Objectives
Myelodysplastic syndrome (MDS) is a highly heterogeneous clonal hematopoietic disorder, characterized by pathological hematopoiesis, peripheral cytopenia and dysplasia of hematopoietic stem and progenitor cells (HSPCs). MDS may malignantly transform into acute myeloid leukemia (AML) in later stage. In recent years, studies have gradually revealed the roles of epigenetic dysregulation, immune imbalance and cytogenetic variations in MDS. Among these factors, cytogenetic abnormalities not only contribute to disease progression but also serve as key prognostic indicators with significant clinical value in MDS diagnosis and treatment. Trisomy 8 is the most common karyotypic abnormality in MDS and may influence the 3D chromatin organization within HSPCs, leading to functional alterations in the genome and changes in chromatin epigenetic states. But the extent to which trisomy 8 drives 3D chromatin structural changes remains unclear. This study aims to investigate the pathogenic mechanisms of trisomy 8 in MDS by examining the relationship between karyotypic abnormalities, 3D chromatin structural alterations, gene expression changes and HSPC dysfunction. We will analyze the impact of trisomy 8 on HSPC function, mitochondrial metabolic homeostasis and 3D genome spatial organization.
Methods
We first reprogrammed bone marrow mononuclear cells (BMMNCs) from two MDS patients to generate induced pluripotent stem cell (iPSC) by Yamanaka factors in vitro. The pluripotent of iPSC was verified through FACS and immunofluorescence. Next，we induced iPSC with different karyotypes to hematopoietic differentiation to form embryoid bodies and assessed the proportion of HSPC markers, CFU ability, engraftment ability and myeloid/erythroid differentiation capacity. Mitochondrial function was assessed by measuring mitochondrial membrane potential, ATP level and ROS level. Apoptosis-related markers were analyzed using FACS to assess the impact of mitochondrial dysfunction on apoptosis. We also employed Hi-C to analyze chromatin conformation and genome interactions in iHSPCs, while RNA-seq was used to examine the effects of trisomy 8 on gene expression patterns and functional pathways. The integration of these multi-omics data facilitated the systematic analysis of how trisomy 8 influences 3D chromatin structure and its molecular regulatory network, ultimately constructing a landscape of transcriptomic and chromatin structural.
Results
1.MDS patients (MDS1 and MDS2) exhibited varying degrees of abnormal leukocyte，hemoglobin and reticulocyte. Chromosomal analysis revealed that MDS1 had a karyotype of 47,XX,+8[20], while MDS2 had a mosaic karyotype of 47,XX,+8[16]/46,XX[4]. Both patients had moderate and low IPSS-R scores, suggesting a strong correlation between trisomy 8 and MDS pathogenesis.
2.BMMNCs were isolated using Ficoll separation and iPSC models were established through Yamanaka factor-mediated reprogramming. Flow cytometry was used to obtain iPSC clones with normal karyotype (MDS-nk) and trisomy 8 karyotype (MDS-tr8) and karyotypic stability was confirmed by G-banding and FISH analysis. Flow cytometry and immunofluorescence staining confirmed the pluripotency of all patient-derived iPSC lines.
3.Upon hematopoietic differentiation, iHSPCs derived from MDS-tr8 exhibited significantly reduced expression of surface markers CD31, CD34, CD43 and CD45 compared to MDS-nk. Additionally, colony-forming ability, engraftment potential and myeloid/erythroid differentiation capacity were all impaired, indicating that trisomy 8 disrupts HSPC development and hematopoietic function.
4.Trisomy 8 led to a decrease in mitochondrial membrane potential in iPSCs, while ATP levels remained unchanged, but calcium homeostasis was disrupted. ROS levels were significantly elevated，resulting in oxidative stress. FACS analysis showed increased expression of apoptosis markers Fas and Caspase-3 in MDS-tr8，indicating that mitochondrial dysfunction promotes apoptosis.
5.Integrated Hi-C and RNA-seq revealed that trisomy 8 increased cis-chromosomal interactions and altered inter-chromosomal interaction patterns, particularly enhancing interactions on chromosome 8. We also observed that structural changes in A/B compartments and topologically associating domains (TAD). Additionally, transcriptomic profiling showed altered gene expression associated with oncogenic signaling pathways, suggesting that trisomy 8 may contribute to MDS progression by altering 3D genome architecture and activating oncogenic pathways.
Conclusions
This study is the first to integrate iPSC models with multi-omics approaches to comprehensively elucidate the pathogenic mechanisms of trisomy 8 in MDS. We uncovered the synergistic effects of 3D genome reorganization and mitochondrial metabolic dysregulation in MDS pathogenic. We summarized several mechanisms by which trisomy 8 drives the pathogenic process of MDS as follows:
1.Hematopoietic function: Trisomy 8 impairs HSPC differentiation and self-renewal, leading to ineffective hematopoiesis and peripheral cytopenia.
2.Metabolic regulation: Mitochondrial membrane potential collapse, ROS accumulation and metabolic imbalance trigger apoptotic signaling, exacerbating bone marrow failure.
3.Genomic architecture: 3D genome reorganization, including TAD compaction and A/B compartment switching, reshapes chromatin accessibility and activates oncogenes on chromosome 8.
4.Molecular network: Changes in gene expression patterns, particularly in CTHRC1, ENPP2 and CCN3, promoting clonal evolution and disease progression.
