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The use of next-generation sequencing instruments to study hematological malignancies generates a tremendous amount of sequencing data. This leads to a challenging bioinformatics problem to store, manage, and analyze terabytes of sequencing data, often generated from extremely different data sources. Our project is mainly focused on sequence analysis of human cancer genomes, in order to identify the genetic lesions underlying the development of tumors. However, the automated detection procedure of somatic mutations and the statistical testing procedure to identify genetic lesions are still an open problem. Therefore, we propose a computational procedure to handle large-scale sequencing data in order to detect exonic somatic mutations in a tumor sample. The proposed pipeline includes several steps based on open-source software and the R language: alignment, detection of mutations, annotation, functional classification, and visualization of results. We analyzed Illumina whole-exome sequencing data from five leukemic patients and five paired controls plus one colon cancer sample and paired control. The results were validated by Sanger sequencing.

BIM is a proapoptotic member of the Bcl-2 family. Here, we investigated the epigenetic status of the BIM locus in NPM/ALK+ anaplastic large cell lymphoma (ALCL) cell lines and in lymph node biopsies from NPM/ALK+ ALCL patients. We show that BIM is epigenetically silenced in cell lines and lymph node specimens and that treatment with the deacetylase inhibitor trichostatin A restores the histone acetylation, strongly upregulates BIM expression, and induces cell death. BIM silencing occurs through recruitment of MeCP2 and the SIN3a/histone deacetylase 1/2 (HDAC1/2) corepressor complex. This event requires BIM CpG methylation/demethylation with 5-azacytidine that leads to detachment of the MeCP2 corepressor complex and reacetylation of the histone tails. Treatment with the ALK inhibitor PF2341066 or with an inducible shRNA targeting NPM/ALK does not restore BIM locus reacetylation; however, enforced expression of NPM/ALK in an NPM/ALK-negative cell line significantly increases the methylation at the BIM locus. This study demonstrates that BIM is epigenetically silenced in NPM/ALK-positive cells through recruitment of the SIN3a/HDAC1/2 corepressor complex and that NPM/ALK is dispensable to maintain BIM epigenetic silencing but is able to act as an inducer of BIM methylation

The application of next-generation sequencing instruments generates a tremendous amount of sequencing data. This leads to a challenging bioinformatics problem to store, manage and analyze terabytes of sequencing data often generated from extremely different data-sources. Our project is mainly focused on the sequence analysis of human cancer genomes, in order to identify the genetic lesions underlying the development of tumors. However, the automated detection procedure of somatic mutations and a statistical based testing procedure to identify genetic lesions are still an open problem. Therefore, we propose a computational procedure to manage large scale sequencing data in order to detect exonic somatic mutations in a tumor sample. The proposed pipeline includes several steps based on open-source softwares and R language: alignment, detection of mutations, annotation, functional classification and visualization of results. We analyzed whole exome sequencing data from 3 leukemic patients and 3 paired controls plus 1 colon cancer sample and paired control. The results were validated by Sanger sequencing. © Springer-Verlag 2012.

BIM is a proapoptotic member of the Bcl-2 family. The BIM promoter can be the target of epigenetic silencing in various types of cancer. Here, we investigated the epigenetic status of BIM locus in NPM-ALK+ Anaplastic Large Cell Lymophoma (ALCL) cell lines and in lymph node biopsies form NPM-ALK+ ALCL patients. In all the cell lines tested, the BIM 5’UTR was densely methylated. Conversely, only very limited evidence of methylated lymphocytes from healthy donors. Treatment with the demethylating agent 5-azacytidine led to 5’UTR demethylation and BIM upregulation. By Chromatin Immunoprecipitation experiments, we also showed that BIM silencing occurs through recruitment of MeCP2 and of the SIN3a/Histone Deacetylase (HDAC) 1/2 co-repressor complex. BIM downregulation is associated with protection from apoptosis. Treatment with the deacetylase inhibitor Trichostatin-A restores the acetylation, upregulates BIM expression and induces massive cell death. Finally, the recruitment of the MeCP2/SIN3/HDAC1-2 silencing complex relies on BIM CpG methylation. Demethylation of BIM CpG island with 5-azacytidine leads to the detachment of the MeCP2 corepressor complex and to the reacetylation of the histone tails. Crizotinib is a selective ATP-competitive small-molecule inhibitor of both ALK tyrosine kinases and their oncogenic variants (e.g. mutations, fusion proteins) and c-MET/ HGF receptor which are implicated in the progression of several cancers. In this study we also demonstrated that NPM-ALK however, is not directly involved in determining the epigenetic status of Bim locus in ALCL NPM-ALK+. This study shows that, the epigenetic therapy such as demethylating agents and HDACi, in association with ALK inhibitor, Crizotinib, treatment could act synergistically, inducing massive apoptosis in ALCL by reactivating BIM expression and by NPM/ALK inactivation respectively.

Chronic myeloid leukemia (CML) is caused by the BCR-ABL hybrid gene. The molecular mechanisms leading from chronic phase (CP) to blast crisis (BC) are not understood. However, both the presence and the levels of BCR-ABL seem to be important for CML progression. BCR-ABL is under the transcriptional control of BCR promoter. Here we focused on the gene expression control of BCR and BCR-ABL upon myeloid differentiation in healthy donors (HDs), CP and BC patients. As previously reported, BCR-ABL is downregulated during myeloid maturation in CP patients. A similar pattern was detected for BCR (but not for ABL) in CP-CML and in HD, thus suggesting that the two genes may be under a similar transcriptional control. In BC this mechanism is similarly impaired for both BCR-ABL and BCR. These data indicate the presence of an 'in trans' deregulated transcription of both BCR and BCR-ABL promoters, associated with CML progression.

Glucocorticoids play a critical role in the therapy of lymphoid malignancies, including pediatric acute lymphoblastic leukemia (ALL), although the mechanisms underlying cellular resistance remain unclear. We report glucocorticoid resistance attributable to epigenetic silencing of the BIM gene in pediatric ALL biopsies and xenografts established in immune-deficient mice from direct patient explants as well as a therapeutic approach to reverse resistance in vivo. Glucocorticoid resistance in ALL xenografts was consistently associated with failure to up-regulate BIM expression after dexamethasone exposure despite confirmation of a functional glucocorticoid receptor. Although a comprehensive assessment of BIM CpG island methylation revealed no consistent changes, glucocorticoid resistance in xenografts and patient biopsies significantly correlated with decreased histone H3 acetylation. Moreover, the histone deacetylase inhibitor vorinostat relieved BIM repression and exerted synergistic antileukemic efficacy with dexamethasone in vitro and in vivo. These findings provide a novel therapeutic strategy to reverse glucocorticoid resistance and improve outcome for high-risk pediatric ALL. (Blood.2010;116(16):3013-3022)