Functional Analysis of the ATM-p53-p21 Pathway in the LRF CLL4 Trial: Blockade at the Level of p21 Is Associated with Short Response Duration

Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Liverpool, United Kingdom.
Clinical Cancer Research (Impact Factor: 8.72). 06/2012; 18(15):4191-200. DOI: 10.1158/1078-0432.CCR-11-2936
Source: PubMed


This study sought to establish whether functional analysis of the ATM-p53-p21 pathway adds to the information provided by currently available prognostic factors in patients with chronic lymphocytic leukemia (CLL) requiring frontline chemotherapy.
Cryopreserved blood mononuclear cells from 278 patients entering the LRF CLL4 trial comparing chlorambucil, fludarabine, and fludarabine plus cyclophosphamide were analyzed for ATM-p53-p21 pathway defects using an ex vivo functional assay that uses ionizing radiation to activate ATM and flow cytometry to measure upregulation of p53 and p21 proteins. Clinical endpoints were compared between groups of patients defined by their pathway status.
ATM-p53-p21 pathway defects of four different types (A, B, C, and D) were identified in 194 of 278 (70%) samples. The type A defect (high constitutive p53 expression combined with impaired p21 upregulation) and the type C defect (impaired p21 upregulation despite an intact p53 response) were each associated with short progression-free survival. The type A defect was associated with chemoresistance, whereas the type C defect was associated with early relapse. As expected, the type A defect was strongly associated with TP53 deletion/mutation. In contrast, the type C defect was not associated with any of the other prognostic factors examined, including TP53/ATM deletion, TP53 mutation, and IGHV mutational status. Detection of the type C defect added to the prognostic information provided by TP53/ATM deletion, TP53 mutation, and IGHV status.
Our findings implicate blockade of the ATM-p53-p21 pathway at the level of p21 as a hitherto unrecognized determinant of early disease recurrence following successful cytoreduction.

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    • "We have recently demonstrated that TP53 deletion/mutation in CLL cells is accompanied by under-expression of mRNAs encoding TP53 and other genes on chromosome 17p (Lin et al, 2013), most likely as a direct result of allelic loss. Since TP53 dysfunction can arise through mechanisms other than TP53 mutation/deletion (Pettitt et al, 2001; Jones et al, 2004; Romanov et al, 2005; Lin et al, 2012), we sought to establish whether TP53 mRNA is under-expressed in some cases of CLL with wild-type TP53 resulting in adverse outcome due to TP53 dysfunction. To address this question, TP53 mRNA levels were quantified in a large cohort of unstimulated CLL samples and correlated with other clinical and laboratory variables, including 13q14 deletion and levels of MIR15A, MIR16-1 and MIR125B. "
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    ABSTRACT: This study was conducted to investigate the possibility that TP53 mRNA is variably expressed in chronic lymphocytic leukaemia (CLL) and that under-expression is associated with TP53 dysfunction and adverse outcome. Although TP53 mRNA levels did indeed vary among the 104 CLL samples examined, this variability resulted primarily from over-expression of TP53 mRNA in 18 samples, all of which lacked TP53 deletion/mutation. These patients had higher lymphocyte counts and shorter overall and treatment-free survival times compared to cases with low TP53 mRNA expression and no TP53 deletion/mutation. Furthermore, TP53 mRNA levels did not correlate with levels of TP53 protein or its transcriptional target CDKN1A. We speculated that the adverse outcome associated with TP53 mRNA over-expression might reflect variation in levels of MIR15A and MIR16-1, which are encoded on chromosome 13q14 and target TP53 and some oncogenes including BCL2. In keeping with our hypothesis, 13q14 copy number and levels of MIR15A/MIR16-1 correlated positively with one another but negatively with levels of TP53 mRNA and BCL2 mRNA. Our findings support a model in which loss of MIR15A/MIR16-1 at chromosome 13q14 results in adverse outcome due to de-repression of oncogenes such as BCL2, and up-regulation of TP53 mRNA as a bystander effect.
    British Journal of Haematology 07/2014; 167(3). DOI:10.1111/bjh.13043 · 4.71 Impact Factor
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    • "(D) Results of the FACS-based assay are shown in a color format using MultiExperiment Viewer. Values lower than the previously established cut-off(Lin et al, 2012) for given parameters were assigned green. Values higher than previously established cut-off were assigned red. "

    British Journal of Haematology 06/2014; 167(4). DOI:10.1111/bjh.13006 · 4.71 Impact Factor
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    • "In this respect, a recent study has demonstrated that a strong induction of p21 by genotoxic agents was predictive of better prognosis in B-CLL [22]. Conversely, a lack of induction of p21 was observed in B-CLL patient samples showing a worse prognosis even in the presence of an integral activation of p53 [22]. Therefore, in order to functionally elucidate the role of p21 in mediating the anti-leukemic activity of DCA+Nutlin-3, we utilized siRNAs to attenuate p21 expression. "
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    ABSTRACT: The anti-leukemic activity of the mitochondria-targeting small molecule sodium dichloroacetate (DCA), used alone and in association with the small molecule inhibitor of the p53/MDM2 interaction Nutlin-3, was analyzed in primary B-chronic lymphocytic leukemia (B-CLL) samples (n=22), normal peripheral blood cells (n=10) and in p53wild-type EHEB, JVM-2, JVM-3 B lymphoblastoid cell lines. DCA exhibited a dose-dependent anti-leukemic activity in both primary B-CLL and B leukemic cell lines with a functional p53 status and showed a synergistic cytotoxic activity when used in combination with Nutlin-3. At the molecular level, DCA positively regulated p53 activity, as documented by post-transcriptional modifications of p53 protein and synergized with Nutlin-3 in increasing the expression of the p53-target genes MDM2, PUMA, TIGAR and in particular p21. The potential role of p21 in mediating the DCA+Nutlin-3 anti-leukemic activity was underscored in knocking-down experiments. Indeed, transfection of leukemic cells with p21 siRNAs significantly decreased the DCA+Nutlin-3-induced cytotoxicity. Taken together, our data emphasize that DCA is a molecule that merits to be further evaluated as a chemotherapeutic agent for B-CLL, likely in combination with other therapeutic compounds.
    Oncotarget 05/2014; 5(12). DOI:10.18632/oncotarget.2018 · 6.36 Impact Factor
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