Zrsr2 and Tet2 cooperation drives myelodysplastic neoplasms: functional and molecular insights into splicing and epigenetic crosstalk

Myelodysplastic neoplasms (MDS) are a heterogeneous group of clonal hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis, peripheral blood cytopenias, and dysplastic myeloid cell morphology in both blood and bone marrow. These clinical features reflect underlying genetic alterations, including chromosomal abnormalities and somatic mutations. Among the most frequent genetic lesions in MDS are mutations in RNA splicing factors and epigenetic regulators. Approximately 25% of patients harbor concurrent mutations in these pathways, suggesting cooperative roles in disease pathogenesis. In particular, mutations in the splicing factor ZRSR2 frequently co-occur with alterations in the epigenetic regulator TET2, although the functional impact of this interaction remains poorly understood. To investigate this, we generated Zrsr2 and Tet2 mutant mice using CRISPR/Cas9 and conducted a comprehensive hematopoietic characterization. This included analyses of peripheral blood (PB), bone marrow (BM), and spleen cells, complete blood counts, PB and BM cytomorphology, spleen histology, and immunophenotyping of BM hematopoietic stem and progenitor cells (HSPCs), myeloerythroid progenitors, B cells, and additional populations from PB and spleen. An aging cohort (~24 months) was also assessed to evaluate age-related hematopoietic changes. HSPC function was evaluated in vitro using colony-forming unit (CFU) assays and in vivo through transplantation experiments with both sorted HSPCs and unfractionated BM. To elucidate the underlying mechanisms, we performed transcriptome and DNA methylation profiling, identifying alterations in gene expression, splicing, and epigenetic regulation. These findings were validated using complementary techniques such as Western blotting, RT-PCR, and others. As part of a collaborative project in Krakow, Poland, we further refined HSC immunophenotyping using the novel surface marker Neogenin-1 (Neo-1). In addition, a novel therapeutic candidate based on CDK8/CD19 inhibition was evaluated in vitro using this disease model. Our findings demonstrate that the cooperation between Zrsr2 and Tet2 mutations disrupts HSPC homeostasis by expanding LT-HSC and MPP2 populations, and impairs myeloerythroid differentiation, ultimately driving MDS development. Although only a subset of young adult mice (25%) exhibited disease features, aging markedly exacerbated the phenotype: up to 86% of aged double-mutant mice developed peripheral cytopenias, particularly thrombocytopenia and anemia, along with multilineage dysplasia and splenomegaly with extramedullary hematopoiesis. Notably, 16% of the animals progressed to secondary acute myeloid leukemia (sAML), underscoring the role of aging in disease evolution. Transplantation experiments confirmed that these hematopoietic defects are cell-autonomous and associated with impaired HSC engraftment. Transcriptomic and splicing analyses revealed dysregulation of key pathways, including MAPK signaling and the Fanconi anemia pathway, implicating inflammatory dysregulation and defective DNA repair. Consistent with this, we detected elevated levels of pro-inflammatory cytokines in the plasma of mice displaying MDS features. Moreover, splicing alterations in several MAPK components converged on MAPK14, a key mediator of inflammatory responses and definitive erythropoiesis in mice. Preliminary in vitro experiments also suggest that CDK8/CDK19 inhibition may provide therapeutic benefit by modulating proliferation and promoting erythroid differentiation. Altogether, this thesis demonstrates that concurrent Zrsr2 mutation and Tet2 loss are sufficient to drive MDS development in mice. In aged animals, disease features appeared with a higher penetrance and one mouse demonstrated progression to sAML, highlighting the impact of aging on disease evolution. Mechanistically, these alterations lead to dysregulated gene expression, aberrant mRNA splicing, and disrupted DNA methylation affecting key biological pathways. Notably, the MAPK14 kinase appears as the most affected target by aberrant splicing. This work provides novel insights into the pathogenesis of the spliceosome/epigenetic MDS subgroup and establishes a robust preclinical model to investigate disease mechanisms and evaluate emerging therapeutic strategies.