The PHD finger protein 6 (PHF6) mutations frequently occurred in hematopoietic malignancies. Although the R274X mutation in PHF6 (PHF6R274X) is one of the most common mutations identified in T cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) patients, the specific role of PHF6R274X in hematopoiesis remains unexplored. Here, we engineered a knock-in mouse line with conditional expression of Phf6R274X-mutated protein in the hematopoietic system (Phf6R274X mouse). The Phf6R274X mice displayed an enlargement of hematopoietic stem cells (HSCs) compartment and increased proportion of T cells in bone marrow. More Phf6R274X T cells were in activated status than control. Moreover, Phf6R274X mutation led to enhanced self-renewal and biased T cells differentiation of HSCs as assessed by competitive transplantation assays. RNA-sequencing analysis confirmed that Phf6R274X mutation altered the expression of key genes involved in HSC self-renewal and T cell activation. Our study demonstrated that Phf6R274X plays a critical role in fine-tuning T cells and HSC homeostasis.

Transforming growth factor β1 (TGF-β1) plays a role in the maintenance of quiescent hematopoietic stem cells (HSCs) in vivo. We asked whether TGF-β1 controls the cell cycle status of HSCs in vitro to enhance the reconstitution activity. To examine the effect of TGF-β1 on the HSC function, we used an in vitro culture system in which single HSCs divide with the retention of their short- and long-term reconstitution ability. Extensive single-cell analyses showed that, regardless of its concentration, TGF-β1 slowed down the cell cycle progression of HSCs but consequently suppressed their self-renewal potential. Cycling HSCs were not able to go back to quiescence with TGF-β1. This study revealed a negative role of TGF-β1 in the regulation of the HSC number and reconstitution activity.

Acute myeloid leukemia (AML) is a major hematopoietic malignancy characterized by the accumulation of immature and abnormally differentiated myeloid cells in bone marrow. Here with in vivo and in vitro models, we demonstrate that the Plant homeodomain finger gene 6 (PHF6) plays an important role in apoptosis and proliferation in myeloid leukemia. Phf6 deficiency could delay the progression of RUNX1-ETO9a and MLL-AF9-induced AML in mice. PHF6 depletion inhibited the NF-κB signaling pathways by disrupting the PHF6-p50 complex and partially inhibiting the nuclear translocation of p50 to suppress the expression of BCL2. Treating PHF6 over-expressed myeloid leukemia cells with NF-κB inhibitor (BAY11-7082) significantly increased their apoptosis and decreased their proliferation. Taken together, in contrast to PHF6 as a tumor suppressor in T-ALL as reported, we found that PHF6 also plays a pro-oncogenic role in myeloid leukemia, and thus potentially to be a therapeutic target for treating myeloid leukemia patients.

Ras homolog enriched in brain (Rheb1) is a small GTPase and is known to be a direct activator of mTORC1. Dysregulation of Rheb1 has been shown to impair the cellular-energetic state and cell homeostasis. However, the role of Rheb1 in monocytes/macrophages differentiation and maturation is not clear. Here, we investigate the role of Rheb1 in mouse myelopoiesis using a Rheb1 conditional deletion murine model. We found that the absolute number of macrophages decreased in the bone marrow (BM) of Rheb1-deficient mice. Loss of Rheb1 inhibited the monocyte-to-macrophage differentiation process. Additionally, Rheb1 deletion reduced phagocytosis ability of macrophages by inhibiting the mTORC1 signaling pathway. Furthermore, 3BDO (an activator of mTORC1) rescued the phagocytosis ability of Rheb1-deficient macrophages. Thus, Rheb1 is critical for macrophage production and phagocytosis and executes these activities possibly via mTORC1-dependent pathway.

Normal hematopoiesis can be disrupted by the leukemic bone marrow microenvironment, which leads to cytopenia-associated symptoms including anemia, hemorrhage and infection. Thrombocytopenia is a major and sometimes fatal complication in patients with acute leukemia. However, the mechanisms underlying defective thrombopoiesis in leukemia have not been fully elucidated. In the steady state, platelets are continuously produced by megakaryocytes. Using an MLL-AF9-induced acute myeloid leukemia mouse model, we demonstrated a preserved number and proportion of megakaryocyte-primed hematopoietic stem cell subsets, but weakened megakaryocytic differentiation via both canonical and non-canonical routes. This primarily accounted for the dramatic reduction of megakaryocytic progenitors observed in acute myeloid leukemia bone marrow and a severe disruption of the maturation of megakaryocytes. Additionally, we discovered overproduction of interleukin-4 from bone marrow endothelial cells in acute myeloid leukemia and observed inhibitory effects of interleukin-4 throughout the process of megakaryopoiesis in vivo Furthermore, we observed that inhibition of interleukin-4 in combination with induction chemotherapy not only promoted recovery of platelet counts, but also prolonged the duration of remission in our acute myeloid leukemia mouse model. Our study elucidates a new link between interleukin-4 signaling and defective megakaryopoiesis in acute myeloid leukemia bone marrow, thereby offering a potential therapeutic target in acute myeloid leukemia.