Predicting resistance to chemotherapy in chronic lymphocytic leukemia - towards a role of PLK2 and miR-27 in oncogenesis

Abstract

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western world, but its etiology has not yet been completely elucidated. Purine nucleoside analogs (PNAs) are among the most effective drugs used in CLL treatment. However, resistance remains a major challenge in clinical management of CLL. The major objectives of this thesis were to better understand the mechanisms of resistance to PNAs in CLL as well as the pathogenesis of the disease, and to find new strategies to predict chemoresistance. In order to identify new mechanisms of chemoresistance, we used genome-wide expression profiling to compare the response to PNAs of chemosensitive and chemoresistant CLL samples. In chemosensitive cells, PNAs predominantly increased the expression of p53-dependent genes, among which PLK2 was the most highly up-regulated at an early time point. Conversely, in chemoresistant samples, p53-dependent and PLK2 responses were abolished. Using quantitative real-time PCR, we confirmed that PNAs significantly increased PLK2 mRNA expression in chemosensitive, but not in chemoresistant CLL samples. The analysis of a larger cohort of CLL patients showed that in vitro cytotoxicity induced by PNAs correlated well with PLK2 mRNA induction. Therefore, we proposed that testing PLK2 induction in response to PNAs might be a new strategy to investigate the integrity of the p53/DNA damage pathway in CLL and to predict clinical sensitivity to these drugs. This study was completed by a review of the current knowledge of p53 functional analysis in CLL. The next step was to investigate the potential role of PLK2 induction in the mechanism of action of PNAs. Unfortunately, we could not reliably detect Plk2 protein in CLL cells, either because of insufficient sensitivity and specificity of the antibodies used for its detection or because of its low expression level. Comparison with other cancer cell lines suggested that PLK2 mRNA and protein expression levels would be rather low in CLL cells. PLK2 has been described as a tumor suppressor gene in some B-cell malignancies. Hence, we analyzed whether PLK2 mRNA and protein levels might be reduced by microRNAs (miRNAs). Among all the miRNAs predicted to target PLK2, we focused on miR-27a/b because they are produced from the same primary transcripts as miR-23 and miR-24 family members, which are up-regulated in CLL patients with poor prognosis. We have shown that miR-27a/b can target the 3’-untranslated region of PLK2 mRNA and that they can reduce endogenous PLK2 mRNA levels. However, only a small subset of CLL cases showed elevated miR-27a/b levels, indicating that these miRNAs are most likely not responsible for the down-regulation of PLK2 in the majority of CLL cases. In addition, we have demonstrated that miR-27 expression correlated well with those of miR-23 and miR-24, suggesting that elevated miR-27 expression might also be associated with unfavorable prognostic factors in CLL. In the last part of this thesis, we investigated another predicted target of miR-27a/b, FBXW7, which might play a role in CLL biology by promoting Notch1 signaling activation. This study was performed in epithelial cancer cell lines for technical reasons. In this cell model, we showed that miR-27a/b target FBXW7 and indirectly regulate the abundance of the activated form of Notch1, but their effects on Notch1 target genes are still unclear. Further studies are required to determine whether these observations might be relevant for CLL biology.(SBIM 3) -- UCL, 201