Telomeres and telomerase and their functional applications in myeloproliferative neoplasms and acute myeloid leukemia

Sammanfattning: Myeloproliferative neoplasms (MPNs) are a group of diseases characterized by hyperproliferation in the myeloid lineages of the bone marrow, leading to increased levels of circulating mature blood cells from one or more lineages. MPNs consist of polycythemia vera, essential thrombocythemia and primary myelofibrosis. Several recurrent mutations are seen in MPNs, most of them resulting in an activation of the JAK/STAT signaling pathway. Telomeres are non-coding repetitive sequences of DNA located at the end of chromosomes. The telomeres are shortened with every cell division and when they become critically short, the cells enter a permanent growth arrest state called replicative senescence. When the telomeres are very short, the cells become genetically unstable and are more susceptible to genetic aberrations. There is accumulating evidence for a role for telomere dysregulation in the pathogenesis of MPNs and AML, and the overall aim of the studies included in this thesis was to better define this role. To clarify a potential dysregulation of telomeres and telomere associated proteins in MPNs, we studied the telomere length (TL), TERT expression and expression of telomere associated proteins in 81 patients with MPNs and 43 healthy controls. We found that patients with MPNs have shorter telomeres in their granulocytes compared to that of healthy controls, but also compared to the patients’ own lymphoid cells. There was no difference in TERT expression between patients and controls. The expression of two positive regulators of TL was lower, and the expression of two negative regulators of TL was higher in patients with MPNs. The dysregulation of telomere binding proteins may contribute to the telomere shortening seen in MPNs. Genetic variants in the TERT locus are implicated in susceptibility to cancer and other diseases. Recent reports also revealed an association between the SNP TERT rs2736100_CC genotype and the risk of developing MPNs in Caucasian populations. We genotyped patients and healthy controls from Sweden and China and found that the TERT rs2736100_C allele is associated with an increased risk of MPN development in both populations. The association of the C-allele with an increased risk of MPNs was only seen in male MPN patients, who generally have a worse outcome than female MPN patients. Moreover, the Chinese healthy population had significantly lower frequency of the TERT rs2736100_C allele compared to their Swedish counterpart, which may contribute to the lower MPN incidence seen in China compared to that in Europe. Patients with the TERT rs2736100_CC had the highest TERT expression, which may make them more susceptible to develop MPNs. The evolution of an acute promyelocytic leukemia (APL)-clone in a patient with a very late relapse was studied to distinguish between a relapse of the first APL, a secondary APL or a second de novo APL, and thereby guide future treatment decisions. Based on identical breakpoints of the PML-RARα gene, but differences in genetic aberrations and mutations in the FLT3-gene, we conclude that the patient most likely suffered a true relapse of her initial APL. We hypothesize that the PML-RARα- bearing pre-leukemic clone survived the initial chemotherapy and did not develop into an APL until seventeen years later, when the clone acquired another FLT3 mutation and other genetic aberrations. JAK2 inhibitors have proven effective in reducing symptoms and splenomegaly in patients with myelofibrosis, but they do not eliminate the disease initiating clones. A telomerase inhibitor is in clinical trials for MF with promising results, but with severe myelosuppression as a side-effect. We studied the effect of the JAK2 inhibitor LY2784544 in combination with the telomerase inhibitor GRN163L in a JAK2V617F –bearing erythroleukemia cell line. The combination had a larger effect on viability and number of cells than either of the drugs alone. Treatment with LY2784544 alone increased the fraction of HEL cells expressing the stem cell marker CD34, an effect that was partially mediated by an up-regulation of the transcription factor KLF4. Importantly, accumulation of CD34 positive cells was not seen after combined LY2784544/GRN163 treatment. This suggests that combining JAK2- and telomerase inhibition may have a therapeutic benefit, and that KLF4 may be a potential therapeutic target in MPNs. Furthermore, JAK2 inhibition reduced the telomerase activity, indicating a direct effect of JAK/STAT signaling on telomere regulation in MPNs.