Quantitative relationship between functionally active telomerase and major telomerase components (hTERT and hTR) in acute leukaemia cells

Functionally active telomerase is affected at various steps including transcriptional and post-transcriptional levels of major telomerase components (hTR and human telomerase reverse transcriptase (hTERT)). We therefore developed a rapid and sensitive method to quantify hTERT and its splicing variants as well as the hTR by a Taqman real-time reverse transcriptase–polymerase chain reaction to determine whether their altered expression may contribute to telomere attrition in vivo or not. Fresh leukaemia cells obtained from 38 consecutive patients were used in this study. The enzymatic level of telomerase activity measured by TRAP assay was generally associated with the copy numbers of full-length hTERT+α+β mRNA (P=0.0024), but did not correlate with hTR expression (P=0.6753). In spite of high copy numbers of full-length hTERT mRNA, telomerase activity was low in some cases correlating with low copy numbers of hTR, raising the possibility that alteration of the hTR : hTERT ratio may affect functionally active telomerase activity in vivo. The spliced nonactive hTERT mRNA tends to be lower in patients with high telomerase activity, suggesting that this epiphenomenon may play some role in telomerase regulation. An understanding of the complexities of telomerase gene regulation in biologically heterogeneous leukaemia cells may offer new therapeutic approaches to the treatment of acute leukaemia

HAPLOINSUFFICIENCY OF TIE2 IN MUTATED BLOOD CELLS SUPPRESS ANGIOGENESIS IN THE BONE MARROW AND INHIBIT PROGRESSION OF MDS

Introduction: Tie2 is a receptor tyrosine kinase and regulates angiogenesis and vascular quiescence. Given that Tie2 modulates microvascular density in cancer, we hypothesized that deletion of Tie2 in blood cells can inhibit progression of myelodysplastic syndrome (MDS). We attempted to understand the role of Tie2 in development of MDS by using an Ezh2/Tet2 double knock out (DKO) mouse model. Methods: We transplanted bone marrow (BM) cells isolated from Cre-ERT2 mice, Tie2flox/wt; Cre-ERT2 mice, Ezh2flox/flox; Tet2flox/flox; Cre-ERT2 mice, Ezh2flox/flox; Tet2flox/flox; Tie2flox/wt; Cre-ERT2 mice and Ezh2flox/flox; Tet2flox/flox; Tie2flox/flox; Cre-ERT2 mice into lethally-irradiated Ly5.1+ recipient mice. Ezh2, Tet2 and Tie2 genes were deleted by administration of tamoxifen one month post the transplantation. Results: We found that Ezh2−/−Tet2−/- DKO, Ezh2−/−Tet2−/- Tie2+/− (DKOTie2+/−) and Ezh2−/−Tet2−/- Tie2−/− TKO mice all developed MDS and MDS/MPN, showing anemia and dysplastic cells in the peripheral blood (PB) and the BM; however, DKOTie2+/− mice showed significantly longer survival than did DKO mice and TKO mice. While DKO mice showed deformed CD31+ endothelial cells and increased vascular density in the BM, DKOTie2+/− mice mitigated the altered vascular formation in the BM. RNA-sequencing revealed that DKOTie2+/− stem cells repressed expression of genes involved in interferon, cell cycles and angiogenesis, compared to DKO stem cells, suggesting that the haploinsufficiency of Tie2 impaired the property of MDS cells to drive angiogenesis in the BM, resulting in the delayed development of MDS. Conclusions: We are now working on the molecular mechanism of how the Tie2 gene in blood cells modulates the angiogenesis to drive the progression of MDS

The oncogenic role of the ETS transcription factors MEF and ERG.

Several ETS transcription factors, including MEF/ELF4 and ERG, can function as oncogenes and are overexpressed in human cancer. MEF cooperates in tumorigenesis in retroviral insertional mutagenesis-based mouse models of cancer and MEF is overexpressed in human lymphoma and ovarian cancer tissues via unknown mechanisms. ERG (Ets related gene) overexpression or increased activity has been found in various human cancers, including sarcomas, acute myeloid leukemia and prostate cancer, where the ERG gene is rearranged due to chromosomal translocations. We have been examining how MEF functions as an oncogene and recently showed that MEF can cooperate with H-Ras(G12V) and can inhibit both p53 and p16 expression thereby promoting transformation. In fact, in cells lacking p53, the absence of Mef abrogates H-Ras(G12V)-induced transformation of mouse embryonic fibroblasts, at least in part due to increased p16 expression. We discuss the known mechanisms by which the ETS transcription factors MEF and ERG contribute to the malignant transformation of cells

The mef/elf4 transcription factor fine tunes the DNA damage response.

The ATM kinase plays a critical role in initiating the DNA damage response that is triggered by genotoxic stresses capable of inducing DNA double-strand breaks. Here, we show that ELF4/MEF, a member of the ETS family of transcription factors, contributes to the persistence of \u3b3H2AX DNA damage foci and promotes the DNA damage response leading to the induction of apoptosis. Conversely, the absence of ELF4 promotes the faster repair of damaged DNA and more rapid disappearance of \u3b3H2AX foci in response to \u3b3-irradiation, leading to a radio-resistant phenotype despite normal ATM phosphorylation. Following \u3b3-irradiation, ATM phosphorylates ELF4, leading to its degradation; a mutant form of ELF4 that cannot be phosphorylated by ATM persists following \u3b3-irradiation, delaying the resolution of \u3b3H2AX foci and triggering an excessive DNA damage response. Thus, although ELF4 promotes the phosphorylation of H2AX by ATM, its activity must be dampened by ATM-dependent phosphorylation and degradation to avoid an excessive DNA damage response