT03877809) clinical trial. The genotypes of the recruited patients have already been reported by Zuccato et al. [45]. The quantification of AHSP mRNA was performed using β-actin as internal reference sequence, as elsewhere described. * = p < 0.05 (statistically significant). Data and relative analyses were generated using the Prism Software v9.02 and paired t-test.

Figure 5

Pictorial representation of the proposed mechanism of action of sirolimus (rapamycin). Sirolimus induces increased expression of γ-globin genes and increased production of HbF (A) [35,36,43]. In addition, sirolimus induced increased expression of the ULK1 gene, leading to induction of autophagy [19,20] (B) and increased expression of the AHSP gene, leading to stabilization of the free α-globin (C) [Figure 2 and Figure 4] (C). Induction of fetal hemoglobin and autophagy cooperate in reducing the excess of free α-globin (D). AHSP-dependent stabilization of the free α-globin, and ULK1/autophagy-dependent reduction of the excess of free α-globin might concur in the reduction of ineffective erythropoiesis.