Physiological MplW514L expression in hematopoietic stem cell causes an essential thrombocythemia and progressive myelofibrosis
Shujing Zhang, Jingjing Liu, Yuan Li, Yi Wang, Lingling Wang, Miaomiao Xu, Yanxia Li, Ge Dong, Shanshan Wang, Yanmei Li, Zhigang Cai, Baobing Zhao
Shujing Zhang, Jingjing Liu, Yuan Li, Yi Wang, Lingling Wang, Miaomiao Xu, Yanxia Li, Ge Dong, Shanshan Wang, Yanmei Li, Zhigang Cai, Baobing Zhao
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Research Article Hematology Oncology

Physiological MplW514L expression in hematopoietic stem cell causes an essential thrombocythemia and progressive myelofibrosis

Abstract

Typ515 (W515) mutations in the protein MPL are one of the key driver mutations promoting BCR-ABL-negative myeloproliferative neoplasms (MPNs), but, to our knowledge, their effects on hematopoietic stem cells (HSCs) and MPN-related hematological abnormalities have not been studied in physiological contexts. Here, we established a MplW514L knock-in mouse model, which largely mimics human MPLW515L mutation during hematopoiesis. The mutant mice developed an essential thrombocythemia–like (ET-like) MPN phenotype, displaying excess megakaryopoiesis and thrombocytosis and progressive myelofibrosis. Mechanistically, we observed that the MplW514L-conditioned HSC compartment had a unique disease-initiating capacity; however, it did not exhibit a obvious advantage of competitive repopulation over the WT control. Notably, single-cell analysis and flow cytometry profiles support that MplW514L expression led to a significant expansion of megakaryocyte-biased stem cell fate within the HSC pool. Finally, JAK2 inhibitor treatment phenotypically alleviated the ET signs but failed to eliminate the disease-initiating HSCs. These findings underscore the etiology of physiological expression of the MPLW515L mutation in HSCs and also provide a valuable in vivo model to evaluate potential therapeutic options for patients with MPLW515L-positive MPN.

Authors

Shujing Zhang, Jingjing Liu, Yuan Li, Yi Wang, Lingling Wang, Miaomiao Xu, Yanxia Li, Ge Dong, Shanshan Wang, Yanmei Li, Zhigang Cai, Baobing Zhao

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Figure 6

MplW514L mice exhibited megakaryocytic skewing in the myeloid progenitor compartment.

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MplW514L mice exhibited megakaryocytic skewing in the myeloid progenitor...
(A) Representative flow cytometric analysis of LK, CMP, GMP, MEP, MKP, Pre Meg-E, and Pre CFU-E in bone marrow of the indicated mice at 10 months. (B and C) Quantification of the proportions of indicated cell population in A. Data are presented as mean ± SD, with each dot representing one mouse. Data shown were representative of 2 independent experiments. P values were determined by 2-tailed unpaired Student’s t test. (D) Schematic summarizing effects of MplW514L mutation on hematopoietic progenitor compartment as in A. Black arrows represent normal differentiation, while red arrows indicated the enhanced megakaryocyte-biased lineage commitment. (E) Pairwise Spearman correlation of genes in MEPs from scRNA-seq data (Figure 4A), showing bias toward MkP-associated modules in MplW514L mice. (F) CellOracle-based evaluation of eigenvector centrality in MEP cells in E. Regulatory network analysis was performed using CellOracle to assess eigenvector centrality scores of genes in MEP cells across 4 groups, indicating the relative influence of individual genes within the inferred gene regulatory networks. (G) Pathway enrichment analysis of differentially expressed genes in MEP cells in E. (H) Statistical analysis of CD41+ cells from the cultured bone marrow cells from MplW514L mice and their WT littermates. Lineage-negative cells were isolated and cultured in the medium with or without thrombopoietin (TPO) for 3 days. Data were obtained from 3 independent experiments and presented as mean ± SD. P values were determined by 2-way ANOVA with Šidák’s multiple comparisons test. (I) Fibrosis-promoting evaluation of the indicated megakaryocyte progenitor (MkP) from the scRNA-seq datasets in Figure 4A, using the AddModuleScore function in Seurat. All gene sets are described in Supplemental Table 4. P values were determined by 1-way ANOVA with Tukey’s multiple comparisons test. **P < 0.01.