Katia Basso

Reverse engineering of regulatory networks in human B cells

Cellular phenotypes are determined by the differential activity of networks linking coregulated genes. Available methods for the reverse engineering of such networks from genome-wide expression profiles have been successful only in the analysis of lower eukaryotes with simple genomes. Using a new method called ARACNe (algorithm for the reconstruction of accurate cellular networks), we report the reconstruction of regulatory networks from expression profiles of human B cells. The results are suggestive a hierarchical, scale-free network, where a few highly interconnected genes (hubs) account for most of the interactions. Validation of the network against available data led to the identification of MYC as a major hub, which controls a network comprising known target genes as well as new ones, which were biochemically validated. The newly identified MYC targets include some major hubs. This approach can be generally useful for the analysis of normal and pathologic networks in mammalian cells.

BCL6 interacts with the transcription factor Miz-1 to suppress the cyclin-dependent kinase inhibitor p21 and cell cycle arrest in germinal center B cells

The BCL6 proto-oncogene encodes a transcriptional repressor that is required for germinal center formation and has been linked to lymphomagenesis. BCL6 functions by directly binding to specific DNA sequences and suppressing the transcription of target genes. Here we report an alternative mechanism by which BCL6 controls the transcription of genes lacking a BCL6 binding site and show that this mechanism was required for the prevention of tumor suppressor p53–independent cell cycle arrest in germinal center B cells. BCL6 interacted with the transcriptional activator Miz-1 and, via Miz-1, bound to the promoter and suppressed transcription of the cell cycle arrest gene CDKN1A. Through this mechanism, BCL6 may facilitate the proliferative expansion of germinal centers during the normal immune response and, when deregulated, the pathological expansion of B cell lymphomas.

Gene expression analysis of peripheral T cell lymphoma, unspecified, reveals distinct profiles and new potential therapeutic targets

Peripheral T cell lymphoma, unspecified (PTCL/U), the most common form of PTCL, displays heterogeneous morphology and phenotype, poor response to treatment, and poor prognosis. We demonstrate that PTCL/U shows a gene expression profile clearly distinct from that of normal T cells. Comparison with the profiles of purified T cell subpopulations (CD4+, CD8+, resting [HLA-DR-], and activated [HLA-DR+]) reveals that PTCLs/U are most closely related to activated peripheral T lymphocytes, either CD4+ or CD8+. Interestingly, the global gene expression profile cannot be surrogated by routine CD4/CD8 immunohistochemistry. When compared with normal T cells, PTCLs/U display deregulation of functional programs often involved in tumorigenesis (e.g., apoptosis, proliferation, cell adhesion, and matrix remodeling). Products of deregulated genes can be detected in PTCLs/U by immunohistochemistry with an ectopic, paraphysiologic, or stromal location. PTCLs/U aberrantly express, among others, PDGFRalpha, a tyrosine-kinase receptor, whose deregulation is often related to a malignant phenotype. Notably, both phosphorylation of PDGFRalpha and sensitivity of cultured PTCL cells to imatinib (as well as to an inhibitor of histone deacetylase) were found. These results, which might be extended to other more rare PTCL categories, provide insight into tumor pathogenesis and clinical management of PTCL/U.

Roles of BCL6 in normal and transformed germinal center B cells

Summary:  BCL6 is a transcriptional repressor required in mature B cells during the germinal center (GC) reaction. Multiple mechanisms act coordinately to timely modulate BCL6 expression at transcriptional and post‐transcriptional levels. BCL6 prevents premature activation and differentiation of GC B cells and provides an environment tolerant of the DNA breaks associated with immunoglobulin gene remodeling mechanisms involved in the production of high‐affinity antibodies of different isotypes. The critical functions exerted by BCL6 during normal B‐cell development can be hijacked by the malignant transformation process. Indeed, BCL6 is targeted by genetic aberrations and acts as an oncogene in GC‐derived lymphomas. The aberrations affecting BCL6 interfere with the multiple levels of regulation that grant a fine tuning of BCL6 expression and activity in physiologic conditions. This review summarizes the current knowledge on BCL6 function and its role in lymphomagenesis.

Simple diagnostic assay for hairy cell leukaemia by immunocytochemical detection of annexin A1 (ANXA1)

A marker capable of distinguishing with certainty between hairy cell leukaemia and other B-cell malignant disease would be of great diagnostic value. Through gene expression profiling, annexin A1 (ANXA1) has been identified as a gene that is upregulated in hairy cell leukaemia. We did immunostaining of 500 B-cell tumours with a specific anti-ANXA1 monoclonal antibody and showed that ANXA1 protein expression is specific to hairy cell leukaemia. Immunocytochemical detection of ANXA1 represents a simple, inexpensive, highly sensitive and specific (100%) assay for diagnosis of hairy cell leukaemia. This assay will be especially useful in distinguishing hairy cell leukaemia from splenic lymphoma with villous lymphocytes and variant hairy cell leukaemia, both of which usually respond poorly to treatments that are effective in hairy cell leukaemia.

A human B-cell interactome identifies MYB and FOXM1 as master regulators of proliferation in germinal centers

Assembly of a transcriptional and post‐translational molecular interaction network in B cells, the human B‐cell interactome (HBCI), reveals a hierarchical, transcriptional control module, where MYB and FOXM1 act as synergistic master regulators of proliferation in the germinal center (GC). Eighty percent of genes jointly regulated by these transcription factors are activated in the GC, including those encoding proteins in a complex regulating DNA pre‐replication, replication, and mitosis. These results indicate that the HBCI analysis can be used for the identification of determinants of major human cell phenotypes and provides a paradigm of general applicability to normal and pathologic tissues.

A systems biology approach to prediction of oncogenes and molecular perturbation targets in B-cell lymphomas

The computational identification of oncogenic lesions is still a key open problem in cancer biology. Although several methods have been proposed, they fail to model how such events are mediated by the network of molecular interactions in the cell. In this paper, we introduce a systems biology approach, based on the analysis of molecular interactions that become dysregulated in specific tumor phenotypes. Such a strategy provides important insights into tumorigenesis, effectively extending and complementing existing methods. Furthermore, we show that the same approach is highly effective in identifying the targets of molecular perturbations in a human cellular context, a task virtually unaddressed by existing computational methods. To identify interactions that are dysregulated in three distinct non‐Hodgkins lymphomas and in samples perturbed with CD40 ligand, we use the B‐cell interactome (BCI), a genome‐wide compendium of human B‐cell molecular interactions, in combination with a large set of microarray expression profiles. The method consistently ranked the known gene in the top 20 (0.3%), outperforming conventional approaches in 3 of 4 cases.

Promoter Hypermethylation of FANCF Disruption of Fanconi Anemia-BRCA Pathway in Cervical Cancer

Patients with advanced stage invasive cervical cancer (CC) exhibit highly complex genomic alterations and respond poorly to conventional treatment protocols. In our efforts to understand the molecular genetic basis of CC, we examined the role of Fanconi Anemia (FA)-BRCA pathway. Here, we show that FANCF gene is disrupted by either promoter hypermethylation and/or deregulated gene expression in a majority of CC. Inhibition of DNA methylation and histone deacetylases induces FANCF gene re-expression in CC cell lines. FANCF-deregulated CC cell lines also exhibit a chromosomal hypersensitivity phenotype after exposure to an alkylating agent, a characteristic of FA patients. We also show the involvement of BRCA1 gene by promoter hypermethylation or down-regulated expression in a small subset of CC patients. Thus, we have found inactivation of genes in the FA-BRCA pathway by epigenetic alterations in a high proportion of CC patients, suggesting a major role for this pathway in the development of cervical cancer. Thus, these results have important implications in understanding the molecular basis of CC tumorigenesis and clinical management in designing targeted experimental therapeutic protocols.

Genome-wide Identification of Post-translational Modulators of Transcription Factor Activity in Human B-Cells

The ability of a transcription factor (TF) to regulate its targets is modulated by a variety of genetic and epigenetic mechanisms, resulting in highly context-dependent regulatory networks. However, high-throughput methods for the identification of proteins that affect TF activity are still largely unavailable. Here we introduce an algorithm, modulator inference by network dynamics (MINDy), for the genome-wide identification of post-translational modulators of TF activity within a specific cellular context. When used to dissect the regulation of MYC activity in human B lymphocytes, the approach inferred novel modulators of MYC function, which act by distinct mechanisms, including protein turnover, transcription complex formation and selective enzyme recruitment. MINDy is generally applicable to study the post-translational modulation of mammalian TFs in any cellular context. As such it can be used to dissect context-specific signaling pathways and combinatorial transcriptional regulation.

tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma

Sequencing studies from several model systems have suggested that diverse and abundant small RNAs may be derived from tRNA, but the function of these molecules remains undefined. Here, we demonstrate that one such tRNA-derived fragment, cloned from human mature B cells and designated CU1276, in fact possesses the functional characteristics of a microRNA, including a DICER1-dependent biogenesis, physical association with Argonaute proteins, and the ability to repress mRNA transcripts in a sequence-specific manner. Expression of CU1276 is abundant in normal germinal center B cells but absent in germinal center-derived lymphomas, suggesting a role in the pathogenesis of this disease. Furthermore, CU1276 represses endogenous RPA1, an essential gene involved in many aspects of DNA dynamics, and consequently, expression of this tRNA-derived microRNA in a lymphoma cell line suppresses proliferation and modulates the molecular response to DNA damage. These results establish that functionally active microRNAs can be derived from tRNA, thus defining a class of genetic entities with potentially important biological roles.