Premature Termination Codon Mutations in Chronic Lymphocytic Leukemia (CLL): Identification of Potential Tumor Suppressors in CLL Including E-Cadherin.

Abstract

The acquisition of a premature termination codon (PTC) is a mutation that results in premature termination of transcription and degradation of the RNA by the non-sense mediated decay (NMD) pathway. This results in loss of expression of the allele, which harbors the PTC and is frequently the “second hit” in the loss of function of tumor suppressor genes. The NMD pathway can be blocked by protein translation inhibitors like emetine resulting in accumulation of abnormal truncated transcripts with PTC. By combining emetine-induced paralysis of the NMD pathway and microarray technology, a large number of genes were screened for the presence of PTCs in chronic lymphocytic leukemia (CLL) cells. Isolated normal peripheral blood B lymphocytes from two volunteers and five CLL samples were initially analyzed by emetine treatment and microarray. Cells were treated with and without emetine and the RNA was hybridized to Affymetrix 133A chip. Data was analyzed by calculating the fold up-regulation of signal intensity with emetine treatment for all the 23,000 genes on the chip. To take into account any non-specific up-regulation of signal intensity with emetine treatment, the fold induction observed with CLL samples was divided by fold induction obtained from the normal B cell samples to get a Nonsense enrichment index (NEI). Genes with the highest NEI in more than one CLL sample were selected for further study. From this cohort, individual genes were then sequenced and PTC mutations successfully identified in at least three genes; E-cadherin, plakophillin-4 (member of the armadillo family of proteins, a component of desmosomes) and TLE-2 (groucho homolog, a transcriptional repressor that represses transcription by TCF/LEF family members). Because E-cadherin has been described as a potential tumor suppressor whose expression is lost in CLL, we focused on this gene in a bigger cohort of 10 CLL patients. CLL cells were treated with emetine and total RNA was isolated. RNA from emetine treated and untreated cells was analyzed by RT-PCR. Amplified DNA fragments were isolated and sequenced. All 10 patients had an E-cadherin PTC: in 8/10 patients an E-cadherin allele had an exon 11 deletion resulting in premature termination codon and a smaller fragment on the RT-PCR assay. In 2/10 patients, a deletion was identified in exon 13 and 14 both of which were partially deleted resulting in RNA with PTC. Thus in CLL cells from 10 of 10 patients of all clinical stages, an inactivating PTC mutation is present in one of the alleles along with a wild type E-cadherin allele. E-cadherin is a transmembrane glycoprotein that is a component of the adherens junction and plays a role in cell-cell interaction, signaling via the beta-catenin pathway and is a known tumor suppressor. In addition, the plakophillin-4 and TLE-2 genes are negative regulators of the Wnt/catenin pathway and potential loss-of-function PTCs in those genes would further dysregulate this cascade. These data further support the role of E-cadherin as a potential tumor suppressor in CLL and suggest that our novel screen can be used to identify additional tumor suppressors in this disease.