The β-thalassemias are inherited genetic disorders affecting the hematopoietic system and caused by more than 350 mutations of the adult β-globin gene [1, 2]. These genetic mutations cause low or absent production of adult hemoglobin (HbA) [1]. In addition, a clinical parameter affecting the pathophysiology of erythroid cells in β-thalassemia is the excess of free α-globin [3, 4], that is caused by the lack or absent production of the β-globin chains to bind with. This deeply affects the production of the HbA (α₂β₂) tetramer under normal physiological conditions [1]. This is clinically relevant, since the free α-globin molecules are prone to precipitate, causing toxicity to the erythroid cells, interference with red cell maturation [5, 6], and ineffective erythropoiesis, as recently reviewed [7]. Accordingly, several concurrent evidences show that decreased expression of α-globin genes is beneficial for β-thalassemia patients [8, 9]. For instance, using the CRISPR-Cas9 approach, several authors have confirmed that downregulation of α-globin is associated with a milder phenotype of β-thalassemia [10, 11]. This conclusion was also supported in transgenic mice mimicking β-thalassemia [12].

In this context, the activation of two pathways has been described in erythroid cells, able to reduce the excess of free α-globin and/or counteracting its cytotoxicity. The first pathway is the activation of Unc-51-like kinase 1 (Ulk-1) dependent autophagy, as proposed by Lechauve et al. (2019) [13] who found that loss of Ulk-1 gene in β-thalassemic mice reduces autophagic clearance of α-globin in red blood cell precursors and exacerbates the disease phenotype. Systemic treatment with the mTORC1 inhibitor rapamycin reduces α-globin precipitates and lessens the pathology symptoms in β-thalassemic mice via an Ulk1-dependent pathway [13]. Similarly, rapamycin reduces free α-globin accumulation in erythroblasts derived from CD34⁺ cells of β-thalassemic individuals [14, 15]. In agreement with these results, Zurlo et al. (2023) found that rapamycin induces autophagy and increased expression of Ulk-1 mRNA in a cohort of β-thalassemia patients treated with low dosages of sirolimus and participating to the Sirthalaclin NCT03877809 clinical trial [15]. This is important, considering that the autophagic process is able to reduce the excess of free α-globin by activation of a proteasome dependent detoxification process [13].

The second pathway is associated to the biological activity of the α-hemoglobin stabilizing protein (AHSP), a chaperone highly expressed in erythroid cells and involved in counteracting α-globin precipitation and related cytotoxicity [16–18]. In this respect, Zurlo et al. (2024) have published a study demonstrating high expression of AHSP gene in β-thalassemia [19]. Interestingly, AHSP mRNA production is increased in β-thalassemia patients treated with sirolimus and participating to the NCT 03877809 clinical trial (Sirthalaclin). Design and key results of this trial have been described elsewhere [20, 21].

No information is available about the possible co-activation of the Ulk1-and AHSP-dependent pathways in sirolimus-treated erythroid cells from β-thalassemia patients. We here present biochemical and molecular analyses of a β-thalassemia patient to determine whether expression of Ulk-1 and AHSP genes can be co-activated.

The studied case was a male transfusion dependent TM (Thalassemia Major) patient, aged 43 years, with a β⁰39/β⁺IVSI-110 genotype, starting the first blood transfusion when he was 5 months old, and participating to the NCT03877809 (Sirthalaclin) clinical trial. Clinical information regarding the patient is reported in Table 1; information on the treatment with sirolimus (1 mg/day) can be found in Zuccato et al. [21].

Information on biochemical and molecular parameters following sirolimus treatment is reported in Supplementary Table S1. Accordingly with previously reported results [21], increase of γ-globin mRNA (and HbF production) occurred in ErPC isolated from this patient after 90–180 days of treatment with sirolimus. This was associated with a decrease in free α-globin chains [21], and levels of bilirubin, soluble transferrin receptor and ferritin (Supplementary Table S1). Transfusion demand evaluated at the end of treatment (360 days) decreased by 12.48% (for information on the employed methodology see Zuccato et al., 2021) [21]. The range of sirolimus accumulated in blood was 1.5 (V6, 90 days of treatment) and 4.6 (V8, 180 days of treatment) pg/mL; no major side effects and no alteration of the immunophenotype were noted, according with elsewhere reported results [21].

In the Erythroid Precursor Cells (ErPcs) isolated from this patient, the expression of Ulk-1 was higher than 10 fold after 90 days treatment with 1 mg/day sirolimus, compared with Ulk-1 expression of ErPCs isolated from the same patient before the initial treatment with sirolimus and similar to ErPCs from other patients participating to the NCT03877809 clinical trial [15, 21]. In order to verify whether the AHSP gene was upregulated in these ErPCs (upregulating Ulk-1) [15], the expression of AHSP was evaluated by RT-qPCR and by Western blotting. The results are shown in Figure 1.

The results presented in Figure 1 demonstrate that ErPCs isolated at V6 (after 90 days of in vivo treatment with sirolimus) and V8 (after 180 days of in vivo treatment with sirolimus) accumulate a much larger amount of AHSP protein with respect to V2 ErPCs (Figures 1A,B). These data are completely in agreement with the RT-qPCR data shown in Figure 1C, that indicates that the AHSP RNA content at V6 and V8 is 20-fold and 48-fold higher compared to V2, respectively. The data concerning the Ulk-1 mRNA expression are shown in Figure 1D, that confirms that the Ulk-1 mRNA content at V6 and V8 is 16-fold and 18-fold higher with respect to V2, respectively. These data were reproducibly confirmed when different housekeeping sequences (β-actin, GAPDH, RPL13A) were employed as internal controls in the RT-qPCR analyses. This study demonstrates that increase in the production of γ-globin mRNA and HbF in erythroid precursor cells (ErPCs) from patients with β-thalassemia treated with sirolimus [21] might be associated with co-induction of Ulk-1 and AHSP genes.

Accordingly, sirolimus decreases the excess free α-globin [19, 21]. Therefore, the activity of sirolimus in vivo could occur through the induction of HbF and γ-globin genes, the activation of autophagy, associated with the upregulation of Ulk-1, the upregulation of AHSP, and the decrease in excess α-globin and inefficient erythropoiesis (as summarized in Figure 2).

The relevance of the results of this Case Report Study is that the novel data presented support the conclusion that the expression of Ulk-1 and AHSP can be co-upregulated by sirolimus in ErPCs from β-thalassemia patients.

A limitation of the data presented in this Case Report, is that they originate from a single-patient study; therefore, our pilot study needs validation in larger cohorts of β-thalassemia patients. Accordingly, our results are expected to stimulate RT-qPCR analysis on ErPCs isolated from the other patients participating to the Sirthalaclin NCT03877809 and Thala-Rap NCT 04247750 clinical trials [15, 20, 21]. Our results support the concept that increased HbF and γ-globin mRNA content, expression of Ulk-1 and autophagy, increased AHSP gene expression should be considered as key end points for future clinical studies.

Finally, our study might stimulate research efforts focusing on the molecular basis of the co-expression of the Ulk-1 and AHSP genes. In this context, a very interesting possibility for autophagy activation in erythroid cells is based on the modulation of GATA-1, a master regulator of erythropoiesis, also regulating autophagy [22–24]. In this respect, Kang et al. (2012) were able to demonstrate, using Chromatin Immunoprecipitation (ChIP) assays, that GATA-1 directly interacts with regulatory sites of many autophagy genes [24]; these molecular approaches demonstrated that GATA-1 regulates autophagy [24]. Finally, GATA-1 regulates the expression of AHSP gene [25]. Further studies are required to verify this hypothesis in β-thalassemia erythroid cells.