Telomerase in hematological and other malignancies : therapeutic implications

Sammanfattning: The Telomere is a nucleoprotein complex consisting of TTAGGG repeated sequences covered by specialized binding proteins termed shelterins masking the end of linear chromosomes and thereby protecting the end of the chromosome from genomic instability. Telomeres become progressively shortened during cellular replication and acts as a mitotic clock to confer a limited lifespan to normal cells. Telomerase reverse transcriptase (TERT) is responsible for lengthening telomeric DNA. The enzyme is silent in most normal cells due to the transcriptional repression of the TERT gene encoding the telomerase catalytic component. Numerous studies have demonstrated that the induction of TERT and subsequent activation of telomerase is prerequisite to malignant transformation of human cells through a telomere-lengthening mechanism. Moreover, evidence has recently shown that TERT or telomerase possesses many other biological activities contributing to tumor development and progression, therefore targeting TET/telomerase has been suggested as a novel anti-cancer strategy. In the present project we studied telomere-lengthening-dependent and independent activities of TERT in malignant cells and explored therapeutic implications of TERT inhibition combined with other anti-cancer strategies in acute myeloid leukemia (AML) and gastric cancer. Paper I is focused on whether TERT inhibition and telomere dysfunction is involved in the anti- tumor effect of the DNA methyltransferase inhibitor 5-azacytidine (5-AZA). We demonstrated that 5-AZA induced DNA damage and telomere dysfunction in AML cell lines coupled with diminished TERT expression, telomere attrition and cellular apoptosis. The results suggests that 5-AZA-mediated TERT inhibition and telomere dysfunction contributes to its anti-cancer activity. Paper II was aimed to define the relationship between the oncogenic cyclooxygenase (COX2) and TERT and to evaluate the synergetic anti-cancer action of simultaneous COX2 and TERT inhibition. We found that the depletion of TERT led to elevated COX2 expression by activating p38. The COX2 inhibitor celecoxib or TERT inhibition alone was insufficient to affect cell viability, however, the combination synergistically killed cancer cells both in vitro and in vivo. Thus, the combined application of COX2 and telomerase inhibitors may be more efficient in cancer treatment. About 30% of AML patients exhibit somatic mutations of FMS-like tyrosine kinase 3 (FLT3), the majority of which carry internal tandem duplication (ITD) in the juxtamembrane. FLT3 inhibitors have been developed for AML treatment. In paper III, we determined whether FLT3-ITD regulated TERT expression and whether TERT affected the therapeutic efficacy of FLT3 inhibitors. We found that the FLT3 inhibitor PKC412 down-regulated TERT expression in a MYC-dependent manner, while ectopic expression of TERT attenuated killing efficacy of PKC412 in AML cells.Altogether, our findings demonstrated that the interplay between TERT and FLT3ITD plays important roles in AML carcinogenesis and that FLT3 inhibitors, when combined with TERT inhibition, are more efficient in the induction of AML cell apoptosis. In paper IV, we determined the effect of bortezomib on telomere homeostasis and its functional consequences. Bortezomib treatment inhibited TERT and telomerase expression, dysregulated shelterin proteins and shortened telomeres in AML and gastric cancer cell lines. The disrupted telomere structure triggered DNA damage response and cellular apoptosis. TERT overexpression significantly decreased DNA damage and telomere dysfunction and attenuated apoptosis mediated by bortezomib. Our findings collectively reveal a profound impact of bortezomib on telomere homeostasis/function, and down-regulation of TERT expression and telomere dysfunction induced by bortezomib thus both contributing to its cancer cell killing actions. In summary, the present results provide novel insights of the biological functions of TERT/telomerase in malignant cells. In addition, the finding that the combination of telomerase inhibition with other anti-cancer agents induced a robust and synergistic anti-tumor effect may have future important clinical implications.