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Abstract: S160

Type: Oral Presentation

Session title: Correlating mutations with phenotype in MDS

Background

Splicing factor (SF) and epigenetic genes mutations are the most frequent genetic alterations found in patients with myelodysplastic syndromes (MDS). About 25% of patients present concurrent mutations in such pathways, suggesting a cooperative role in the pathogenic mechanism of MDS. Inactivating mutations in the minor spliceosome factor ZRSR2 and alterations in the epigenetic regulator TET2 are frequently found together and are associated with higher blast percentage in bone marrow (BM), higher risk of AML transformation, and shorter overall survival. The underlying molecular mechanism of disease remains, however, unclear.

Aims

To investigate the functional role of ZRSR2 and ZRSR2-TET2 mutations in hematopoiesis and MDS.

Methods

We generated a Zrsr2 mutant mouse model (Zrsr2^mut) by CRISPR/Cas9. A Zrsr2-Tet2 double mutant model (Zrsr2^mutTet2^mut) was generated by crossing Zrsr2^mut mice with a Tet2 knock-out model (Tet2^mut) previously established in our laboratory. Hematopoietic cell immunophenotype was analyzed by flow cytometry. Peripheral blood (PB) smears were stained for morphologic evaluation. To study hematopoietic stem and progenitor cells (HSPCs) functionality we conducted competitive BM transplantation and colony-forming unit (CFU) assays. Whole transcriptome sequencing of LSK cells was performed to study gene expression and to identify changes in splicing patterns.

Results

Zrsr2^mutTet2^mut mice immunophenotyping indicated a global alteration of hematopoiesis. In BM, Lin^- Sca-1⁺ c-kit⁺ (LSK) cell compartment was expanded, being CD48⁺CD150⁺ (HPC2) cells significantly increased and CD48^-CD150^- (MPP) significantly reduced. Myeloerythroid differentiation was disrupted, showing an increase in the myeloid pre-GM progenitors accompanied by a sharp decrease in GMP. Erythroid progenitors showed a similar behavior, having a significant increase of pre-CFU-E cells and a reduction of CFU-E. CFU assays confirmed a significant increase in myeloid progenitors and an increase of erythroid progenitors in double-mutant mice. Notably, a subset of Zrsr2^mutTet2^mut mice (30%) showed a more striking phenotype, including PB anemia and thrombocytopenia, leukocytosis, splenomegaly, and extramedullary hematopoiesis. Morphologically, peripheral blood cells showed dysplastic features including red blood cell abnormalities and giant platelets.

Transplantation experiments indicated an impairment of Zrsr2^mut cell capacity to repopulate the receptor BM. Conversely, transplantation of Zrsr2^mutTet2^mut cells revealed an enhanced engraftment capacity when compared to WT BM. Immunophenotypic analysis of Zrsr2^mutTet2^mut recipients recapitulated the erythroid phenotype observed in steady-state animals. One out of the five analyzed mice (20%) showed a more marked phenotype, similarly to what happened in non-transplanted mice.

RNA-seq analysis from Zrsr2^mutTet2^mut cells revealed 2952 differentially expressed genes and 43272 differential alternative splicing events in comparison to controls. Exon skipping (ES) was the most common alternative splicing (AS) event (71%) while intron retention (IR) was less common (7%).

Conclusion

Here we report that co-occurring mutations in Zrsr2-Tet2 disturb normal hematopoiesis and cause abnormal myeloid and erythroid differentiation. Thirty percent of these mice displayed some MDS-like abnormalities. Transcriptome analysis revealed an important dysregulation in gene expression accompanied by aberrant mRNA splicing. This study provides new insights into the biology of this MDS subtype and opens the possibility to more in-depth interrogate its molecular disease mechanism.

Keyword(s): Hematopoiesis, Mouse model, Myelodysplasia, Myeloid malignancies