trichostatin-a and Leukemia--Lymphocytic--Chronic--B-Cell

A commonly deleted region in chronic lymphocytic leukemia (CLL) is the 11q22-23 region, which encompasses the ATM gene. Evidence suggests that tumor suppressor genes other than ATM are likely to be involved in CLL with del(11q). A microRNA (miR) cluster including the miR-34b and miR-34c genes is located, among other genes, within the commonly deleted region (CDR) at 11q. Interestingly, these miRs are part of the TP53 network and have been shown to be epigenetically regulated. In this study, we investigated the expression and methylation status of these miRs in a well-characterized cohort of CLL, including cases with/without 11q-deletion. We show that the miR-34b/c promoter was aberrantly hypermethylated in a large proportion of CLL cases (48%, 25/52 cases). miR-34b/c expression correlated inversely to DNA methylation (P = 0.003), and presence of high H3K37me3 further suppressed expression regardless of methylation status. Furthermore, increased miR-34b/c methylation inversely correlated with the presence of 11q-deletion, indicating that methylation and del(11q) independently silence these miRs. Finally, 5-azacytidine and trichostatin A exposure synergistically increased the expression of miR-34b/c in CLL cells, and transfection of miR-34b or miR-34c into HG3 CLL cells significantly increased apoptosis. Altogether, our novel data suggest that miR-34b/c is a candidate tumor suppressor that is epigenetically silenced in CLL.

Topics: Adult; Aged; Aged, 80 and over; Antimetabolites, Antineoplastic; Apoptosis; Azacitidine; Cell Cycle Proteins; Chromosomes, Human, Pair 11; Cohort Studies; Female; Gene Deletion; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Methylation; MicroRNAs; Middle Aged; Promoter Regions, Genetic

The ontogenetic Wnt pathway shows almost no activity in adult tissues. In contrast, chronic lymphocytic leukemia (CLL) cells show constitutionally active Wnt signaling, which is associated with upregulated levels of pathway members such as Wnt3 and lymphoid enhancer-binding factor-1. Functionally, this results in increased resistance to apoptosis. We therefore assumed that targeting members of the pathway could reveal new therapeutic options for the treatment of CLL.. Screening a Wnt compound library with 75 Wnt modulators via ATP assay revealed Trichostatin A as an outstanding substance with strong viability decreasing effects on CLL cells and little effect on healthy peripheral blood mononuclear cells (PBMCs). Further survival analysis was performed via fluorescence-activated cell sorting analysis.. A maximum effect was achieved after 48 h with a wide therapeutic window in contrast to PBMCs (CLL cells: 0.253 µM, PBMCs: 145.22 µM). Trichostatin A induced caspases and acted via a dual mechanism to reveal histone and non-histone targets. Histone targets were displayed in deacetylation inhibition at DNA level, and non-histone targeting was demonstrated by elevated levels of Dickkopf-related protein 1 mRNA. Primary cells of patients with critical mutations such as TP53 or those who had already undergone extensive previous treatment responded well to the treatment. Moreover, the approved histone deacetylase (HDAC) inhibitor suberoylanilidehydroxamic acid (SAHA) was not as effective as Trichostatin A (Trichostatin A: 0.253 µM, SAHA: 7.88 µM). Combining Trichostatin A with established CLL drugs fludarabine or bendamustine showed an additive effect in vitro.. Taken together, Trichostatin A appears to act via a dual anti-HDAC/Wnt mechanism with a high selectivity and efficacy in CLL and therefore warrants further investigation.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 7; Cell Survival; Drug Resistance, Neoplasm; Drug Synergism; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Leukemia, Lymphocytic, Chronic, B-Cell; Leukocytes, Mononuclear; Protein Processing, Post-Translational; Sequence Deletion; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Vorinostat; Wnt Signaling Pathway

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are mature CD5(+) B-cell malignancies with different biological/clinical characteristics. We recently reported an association between different prognostic subgroups of CLL (i.e., IGHV mutated and unmutated) and genomic methylation pattern. However, the relationship between DNA methylation and prognostic markers, such as the proliferation gene expression signature, has not been investigated in MCL. We applied high-resolution methylation microarrays (27,578 CpG sites) to assess the global DNA methylation profiles in 20 MCL (10 each with high/low proliferation signature) and 30 CLL (15 poor-prognostic IGHV unmutated subset #1 and 15 good-prognostic IGHV mutated subset #4) samples. Notably, MCL and each CLL subset displayed distinct genomic methylation profiles. After unsupervised hierarchical clustering, 17/20 MCL cases formed a cluster separate from CLL, while CLL subsets #1 and #4 formed subclusters. Surprisingly, few differentially methylated genes (n = 6) were identified between high vs. low proliferation MCL. In contrast, distinct methylation profiles were demonstrated for MCL and CLL. Importantly, certain functional classes of genes were preferentially methylated in either disease. For instance, developmental genes, in particular homeobox transcription factor genes (e.g., HLXB9, HOXA13), were more highly methylated in MCL, whereas apoptosis-related genes were enriched among targets methylated in CLL (e.g., CYFIP2, NR4A1). Results were validated using pyrosequencing, RQ-PCR and reexpression of specific genes. In summary, the methylation profile of MCL was homogeneous and no correlation with the proliferation signature was observed. Compared to CLL, however, marked differences were discovered such as the preferential methylation of homeobox genes in MCL.

Topics: Apoptosis Regulatory Proteins; Azacitidine; Cell Division; Cell Line, Tumor; CpG Islands; Decitabine; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA, Neoplasm; Female; Gene Expression Regulation, Leukemic; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Genes, Immunoglobulin; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunoglobulin Heavy Chains; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Mantle-Cell; Male; Neoplasm Proteins; Sequence Analysis, DNA; Transcription Factors

E3 ubiquitin ligases catalyze the final step in the ubiquitylation cascade and therefore determine the specificity of this important cellular metabolic pathway. Although first thought to be constitutively active, increasing evidence demonstrates the existence of a wide variety of posttranslational modifications that regulate the activity of these enzymes. Here we show that upon induction of apoptosis the ubiquitin ligase Itch is processed by caspases both in vitro and ex vivo in cells from patients with chronic lymphocytic leukemia (CLL). The specific cleavage site was mapped to residue Asp240. Interestingly, cleavage of Itch by active caspases does not inhibit the catalytic activity of Itch, but results in the loss of an N-terminal Itch fragment that contains a negative regulatory region. Our data suggests that caspase-dependent Itch cleavage might be an important regulator of Itch at the endogenous level under both physiological and stressed conditions.

Topics: Apoptosis; Caspases; Catalysis; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Indoles; Leukemia, Lymphocytic, Chronic, B-Cell; Panobinostat; Repressor Proteins; Stress, Physiological; Substrate Specificity; Tumor Cells, Cultured; Ubiquitin-Protein Ligases

Recently, it has been found that inappropriate expression of microRNAs (miRNAs) is strongly associated with carcinogenesis. In this study, we demonstrated that the expression of miRNAs (miRs) -143 and -145, the levels of which were previously shown to be reduced in colon cancers and various kinds of established cancer cell lines, was also decreased in most of the B-cell malignancies examined, including chronic lymphocytic leukemias (CLL), B-cell lymphomas, Epstein-Barr virus (EBV)-transformed B-cell lines, and Burkitt lymphoma cell lines. All samples from 13 CLL patients and eight of nine B-cell lymphoma ones tested exhibited an extremely low expression of miRs-143 and -145. The expression levels of miRs-143 and -145 were consistently low in human Burkitt lymphoma cell lines and were inversely associated with the cell proliferation observed in the EBV-transformed B-cell lines. Moreover, the introduction of either precursor or mature miR-143 and -145 into Raji cells resulted in a significant growth inhibition that occurred in a dose-dependent manner and the target gene of miRNA-143 was determined to be ERK5, as previously reported in human colon cancer DLD-1 cells. Taken together, these findings suggest that miRs-143 and -145 may be useful as biomarkers that differentiate B-cell malignant cells from normal cells and contribute to carcinogenesis in B-cell malignancies by a newly defined mechanism.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Burkitt Lymphoma; Cell Line, Tumor; Cell Survival; Decitabine; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Leukemia, Lymphocytic, Chronic, B-Cell; Lymph Nodes; Lymphoma, B-Cell; MicroRNAs; RNA, Neoplasm; Tumor Cells, Cultured