A Correlation of Ineffective Erythropoiesis and Dysregulated Pathways in Myelodysplastic Syndromes

Background: Over 90% of patients with myelodysplastic syndromes (MDS) exhibit symptoms of anemia at diagnosis, often after an extended latency period. One of the main causes of anemia is ineffective erythropoiesis, a principal pathophysiological feature of MDS. Our previous studies indicated that the underlying mechanisms of ineffective erythropoiesis include abnormal iron metabolism, premature aging and apoptosis during erythroid differentiation. In this study, the NUP98-HOXD13 (NHD13) transgenic mice model was used to explore signaling pathway alterations from preclinical to severe symptomatic stages of MDS.

Methods: We retrospectively analyzed 783 newly diagnosed primary MDS patients from 2016 to 2023 with pre-diagnosed blood counts. The NHD13 mouse model was used to investigate dynamic changes in signaling pathways related to ineffective erythropoiesis in MDS.

Results: Over a prolonged period before definitive diagnosis, MDS patients gradually experienced slow reductions in HGB levels and increases in MCV. A consistent annual decline in HGB levels, at a rate of 2.78g/L, was observed. In contrast, MCV tended to increase at 0.83fL/year.

Due to the difficulty in obtaining samples from MDS patients before diagnosis, we utilized NHD13 mouse model to mimic human MDS. The peripheral blood counts of NHD13 mice showed similarities to MDS patients, with an increase in MCV followed by a decrease in HB levels as the disease progressed. Based on the severity of anemia and changes in MCV, four time points were selected (6-week without anemia, 12-week with mild anemia, 16-week with obvious anemia, and 20-week with severe macrocytic anemia), corresponding to the pre-disease, early, intermediate and late stages of MDS patients.

We compared the phenotype of NHD13 and WT mice at four time points. At 16-week, NHD13 mice showed a significant reduction in erythroid-committed progenitors (Lineage^-cKit⁺Sca1⁻CD34⁻Fcγ⁻CD71⁺) (P=0.0038). Flow cytometric analysis revealed increased Stage II erythroblasts (P=0.0151) and decreased Stage VI erythrocytes (P=0.0062) in NHD13 mice at 16-week. Additionally, NHD13 mice exhibited increased early-stage and terminal erythroid cells in the spleen, indicating extramedullary hematopoiesis.

To investigate mechanisms underlying signaling pathway alterations associated with the exacerbation of ineffective erythropoiesis in MDS, we collected bulk RNA-sequencing data from erythroid progenitors of NHD13 and WT mice at four time points. GSVA and GSEAbase methods were used to calculate pathway activity scores for each sample across these time points.

It was revealed that the ferroptosis signaling pathway was highly activated in the pre-disease stage but decreased during MDS progression. Cellular senescence and the cell cycle signaling pathways were upregulated during the early stage but decreased in the later phase. In contrast, apoptosis, pyroptosis and inflammasomes signaling pathways were significantly upregulated in the late stage of the disease. Autophagy, mitophagy and immune deficiency pathways remained stable throughout the disease.

Glutathione peroxidase 4 (GPX4) plays a crucial role in preventing ferroptosis, while nuclear receptor coactivator 4 (NCOA4) mediates the autophagic degradation of ferritin, both of which are essential for maintaining iron homeostasis. Concurring with the ferroptosis pathway alterations described above, the expression of GPX4 decreases and NCOA4 increases in erythroid-committed progenitors from NHD13 mice during the early stages. Furthermore, compared to WT mice, transmission electron microscopy analysis showed ferroptosis-specific changes of mitochondria in erythroid cells from NHD13 mice during the early stage of the disease, including mitochondrial iron deposition, mitochondrial swelling and enhanced membrane density. In the later stage, there was an increase in apoptosis among erythroid cells in NHD13 mice.

Conclusions: Our results demonstrated dynamic changes in signaling pathways related to ineffective erythropoiesis during the progression of MDS in animal models. Specifically, ferroptosis pathway was significantly upregulated in the pre-disease stage of the disease, and the expression of senescence and cell cycle-related pathways was upregulated in the early stage. Apoptosis, pyroptosis and inflammasome signaling pathways were activated in the late stage.