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Overexpressed BCL6 (LAZ3) oncoprotein triggers apoptosis, delays S phase progression and associates with replication foci

One of the most frequent genetic abnormalities associated with non Hodgkin lymphoma is the structural alteration of the 5′ non coding/regulatory region of the BCL6 (LAZ3) protooncogene. BCL6 encodes a POZ/Zn finger protein, a structure similar to that of many Drosophila developmental regulators and to another protein involved in a human hematopoietic malignancy, PLZF. BCL6 is a sequence specific transcriptional repressor controlling germinal center formation and T cell dependent immune response. Although the expression of BCL6 negatively correlates with cellular proliferation in different cell types, the influence of BCL6 on cell growth and survival is currently unknown so that the way its deregulation may contribute to cancer remains elusive. To directly address this issue, we used a tetracycline-regulated system in human U2OS osteosarcoma cells and thus found that BCL6 mediates growth suppression associated with impaired Su2009phase progression and apoptosis. Interestingly, overexpressed BCL6 can colocalize with sites of ongoing DNA synthesis, suggesting that it may directly interfere with Su2009phase initiation and/or progression. In contrast, the isolated Zn finger region of BCL6, which binds BCL6 target sequence but lacks transcriptional repression activity, slows, but does not suppress, U2OS cell growth, is less efficient at delaying Su2009phase progression, and does not trigger apoptosis. Thus, for a large part, the effects of BCL6 overexpression on cell growth and survival depend on its ability to engage protein/protein interactions with itself and/or its transcriptional corepressors. That BCL6 restricts cell growth suggests that its deregulation upon structural alterations may alleviate negative controls on the cell cycle and cell survival.

Small deletions occur in highly conserved regions of the LAZ3/BCL6 major translocation cluster in one case of non-Hodgkin's lymphoma without 3q27 translocation

The LAZ3/BCL6 gene encoding a Zinc-finger nuclear protein is altered in Non-Hodgkins Lymphomas (NHLs) by translocations, mutations and/or deletions clustered in its 5′ non coding region, in a 3.3u2009Kbp EcoRI fragment which thus defines the Major Translocation Cluster (MTC). In the present study, we describe at the molecular level the deletions found in the MTC of two (NHL) cases using, (i) DNA obtained from a patient (GUI) with a monosomy 3 and three microdeletions of 101, 22, 25u2009bp in its unique untranslocated 3q27 allele; (ii) a cell line derived from a patient (VAL) carrying a t(3;4) (q27;p11) translocation and a 2.4u2009Kbp deletion in the untranslocated allele. As the MTC is recurrently subject to alterations, we have cloned and sequenced the murine equivalent of the human MTC and promoter region in an attempt to identify sequences well conserved in mammals that may be thus important for the LAZ3/BCL6 gene regulation. We show that the human and mouse 5′ upstream regions of the LAZ3/BCL6 gene although mainly intronic share a particularly high homology of 79% on the overall sequence. Strikingly, the small sequences which are deleted in patient (GUI) are highly conserved (81%, 100% and 92% respectively). Furthermore, they may play a role in the pathogenesis since proteins prepared from B cell lines and HeLa nuclear extracts bind to these sequences in gel retardation assays. Although a large part of this region is intronic, the high conservation of its sequence and the frequency of alterations in NHLs suggest that they are likely to be significant for the regulation of the LAZ3/BCL6 gene.

Dysregulation and overexpression of HMGA2 in myelofibrosis with myeloid metaplasia.

Among cytogenetic studies of patients affected with myelofibrosis with myeloid metaplasia (MMM), a rare chronic myeloproliferative disorder, we found several reports of structural abnormalities of the long arm of chromosome 12. Two MMM patients had a balanced translocation involving 12q: t(4;12)(q32;q15) and t(5;12)(p14;q15), respectively. FISH (fluorescence in situ hybridization) analysis showed that BAC (bacterial artificial chromosome) RP11‐366L20 overlaps the breakpoint in both cases. A gene, HMGA2, most of which is included in that BAC, thus was identified as a potential candidate. Using reserves transcriptase–polymerase chain reaction (RT‐PCR), we looked for expression of HMGA2 in blood mononuclear cells from these 2 patients and demonstrated a transcript in both. Moreover, we found the gene expressed in the hematopoietic cells of 10 of 10 additional patients bearing no 12q anomalies. HMGA2, not expressed in normal subjects, is implicated in benign solid tumors such as lipomas, leiomyomas, and other rare tumors of mesenchymal origin. We postulate that its dysregulation and overexpression in myeloid progenitors contribute also to the pathogenesis of MMM.

Nonrandom fusion of L-Plastin(LCP1) and LAZ3(BCL6) genes by t(3;13)(q27;q14) chromosome translocation in two cases of B-cell non-Hodgkin lymphoma

The LAZ3(BCL6) gene on chromosome band 3q27 is nonrandomly disrupted in B‐cell non‐Hodgkin lymphoma (B‐NHL) by chromosomal translocations clustered within a 3.3‐kb MTC (major translocation cluster) located between the two first noncoding exons. These translocations generally result in the expression of a chimeric mRNA transcript between the LAZ3 gene and sequences derived from the partner chromosome. Using RACE RT‐PCR, we previously demonstrated fusion of LAZ3 with the RhoH/TTF gene, a hemopoietic cell‐specific small GTPase involved in cytoskeleton organization, and with the BOB1/OBF1 gene, a B‐cell–specific coactivator of octamer‐binding transcription factors, following translocations t(3;4)(q27;p13) and t(3;11)(q27;q23), respectively. Here we report the identification of the L‐Plastin(LCP1) gene as a novel LAZ3 partner in chimeric transcripts resulting from a t(3;13)(q27;q14) translocation, in two cases of B‐cell lymphoma. As a consequence of the translocation, the 5′ regulatory region of each gene was exchanged, creating both LCP1‐LAZ3 and reciprocal LAZ3‐LCP1 fusion transcripts in one case, and only a LCP1‐LAZ3 fusion transcript in the other. The 13q14 chromosome region is frequently disrupted in various proliferative disorders, and the LCP1 gene defines a new breakpoint site in this region. This gene encodes an actin‐binding protein and is the second LAZ3 partner gene, with the RhoH/TTF gene, involved in actin cytoskeleton organization. Genes Chromosomes Cancer 26:97–105, 1999.

Detection of somatic quantitative genetic alterations by multiplex polymerase chain reaction for the prediction of outcome in diffuse large B-cell lymphomas.

Genomic gains and losses play a crucial role in the development of diffuse large B-cell lymphoma. This study shows that multiplex polymerase chain reaction of short fluorescent fragments is a reliable method for detecting somatic quantitative genetic alterations in diffuse large B-cell lymphoma. Background Genomic gains and losses play a crucial role in the development of diffuse large B-cell lymphomas. High resolution array comparative genomic hybridization provides a comprehensive view of these genomic imbalances but is not routinely applicable. We developed a polymerase chain reaction assay to provide information regarding gains or losses of relevant genes and prognosis in diffuse large B-cell lymphomas. Design and Methods Two polymerase chain reaction assays (multiplex polymerase chain reaction of short fluorescent fragments, QMPSF) were designed to detect gains or losses of c-REL, BCL6, SIM1, PTPRK, MYC, CDKN2A, MDM2, CDKN1B, TP53 and BCL2. Array comparative genomic hybridization was simultaneously performed to evaluate the sensitivity and predictive value of the QMPSF assay. The biological and clinical relevance of this assay were assessed. Results The predictive value of the QMPSF assay for detecting abnormal DNA copy numbers ranged between 88–97%, giving an overall concordance rate of 92% with comparative genomic hybridization results. In 77 cases of diffuse large B-cell lymphomas, gains of MYC, CDKN1B, c-REL and BCL2 were detected in 12%, 40%, 27% and 29%, respectively. TP53 and CDKN2A deletions were observed in 22% and 36% respectively. BCL2 and CDKN2A allelic status correlated with protein expression. TP53 mutations were associated with allelic deletions in 45% of cases. The prognostic value of a single QMPSF assay including TP53, MYC, CDKN2A, SIM1 and CDKN1B was predictive of the outcome independently of the germinal center B-cell like/non-germinal center B-cell like subtype or the International Prognostic Index. Conclusions QMPSF is a reliable and flexible method for detecting somatic quantitative genetic alterations in diffuse large B-cell lymphomas and could be integrated in future prognostic predictive models.

DNA Replication Progresses on the Periphery of Nuclear Aggregates Formed by the BCL6 Transcription Factor

ABSTRACT The BCL6 proto-oncogene, frequently alterated in non-Hodgkin lymphoma, encodes a POZ/zinc finger protein that localizes into discrete nuclear subdomains. Upon prolonged BCL6 overexpression in cells bearing an inducible BCL6 allele (UTA-L cells), these subdomains apparently coincide with sites of DNA synthesis. Here, we explore the relationship between BCL6 and replication by both electron and confocal laser scanning microscopy. First, by electron microscope analyses, we found that endogenous BCL6 is associated with replication foci. Moreover, we show that a relatively low expression level of BCL6 reached after a brief induction in UTA-L cells is sufficient to observe its targeting to mid, late, and at least certain early replication foci visualized by a pulse-labeling with bromodeoxyuridine (BrdU). In addition, when UTA-L cells are simultaneously induced for BCL6 expression and exposed to BrdU for a few hours just after the release from a block in mitosis, a nuclear diffuse BCL6 staining indicates cells in G1, while cells in S show a more punctate nuclear BCL6 distribution associated with replication foci. Finally, ultrastructural analyses in UTA-L cells exposed to BrdU for various times reveal that replication progresses just around, but not within, BCL6 subdomains. Thus, nascent DNA is localized near, but not colocalized with, BCL6 subdomains, suggesting that they play an architectural role influencing positioning and/or assembly of replication foci. Together with its previously function as transcription repressor recruiting a histone deacetylase complex, BCL6 may therefore contribute to link nuclear organization, replication, and chromatin-mediated regulation.

Cryptic and partial deletions of PRDM16 and RUNX1 without t(1;21)(p36;q22) and/or RUNX1-PRDM16 fusion in a case of progressive chronic myeloid leukemia: a complex chromosomal rearrangement of underestimated frequency in disease progression?

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the presence in leukemic stem cells of the Philadelphia chromosome (Ph) and the formation of the BCR‐ABL1 fusion. Untreated, the disease progresses to accelerate phase and blast crisis in which hematopoietic differentiation has become arrested. CML progression is frequently associated with cytogenetic evidence of clonal evolution, defined as additional chromosomal aberrations. We here report a CML resistant to tyrosine kinase inhibitors that rapidly progressed to blastic phase. At this time, array CGH performed on CD34+ cells revealed cryptic partial deletions of both PRDM16 and RUNX1 and duplication of the der(21) chromosome. These genomic rearrangements were confirmed by FISH with probes targeting the deletion on chromosome 21 (24 kb), and with BAC probes flanking the deletion on 1p36 (220 kb). However, no cryptic t(1;21)(p36;q22) and/or RUNX1‐PRDM16 were detected, suggesting that these deletions are the residual hallmarks of a more complex mechanism of chromosomal rearrangement, as indicated by the additional inversion of the region bounded by 1p36.32 and 1p36.12 breaks. At the molecular level, these abnormalities lead to the overexpression of the PR‐domain negative oncogenic isoform of PRDM16, associated with two deleted copies within the runt domain of C‐teminal aberrant RUNX1. These events are not detectable by conventional cytogenetic and molecular strategies, and may be of underestimated frequency in disease progression.

Quantification of JAK2V617F mutation by next-generation sequencing technology.

complete molecular responses suggests selective targeting of the malignant clone [2]. Earlier clinical studies have likewise demonstrated a beneficial effect in early stage B-CLL using nonpegylated interferon alpha 2a [3]. The diagnosis of concomitant CLL/MPN is a rare event and in a recent study analysis of prognostic markers for CLL demonstrated that in patients with a coexistent MPN, lymphoproliferative disorders follow a clinically indolent course [4,5]. Our patient has achieved a substantial hematologic, molecular and splenic response in his PV after 22 months on low dose Pegasys. Moreover, he has achieved a favorable reduction in the B-cell CLL clonal population and a change in Zap-70 status from indeterminate to negative. As far as we know, this is the first report of treatment of concomitant PV and chronic lymphocytic leukemia with Pegasys and the first to demonstrate a positive therapeutic effect on both disorders.