Development of a Multi-Step Leukemogenesis Model of MLL-Rearranged Leukemia Using Humanized Mice

Mixed-lineage-leukemia (MLL) fusion oncogenes are intimately involved in acute leukemia and secondary therapy-related acute leukemia. To understand MLL-rearranged leukemia, several murine models for this disease have been established. However, the mouse leukemia derived from mouse hematopoietic stem cells (HSCs) may not be fully comparable with human leukemia. Here we developed a humanized mouse model for human leukemia by transplanting human cord blood-derived HSCs transduced with an MLL-AF10 oncogene into a supra-immunodeficient mouse strain, NOD/Shi-scid, IL-2Rγ−/− (NOG) mice. Injection of the MLL-AF10-transduced HSCs into the liver of NOG mice enhanced multilineage hematopoiesis, but did not induce leukemia. Because active mutations in ras genes are often found in MLL-related leukemia, we next transduced the gene for a constitutively active form of K-ras along with the MLL-AF10 oncogene. Eight weeks after transplantation, all the recipient mice had developed acute monoblastic leukemia (the M5 phenotype in French-American-British classification). We thus successfully established a human MLL-rearranged leukemia that was derived in vivo from human HSCs. In addition, since the enforced expression of the mutant K-ras alone was insufficient to induce leukemia, the present model may also be a useful experimental platform for the multi-step leukemogenesis model of human leukemia

Changes of T-lymphocyte subpopulation and differential expression pattern of the T-bet and GATA-3 genes in diffuse large B-cell lymphoma patients after chemotherapy

BACKGROUND AND OBJECTIVE: T cell-mediated immunity plays an important role in enhancing antitumor response.This study aimed to investigate the changes in the T-lymphocyte subpopulation and to characterize the differential expression pattern of corresponding regulatory genes in peripheral blood mononuclear cells (PBMCs) from diffuse large B cell lymphoma (DLBCL) patients before and after chemotherapy. METHODS: A total of 56 DLBCL patients were recruited for analysis of T-cell subset distribution in the peripheral blood using flow cytometry; serum interferon (IFN)-γ and interleukin (IL)-4 levels using enzyme-linked immunosorbent assays; and early growth response protein 1 (EGR-1), T-bet, GATA-binding protein 3 (GATA-3), and transforming growth factor (TGF)-β mRNA levels using quantitative reverse-transcription polymerase chain reaction. Twenty-six healthy subjects served as controls. RESULTS: The percentage of CD3(+)CD4(+)T lymphocytes in peripheral blood from DLBCL patients was significantly decreased, whereas the percentages of CD3(+)CD8(+)T and CD4(+)CD25(+)T cells were significantly increased compared to those in controls (p < 0.05). Serum levels of IFN-γ and IL-4 were also significantly lower in DLBCL patients than those in controls (p < 0.05), and the levels of EGR-1, T-bet, and GATA-3 mRNA in PBMCs were lower (2.69 ± 1.48, 9.43 ± 2.14, and 20.83 ± 9.05 fold, respectively) in DLBCL patients than those in controls. Furthermore, there was a positive association between the levels of EGR-1 and T-bet mRNA (p = 0.001). However, the level of TGF-β mRNA was significantly increased in DLBCL patients, which was inversely associated with the T-bet mRNA level (p = 0.008), but positively associated with the percentage of T regulatory cells in PBMCs (p = 0.011). After three cycles of chemotherapy, the distribution of T-lymphocyte subsets in DLBCL patients were changed, and the levels of EGR-1, T-bet, and GATA-3 mRNA were significantly increased (p < 0.05) compared to those before chemotherapy. CONCLUSIONS: These results demonstrate the changes in T-lymphocyte subpopulations and the altered expression 34 pattern of the corresponding regulatory genes in PBMCs from DLBCL patients after chemotherapy, which are associated with the response of patients to treatment. The preferential expression of the T-bet gene after chemotherapy was closely correlated with the increased expression of the EGR-1 gene and decreased expression of the TGF-β gene

AML1/ETO cooperates with HIF1α to promote leukemogenesis through DNMT3a transactivation

The mechanisms by which AML1/ETO (A/E) fusion protein induces leukemogenesis in acute myeloid leukemia (AML) without mutagenic events remain elusive. Here we show that interactions between A/E and hypoxia-inducible factor 1α (HIF1α) are sufficient to prime leukemia cells for subsequent aggressive growth. In agreement with this, HIF1α is highly expressed in A/Epositive AML patients and strongly predicts inferior outcomes, regardless of gene mutations. Co-expression of A/E and HIF1α in leukemia cells causes a higher cell proliferation rate in vitro and more serious leukemic status in mice. Mechanistically, A/E and HIF1α form a positive regulatory circuit and cooperate to transactivate DNMT3a gene leading to DNA hypermethylation. Pharmacological or genetic interventions in the A/E–HIF1α loop results in DNA hypomethylation, a re-expression of hypermethylated tumor-suppressor p15INK4b and the blockage of leukemia growth. Thus high HIF1α expression serves as a reliable marker, which identifies patients with a poor prognosis in an otherwise prognostically favorable AML group and represents an innovative therapeutic target in high-risk A/E-driven leukemi

AML1/ETO cooperates with HIF1α to promote leukemogenesis through DNMT3a transactivation.

The mechanisms by which AML1/ETO (A/E) fusion protein induces leukemogenesis in acute myeloid leukemia (AML) without mutagenic events remain elusive. Here we show that interactions between A/E and hypoxia-inducible factor 1α (HIF1α) are sufficient to prime leukemia cells for subsequent aggressive growth. In agreement with this, HIF1α is highly expressed in A/E-positive AML patients and strongly predicts inferior outcomes, regardless of gene mutations. Co-expression of A/E and HIF1α in leukemia cells causes a higher cell proliferation rate in vitro and more serious leukemic status in mice. Mechanistically, A/E and HIF1α form a positive regulatory circuit and cooperate to transactivate DNMT3a gene leading to DNA hypermethylation. Pharmacological or genetic interventions in the A/E-HIF1α loop results in DNA hypomethylation, a re-expression of hypermethylated tumor-suppressor p15(INK4b) and the blockage of leukemia growth. Thus high HIF1α expression serves as a reliable marker, which identifies patients with a poor prognosis in an otherwise prognostically favorable AML group and represents an innovative therapeutic target in high-risk A/E-driven leukemia