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

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

Present status and perspectives in functional analysis of p53 in chronic lymphocytic leukemia

Aberrations of TP53 (mutations and/or deletions) are associated with a dismal prognosis in chronic lymphocytic leukemia (CLL). Complete loss of ATM is another mechanism of failed DNA damage response and also associated with poorer prognosis in CLL. However, p53 dysfunction may arise through alternative mechanisms unrelated to structural aberrations (deletion and/or mutation) of TP53 or ATM, and thus be undetectable by traditional DNA-directed approaches (fluorescence in situ hybridization [FISH], sequencing, karyotyping). In order to address the latter changes, and also to better understand the consequences of TP53/ATM aberrations, p53 functional assays have recently been developed. The purpose of dynamic assessment of p53 response in CLL is to carry out a comprehensive analysis of all mechanisms causing p53-deficient phenotype, including those unrelated to genomic aberrations of TP53 and ATM. The present review focuses on the current knowledge of p53 function assays in CLL, including important features such as technical issues, correlation with structural aberrations and clinical value.status: publishe

Present status and perspectives in functional analysis of p53 in chronic lymphocytic leukemia

Aberrations of TP53 (mutations and/or deletions) are associated with a dismal prognosis in chronic lymphocytic leukemia (CLL). Complete loss of ATM is another mechanism of failed DNA damage response and also associated with poorer prognosis in CLL. However, p53 dysfunction may arise through alternative mechanisms unrelated to structural aberrations (deletion and/or mutation) of TP53 or ATM, and thus be undetectable by traditional DNA-directed approaches (fluorescence in situ hybridization [FISH], sequencing, karyotyping). In order to address the latter changes, and also to better understand the consequences of TP53/ATM aberrations, p53 functional assays have recently been developed. The purpose of dynamic assessment of p53 response in CLL is to carry out a comprehensive analysis of all mechanisms causing p53-deficient phenotype, including those unrelated to genomic aberrations of TP53 and ATM. The present review focuses on the current knowledge of p53 function assays in CLL, including important features such as technical issues, correlation with structural aberrations and clinical value

Nucleoside analogs induce proteasomal down-regulation of p21 in chronic lymphocytic leukemia cell lines

Nucleoside analogs (NAs) represent an important class of anticancer agents that induce cell death after conversion to triphosphate derivatives. One of their most important mechanisms of action is the activation of p53, leading to apoptosis through the intrinsic pathway. Classically, the activation of p53 also induces p21 accumulation, which leads to cell cycle arrest at the G1/S transition. In previous work, we observed that 2-chloro-2'-deoxyadenosine (CdA), a NA with high activity in lymphoid disorders, including chronic lymphocytic leukemia (CLL), promotes the G1/S transition in the CLL cell line EHEB at cytotoxic concentrations. This finding led us to investigate the p21 response to NAs in these cells. We show here that CdA, but also fludarabine, gemcitabine, and cytarabine, strongly reduced the p21 protein level in EHEB cells as well as in JVM-2 cells, another CLL cell line. This p21 depletion occurred despite induction of p53 and increase of p21 mRNA and was prevented by proteasome inhibitors. Increase of proteasomal degradation caused by NAs appeared to be ubiquitin-independent. Also, NAs induced in these cells an increase of cyclin-dependent kinase (Cdk2) activity and a monoubiquitination of cell proliferating nuclear antigen (PCNA), two processes that are negatively regulated by p21. These changes were not observed with other p53 activators, like etoposide and nutlin-3a that increased the p21 protein level. In conclusion, our study reveals that NAs can induce an alternative pattern of cellular response in some cell models

Impaired up-regulation of polo-like kinase 2 in B-cell chronic lymphocytic leukaemia lymphocytes resistant to fludarabine and 2-chlorodeoxyadenosine: a potential marker of defective damage response

The functional evaluation of ataxia telangiectasia mutated (ATM) and p53 was recently developed in B-cell chronic lymphocytic leukaemia (B-CLL), a disease in which the response to DNA damage is frequently altered. We identified a novel biomarker of chemosensitivity based on the induction of DNA damage by the purine nucleoside analogues (PNA) fludarabine and 2-chlorodeoxyadenosine (CdA). Using genome-wide expression profiling, it was observed that, in chemosensitive samples, PNA predominantly increased the expression of p53-dependent genes, among which PLK2 was the most highly activated at early time points. Conversely, in chemoresistant samples, p53-dependent and PLK2 responses were abolished. Using a quantitative real time polymerase chain reaction, we confirmed that PNA dose- and time-dependently increased PLK2 expression in chemosensitive but not chemoresistant B-CLL samples. Analysis of a larger cohort of B-CLL patients showed that cytotoxicity induced by PNA correlated well with PLK2 mRNA induction. Interestingly, we observed that failure to up-regulate PLK2 following PNA and chemoresistance were not strictly correlated with structural alterations in the TP53 gene. In conclusion, we propose that testing PLK2 activation after a 24-h incubation with PNA could be used to investigate the functional integrity of DNA damage-response pathways in B-CLL cells, and predict clinical sensitivity to these drugs.status: publishe

Impaired up-regulation of polo-like kinase 2 in B-cell chronic lymphocytic leukaemia lymphocytes resistant to fludarabine and 2-chlorodeoxyadenosine : a potential marker of defective damage response

Summary The functional evaluation of ataxia telangiectasia mutated (ATM) and p53 was recently developed in B-cell chronic lymphocytic leukaemia (B-CLL), a disease in which the response to DNA damage is frequently altered. We identified a novel biomarker of chemosensitivity based on the induction of DNA damage by the purine nucleoside analogues (PNA) fludarabine and 2-chlorodeoxyadenosine (CdA). Using genome-wide expression profiling, it was observed that, in chemosensitive samples, PNA predominantly increased the expression of p53-dependent genes, among which PLK2 was the most highly activated at early time points. Conversely, in chemoresistant samples, p53-dependent and PLK2 responses were abolished. Using a quantitative real time polymerase chain reaction, we confirmed that PNA dose- and time-dependently increased PLK2 expression in chemosensitive but not chemoresistant B-CLL samples. Analysis of a larger cohort of B-CLL patients showed that cytotoxicity induced by PNA correlated well with PLK2 mRNA induction. Interestingly, we observed that failure to up-regulate PLK2 following PNA and chemoresistance were not strictly correlated with structural alterations in the TP53 gene. In conclusion, we propose that testing PLK2 activation after a 24-h incubation with PNA could be used to investigate the functional integrity of DNA damage-response pathways in B-CLL cells, and predict clinical sensitivity to these drugs