Oncogenic NRAS mutations are frequently identified in human myeloid leukemias. In mice, expression of endogenous oncogenic Nras (Nras^G12D/+) in hematopoietic cells leads to expansion of myeloid progenitors, increased long-term reconstitution of bone marrow cells, and a chronic myeloproliferative neoplasm (MPN). However, acute expression of Nras^G12D/+ in a pure C57BL/6 background does not induce hyperactivated granulocyte macrophage colony-stimulating factor signaling or increased proliferation in myeloid progenitors. It is thus unclear how Nras^G12D/+ signaling promotes leukemogenesis. Here, we show that hematopoietic stem cells (HSCs) expressing Nras^G12D/+ serve as MPN-initiating cells. They undergo moderate hyperproliferation with increased self-renewal. The aberrant Nras^G12D/+ HSC function is associated with hyperactivation of ERK1/2 in HSCs. Conversely, downregulation of MEK/ERK by pharmacologic and genetic approaches attenuates the cycling of Nras^G12D/+ HSCs and prevents the expansion of Nras^G12D/+ HSCs and myeloid progenitors. Our data delineate critical mechanisms of oncogenic Nras signaling in HSC function and leukemogenesis. Key Points Nras^G12D/+ induces proliferation and increases self-renewal and myeloid differentiation bias in HSCs. ERK1/2 is constitutively hyperactivated in Nras^G12D/+ HSCs and downregulation of the MEK/ERK signaling attenuates Nras^G12D/+ HSC phenotypes.

Achievement of immunocompetent and therapeutic T lymphopoiesis from pluripotent stem cells (PSCs) is a central aim in T cell regenerative medicine. To date, preferentially reconstituting T lymphopoiesis in vivo from PSCs remains a practical challenge. Here we documented that synergistic and transient expression of Runx1 and Hoxa9 restricted in the time window of endothelial-to-hematopoietic transition and hematopoietic maturation stages in a PSC differentiation scheme (iR9-PSC) in vitro induced preferential generation of engraftable hematopoietic progenitors capable of homing to thymus and developing into mature T cells in primary and secondary immunodeficient recipients. Single-cell transcriptome and functional analyses illustrated the cellular trajectory of T lineage induction from PSCs, unveiling the T-lineage specification determined at as early as hemogenic endothelial cell stage and identifying the bona fide pre-thymic progenitors. The induced T cells distributed normally in central and peripheral lymphoid organs and exhibited abundant TCRαβ repertoire. The regenerative T lymphopoiesis restored immune surveillance in immunodeficient mice. Furthermore, gene-edited iR9-PSCs produced tumor-specific T cells in vivo that effectively eradicated tumor cells. This study provides insight into universal generation of functional and therapeutic T cells from the unlimited and editable PSC source.

Hematopoiesis is a progressive process collectively controlled by an elaborate network of transcription factors (TFs). Among these TFs, GATA2 has been implicated to be critical for regulating multiple steps of hematopoiesis in mouse models. However, whether similar function of GATA2 is conserved in human hematopoiesis, especially during early embryonic development stage, is largely unknown. To examine the role of GATA2 in human background, we generated homozygous GATA2 knockout human embryonic stem cells (GATA2 (-/-) hESCs) and analyzed their blood differentiation potential. Our results demonstrated that GATA2 (-/-) hESCs displayed attenuated generation of CD34(+)CD43(+) hematopoietic progenitor cells (HPCs), due to the impairment of endothelial to hematopoietic transition (EHT). Interestingly, GATA2 (-/-) hESCs retained the potential to generate erythroblasts and macrophages, but never granulocytes. We further identified that SPI1 downregulation was partially responsible for the defects of GATA2 (-/-) hESCs in generation of CD34(+)CD43(+) HPCs and granulocytes. Furthermore, we found that GATA2 (-/-) hESCs restored the granulocyte potential in the presence of Notch signaling. Our findings revealed the essential roles of GATA2 in EHT and granulocyte development through regulating SPI1, and uncovered a role of Notch signaling in granulocyte generation during hematopoiesis modeled by human ESCs.