Expression of Gata1s Followed By Stag2 Deficiency Promotes Abnormal Megakaryocytopoiesis and Induces Lethal Myelofibrosis

Down syndrome (DS) children develop transient abnormal myeloproliferative disease (TAM) in the neonatal period, which usually resolves spontaneously. However, about 20% of the TAM children develop acute megakaryoblastic leukemia (DS-AMKL) by the age of 4 years. Mutations in GATA1, which generates a truncated isoform [GATA1-short (GATA1s)], are found in all cases of TAM and DS-AMKL, while mutations in cohesin-related genes are found in about half of the AMKL cases, suggesting that multistep acquisition of gene mutations contributes to the DS-AMKL pathogenesis. Recent study reported that GATA1s synergizes with other factors, such as chromosome 21-encoded miR-125b and mutations in STAG2, encoding a component of cohesion complex, in leukemogenesis. However, its mechanism remains largely unknown. In this study, we evaluated the pathological impact of the combination of Gata1s and the loss of Stag2 in mice.

We used mouse models in which Gata1 exon 2 was deleted to express Gata1s and X-linked Stag2 was floxed. By crossing these mice with Rosa26-Cre-ERT2 transgenic mice, we generated Gata1s; Stag2^f/f;Cre-ERT2 mice. We transplanted fetal liver cells from Cre-ERT2, Stag2^f/f;Cre-ERT2 or Stag2^f/y;Cre-ERT2, Gata1s;Cre-ERT2, and Gata1s;Stag2^f/f;Cre-ERT2 or Gata1s;Stag2^f/y;Cre-ERT2 mice into lethally irradiated recipient mice. After engraftment of the donor cells, we deleted Stag2 in donor cells by tamoxifen injection 2 weeks after transplantation. The recipient mice engrafted with fetal liver cells from Gata1s and Stag2 knock-out compound mice (GATA1s;Stag2^Δ/Δ) developed progressive pancytopenia and died around 90 to 180 days after Stag2 deletion. Bone marrow (BM) analysis of in GATA1s;Stag2^Δ/Δ mice 1 month after the deletion of Stag2 revealed significantly decreased BM cellularity. Lin^-Sca-1⁺c-Kit⁺ (LSK) hematopoietic stem and progenitor cells (HSPCs) were decreased. Within the myeloid progenitor (MP) compartment, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) were increased, while megakaryocyte/erythroid progenitors (MEPs) were decreased. In contrast, the numbers of megakaryocytic progenitors (MkPs) and premegakaryocyte-erythroid progenitors (PreMegEs) were increased. Erythropoiesis was severely impaired at the later stages of erythroid differentiation, while CD11b⁺ myeloid cells were increased in the BM of GATA1s;Stag2^Δ/Δ mice which were also increased in the Stag2^Δ/Δ mice, too. Hematoxylin-eosin (H&E) staining and silver staining of BM and spleen sections of GATA1s;Stag2^Δ/Δ mice 1 month after the deletion of Stag2 showed increased dysplastic megakaryocytes with marked fibrosis and osteosclerosis. BM fibrosis and osteosclerosis were progressive and became more prominent in moribund mice. All these phenotype were absent or mild in GATA1s and Stag2^Δ/Δ mice, indicating cooperative effects of GATA1s and the loss of Stag2. In competitive repopulation assays, GATA1s;Stag2^Δ/Δ hematopoietic cells were gradually outcompeted by the competitor cells, while Stag2^Δ/Δ cells showed a competitive advantage.

RNA sequencing analysis of of LSK HSPCs revealed enrichment of fibrosis signature genes (Psaila et al., Molecular Cell 2020) in GATA1s;Stag2^Δ/Δ HSPCs but not in single mutant mice. Among differentially expressed genes, Pdgfa, Ccl5, Tgfb1, key inflammatory mediators of myelofibrosis, were up-regulated in GATA1s;Stag2^Δ/Δ HSPCs. These results indicate a synergistic effect of Gata1s and Stag2 loss in promoting abnormal megakaryocytopoiesis which leads to myelofibrosis. Although the GATA1s;Stag2^Δ/Δ mice did not develop leukemia, DS-AMKL is associated with myelofibrosis at a high frequency, suggesting that our mouse model recapitulates a part of the DS-AMKL ontogeny and represents pre-AMKL state. Additional factors necessary for progression to leukemia are currently under investigation.

Ogawa:Novartis: Honoraria, Speakers Bureau; DaiichiSankyo: Speakers Bureau; Nanpu Hospital: Research Funding; Astrazeneca: Speakers Bureau; 2015-239547: Patents & Royalties; The Chemo-Sero-Therapeutic Research Institute: Speakers Bureau; Pfaizer: Speakers Bureau; Otsuka Pharmatheutical: Research Funding; Sysmex: Honoraria; MSD: Speakers Bureau; Clinical Research Support Center Kyushu: Research Funding; ASAHI Genomics: Current equity holder in publicly-traded company; Kirin/Chugai: Speakers Bureau; Esai Pharmatheutical: Consultancy; Chordia Threapeutics: Consultancy, Current equity holder in publicly-traded company, Research Funding; The Mitsubishi foundation: Honoraria; Astellas: Speakers Bureau; 2014-191287: Patents & Royalties; 62/187386 (US01): Patents & Royalties; 15/353395 (US03): Patents & Royalties; PCT/JP2014/062112 (WO01): Patents & Royalties; 2013-096582 (JP01): Patents & Royalties; 2013-526957 (JP02): Patents & Royalties. Iwama:Daiichi-Sankyo Pharmatheutical: Research Funding; Nissan Chemical Indistries: Research Funding.