Loss of RNA-binding protein CELF2 promotes acute leukemia development via FAT10-mTORC1.

RNA-binding proteins (RBPs) are critical regulators for RNA transcription and translation. As a key member of RBPs, ELAV-like family protein 2 (CELF2) has been shown to regulate RNA splicing and embryonic hematopoietic development and was frequently seen dysregulated in acute myeloid leukemia (AML). However, the functional role(s) of CELF2 in hematopoiesis and leukemogenesis has not been fully elucidated. In the current study, we showed that Celf2 deficiency in hematopoietic system led to enhanced HSCs self-renewal and differentiation toward myeloid cells in mice. Loss of Celf2 accelerated myeloid cell transformation and AML development in MLL-AF9-induced AML murine models. Gene expression profiling integrated with RNA immunoprecipitation sequencing (RIP-Seq), together with biochemical experiments revealed that CELF2 deficiency stabilizes FAT10 mRNA, promotes FAT10 translation, thereby increases AKT phosphorylation and mTORC1 signaling pathway activation. Notably, combination therapy with a mTORC1 inhibitor (Rapamycin) and a MA9/DOTL1 inhibitor (EPZ-5676) reduced the leukemia burden in MLL-AF9 mice lacking Celf2 in vivo. Our study elucidated a novel mechanism by which the CELF2/FAT10-AKT/mTORC1 axis regulates the proliferation of normal blood cells and the development of AML, thus providing potential therapeutic targets for myeloid leukemia suppression.

PHF6 maintains acute myeloid leukemia via regulating NF-κB signaling pathway.

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.

R274X-mutated Phf6 increased the self-renewal and skewed T cell differentiation of hematopoietic stem cells.

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.

Rapamycin Promotes the Expansion of Myeloid Cells by Increasing G-CSF Expression in Mesenchymal Stem Cells.

Rapamycin, also known as sirolimus, an inhibitor of mammalian target of rapamycin (mTOR), is a regulatory kinase responsible for multiple signal transduction pathways. Although rapamycin has been widely used in treating various hematologic diseases, the effects of rapamycin are still not fully understood. Here we found that both oral and intraperitoneal administration of rapamycin led to the expansion of myeloid lineage, while intraperitoneal administration of rapamycin impaired granulocyte differentiation in mice. Rapamycin induced bone marrow mesenchymal stem cells to produce more G-CSF in vitro and in vivo, and promoted the myeloid cells expansion. Our results thus demonstrated that intraperitoneal administration of rapamycin might promote the expansion of myeloid lineage while impair myeloid cell differentiation in vivo.

PHF6 and JAK3 mutations cooperate to drive T-cell acute lymphoblastic leukemia progression.

T-cell acute lymphoblastic leukemia (T-ALL) is a malignant hematologic disease caused by gene mutations in T-cell progenitors. As an important epigenetic regulator, PHF6 mutations frequently coexist with JAK3 mutations in T-ALL patients. However, the role(s) of PHF6 mutations in JAK3-driven leukemia remain unclear. Here, the cooperation between JAK3 activation and PHF6 inactivation is examined in leukemia patients and in mice models. We found that the average survival time is shorter in patients with JAK/STAT and PHF6 comutation than that in other patients, suggesting a potential role of PHF6 in leukemia progression. We subsequently found that Phf6 deficiency promotes JAK3M511I-induced T-ALL progression in mice by inhibiting the Bai1-Mdm2-P53 signaling pathway, which is independent of the JAK3/STAT5 signaling pathway. Furthermore, combination therapy with a JAK3 inhibitor (tofacitinib) and a MDM2 inhibitor (idasanutlin) reduces the Phf6 KO and JAK3M511I leukemia burden in vivo. Taken together, our study suggests that combined treatment with JAK3 and MDM2 inhibitors may potentially increase the drug benefit for T-ALL patients with PHF6 and JAK3 comutation.

Rheb1-Deficient Neutrophils Promote Hematopoietic Stem/Progenitor Cell Proliferation via Mesenchymal Stem Cells.

Myeloid cells have been identified as hematopoietic stem cell (HSC)-regulating cells. However, the mechanisms by which myeloid cells regulate the function of HSCs are not fully defined. Our previous study indicated that the HSCs are over-expanded in Vav1-Cre;Rheb1 f l/fl mice. Here, using in vivo and in vitro models, we found that Rheb1-deficient neutrophils remodeled the bone marrow environment and induced expansion of HSCs in vivo. Further studies showed that loss of Rheb1 impaired neutrophils' ability to secrete IL-6, led mesenchymal stem cells (MSCs) to produce more SCF, and promote HSC proliferation. We further found that IL-6 suppressed SCF mRNA expression in human MSCs. Interesting, the high level of IL-6 was also related with poor survival of chronic myeloid leukemia (CML) patients, and higher expression of IL-6 in CML cells is associated with the lower expression of SCF in MSCs in patients. Our studies suggested that blocking IL-6 signaling pathway might stimulate MSCs to secrete more SCF, and to support hematopoietic stem/progenitor cells proliferation.

Interleukin-1ß inhibits normal hematopoietic expansion and promotes acute myeloid leukemia progression via the bone marrow niche.

Enhanced interleukin-1ß (IL-1ß) signaling is a common event in patients with acute myeloid leukemia (AML). It was previously demonstrated that chronic IL-1ß exposure severely impaired hematopoietic stem cell (HSC) self-renewal capability in mice and promoted leukemia cell growth in primary AML cells. However, the role of IL-1ß in the murine bone marrow (BM) niche remains unclear. Here, we explored the role of IL-1ß in the BM niche in Il-1r1-/- mice, chronic IL-1ß exposure mice and mixed lineage leukemia-AF9 fusion gene (MLL-AF9)-induced AML mice models. We demonstrated that IL-1R1 deficiency did not affect the function of HSCs or niche cells under steady-state conditions or during transplantation. Chronic exposure to IL-1ß decreased the expansion of Il-1r1-/- hematopoietic cells in Il-1r1+/+ recipient mice. These results indicated that IL-1ß exposure impaired the ability of niche cells to support hematopoietic cells. Furthermore, we revealed that IL-1R1 deficiency in niche cells prolonged the survival of MLL-AF9-induced AML mice. The results of our study suggest that inhibition of the IL-1ß/IL-1R1 signaling pathway in the niche might be a non-cell-autonomous therapy strategy for AML.