Inhibition of casein kinase 2 induces cell death in tyrosine kinase inhibitor resistant chronic myelogenous leukemia cells.

Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR-ABL oncogene. Despite the high performance of treatment with tyrosine kinase inhibitors (TKI), about 30% of patients develop resistance to the therapy. To improve the outcomes, identification of new targets of treatment is needed. Here, we explored the Casein Kinase 2 (CK2) as a potential target for CML therapy. Previously, we detected increased phosphorylation of HSP90ß Serine 226 in patients non-responding to TKIs imatinib and dasatinib. This site is known to be phosphorylated by CK2, which was also linked to CML resistance to imatinib. In the present work, we established six novel imatinib- and dasatinib-resistant CML cell lines, all of which had increased CK2 activation. A CK2 inhibitor, CX-4945, induced cell death of CML cells in both parental and resistant cell lines. In some cases, CK2 inhibition also potentiated the effects of TKI on the cell metabolic activity. No effects of CK2 inhibition were observed in normal mononuclear blood cells from healthy donors and BCR-ABL negative HL60 cell line. Our data indicate that CK2 kinase supports CML cell viability even in cells with different mechanisms of resistance to TKI, and thus represents a potential target for treatment.

Simplifying procedure for prediction of resistance risk in CML patients - Test of sensitivity to TKI ex vivo.

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL have dramatically improved chronic myeloid leukemia therapy. While imatinib remains to be the first line therapy, about 30% of patients develop resistance or intolerance to this drug and are recommended to switch to other TKIs. Nilotinib and dasatinib are currently implemented into the first line therapy and other inhibitors have already entered the clinical practice. This opens further questions on how to select the best TKI for each patient not only during the therapy but also at diagnosis. The individualized therapy concept requires a reliable establishment of prognosis and prediction of response to the available TKIs. We tested the ex vivo sensitivity of patient primary leukocytes to imatinib, nilotinib and dasatinib - two concentrations of each inhibitor for 48h incubation - and we evaluated the usefulness of such tests for the clinical practice. Besides reflecting the actual sensitivity to the therapy, our optimized simple tests were able to predict the outcome in 90/87% of patients, for the next 12/24months, respectively. According to these results, the presented ex vivo testing could help clinicians to select the appropriate drug for each patient at diagnosis and also at any time of the therapy.

Hsp90 - a potential prognostic marker in CML.

Heat shock proteins (Hsp) are important for the stability and function of cell proteins and thus for cell survival under physiological as well as stress conditions. Hsps were also reported to play an important role in tumorogenesis including leukemias. In this study we followed up Hsp70 and 90 protein levels in samples from patients with chronic myeloid leukemia (CML) to evaluate these Hsps with regard to their ability to characterize the disease status and disease prognosis. We analyzed 68 samples of total leukocytes of CML patients with different response to therapy with tyrosine kinase inhibitors. The results of Western blot analyses showed that the level of Hsp70 did not change in the course of the disease and did not correlate with response to therapy. In contrast, Hsp90 levels showed good correlation with the disease state. Patients with good response to therapy (major molecular response-MMR, molecular remission-CMR) had low expression levels of Hsp90, similar to those in healthy individuals. High Hsp90 levels were found in patients with resistance to therapy (hematological relapse-HR, accelerated phase or blast crisis), and in leukemic cell line K562. The results of the study suggested that not the kinetics but the particular level of Hsp90 at any time point since therapy start is of prognostic value: Hsp90 level above 0.27 significantly predicted poor response to TKI therapy (relapse, progression) and the level below 0.085 good response (MMR, CMR). In conclusion, Hsp90 level in total leukocytes could serve as a risk factor at diagnosis as well as during therapy and might help in clinical decision making especially in cases where BCR-ABL monitoring is of low predictive value. Our data suggest that high expression of HSP90 contributes to the aggressivity of the disease and should be considered as an important target for specialized CML therapy.

Expression of four major WT1 splicing variants in acute and chronic myeloid leukemia patients analyzed by newly developed four real-time RT PCRs.

Although the mechanism of action of leukemic oncogene Wilms' tumor gene 1 (WT1) remains unclear, WT1 has already been used in monitoring of patients with acute myeloid leukemia (AML) and it is being tested for immunotherapy. More detailed understanding of the role of WT1 in leukemia may improve its utilization. At least 36 isoforms may be produced. Four major variants denoted as -5/-KTS, -5/+KTS, +5/-KTS and +5/+KTS are produced by combining splicing of exon 5 and KTS sequence. In this study, we report applicability of newly developed real-time RT PCRs enabling for the first time full quantification of the four major WT1 splicing variants. Following careful optimization and testing of quantification reliability of four assays, we analyzed 34 samples of patients with AML and 12 samples of patients with chronic myeloid leukemia (CML) at the time of diagnosis. Analyses of five more CML patients provided insight into WT1 variants expression kinetics. We found predominance of +5/+KTS in both diagnoses. Comparison of WT1 variant expression in AML and CML patients' groups differing in response to therapy suggested possible importance of particular WT1 variant levels as markers of further disease course.

Expression patterns of microRNAs associated with CML phases and their disease related targets.

BACKGROUND: MicroRNAs are important regulators of transcription in hematopoiesis. Their expression deregulations were described in association with pathogenesis of some hematological malignancies. This study provides integrated microRNA expression profiling at different phases of chronic myeloid leukemia (CML) with the aim to identify microRNAs associated with CML pathogenesis. The functions of in silico filtered targets are in this report annotated and discussed in relation to CML pathogenesis. RESULTS: Using microarrays we identified differential expression profiles of 49 miRNAs in CML patients at diagnosis, in hematological relapse, therapy failure, blast crisis and major molecular response. The expression deregulation of miR-150, miR-20a, miR-17, miR-19a, miR-103, miR-144, miR-155, miR-181a, miR-221 and miR-222 in CML was confirmed by real-time quantitative PCR. In silico analyses identified targeted genes of these miRNAs encoding proteins that are involved in cell cycle and growth regulation as well as several key signaling pathways such as of mitogen activated kinase-like protein (MAPK), epidermal growth factor receptor (EGFR, ERBB), transforming growth factor beta (TGFB1) and tumor protein p53 that are all related to CML. Decreased levels of miR-150 were detected in patients at diagnosis, in blast crisis and 67% of hematological relapses and showed significant negative correlation with miR-150 proved target MYB and with BCR-ABL transcript level. CONCLUSIONS: This study uncovers microRNAs that are potentially involved in CML and the annotated functions of in silico filtered targets of selected miRNAs outline mechanisms whereby microRNAs may be involved in CML pathogenesis.

MicroRNA-451 in chronic myeloid leukemia: miR-451-BCR-ABL regulatory loop?

Chronic myeloid leukemia (CML) is caused by constituve activity of BCR-ABL tyrosine kinase. Despite of high efficiency of imatinib, selective BCR-ABL inhibitor, about 30% of patients develop resistance. Novel markers and targets for therapy are thus necessary. MicroRNAs are small intereference RNAs whose role in physiological and malignant hematopoiesis has been shown. This study is focused on miR-451 in CML. Following our observation of miR-451 downregulation in CML, we further show its relation to BCR-ABL activity. Our data together with current literature indicate a more complex relationship of miR-451 and BCR-ABL in CML.

High-resolution melt curve analysis: initial screening for mutations in BCR-ABL kinase domain.

Mutations in BCR-ABL kinase domain are associated with resistance to tyrosine kinase inhibitors in some patients with chronic myeloid leukemia. Therefore, mutation detection becomes essential in such patients. We aimed to apply high-resolution melt curve analysis (HRM) for a rapid screening prior to sequencing to select only mutation positive samples. One hundred and one samples with different mutations and mutational ratios were used for HRM testing. HRM results of 100/101 samples were concordant with sequencing data. We found HRM as a suitable and sensitive method for initial rapid screening of BCR-ABL KD mutations to sequence only positive samples.