| ObjectiveAs a key member of LEF/TCF family, LEF1plays critical roles in regulating cellproliferation, differenciation and death. Previous studies have identified the aberrantactivation of lymphoid enhancer-binding factor-1(LEF1) in chronic lymphocyticleukemia (CLL). Recent investigation showed that LEF1could enhance cell survivalby inhibiting the necroptotic pathway in CLL cells. Ethacrynic acid (EA) has beenfound to be cytotoxic to CLL cells by targeting LEF1. The current research aims toidentify the prognostic effect of LEF1on CLL patiens, investigate the mechanism ofEA killing CLL cells, and find out whether EA could enhance the cytotoxicity ofchemotherapeutic agents to CLL cells.MethodsWe determined LEF1mRNA and protein expression levels by realtime PCR andwestern blot, respectively. SiRNAs were transferred to primary CLL cells to suppressthe expression of LEF1and its target gene. MTT was used to evaluate the viability ofCLL cells. The primary CLL cells were treated with EA, and the expression level ofLEF1and its target gene were examined after treatment using realtime PCR andwestern blot. Chromatin immunoprecipitation (ChIP) assay was performed toexamine the effect of EA on the function of LEF1and investigate the target of EA in CLL cells.Results:LEF1was significantly up-regulated in both MBL and CLL patients comparedwith normal B cells (P<0.0001). High expression of LEF1is related to the unmutatedstatus of IGHV in CLL patients (P=0.0002). Treatment-free survival (TFS) andoverall survival (OS) were much longer in CLL patients with low LEF1expressionthan in those with high LEF1levels (TFS: P=0.0356; OS: P=0.003). In CLL patientswith p53abnormalities, high expression of LEF1is related to inferior OS (P=0.0066).EA induced both apoptosis and necroptosis in primary CLL cells in vitro. EA inhibitsthe recruitment of LEF1to DNA promoters and restores the expression ofcylindromatosis (CYLD) in CLL cells. Inhibition of CYLD expression could protectCLL cells from the cytotoxicity of EA. Furthermore, the CLL cell viabilitiesdecreased signficantly when fludarabine/cyclophosphamide was added with EA.ConclusionsLEF1expression is aberrantly up-regulated in MBL and CLL cells. LEF1is anadverse prognostic factor for CLL patients. EA induced both apoptosis andnecroptosis in CLL cells. The dysfunction of LEF1and restoration of CYLDexpression contribute to the cytotoxic effect of EA on CLL cells. EA also enhancedthe cytotoxicity of both fludarabine and cyclophosphamide against CLL cells in vitro. ObjectiveChronic lymphocytic leukemia (CLL) is characterized by the clonalaccumulation and expansion of morphologically mature B-lymphocytes in bonemarrow and secondary immune organs. Despite those new agents coming up in recentyears, CLL is still incurable. CLL is highly variable and heterogeneous in clinicalcourses and outcomes. Several independent prognostic markers have been identifiedin CLL in recent years. The mutation status of immunoglobulin heavy-chainvariable-region (IGHV) gene is one of the most stable prognostic factors for CLL.The ummutated status of IGHV predicts unfavorable outcome of CLL patients.Cylindromatosis (CYLD) is recently identified as a tumor suppressor. As andeubiquitinase, CYLD regulates a variety of tumor-related signaling pathways bydeubiquitinasing target proteins. CYLD plays a regulatory part in cell survival anddeath. In the present study, we investigated the prognostic effect of CYLD on CLLpatients by measuring the CYLD mRNA expression in125CLL patients.MethodWe determined the expression of CYLD mRNA in125CLL patients byquantitative real-time PCR analysis, and investgated the potential correlation amongCYLD mRNA expression and age, clinical stages, peripheral lymphocyte counts,immunophenotypic features, cytogenetic features, IGHV mutation status, p53abnormalities, and NOTCH1mutations of CLL patients. We also assessed theprognostic effect of CYLD on treatment-free survival (TFS) and overall survival (OS)of CLLpatients.ResultsCYLD mRNA expression was dramatically suppressed in CLL cells compared tonormal CD19+B cells (P <0.0001). The mean expression level of CYLD in CLL cells is0.038±0.004, while the mean expression level of CYLD in normal CD19+B cells is0.155±0.019. The125CLL patients included90males and35females with a medianage of60years (range31-92years). According to Rai stage classification,83patietnswere in stage0-2,42in stage3-4. According to Binet stage classification,59patientswere in stage A,28in stage B, and38in stage C. Among82patients with mutatedIGHV, the mean expression level of CYLD was0.045±0.005. In43patients withummutated IGHV, the mean expression level of CYLD was merely0.025±0.004. Theresults demonstrated that CYLD expression is related to the mutation status of IGHV(P=0.0018). Among32patients with positive CD38expression, the mean expressionlevel of CYLD was0.027±0.005. In the other73patients with negative CD38expression, the mean expression level of CYLD was0.041±0.005. CYLD expressionis also associated to the expression of CD38(P=0.0499). No statistically significantcorrelation has been found among CYLD expression level and age, clinical stages,peripheral lymphocyte counts, cytogenetic features, p53abnormalities, and NOTCH1mutation (P>0.05). We found a trend for high CYLD expressers to show longer OSthan low CYLD expressers (P=0.0534). The median survival time for high CYLDexpressers was not reached, while the median survival time for low CYLD expresserswas150months. No correlation between CYLD expression and TFS has beenidentified. For CLL patients with mutated IGHV, we found that high CYLDexpression was associated with longer OS without reaching statistical significance(P=0.0547).ConclusionsCYLD expression is down-regulated in CLL cells compared to normal B cells.High CYLD expression was strongly associated with IGHV mutated status and CD38negativity. Low CYLD expression also predicts a trend towards inferior OS in CLLpatients. |
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