Activation of EVI1 transcription by the LEF1/ß-catenin complex with p53-alteration in myeloid blast crisis of chronic myeloid leukemia.

The presence of a BCR-ABL1 fusion gene is necessary for the pathogenesis of chronic myeloid leukemia (CML) through t(9;22)(q34;q11) translocation. Imatinib, an ABL tyrosine kinase inhibitor, is dramatically effective in CML patients; however, 30% of CML patients will need further treatment due to progression of CML to blast crisis (BC). Aberrant high expression of ecotropic viral integration site 1 (EVI1) is frequently observed in CML during myeloid-BC as a potent driver with a CML stem cell signature; however, the precise molecular mechanism of EVI1 transcriptional regulation during CML progression is poorly defined. Here, we demonstrate the transcriptional activity of EVI1 is dependent on activation of lymphoid enhancer-binding factor 1 (LEF1)/ß-catenin complex by BCR-ABL with loss of p53 function during CML-BC. The activation of ß-catenin is partly dependent on BCR-ABL expression through enhanced GSK3ß phosphorylation, and EVI1 expression is directly enhanced by the LEF1/ß-catenin complex bound to the EVI1 promoter region. Moreover, the loss of p53 expression is inversely correlated with high expression of EVI1 in CML leukemia cells with an aggressive phase of CML, and a portion of the activation mechanism of EVI1 expression is dependent on ß-catenin activation through GSK3ß phosphorylation by loss of p53. Therefore, we found that the EVI1 activation in CML-BC is dependent on LEF1/ß-catenin activation by BCR-ABL expression with loss of p53 function, representing a novel selective therapeutic approach targeting myeloid blast crisis progression.

Activation of NOTCH signaling impedes cell proliferation and survival in acute megakaryoblastic leukemia.

The genetic lesions that drive acute megakaryoblastic leukemia (AMKL) have not been fully elucidated. To search for genetic alterations in AMKL, we performed targeted deep sequencing in 34 AMKL patient samples and 8 AMKL cell lines and detected frequent genetic mutations in the NOTCH pathway in addition to previously reported alterations in GATA-1 and the JAK-STAT pathway. Pharmacological and genetic NOTCH activation, but not inhibition, significantly suppressed AMKL cell proliferation in both in vitro and in vivo assays employing a patient-derived xenograft model. These results suggest that NOTCH inactivation underlies AMKL leukemogenesis. and NOTCH activation holds the potential for therapeutic application in AMKL.

Highly efficient Runx1 enhancer eR1-mediated genetic engineering for fetal, child and adult hematopoietic stem cells.

A cis-regulatory genetic element which targets gene expression to stem cells, termed stem cell enhancer, serves as a molecular handle for stem cell-specific genetic engineering. Here we show the generation and characterization of a tamoxifen-inducible CreERT2 transgenic (Tg) mouse employing previously identified hematopoietic stem cell (HSC) enhancer for Runx1, eR1 (+24 m). Kinetic analysis of labeled cells after tamoxifen injection and transplantation assays revealed that eR1-driven CreERT2 activity marks dormant adult HSCs which slowly but steadily contribute to unperturbed hematopoiesis. Fetal and child HSCs that are uniformly or intermediately active were also efficiently targeted. Notably, a gene ablation at distinct developmental stages, enabled by this system, resulted in different phenotypes. Similarly, an oncogenic Kras induction at distinct ages caused different spectrums of malignant diseases. These results demonstrate that the eR1-CreERT2 Tg mouse serves as a powerful resource for the analyses of both normal and malignant HSCs at all developmental stages.

Super-enhancers for RUNX3 are required for cell proliferation in EBV-infected B cell lines.

Epstein-Barr virus nuclear antigens 2 (EBNA2) mediated super-enhancers, defined by in silico data, localize near genes associated with B cell transcription factors including RUNX3. However, the biological function of super-enhancer for RUNX3 gene (seR3) remains unclear. Here, we show that two seR3s, tandemly-located at 59- and 70-kb upstream of RUNX3 transcription start site, named seR3 -59h and seR3 -70h, are required for RUNX3 expression and cell proliferation in Epstein-Barr virus (EBV)-positive malignant B cells. A BET bromodomain inhibitor, JQ1, potently suppressed EBV-positive B cell growth through the reduction of RUNX3 and MYC expression. Excision of either or both seR3s by employing CRISPR/Cas9 system resulted in the decrease in RUNX3 expression and the subsequent suppression of cell proliferation and colony forming capability. The expression of MYC was also reduced when seR3s were deleted, probably due to the loss of trans effect of seR3s on the super-enhancers for MYC. These findings suggest that seR3s play a pivotal role in expression and biological function of both RUNX3 and MYC. seR3s would serve as a potential therapeutic target in EBV-related widespread tumors.