Co-Induction of ULK-1 and AHSP mRNAs in Erythroid Precursor Cells Isolated From a Sirolimus-Treated β-Thalassemia Patient: A Case Report Study

Abstract

Introduction: The β-thalassemias are inherited genetic disorders affecting the hematopoietic system and caused by mutations of the adult β-globin gene, leading to low or absent production of adult hemoglobin. In addition, an excess of free α-globin is associated with ineffective erythropoiesis. In fact, the free α-globin molecules are prone to precipitate, causing toxicity to the erythroid cells, and interference with red cell maturation. In order to counteract the detrimental effects of the excess of α-globin, two pathways might be activated in β-thalassemia erythroid cells, i.e. Unc-51-like kinase 1 (Ulk-1)-mediated induction of autophagy and increased expression of the α-hemoglobin stabilizing protein (AHSP).

Case presentation: The studied case was a male transfusion dependent TM (Thalassemia Major) patient, aged 43 years, with a β⁰39/β⁺IVSI-110 genotype (XmnI polymorphism: -/-), starting the first blood transfusion when he was 5 months old, and participating to the NCT03877809 (Sirthalaclin) clinical trial.

Methods: Expression of AHSP and Ulk-genes in Erythroid precursor cells (ErPCs) was studied by Reverse transcription (RT)-qPCR and Western blotting ErPCs were isolated from the propositus after 90 and 180 days of treatment with sirolimus.

Results and discussion: This study demonstrates for the first time that increase in the production of γ-globin2 mRNA and HbF in ErPCs from a patient with β-thalassemia treated with sirolimus might be associated with co-induction of Ulk-1 and AHSP genes.

Keywords: AHSP; Ulk-1; autophagy; sirolimus; β-thalassemia.

FIGURE 1

Expression of AHSP (A–C) and Ulk-1 (D) genes in ErPCs isolated from the propositus after 90 and 180 days of treatment with sirolimus as elsewhere reported [21]. (A,B) Western blotting analysis. Autoradiograms (the uncut version of the gels is shown in Supplementary Figure S1) are shown in panel (A); the densitometric analysis is shown in panel (B). (C,D) RT-qPCR analysis showing the relative content of AHSP (C) and Ulk-1 (D) mRNAs (internal control: GAPDH). The protocols and antibodies used for the Western blotting shown in (A) have been reported in Zurlo et al. (2024) [19]. The protocols and PCR primers for the RT-qPCR analyses shown in (C,D) have been reported in Zurlo et al. (2023) [15] (for Ulk-1 mRNA) and Zurlo et al. (2024) [19] (for AHSP mRNA).

FIGURE 2

Pictorial representation of the proposed mechanism of action of sirolimus (rapamycin), based on the results of the present Case Report Study. Sirolimus induces an increase in the expression of γ-globin genes (Supplementary Table S1) and an increased HbF production [21]. Furthermore, sirolimus induced an increase in the expression of the AHSP gene (Figures 1A–C), possibly leading to stabilization of free α-globin and an increase in the expression of the Ulk-1 gene (Figure 1D), possibly leading to the induction of autophagy. Induction of fetal hemoglobin and autophagy co-operate in reducing excess free α-globin. AHSP-dependent stabilization of free α-globin and Ulk-1/autophagy-dependent reduction of excess free α-globin might contribute to the reduction of ineffective erythropoiesis.